HLA typing:
Identification of a foreign HLA by recipient T cell can trigger an immune response against the allograft.
Types :
1- Serological typing: sera with antibodies to multiple Known HLA alleles are used, to this T lymphocytes of the recipient are added with complement and dye. It gives rapid results. Which leads to less cold ischemia times.
2- Molecular typing:Sequence-specific primer PCR: PCR HLA typing is highly specific with identification of specific allele with no cross-reactivity. HLA antibody screen
Preformed anti-HLA antibodies (DSA) may occur as a result of previous sensitization as in ; blood transfusion, and prior transplantation. they can cause positive crossmatch, so refuse this donor. DSA low titres can make ABMR. 1- Cytotoxic antibody screening
A group of donors cells . Recipient serum is mixed with donor lymphocyte along with complement and dye. If the serum contains antibody, it will bind cell surface antigen causing complement activation results in cell death and dye enter the cell. The percentage of cell death represents as PRA. false positive results can occur as in non-HLA antibodies, autoantibodies.false -ve if low AB titres. 2- Solid phase antibody screening: I. ELISA platform contains purified HLA molecules that bind to individual HLA antibody after the addition of recipient serum. II- single antigen beads: (SAB)
Beads coated with varible class I or II HLA antigens are added to fluorescent dye conjugated microbead to detect the HLA antibody in the patient serum. The fluorescence detected by flow cytometer or via the single antigen beads (SAB) Luminex platform. Positive beads represent PRA percent which ; represents a probability of a positive crossmatch. cPRA is based on unacceptable HLA antigens- to which the patient has been sensitized to. High c PRA carries high probability of positive crossmatch.
SAB can identify anti-HLA antibodies for all common and numerous rare alleles.
Solid phase assays can detect both complement and non-complement binding antibodies. They are so sensitive. But ; false negative results can occur in: Sera with high anti-HLA antibodies titer give negative results when tested neat, while react positive after 1:10 dilution. This called prozone phenomenon, Presence of IgM antibodies, Drugs like IVIG , Epitope sharing. Crossmatching (XM)
Presence of DSAs represented as a positive crossmatch was contraindicated to transplantation. it is insufficient to identify all relevant antibodies, and may exclude potential donor unnecessarily, to avoid this; solid phase test should be used together with crossmatch. 1- Complement dependent cytotoxicity crossmatch
Positive test as determined by number of destroyed cells, points to the presence of DSA. It is done for both B and T lymphocytes. In the presence of low titre DSA the sensitivity of the test is low. False negative can occur in low titre antibodies. While false positive can occur with autoantibodies, nonHLA antibodies. 2- Flow cytometry crossmatch:
It detects DSA whether complement fixing or non-complement fixing. Recipient serum is mixed with donor lymphocytes . With the use of flow-cytometry, B and T lymphocytes can be determined and their DSA individually determined. It is highly sensitive but not available widely. Virtual crossmatching (VXM): both donor HLA typing and solid phase screening are utilized together to predict true in vitro crossmatch results. Its result changes over time, so it should be done within 3 – 6 months. It does not identify Null HLA alleles
Immunological recipient risk :due to prior transplantation, recipient age, DSA, sex, original disease: High immunological risk:
High titres of DSA at time of transplantation . Treatment with desensitization protocols. Intermediate risk:
Low titre of DSA. Treatment : intensification of the immunosuppressive drugs and post transplant monitoring of DSA Standard risk: No DSA , for standard conventional immunosuppression therapy.
Ramy Elshahat
2 years ago
Human leukocyte antigen typing and crossmatch: A comprehensive review there are two main methods for HLA typing 1. Serology method 2. Molecular method. 1)Serology method In which Recipient’s lymphocytes (which carries unknown antigens) are mixed with antibodies to known HLA antigens, dye and complement in a tray. If the antigen reacted with the specific corresponding antibody, cell lysis occurs mediated by complement system. Dead cells are stained by the dye and identified by phase contrast microscope then HLA typing is diagnosed. Advantages: · rapid HLA typing which reflect in reducing cold ischemia time in cadaveric transplantation. · Able to diagnose “null” HLA alleles Disadvantages: · Unable to diagnose all HLA alleles. · Cross reactivity with CREG specially in using nonspecific broad antibodies. · Can’t be use in diagnosis epitopic analysis. That’s why most of HLA labs now using molecular methods. 2)Molecular method. HLA typing on nuclear level which translated by m-RNA to HLA antigens on cell surface which done by 1. Sequence specific primer PCR. -HLA related DNA sequences on chromosome 6 is diagnosed by specific manufactured primer. 2. Sequence specific oligo-nucleotide probes. -HLA related DNA sequencing diagnosed by oligo-nucleotide probes tagged by specific fluorescent dye. 3. Direct DNA sequencing. -direct diagnosis of the DNA sequencing. Advantages: · Specific · Can be used in diagnosis of epitopic matching specially in high resolution typing. Disadvantages: · Unable to identify new alleles · It takes time which means prolonged ischemia time Crossmatch for antibodies screening. 2 main methods for antibodies screening: Wet crossmatch which means actual mixing donor’s lymphocytes which carries HLA typing with recipient’s serum in which Ab is present. 1. Wet crossmatch includes (CDCXM and FCXM) 2. Solid phase crossmatch which means mixing recipient’s serum with beads carrying different HLA typing. 1)Wet crossmatch: A) Cytotoxic antibody screening: It’s one of the wet crossmatch modalities in which recipient’s serum (containing Ab) is mixed with donor’s lymphocytes (carrying HLA) along with complement and dye in a tray. Positive results are considered if cell death occurred and uptake of the dye. The result is express as percentage against both B cell (which carries both class I, II) and T cell (which carries only class I) Advantages: Specific in detection in complement fixing antibodies. Disadvantages: · less sensitive give you false negative due to low antibody titer. · False positive due to non-HLA antibodies, autoantibodies, some drugs like Rituximab (can be avoided by adding pronase) and nonspecific Ig M antibodies (which can be avoided by adding DTT). · Can’t identify antibody specificities and unacceptable antigens. b) Flow cytometry crossmatch. (FCXM) it’s wet crossmatch in which recipient serum (containing antibodies) mixed with donor’s lymphocytes then anti-anti HLA antibodies are added if some positive reactions are presents it will be diagnosed by florescent tagged antibodies. Results are diagnosed by flowcytometry and expressed by MCS. Advantages: -very sensitive can diagnose all antibodies even non-HLA antibodies and non-complement fixing Ab which may not carry harmful effect on the graft. Disadvantages: -non specific 2)Solid phase antibody screening. I. ELISA. It’s type of solid phase crossmatch in which purified HLA antigen mixed with recipient serum. Enzyme conjugated antibodies to IgG is added. Antigen antibody positive reaction is identified by optical density reading. II. mixed microbead platform/ single -antigen beads. it’s type of solid phase crossmatch in which latex beads coated with class I or II HLA antigens are mixed with recipient serum and used for antibody screening. Fluorescent dye is added and AB are detected via flowcytometry. Advantage: -it gives specific list of antibodies and unacceptable antigens. Disadvantage: -false positive cause by denaturated proteins -false negative cause by prozone phenomenon. Risk assessment in renal transplant patients: Depend on multiple factors like age of recipient, sex ,1ry kidney disease, HLA mismatch and DSA Specific definition and risk stratifications differs from transplant center to another The more HLA mismatch and the Prescence of DSA consider immunological risk and associated with increase risk of rejection, poor long term and short-term graft survival.
Wael Jebur
2 years ago
This article is displaying the different methods performed to outline the immunologic risk pertaining to kidney transplantation elucidating on the HLA typing and anti-HLA antibody detection methods .It started by showcasing a clinical vignette of SLE case with end stage kidney disease tested positive with CDC and negative with FCMX to both T and B cells. and SAB Luminex did not reveal any antibodies. He don’t have a positive history of blood transfusion. This contradicting result was stemmed from the presence of non HLA antibodies binding to the FC region. The HLA A, B and DR are the major determinants of Allo-sensitization, with DR antigen has the major impact than HLA A and B, One study suggested that the DR mismatch affect the the outcome in the first 6 months. And the HLA B effect is usually after 2 years.
6 antigens mismatch is reflecting 65% higher risk of rejection in comparison to 13% with 1 antigen mismatch.
Taboo mismatches:
Seven specific HLA mismatch combinations were associated with reduced renal allograft survival.
one third of waitlisted patients are having anti-HLA antibodies. They are usually stemming from previous transplantation, blood transfusion or pregnancy.
quantification and specification of DSAs are important to be stratify further management.
Notwithstanding negative cross match, even low DSAs titer might portend a deleterious consequences.
Methods to overcome the DSAs barrier entail desensitization, paired kidney donation and acceptable mismatches.
Jamila Elamouri
2 years ago
Human leukocyte antigen typing and crossmatch: A comprehensive review
Understanding crossmatch and HLA typing for kidney transplantation and applying it in clinical practice is an important step to achieving a good outcome
HLA typing:
Identification of a foreign HLA by the recipient T cell can trigger an immune response with activation of complement cascade directed against the allograft causing its rejection. Therefore; HLA typing is a key step in transplantation.
There are serologic and molecular typing.
1- Serological typing
Tray wells containing sera with antibodies to multiple Known HLA alleles are used, to this T lymphocytes of the recipient are added with complement and dye. If antigen on T-cell reacts to antibody in the well, complement activates and cell lysis occurs and dye enters the cells. coloured cells identified by polarized microscope. By elimination and comparison of positive wells, the HLA type is assigned. Advantage:
· It gives rapid results. Which leads to less cold ischemia times. · Differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression. This allele called null HLA are of less immunological significance. Disadvantages: 1- As the HLA is polymorphic, sera with antibody specificities that are capable of identifying the ever-growing number of HLA alleles are lacking. For example; the HLA-Cw, DQ and DP antigen that may have clinically significant effects on the transplantation, their serologic assays are scare. 2- Serologic methods do not exactly detect differences in HLA protein small amino acids that may be antigenic and trigger potent immunological responses.
2- Molecular typing: done by: · Sequence-specific primer polymerase chain reaction: in this method, recipient DNA is amplified in several wells. Each has a primer that is integral to specific HLA alleles. Amplification of the DNA occurs if meets the complementary DNA probe. Which then implanted into an agarose gel and undergoes electrophoresis, ultimately appearing as a band. By matching the primer that cause amplification to DNA sequences of several candidate alleles, the HLA typing is assigned. · Sequence specific oligonucleotide probes: Probes with oligonucleotide complementary to specific DNA segments of different alleles is mixed with amplified DNA. Then by fluorescent tags, unique HLA alleles are identified. HLA type is then assigned using available sequences. · Direct DNA sequencing: This method allows for a precise gene sequence. HLA typing is determined by comparison.
PCR HLA typing is highly specific with identification of specific allele with no cross-reactivity. Cross-reactivity is the identification of an allele similar to the allele of concern.
The new alleles that not on the HLA sequence databank will fail to be identified by this method.
The dissimilarity in the HLA antigen contributes to the alloimmune burden.
Recently, with the use of potent immunosuppression, the HLA mismatching in deceased donor kidney transplants become less significant. HLA antibody screen
Preformed anti-HLA antibodies (DSA) may occur as a result of previous sensitization during blood transfusion, pregnancy, and prior transplantation. they can cause positive crossmatch, leading to exclusion of the donor. DSA even in low titres can lead to antibody mediated rejection. Still transplantation in sensitized patients is possible by using strategies such as desensitization, paired exchange and acceptable mismatching. 1- Cytotoxic antibody screening
A group of donors cells that represent the population of potential deceased donors are selected. Recipient serum is mixed with donor lymphocyte along with complement and dye. If the serum contains antibody, it will bind cell surface antigen causing complement activation results in cell death and dye enter the cell.The percentage of cell death represents as PRA, which is a tool that can estimate the risk of recipient to have a positive crossmatch to a likely donor organ taken from a similar population. Limitations of cytotoxic crossmatch:
The cell panels which are commercially produced, may not represent the population truly, causing PRA percent numercally is not correlate with the type or amount of antibody. Significant false positive results can occur as a result to non-HLA antibodies, autoantibodies, and nonspecific IgM antibodies. False negative results can be if there is low antibody titres leading to miss of significant antibody.
Because; the well contains several antigens, the specificity of these antibodies cannot be determined as well the unacceptable antigens 2- Solid phase antibody screening:
Soluble or recombinant HLA molecules are used. I. Enzyme-linked immunosorbent assay platform:
ELISA platform contains purified HLA molecules that bind to individual HLA antibody after the addition of recipient serum, enzyme conjugated antibodies to IgG is then added to detect the presence of HLA antibody in the serum which is bound to the antigen. II- Microbead platform/single antigen beads: (SAB)
Beads coated with varible class I or II HLA antigens are added to fluorescent dye conjugated microbead to detect the HLA antibody in the patient serum. The fluorescence detected by flow cytometer or via the single antigen beads (SAB) Luminex platform. Positive beads represent PRA percent.
SAB specificities are compared with HLA frequencies in the donor population to determine the cPRA which estimate the possibility of a positive crossmatch/DSA to a randomly selected donor. PRA represents a probability of a positive crossmatch. cPRA is based on unacceptable HLA antigens- to which the patient has been sensitized to. It is calculated from HLA antigen frequencies among about twelve thousand donors and used for allocation of kidney offers. High c PRA carries high probability of positive crossmatch.
SAB can identify anti-HLA antibodies for all common and numerous rare alleles.
Solid phase assays can detect both complement and non-complement binding antibodies. They are so sensitive, can detect antibody with low titre which may exclude a potential donor.
As the list of HLA alleles is always growing, complete spectrum of the specific HLA antigens cannot be fully presented on the solid phase assays.
False negative results can occur in: 1- Sera with high anti-HLA antibodies titer give negative results when tested neat, while react positive after 1:10 dilution. This called prozone phenomenon. Which occurs due to C1 effect that compete with the antibodies binding to HLA molecules. 2- Presence of IgM antibodies. Resolved with DTT and serum dilution. 3- Drugs like IVIG 4- Epitope sharing. The HLA antigens on the beads share mutual antibody binding epitopes causing same antibodies bind to more than one bead. resulting in reduction in the MFI on a single bead. Crossmatching (XM) Presence of DSAs represented as a positive crossmatch was contraindicated to transplantation. it is insufficient to identify all relevant antibodies, and may exclude potential donor unnecessarily, to avoid this; solid phase test should be used together with crossmatch results to identify those that are immunologically relevant. 1- Complement dependent cytotoxicity crossmatch Positive test as determined by number of destroyed cells, points to the presence of DSA. It is done for both B and T lymphocytes. In the presence of low titre DSA the sensitivity of the test is low. That can be increased by increasing the incubation time, addition of AHG-enhanced method or use of additional wash steps. False negative can occur with low titre antibodies. While false positive can occur with autoantibodies, IgM/IgG HLA or nonHLA antibodies. 2- Flow cytometry crossmatch:
It detects DSA whether complement fixing or non-complement fixing. Recipient serum is mixed with donor lymphocytes and then tagged with fluorochrome conjugated anti-IgG antibody. With the use of flow-cytometry, B and T lymphocytes can be determined and their DSA individually determined. It is highly sensitive but not available widely, so its role in assessing the immunological risk is still unclear. Virtual crossmatching (VXM):
In this, both donor HLA typing and solid phase screening are utilized together to predict the true in vitro crossmatch results.
The data is used to predict the true in vitro crossmatch results by “mixing” identified antibody specificities of recipient serum with donor HLA antigens. It gives rapid result so can shorten the wait times. VXM allows physicians to consider donor organs that would not otherwise be available. Its result changes over time, therefore it should be done considering all available serum results including at least one recent within 3 – 6 months.
False positive results that may lead to wrong exclusion of potential donors, can occur in presence of:
1- Low titer DSA
2- Non-complement fixing antibodies
False negative results:
1- HLA donor antigens cannot be correctly represented, because they have been classed differently.
Disadvantages:
· Its result is not fully accurate and actual crossmatch must be done.
· It does not identify Null HLA alleles that can carry a significant risk if misidentified.
Defining recipient risk based on pre-transplant donor crossmatch and antibody screening: High immunological risk:
High titres of DSA at time of transplantation for mismatched donor HLA. Treatment with desensitization Intermediate risk Low titre of DSA, and historic DSA is not detectable. Treatment option; intensification of the immunosuppressive drugs and post –transplant monitoring of DSA Standard risk:
No evidence of DSA
Nasrin Esfandiar
3 years ago
· This article discuses about an interesting case of SLE. Recurrence rate of lupus in transplant kidneys is low. A 30 y/o man with SLE has been doing hemodialysis since 5 year ago, candidate for receiving kidney from a deceased donor 100 mismatch, with B cell CDC XM positive while FCXM and Luminex-SAB negative. To interpret this case, different method of HLA-typing and XM is necessary.
· HLA typing methods:
Serological: Donors lymphocytes are added to Trays well with special HLA Abs. If donor’s HLA is the same with lymphocytes will lysis by complement and dye enters cells. This is a quick method and so suitable for deceased donors.
Limitation: serologic assay is limited for HLA-CW and DQ, DP. Molecular method uses PCR-SSP and SSO probes and DNA sequencing.
HLA-DR, HLA-B and HLA-A are important in alloimmune burden, respectively. Higher number of HLA mismatches are associated with higher risk of graft failure. Six HLA mismatches causes 64%risk while for one HLA mismatch risk is 13%.
· Screening for HLA:
One third of in waiting list have anti-HLA Abs which if it is DSA, will be important.
· Two type methods:
· Cell based or cytotoxic method: There are thirty to forty potential donor’s lymphocytes in each panel. After adding recipient’s serum and washing, complement and dye is added. If patient has antibody, uptake of dye due to cell death will occur. Its grade is expressed as PRA and indicates risk for positive XM.
False positive: Non-HLA Abs, Auto antibodies, nonspecific IgM antibodies.
False negative: Non-complement fixing antibodies and low titer antibodies.
· Solid phase assays: ELISA and Microbeads platform or single-antigen beads.
· Luminex-SAB: In this method, there are different class I or II HLA antigens on a bead. If added recipient’s serum contains anti-HLA Abs, then by adding fluorescent dye, anti-HLA Abs can be detected by Flow cytometer (Flow PRA) or Luminex –SAB. This method can determine a complete list of specific anti-HLA antibodies. The HLA frequency in the donor population is compared to determine CPRA which is the best estimate of having a positive cross match with a random donor. It is calculated among twelve thousand kidney donors in the USA and indicates the proportion of donors who express these unacceptable antigens. Sensitized recipients would be higher on the list. SAB assays could be available in 3-4 h and virtual XM could be done to determine pre-transplant DSA.
· Limitation for SAB:
High sensitivity for detection of anti-HLA Abs even if they are insufficient to cause positive XM. Both complement fixing and non-complement fixing Abs are detected but non-HLA Abs won’t be detected by this method. Increasing list of HLA alleles is another limitation.
Prozone phenomenon: False negative results are seen would be positive results due to effect of C1 or IgM Abs. IgM Abs could be removed by adding DTT. Some serum proteins and IVIg and epitope sharing would cause false negative results.
· Cross match (XM): it is used to determine if recipient has antibodies against donor’s Ag.
1-T cell cytotoxic XM: 20% False positive and 4% False negative result.
CDC-XM could be done for both B cell and T cells.
If antibody is low titer, its sensitivity would be low which increases by adding AHG or high incubation time and more washing.
False positive CDC-XM: Autoantibodies, IgM HLA or non-HLA Abs.
False negative CDC-XM: Low titer antibodies
2-FCXM: This method detects DSA without correlation with complement –fixing. Recipient’s serum is mixed with donor’s lymphocytes then tagged with a flour-chrome conjugated with anti-IgG Abs. This method is more sensitive than CDC-XM.
3-VXM: This method predicts result of a real XM by using donors HLA typing and recipients Luminex-SAB. Luminex should be repeated after pregnancy, Tx or blood transfusion and every 3-6 months to interpret correctly, all previous Luminex results should be considered. False positive VXM: Low titer Abs or non-complement fixing Abs and null-alleles.
False negative results: Because of different classification for donors HLA. VXM should be confirmed by a real XM before transplantation.
· Risk defining:
High immunological risk: High titers of DSA are present at the time of TX which leads to hyper acute rejection.
Intermediate risk: There are low titer DSA at the time of TX but no historic DSA is detected and needs intensified immunosuppression and then immunological monitoring after TX.
Standard risk: There are no DSA at the time of TX.
ahmed saleeh
3 years ago
Renal transplantation is the best for ESKD patients .
Lupus nephritis can recur in quarter of tge pts after Tx .
* HLA typing methods :
Serological typing (and remember the null alleles)
Molecular methods
A.PCR using primers
B. Oligonucleotid probes
C. Direct DNA sequencing
* HLA antibody Screening
Sensitization due to Pregnancy , blood Tx , prior Tx
A. CDC
B . Solid phase screening using ELISA or SAB
N.B SAB : artefact is improved by dilution method , DDT
and also need to be mentioned the prozone phenomenon and The IVIG effect on SAB results
*Cross matching
A. CDC
B. Flow cytometry
*Virtual Cross match not 100% accurate can give false positive or false negative
Defining risk
A. HIGH Risk
B. Intermediate Risk
C. Standard Risk
Conclusion
Tx immunology is very important in successful Tx outcome
Manal Malik
3 years ago
Renal transplantation remains the best modalities of RRT for ESRD patients. HLA TYPING: HLA typing is essential step in renal transplant as reorganization of foreign HLA by recipient T lymphocyte will trigger an immune response HLA typing perform serologic or molecular typing methods Serological typing: Recipient lymphocytes are introduce into the tray wells contracting sera ,complement and dye, then the cell death take place as a result of complement activation .finally the cell death identify under phase contrast microscopy Advantage: Ø the results are available in short time Ø decrease cold ischemia time Ø able to identify null HLA alleles Disadvantage:
unable to identify the HLACw,DQ, and DP antigen that will affect the allograft outcome.
Molecular typing: there are different methods to apply molecular typing:
Sequence-specific primer polymerase chain reaction:
HLA typing is perform by matching the amplification product of subject DNA to the DNA sequences of other patients alleles.
Sequence specific oligonucleotide probes:
Subject amplified DNA is mixed with oligonucloetide probes. HLA alleles recognized by fluorescent tags. Precise nucloetide order identified via sequencing.
Direct DNA sequence determine the order of neuclotide in the gene interest
ADDVANGES OF MOLECULAR TYPING
1-Clearly identify difference in HLA Ag between donor and recipient
2-offer details to amino acid level ,epitopes in HLA typing
3-higly specificity detect specific alleles
Disadvantage: 1-not identify new alleles on currently in HLA sequence HLA-A, HLA-B,and HLA-DR are most important antigens in kidney transplantation.so it has significant impact on allograft survival HLA mismatching in deceased donor kidney transplantation is of lesser significance due to use of potent immunosuppression HLA ANTIBODY SCREENING:
Cytotoxic (cell-based) antibody screening
Recipient serum is mixed with cell donor lymphocytes along with complement and dye, In a tray complement activation result in cell death and uptake of the dye so the result is express as percentage panel reactive(PRA) Advantage: risk of positive cross matching of a given recipient Disadvantage:
PRA percent so no change in type or amount of antibody
not truely represent the population
false positive result due to non HLA antibodies, auto antibodies and non specific Abs
false negative result
can not identify some of antibodies specificities and unacceptable antigens
Solid phase antibody screening
use soluble or recombinant HLA molecule in this method instead of lymphocyte target: a) Enzyme-linked immunosorbent assay platform:
depend on using HLA glycoproteins fixed into microtiter well then add serum to wells and wash it to estimate DSA b) single antigen beads(SAB):
It is more sensitive than CDC and FC
the SAB Luminex assay can suspect to some artifact although can be overcome by delusion or add DTT
reduction in MFI can occur due to HLA antigens bounding
prevent hyperacute rejection
detect DSA
positive result is contraindicated to transplantation
identify specific recipient antibody to the donor
it has false negative and positive results
need solid phase antibody test to identify those that are immunological relevant
CROSSMATCHING (XM)
Complement-dependent cytotoxicity crossmatch
Ø positive test is considerable number of lymphocytes are destroyed after the incorporation of complement
Ø less sensitive than the other test
Ø false negative result and false positive result
Ø CDC -XM can be done for B &T lymphocytes
Flow cytometry crossmatch:
In this method recipient serum is mixed with donor lymphocytes and then tagged with a fluorochrome conjugated anti IgG antibody.
Advantage:
Ø More sensitive test
Disadvantage:
Ø Different methods used between different laboratories can result in a different results
Ø Assessing immunological risk still not clear. Virtual cross matching:
Both donor HLA typing and solid phase antibody screening are used together
The data is used to forecast the actual in vitro crossmatch results by “mixing” identified antibody specificities of recipient serum with donor HLA antigens
Advantage:
Decrease waiting time for sensitized patients
Disadvantage:
False -ve results
False +ve results
Not identify the HLA null alleles which will affect future transplant
Defining risk: High immunological risk:
High HLA DSA at time of transplantation
Lead to hyper acute rejection
Desensitization can reduce the risk of hyper acute rejection
Intermediate immunological risk:
Decrease titre of DSA at time of transplant
Transplant can be carry on but need intensified immunosuppression and immunological monitoring in the post transplant period
Standard immunological risk:
No HLA DSA
Immunological risk assessment pretransplant based on :
Immunological assessment for prerenal transplant is crucial for successful graft outcome
Detect DSA by molecular technique will prevent false exclusion of some donors
Some autoimmune disease and oral medication can cause false +ve crossmatching.
Ahmed Omran
3 years ago
HLA typing is an essential part of renal transplantation.
HLA typing include meth0ds : Serological
Recipient lymphocytes are mixed with sera with different known HLA alleles . HLA Cw ,DQ and DP are not recognized by that method. Molecular Sequence specific PCR: DNA from subject is amplified in several wells with primes which are complimentary to HLA alleles Sequence specific oligonucleotide Probes; certain order of nucleotides of gene of concern is determined. Direct DNA sequencing-: detects significant alleles and specific alleles without cross reactivity HLA NTIBODY SCREENING
Cytotoxic antibody screening
Recipient serum is mixed with 30-40 donor lymphocytes , dye and Complement . Results are shown as PRA Solid phase antibody screening
Using recombinant HLA molecules instead of lymphocytes ELISA platform Microbead platform
Estimating PRA ; more sensitive than ELISA. c PRA determines unacceptable antigens CROSS MATCHING
Complement dependent Cytotoxic cross match:It can be processed for B and T lymphocytes
Flow cytometry: recipient serum is added to donor lymphocytes and Fluorescence for tagging is used. DSA detected independent of compliment fixation .
Virtual cross match-:combination of Solid phase antibody screening and HLA Typing; predicting suitable donors. Immunological Risk Groups are high,intermediate and low groups according to DSA levels and history of sensitization.
MOHAMED Elnafadi
3 years ago
HLA TYPING :
HLA typing play essential role in renal transplantation, as recognition of foreign HLA by recipient T lymphocytes will stimulate an immune response. 1. Serology method
a tray containing sera with antibodies to a multitude of known HLA alleles is used recipient lymphocytes are introduced into the tray wells contacting sera, complement and dye. In tray wells where antibodies can bind to the antigens on the surface of lymphocytes;
complement is activated. results in complement pathways triggered resulting in cell death, ultimately allowing the dye to enter the cell. Tray wells with significant cell death are then identified under phase contrast microscopy.
This method also offers the ability to differentiate HLA alleles
Disadvantages he HLA-Cw, DQ, and DP antigen have clinically significant effects on the outcomes of allografts. However, serologic assays are scarce for these loci. 2. Molecular method ( Sequence-specific primer polymerase chain reaction )
extracted DNA from the subject is amplified in several wells. Each well has primers that are complementary to specific HLA alleles.DNA probes are complementary to the specific sequence of the HLA molecule, an amplification product is formed. This is then instilled into an agarose gel and undergoes electrophoresis where they appear as a band. ( equence specific oligonucleotide probes )
Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles. Unique HLA alleles are then identifiedusing fluorescent tags. ( Direct DNA sequencing)
determines the precise order of nucleotides in the gene of interest.
Using published HLA allele sequences, HLA type is subsequently assigned by comparison.
. HLA ANTIBODY SCREENING
methods used for HLA antibody screening Cytotoxic (cell-based) antibody screening
set of cell donors are randomly selected to be representative of a population. This should be representative of the population of potential deceased donors. Each panel consists of around 30 to 40 different donor lymphocytes.
false positive results can be produced due to nonHLA antibodies, autoantibodies and nonspecific IgM antibodies. false negative results complement dependent method requires higher antibody titres to be activated. Solid phase antibody screening
recombinant HLA molecules instead of lymphocytes targets – as lymphocytes present
both HLA as well as non-HLA molecule. Enzyme-linked immunosorbent assay platform
purified HLA molecules are applied toenzyme-linked immunosorbent assay (ELISA) platforms and will bind individually to HLA antibody after the addition of recipient serum nzyme conjugated antibodies to IgG (human) is then added to detect the presence of HLA antibody in the serum which is bound to the antigen. Detection is performed by optical density reading. Microbead platform/single-antigen beads
Pooled panel beads with several different class Ⅰ or Ⅱ HLA antigens on a bead
Microbead that is fluorescent dye conjugated detect the presence of HLA antibody in the serum which is bound to the antigen. solid phase antibody screening addresses detect both complement and non-complement binding simultaneously. More sensitive detecting antibody that is below the threshold associated with a positive crossmatch. CROSSMATCHING (XM) Complement-dependent cytotoxicity crossmatch
positive if a considerable number of lymphocytes are destroyed after the incorporation of complement when significant DSA has been bound to the cell surface.done for B and T lymphocytes. Sensitivity is limited. Flow cytometry crossmatch
detects DSA independent of complement fixation
detects the presence or lack of IgG DSA on donor lymphocytes.recipient serum is mixed with donor lymphocytes and then tagged with a fluorochromeconjugated anti-IgG antibody.
more sensitive Virtual crossmatch
both donor HLA typing and solid phase antibody screening are utilised together. It is not precisely a crossmatch in the sense of mixing serum and lymphocytes. The data is used to forecast the actual in vitro crossmatch results by “mixing” identified antibody specificities of recipient serum with donor HLA antigens.
VXM can lead to shorter wait times and improved outcomes for sensitised transplant recipients.
permits transplant physiciansto consider donor organs that would not otherwise be
available by means of a prospective crossmatch strategy,and thereby, allows to consider a potentially positive crossmatch a risk factor for donor selection.
Wee Leng Gan
3 years ago
Comprehensive review for HLA typing and crossmatch.
Proper HLA typing and crossmatch ensure good clinical outcome in renal transplant patient. HLA typing method
1. Serology method
2. Molecular method.
1) Serology method
Main concept :
Recipient blood lymphoctes containing antigen mix with control sera with antibodies of know HLA alleles , dye and complement in a tray. Complement activation through antigen antibody reaction resulted cell death which stained by the dye. The death cell is identified by phase contrast microscope.
Advantages :
I. Short process time for HLA typing.
II. Reduce cold ischemia time.
III. Able to idenitfy “null” HLA alleles.
Disadvantages:
I. Not able to identify all HLA alleles, especially HLA- Cw , DQ, DP antigen.
II. Unable to detect different HLA small amino acids.
Outcome : Not popular due to its limitation and more advance HLA typing method.
2) Molecular method.
Main concept :
A) Sequence specific primer PCR.
-HLA typing is perform by matching the amplification product of subject DNA to the DNA sequences of other candidate alleles.
B) Sequence specific oligonycleotide probes.
-Subject amplified DNA is mixed with oligonucloetide probes. HLA alleles recognized by fluorescent tags. Precise nucloetide order identified via sequencing.
C) Direct DNA sequencing.
-Determine precise order of nucleotides in a gene directly.
Advantages :
I. Highly specific.
II. Provide clear indentification of HLA antigens between donor and recipient.
III. Provide insight to future mismatch by assesing donor recipient antigens, epitopes and amino acids sequence of HLA typing.
Disadvantages:
I. Unable to identify new alleles not currently available on the HLA sequence databank.
Outcome : Prefer method for HLA typing due to high accuracy.
Key notes
1) Mismatch for different HLA antigens provide different clinical outcomes. According to Eurotransplant and old UK transplant data the clinical impact of HLA mismatch is greatest for HLA-DR followed by HLA-A or HLA-B.
2) Taboo mismatch indicate 7 specific HLA mismatch which result in poor renal allograft survival.
HLA Antibody screening.
A) Cytotoxic antibody screening.
Concept :
Recipient serum is mixed with “cell donor” lymphocytes along with complement and dye in a tray. Complement activation resulted in cell death and uptake of the dye. The result is express as percentage panel reactive body ( PRB )
Advantages :
I. Approximation the risk of positive corssmatch of a given recipient.
Disadvantages:
I. Cell panel used may not represent the true population.
II. False positive due to non HLA antibodies , autoantibodies and nonspecific Ig M antibodies.
III. False negative due to low antibody titre.
IV. Unable to identify complete lists of antibody specificities and unacceptable antigens.
B) Solid phase antibody screening.
I. ELISA .
Concept :
Adding purified HLA antigen bind to HLA antibody after adding recipient serum. Enzyme conjugated antibodies to Ig G is added. Antigen antibody reaction is identify by optical density reading.
II. Microbead platform/ single -antigen beads.
Concept:
Pooled panel beads coated with class I or II HLA antigens are used for antibody screening. Fluorescent dye is added and can be detetcted via FLOW PRA or SAB. C PRA is obtained by comparing the HLA frequencies in the donor population. Best estimate positive crossmatch.
Key notes
1. C PRA : Detect antibody in recipeint via following methods. Serological, Sold phase assay ( ELISA, FLOW Cytometry, SAB )
2. PRA : Recipient’s serum is examined in a pool of local donar antigens via complement dependent cytotoxicity .
3. Microbead assays (PRA , SAB Luminex ) 10% more sensitive for lower antibody then ELISA.
4. ELISA 10% more sensitive then AHG.
5. SAB assays results available in 3-4 hours.
6. SAB enable VXM to identify DSA before transplant. Provide extra benefit for organ allocation and risk stratification.
7. SAB allow identification of rare antigens and alleles beside the common one.
8. SAB -Luminex susceptible for prozone phenomenon.
Crossmatching.
Crossmatch via PRA able to identify specific recipient antibody to donor.
3 methods
1) Complement dependent cytotoxicity corssmatch.
Concept : Activated complement causing B or T lyphocytes lysis which indicate that significant DSA boubd to the cell surface. Sensitivity dependant on antibody titre, but this can be overcome by increasing incubation duration, AHG enhanced method and additional wash steps.
2) Flow cytometry crossmatch. (FCXM)
Concept: Accurately detect Ig G DSA on donor lymphocytes idependent of complement fixation. DSA towards B and T lymphoctes can be identified seperately. Provide huge variation of results among different laboratories. Not widely available.
3) Virtual crossmatching. (VXM)
Concept : Applying both donor HLA typing and solid phase antibody screening. Recipient serum which contain identified antibody is mix with donor HLA antigens. Reduce the waiting times and improve outcomes for sensitised transplant recipients. Prosspective crossmatch allows to consider probable positive crossmatch a risk for donor selection. However, actual crossmatch is needed as VXM is not highly accurate.
Risk assessment in renal transplant patients
1) High Immunological Risk
-High antibodies specific for mismatch donor HLA upon transplantation.
-risk of hyperacute rejection.
-Generally presence of DSA precludes transplantation. However, role of pre transplant desensitisation regimens being reported.
2) Intermediate Immunological Risk
-Low DSA titre and historic DSA not detected upon transplantation.
-May proceed with transplant with intensified immunosuppression.
3) Standard Immunological risk
-No evidence of donor HLA sensitisation.
Mohamed Essmat
3 years ago
HLA matching has a significant effect on graft survival. Serologic techniques: Quick results which decrease ischemia time in deceased donor transplant determine “null” HLA alleles (of low immunological significance as has identifiable DNA sequence with no cell surface antigen expression) but the available sera can’t identify all HLA alleles , can’t identify HLA-cw, DQ and DP antigens which are clinically significant, can’t detect small amino acids in HLA proteins which may be antigenic so not used now.
Molecular techniques: Sequence-specific primer PCR Sequence specific oligonucleotide probes-Direct DNA sequencing detect significant alleles and differentiate between donor and recipient at level of antigens, epitopes and amino acids but can’t identify new alleles that are not on primers used in HLA-typing. HLA antibody screening Detection of preformed anti-HLA antibodies is important as they increase the immunological risk of graft failure. Cytotoxic antibody screening: Expressed as percentage PRA, used to estimate risk of recipient having positive cross match but depends on commercially available cell panel which may not represent the population HLA frequency and racial differences can’t be considered Can’t determine antibody specificity and unacceptable antigens. Solid phase antibody screening: Use recombinant HLA molecules instead of lymphocytes and identify antibody specificity of recipient serum Cross-matching: Identifies if a recipient has antibodies to specific donor.
CDC-XM Done for B and T lymphocytes False negative results due to low titer antibodies not detected False positive results due to detection of autoantibodies, IgM/IgG HLA or non HLA antibodies.
FCXM Recipient serum mixed with donor lymphocytes Identify T and B lymphocytes with no need for complement fixation more sensitive than CDC and detect IgG DSA on donor lymphocytes
Virtual crossmatch: Donor HLA typing and solid phase antibody screening are used together to predict the actual crossmatch in vitro Leads to shorter waiting time, better outcome in sensitized recipients and better donor selection as predict potentially positive crossmatch. should include all serum results with recent sample as antibodies presence, titer and specificity vary with time especially with sensitizing events but false positive results due to low titer antibodies (clinically insignificant but detected by SAB) and/or non-complement binding antibodies. False negative results as all potential HLA donor antigens may be not correctly represented. Defining immunological risk: High High titer of circulating DSA
Intermediate low titer of DSA Historic DSA not detectable Acceptable transplant with intensified immunosuppression and close monitoring post-transplant
Standard No evidence of sensitization to HLA of the donor
Mohammed Sobair
3 years ago
Crossmatch methods and human leukocyte antigen (HLA) typing play a pivotal role in
improving organ allocation and afford better matches to recipients.
HLA TYPING:
HLA typing is a crucial step in renal transplantation, as recognition of foreign HLA by
recipient T lymphocytes would trigger an immune response. HLA laboratories currently
perform serologic as well as molecular typing methods.
Serological typing:
Recipient lymphocytes are introduced into the tray wells contacting sera, complement
and dye.
In tray wells where antibodies can bind to the antigens on the surface of lymphocytes;
complement is activated. This results in complement pathways triggered resulting in cell
death, ultimately allowing the dye to enter the cell. Tray wells with significant cell death
are then identified under phase contrast microscopy.
Advantage
Short time.
Disadvantage:
Lack of sera with antibody specificities that are capable of identifying the ever-growing
number of HLA not readily detect differences in HLA protein small amino.
Molecular typing:
Sequence-specific primer polymerase chain reaction.
Direct DNA sequencing: This method determines the precise order of nucleotides in the
gene of interest.
Sequence specific oligonucleotide probes.
Advantage:
Identify differences in HLA antigen between donor and recipient. Often with detail to the
amino acid level that can provide insight to the risk accompanying mismatched donor-
recipient antigens, epitopes and amino acid.
HLA typing based on polymerase chain reaction (PCR) is highly specific where specific
allele Where specific alleles are identified with no cross-reactivity.
Disadvantage: it poses is that new alleles not currently on the HLA sequence databank
will fail to be identified.
HLA ANTIBODY SCREENING:
Third of patients who are waitlisted for transplantation may have a degree of anti-HLA
antibodies detected. Preformed antibodies increase the chances of immunological
failure of the allograft e chances of immunological failure of the allograft by causing
positive crosshatches and, thereby, result in the exclusion of donors.
Methods used for HLA antibody screening:
Cytotoxic (cell-based) antibody screening:
A set of cell donors are randomly selected to be representative of a population.
Each panel consists of around 30 to 40 different donor lymphocytes.
Recipient serum is mixed with “cell donor” lymphocytes in individual wells along with
complement and dye. The serum contains antibodies that bind to the cell surface with
adequate density complement pathways are activated which results in cell death and
uptake of the dye.
Degree of cytotoxicity is expressed as percentage PRA (panel reactive antibody). It is a
tool that can be employed to approximate the risk of a given recipient of having a
positive crossmatch.
Disadvantage:
PRA percent can be different numerically without a corresponding change in the type or
amount of antibody.
This largely depends on the cell panel used which are commercially produced and may
not truly represent the population.
HLA frequencies and racial differences need to be factored in but cannot be done.
False positive results can be produced due to non HLA antibodies, autoantibodies and
nonspecific IgM antibodies.
False negative results are possible as this is purely complement dependent that
requires higher antibody titers to be activated, the lack of a complement activation
simply due to low titers allows a true antibody to be hidden.
Solid phase antibody screening:
Use soluble or recombinant HLA molecules: it’s either
Enzyme-linked immunosorbent assay platform:
HLA molecules are applied to ELISA platforms and will bind individually to HLA antibody
after the addition of recipient serum. Enzyme conjugated antibodies to IgG (human) is
then added to detect the presence of HLA antibody in the serum which is bound to the
antigen. Detection is performed by optical density
Microbeads platform/single-antigen beads:
Pooled panel beads with several different class Ⅰ or Ⅱ HLA antigens on a bead yield a
positive or negative result and are utilized for screening. Microbeads that is fluorescent
dye conjugated is then added to detect the presence of HLA antibody in the serum
which is bound to the antigen.
Fluorescence detection can be done traditionally using:
A flow cytometer (Flow PRA®) or
Via the single-antigen beads (SAB) Luminex® platform.
These estimate PRA by the proportion of positive beads.
Advantage:
Better discriminate immunologically relevant positive crossmatches from false-positive
results when traditional cell based methods are complemented with solid phase assays
Microbeads assays (both Flow PRA® and Luminex®) are ten percent more sensitive for
lower titer antibody than ELISA.
Assays are specific for anti-HLA antibodies.
SAB assays are rapid with results available 3-4hrs.
SAB assays permit identification of anti-HLA antibodies for all common and numerous
rare antigens and alleles. to eleven HLA loci.
Disadvantage:
Being too sensitive they can detect antibody that is below the threshold associated with
a positive crossmatch. The detected antibody may preclude a potential donor.
With growing list of HLA alleles, the complete spectrum of unique HLA antigens cannot
be fully presented on solid phase assays.
CROSSMATCHING:
Either:
Complement-dependent cytotoxicity crossmatch:
The complement-dependent cytotoxicity crossmatch is interpreted as positive if a
considerable number of lymphocytes are destroyed after the incorporation of
complement. This suggests that a significant DSA has been bound to the cell surface.
Complement-dependent cytotoxicity crossmatch (CDC-XM) can be done for B and T
lymphocytes.
Sensitivity is limited if the relevant antibody is in low titres, but this can be overcome by
increasing the incubation time, the use of the AHG-enhanced method as well as
additional wash steps.
Low titre antibody resulting in false negatives. CDC-XM can also give false positives by
detecting autoantibody, IgM/IgG HLA or non-HLA.
Flow cytometry crossmatch:
FCXM detects DSA independent of complement fixation:
Recipient serum is mixed with donor lymphocytes and then tagged with a fuorochromes
conjugated anti-IgG antibody.
Virtual cross matching:
In VXM, both donor HLA typing and solid phase antibody screening are utilized together.
By “mixing” identified antibody specificities of recipient serum with donor HLA antigens.
DEFINING RISK:
High immunological risk:
High titres of circulating antibodies specific for mismatched donor HLA (DSA).
Can lead to hperacute rejection. The presence of DSA precludes transplantation.
However, there are reports of innovative pre-transplant desensitization regimens to
reduce this risk.
Intermediate immunological risk:
There is a low titer of DSA, and historic DSA is not detectable.
It may be acceptable to consider intensified immunosuppression as well as
immunological monitoring in the post-transplant period.
Standard immunological risk:
There is no evidence of donor directed sensitization.
nawaf yehia
3 years ago
Renal transplantation Remains the best option for patients with end-stage renal disease, however, careful selection and preparation must be carried out to improve the success of these of this treatment modality.
The important steps to ensure maximal matching between the donor and recipient are :
First. HLA typing
Second. HLA antibody screening. this is mainly to look for donor specific antibodies. DSA.
Third. Cross-matching.
Regarding th first step HLA typing. This can be done by either serologic methods, or molecular typing methods.
for the serological typing, It includes the use of commercially available sera containing antibodies to a multitude of known HLA alleles , along with complement use .Recipient lymphocytes are introduced into the tray which contain the serum And when antibodies bind to antigens on the surface of lymphocytes, then the complement is is activated ultimately leading to cell death and this can be identified on the phase contrast microscopy.
The key benefit of serologic typing is that the results are available in a short period, which is particularly important in case of deceased donor kidney transplantation. Which means less cold ischemia time. This method. Also. Helps to differentiate HLA alleles. That have identifiable, DNA sequences. But no cell surface antigen expression. Termed as the null HLA alleles.
The drawback of this method is the lack of Sera with antibody specificity that are capable of identifying the ever-growing number of HLA alleles. Another drawback of this method. Is that the HLA Cw , DQ. DP. Antigens which May have clinically significant effects on the outcomes of allograft. However, these assays are scars for these loci. The third drawback is that serologic methods Do not readily detect differences in HLA protein small amino acids,these may be antigenic enough to trigger potent immunological response.
The other method is molecular typing. And here we have three ways first. Sequence-specific primer, polymerase chain reaction. Second sequence specific oligo nucleotide probes ,Third is direct DNA sequencing.
It can readily and clearly identified differences in HLA antigens between donor and recipient. HLA typing based on PCR is highly specific
The Three Most important HLA antigens Considered in kidney Transplantation are HLA-A, HLA-B and HLA DR. Where the fewer of the mismatches, the better the match between the donor and the recipient. And the better Transplant outcome.
HLA antibody screening.
Almost a third of patients who are on the waiting list for kidney transplantation. Art sensitized. Which means having HLA antibodies which usually occur in three instances :pregnancy. Post blood, transfusion. And in case of Prior transplantation.
The presence of preformed antibodies, increases the chances of immunological failure of The graft. And By causing positive cross-match , thereby leading to exclusion of the donor.
Methods used for HLA. Antigens. Screening are the following first cytotoxic antibody screening. 2nd is solid phase antibody screening.
Where in the first the Recipient,s Serum is mixed with donor lymphocytes. And the degree of cytotoxicity is expressed as percentage panel reactive antibody (PRA)
The limitations of this of this method are that PRA percent can be different numerically without a corresponding change in the type or amount of antibody.
False positive results. Can be due to non HLA antibodies autoantibodies or nonspecific IGM antibodies. Whereas false negative results Can occur cases of low antibody titers Where are the complement Is not activated.
Solid phase screening
Here it includes the use of recombinant HLA molecules. Instead of using lymphocytes targets, variants of this method.
1)Enzyme-linked immunosorbent assay platform.
2)microbead platform. Or called single antigen Beads. :the beads are individually coated with class 1 or 2 HLA, antigens of an individual patient. And as the fluorescent dye is used in this method , So fluorescence detection can be done traditionally using a flow cytometerOr with single antigen beads luminex. Platform.
The importance of single antigen bead Is thatIt can Detect A list of distinct antibody specificities that are subsequently compared with the HLA frequencies in the donor population, to determine the calculated panel reactive antibody cPRA ,This yields, the best estimate of the likelihood of a positive cross-match.
Regarding the difference between pra and cpra.
in traditional PRA a high value translated to a high probability of a positive cross-match. Where in cPRA is based on unacceptable HLA, antigens that The patient has been sensitized to , cPRA estimate the proportion of donors with whom a particular recipient would be incompatible.
Despite the benefits of the solid phase antibodies. Some limitations are there ; Being too sensitive, they can detect antibod That is below the threshold associated with the positive cross-match. Also that non HLA antibodies Cannot be accounted for utilizing this method solely. Moreover, this single antigen bead luminex essay has been shown to be susceptible to an artifact known as the protozoan phenomenal. Where sera with high anyi HLA antibody titers give negative results when tested neat, but become strongly positive after dilution , also a false positive result can occur with igM antibodies binding to the beads as it can also occur with the use of IVIG therapy.
, cross-matching
We have three ways. First complement dependent cytotoxicitycross-match
Complement dependent cytotoxicity, cross-match can be done for B and Tlymphocytes. It’s sensitivity is limited if the relevant antibody Is in low titer, But this can be overcome by increasing the incubation time and with the use of anti-human globulin enhanced method. The CDC cross-match also can give false positive results by detecting autoantibodies, IgM. Antibody or non HLA and antibodies.
The second way is flow cytometry cross-match.
This method is more sensitive than the complement dependent cytotoxicity Cross-match. Third is virtual cross-match.
In which both donor HLA typing and solid phase antibody screening are utilized together.
The data is used to forecast the actual in vitro cross match results by mixing identified antibody specificities of recipients Serum with donor HLA, antigens, the use of virtual cross-match can lead to short-term, wait time and improved outcomes for sensitized transplant Recipients.
It’s important to notice that titres, specificities and presence or absence of antibodies could significantly vary over time ; Thus.The use of antibody specificity. From serum samples earlier than six months, could not predict a cross match with certainty. However, results from virtual cross-match are not 100% accurate and current practice mandates The actual cross-match to be performed as well.
virtual cross-match does not identify HLA. Null allele is not a jelly and leads. Our ones have identifiable, DNA sequences with more connect typing, but not express HLA products on sale surface.
Defining the risk.
So, we have three categories of the immunological risk in the kidney transplantation.
First high immunological risk ; here at the type of transplantation, there are high titers of circulating. Antibodies specific for mismatched donor DSA. This can lead to hyperacute rejection. Intermediate risk : low DSA titres , need intensified immunosuppression with close monitoring Standard immunological risk. Where there is no evidence of donor directed sensitization to HLA .
in conclusion :Interpretation and clinical application of transplant Immunology are crucial steps to successful outcome.
Dalia Ali
3 years ago
Renal transplantation remains the best option for patients suffering from end stage renal
disease (ESRD). Given the worldwide shortage of organs and growing population of patients with ESRD
HLA TYPING
is a important step in renal transplantation, as recognition of foreign HLA by recipient T lymphocytes would trigger an immune response.
HLA laboratories currently perform serologic as well as molecular typing methods.
HLA laboratories currently perform serologic as well as molecular typing methods.
recipient lymphocytes are introduced into the tray wells contacting sera, complement and dye.
When antibodies bind to the antigens on the surface of lymphocytes the complement is activated resulting in cell death and dye to enter the cell. Tray wells with significant cell death are then identified under phase contrast microscopy. Through a process of comparison and elimination of positive wells the HLA type is assigned.
Advantages
results are available in a short period.
This is important in deceased donor renal transplantation because decrease cold ischemia times.
This method also offers the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression. These alleles termed “null” HLA alleles are of less immunological significance
Disadvantages
lack of sera with antibody specificities that are capable of identifying the ever-growing number of HLA alleles
The HLA-Cw, DQ, and DP antigen have clinically significant effects on the outcomes of allografts. However, serologic assays are scarce for these loci.
serologic methods do not readily detect differences in HLA protein small amino acids. These may be antigenic enough to trigger potent immunological responses
Molecular typing
Sequence-specific primer polymerase chain reaction
In this approach extracted DNA from the subject is amplified in several wells. Each well has primers that are complementary to specific HLA alleles.
Sequence specific oligonucleotide probes
Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles. Unique HLA alleles are then identified using fluorescent tags.
Direct DNA sequencing
This method determines the precise order of nucleotides in the gene of interest. Using published HLA allele sequences, HLA type is subsequently assigned by comparison.
Molecular typing regardless of the method can identify differences in HLA antigen between donor and recipient. Often with detail to the amino acid level that can provide insight to the risk accompanying mismatched donor-recipient antigens, epitopes and amino acid.
HLA typing based on polymerase chain reaction (PCR) is highly specific where specific alleles are identified with no cross-reactivity.
disadvantage
it poses is that new alleles not currently on the HLA sequence databank will fail to be identified.
HLA ANTIBODY SCREENING
detection of anti-HLA antibodies is important , when the crossmatch is negative, even low titres of DSA can lead to early as well as late antibody mediated rejection
For sensitised patients, successful transplantation is possible by using strategies such as desensitisation, paired exchange and acceptable mismatching
Cytotoxic (cell-based) antibody screening
recipient serum is mixed with “cell donor” lymphocytes in individual wells along with complement and dye. Where the serum contains antibodies that bind to the cell surface with adequate density complement pathways are activated which results in cell death and uptake of the dye
false positive results
can be produced due to nonHLA antibodies, autoantibodies and nonspecific IgM antibodies.
false negative results
complement dependent method requires higher antibody titres to be activated
Solid phase antibody screening
Enzyme-linked immunosorbent assay platform
purified HLA molecules are applied to enzyme-linked immunosorbent assay (ELISA) platforms and will bind individually to HLA antibody after the addition of recipient serum
Enzyme conjugated antibodies to IgG (human) is then added to detect the presence of HLA antibody in the serum which is bound to the antigen. Detection is performed by optical density reading.
Microbead platform/single-antigen beads
Pooled panel beads with several different class Ⅰ or Ⅱ HLA antigens on a bead
Microbead that is fluorescent dye conjugated detect the presence of HLA antibody in the serum which is bound to the antigen.
solid phase antibody screening addresses detect both complement and non-complement binding simultaneously. More sensitive detecting antibody that is below the threshold associated with a positive crossmatch.
CROSSMATCHING (XM)
Complement-dependent cytotoxicity crossmatch
the complement-dependent cytotoxicity crossmatch is considered as positive if a considerable number of lymphocytes are destroyed after the incorporation of complement when significant DSA has been bound to the cell surface.
Complement-dependent cytotoxicity crossmatch (CDC-XM) done for B and T lymphocytes. Sensitivity is limited when there is in low antibodies titres, this can be overcome by increasing the incubation
Time and use of the AHG-enhanced method as well as additional wash steps
this method could miss low titre antibody resulting in false negatives. CDC-XM can also give false positives by detecting autoantibody, IgM/IgG HLA or non-HLA.
Flow cytometry crossmatch
Flow cytometry crossmatch (FCXM) detects DSA independent of complement fixation
detects the presence or lack of IgG DSA on donor lymphocytes.
The recipient serum is mixed with donor lymphocytes and then tagged with a fluorochromeconjugated anti-IgG antibody.
Compared to complement-dependent cytotoxicity crossmatch FCXM give greater sensitivity
Virtual crossmatch
in vitro crossmatch results by “mixing” identified antibody specificities of recipient serum with donor HLA antigens
The use of VXM can lead to shorter wait times and improved outcomes for sensitised transplant recipients
VXM should, be done on all available serum results including at least one recent within less than 3-6 mo for a given patient because the titers , specificities, and presence or absence of antibodies could significantly vary over time.
False positive results of VXM may arise where there are significantly low titre and/or non-complement binding antibodies, thereby, resulting in the wrong exclusion of potential donors
Conclusion
Interpretation and clinical application of transplant immunology are crucial steps to a successful outcome.
Abdullah Raoof
3 years ago
Hla typing
Three important steps for successful transplantation are
1- HLA TYPING
2- DSA SCREENING
3- CROSSMATING
1- HLA typing is crucial step in successful kidney transplantation .
There is two main method of HLA typing
A-Serological typing
a tray containing sera with antibodies ( commercially available )
to a multitude of known HLA alleles is used. For typing, recipient lymphocytes are introduced into the tray wells contacting sera, complement and dye. In tray wells where antibodies can bind to the antigens on the surface of lymphocytes; complement is activated. This results in complement pathways triggered resulting in cell death, ultimately allowing the dye to enter the cell. Tray wells with significant cell death are then identified under phase contrast microscopy .
advantages : – the result is available in short time , which means less ischemia in deseased kidney transplantation .
disadvantages :- un availability of ab against overgrowing number of HLA allels .
B -Molecular typing a. Sequence-specific primer polymerase chain reaction . b. Sequence specific oligonucleotide probes. c. Direct DNA sequencing. 2- HLA ANTIBODY SCREENING
– About 1/3 of of waitlist patients are DSA positive .
– Sensitization occur by (blood transfusion , pregnancy ,previous transplan )
– Even low titre of DSA may lead to early as well as to late rejection .
– For sensitized patients transplantation is possible by
Ø Desensitization
Ø Paired exchanged
Ø Acceptable mismatching 2-1 Cytotoxic (cell-based) antibody screening
The method is similar to that of serologic typing howeverhere recipient serum (instead of commercially prepared ANT HLA ab ) is mixed with “cell donor” lymphocytes
in individual wells along with complement and dye.
It is a tool by which we can approximate a risk of positive CM .
false positive results can be produced due to
Ø non-HLA antibodies,
Ø autoantibodies
Ø nonspecific IgM antibodies.
false negative results are possible simply due to
Ø low antibody
Ø complete lists of antibody specificities and unacceptable antigens cannot be identified as there are several antigens in each well
2-2 Solid phase antibody screening
This method employs soluble or recombinant HLA molecules instead of lymphocytes.
The variants of these methods are
A-Enzyme-linked immunosorbent assay platform:
In this method, purified HLA molecules are applied to enzyme-linked immunosorbent assay (ELISA) platforms and will bind individually to HLA antibody after the addition of recipient serum[. Enzyme conjugated antibodies to IgG (human) is then added to detect the presence of HLA antibody in the serum which is bound to the antigen. Detection is performed by optical density reading.
B- Microbead platform/single-antigen beads:
Pooled panel beads with several different class Ⅰ or Ⅱ HLA antigens on a bead yield a positive or negative result and are utilised for screening .
Fluorescence detection can be done traditionally using
a flow cytometer (Flow PRA) or via the single-antigen beads (SAB) Luminex platform.
These estimate PRA by the proportion of positive beads.
SAB are individually coated with a single HLA antigen and yield a list of distinct antibody specificities.
PRA estimates the probability of positive XM by using small no of antigens .
cPRA is the same as PRA , but using large no of antigens ( 10,000- 12,000) by using computer .it is useful in allocation of kidney and pancrease .
microbeads assay is more sensitive than ELISA method which is more sensitive than cell based cytotoxic method .
SAB is specific and sensitive and available within 3-4 hours .
Solid phase diadvantages
1. They detect both complement and non-complement binding simultaneously.
2. they can detect antibody that is below the threshold associated with a positive crossmatch which can preclude a potential donor.
3. Cannot detect Non-HLA antibodies .
In the SAB – Luminex® assay the very high titre AB may give negative result( Prozone phenomenon) which needs to be diluted after (1:10 dilution) to give the correct result .
Also to avoid a false positive result from IgM AB the sample needs to be treated by dithiothreitol (DTT) which removes IgM from sample .
3- CROSSMATCHING (XM) 3-1 Complement-dependent cytotoxicity crossmatch Positive XM is contraindication to transplantation .
Similar to cytotoxic assay the complement-dependent cytotoxicity crossmatch is interpreted as positive if a considerable number of lymphocytes are destroyed after the incorporation of complement . This suggests that a significant DSA has been bound to the cell surface. Complement-dependent cytotoxicity crossmatch (CDC-XM) can be done for B and T lymphocytes.
Sensitivity is limited if the relevant antibody is in low titres, but this can be overcome by
Ø increasing the incubation time .
Ø the use of the AHG-enhanced method .
this method has low sensitivity which may miss the low titre antibody which is clinically significant .
3-2 Flow cytometry crossmatch
Flow cytometry crossmatch (FCXM) detects DSA independent of complement fixation. It precisely detects the presence or lack of IgG DSA on donor lymphocytes.
In this method, recipient serum is mixed with donor lymphocytes and then tagged with a fluorochromeconjugated anti-IgG antibody.
Several antibodies with separate fluorochromes particular to B and T lymphocyte
surface proteins can be added .
With the useof flow-cytometry, B and T lymphocytes can be readily identified and have their DSA individually interrogated.
It has high sensitivity .
3-3 – Virtual crossmatching
In virtual crossmatch (VXM), both donor HLA typing and solid phase antibody screening are utilised together.
It is not precisely a crossmatch in the sense of mixing serum and lymphocytes .
The data is used to forecast the actual in vitro crossmatch results by “mixing” identified antibody
specificities of recipient serum with donor HLA antigens .
The use of VXM can lead to shorter wait times and improved outcomes for sensitised transplant recipients .
The results from VXM are not a hundred percent accurate and current practice mandates an actual
crossmatch be performed as well .
DEFINING RISK High immunological risk
there are high titres of circulating antibodies specific for mismatched donor HLA (DSA).
This can lead to hyperacute rejection.
Thepresence of DSA precludes transplantation. But this risk can be reduced by desensitization .
Intermediate immunological risk
There is a low titer of DSA, and historic DSA is not detectable. It may be acceptable
to consider intensified immunosuppression as well as immunological monitoring in the post-transplant period. Standard immunological risk
Where there is no evidence of donor directed sensitisation
to HLA. Transplantation can be done even without induction therap in low risk patients .
AMAL Anan
3 years ago
*HLA typing is a crucial step in renal
transplantation, as recognition of foreign HLA by recipient T lymphocytes would trigger an immune response.
*** Serological typing:
-a tray containing sera with antibodies to a multitude of known HLA alleles is used.
-For typing, recipient lymphocytes are introduced into the tray wells contacting sera, complement and dye. In tray wells where antibodies can bind to the antigens on the surface of lymphocytes; complement is activated. This results in complement pathways triggered resulting in cell death, ultimately allowing the dye to enter the cell.
-The key benefit of serologic typing is that results are available in a short period. This is particularly important in deceased donor renal transplantation. Quick results mean less cold ischemia times.
-This method also offers the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression.
-serologic methods do not readily detect differences in HLA protein small amino acids .
***Molecular typing :
-Sequence- specific primer polymerase chain reaction.
– Sequence specific oligonucleotide probes.
-Direct DNA sequencing.
*** HLA antibodies screening:
-Sensitised patients who are pregnant, post blood transfusions and prior transplantation which causes positive crossmatch and increase chance of immunological failure .
-Low titres DSA even with negative crossmatch cause early and late antibody mediated rejection.
For successful transplantation ; desensitisation , paired exchange and acceptable mismatching must be planed .
>>>Screening by :
-Cytotoxic ( cell-based ) antibody screening.
-Solid phase antibody screening.
-Enzyme-linked immunosorbent assay platform.
-Microbead platform /single-antigen beads.
***CROSSMATCHING (XM):
-The presence of donor-specific cytotoxic antibodies depicted as a positive crossmatch was a contraindication to transplantation. With PRA that identifies several antibodies to a potential cluster of donors, the crossmatch will identify if a recipient had antibodies to a specific donor of interest.
-The solid-phase antibody test should be used together with crossmatch results to identify those that are immunologically relevant.
••• Complement-dependent cytotoxicity crossmatch :
-It is interpreted as positive if a considerable number of lymphocytes are destroyed after the incorporation of complement.This suggests that a significant DSA has been bound to the cell surface. Complement-dependent cytotoxicity crossmatch (CDC-XM) can be done for B and T lymphocytes. Sensitivity is limited if the relevant antibody is in low titres, but this can be overcome by increasing the incubation time, the use of the AHG-enhanced method as well as additional wash steps. The complement fixing antibody to anti-human immunoglobulin (AHG) will bind to any DSA present on lymphocytes.
••• Flow cytometry crossmatch :
It detects DSA independent of complement fixation. It precisely detects the presence or lack of IgG DSA on donor lymphocytes. In this method, recipient serum is mixed with donor lymphocytes and then tagged with a fluorochrome- conjugated anti-IgG antibody.
••• Virtual crossmatching :
– In virtual crossmatch (VXM), both donor HLA typing and solid phase antibody screening are utilised together. It is not precisely a crossmatch in the sense of mixing serum and lymphocytes.
– The use of VXM can lead to shorter wait times and improved outcomes for sensitised transplant recipients. The speed of results generated allows a VXM to be performed at the time of donor identification owing to the fact that there is progressively sensitive and specific flow cytometry technology.
*** DEFINING RISK :
•••High immunological risk :
At the time of transplantation, there are high titres of circulating antibodies specific for mismatched donor HLA (DSA). This can lead to hyperacute rejection. The presence of DSA precludes transplantation. However, there are reports of innovative pre-transplant desensitisation regimens to reduce this risk.
•••Intermediate immunological risk :
At the time of transplantation, there is a low titer of DSA, and historic DSA is not detectable. It may be acceptable to consider intensified immunosuppression as well as immunological monitoring in the post-transplant period.
••• Standard immunological risk
Where there is no evidence of donor directed sensitisation to HLA.
REFERENCES :
1.Mojcik CF, Klippel JH. End-stage renal disease and systemic lupus erythematosus. Am J Med 1996; 101: 100-107 [PMID: 8686702 DOI: 10.1016/S0002-9343(96)00074-5]
Nossent HC, Swaak TJ, Berden JH. Systemic lupus erythematosus after renal transplantation: patient and graft survival and disease activity. The Dutch Working Party on Systemic Lupus Erythematosus. Ann Intern Med 1991; 114: 183-188 [PMID: 1984742 DOI: 10.7326/0003-4819-114-3-183]
Ponticelli C, Moroni G. Renal transplantation in lupus nephritis. Lupus 2005; 14: 95-98 [PMID: 15732296 DOI: 10.1191/09612033 05lu2067oa]
Contreras G, Mattiazzi A, Guerra G, Ortega LM, Tozman EC, Li H, Tamariz L, Carvalho C, Kupin W, Ladino M, LeClercq B, Jaraba I, Carvalho D, Carles E, Roth D. Recurrence of lupus nephritis after kidney transplantation. J Am Soc Nephrol 2010; 21: 1200-1207 [PMID: 20488956 DOI: 10.1681/asn.2009101093] Stone JH, Amend WJ, Criswell LA. Outcome of renal transplantation in systemic lupus erythematosus. Semin Arthritis Rheum 1997; 27: 17-26 [PMID: 9287386 DOI: 10.1016/ S0049-0172(97)80033-9]
Costenbader KH, Feskanich D, Stampfer MJ, Karlson EW. Reproductive and menopausal factors and risk of systemic lupus erythematosus in women. Arthritis Rheum 2007; 56: 1251-1262 [PMID: 17393454 DOI: 10.1002/art.22510]
Gilbert EL, Ryan MJ. Estrogen in cardiovascular disease during systemic lupus erythematosus. Clin Ther 2014; 36: 1901-1912 [PMID: 25194860 DOI: 10.1016/j.clinthera.2014.07.021]
von Andrian UH, Mackay CR. T-cell function and migration. Two sides of the same coin. N Engl J Med 2000; 343: 1020-1034 [PMID: 11018170 DOI: 10.1056/nejm200010053431407]
Tinckam KJ. Basic histocompatibility testing methods. In: Chandraker A, editor. Core concepts in renal transplantation. New York: Springer Science + Business Media, LLC, 2012: 21-42 Robinson J, Waller MJ, Parham P, de Groot N, Bontrop R, Kennedy LJ, Stoehr P, Marsh SG. IMGT/HLA and IMGT/MHC: sequence databases for the study of the major histocompatibility complex. Nucleic Acids Res 2003; 31: 311-314 [PMID: 12520010 DOI: 10.1093/nar/gkg070]
Claas FH, Witvliet MD, Duquesnoy RJ, Persijn GG, Doxiadis II. The acceptable mismatch program as a fast tool for highly sensitized patients awaiting a cadaveric kidney transplantation: short waiting time and excellent graft outcome. Transplantation 2004; 78: 190-193 [PMID: 15280676 DOI: 10.1097/01. TP.0000129260.86766.67].
Weam Elnazer
3 years ago
When it comes to kidney transplantation, HLA typing is a critical component of the process. HLA interpretations may be tricky, and a variety of modalities can be utilized for this aim, each with its own set of advantages and disadvantages.
The following are examples of HLA typing methods:
1- Serological research
In this procedure, the recipient cells are mixed with sera carrying various HLA alleles that have been identified before. Null alleles may be readily excluded from a population. These approaches do not distinguish between HLA Cw, DQ, and DP.
2- The molecular level
These are some examples:
In this procedure, DNA from the individual is amplified in many wells containing Primes that are complementary to HLA alleles, and the result is a single amplicon of DNA.
The use of sequence-specific oligonucleotide probes allows for the determination of the precise order of nucleotides in a gene of interest.
Direct DNA sequencing has the advantage of being able to identify both significant alleles and particular alleles without the need for cross-reactivity.
SCREENING OF THE HLA ANTIBODY
Antibody screening for cytotoxic activity
This recipient serum is combined with 30-40 donor lymphocytes, dye, and Compliment to create a unique mixture. The results are provided in the form of PRA.
Antibody screening using solid-phase technology
Instead of lymphocytes, it makes use of recombinant HLA molecules.
ELISA is a platform for research.
Platform made of microbeads
PRA is estimated using this method, which is more sensitive than ELISA. The cPRA detects antigens that are deemed undesirable.
MATCHING CROSSWORDS
Dependent on the compliment It is possible to do a cytotoxic crossmatch on both B and T cells.
Transfection with recipient serum and the addition of fluorescent tags to donor cells is performed using flow cytometry. It is capable of detecting DSA in the absence of complement fixation.
The virtual crossmatch is a mix of solid-phase antibody screening and HLA typing that provides a virtual cross match.
Risk stratification categories: According to on immunological risk, Gebel et al. proposed the following risk stratification categories in prospective renal transplant patients:
High level of immunological hazard
These individuals exhibit high titers of circulating DSA that is specific for mismatched donor HLA, which indicates that they are infected. Due to the high risk of acute rejection in the presence of DSA, such individuals are often barred from receiving a transplant in most cases.
Immunological hazard of intermediate severity
In this group, the DSA titer at the time of transplantation is low, and there is no identifiable historical DSA. In these individuals, intensified immunosuppression, as well as immunological surveillance throughout the post-transplant period, are essential.
Immunological hazard as defined by standard practice
There is no indication of DSA in this case, and the pre-transplant immunological risk assessment is based on the findings of the donor cross-match and antibody screening tests.
Professor Ahmed Halawa
Admin
3 years ago
Thank you all
I enjoyed reading your comments.
saja Mohammed
3 years ago
Summarize the article
====================
This article provides a case-based review of the different methods used for HLA -typing and quantification of the DSAs ,also facilitate the assessment of the patient immunological status. and help in risk stratification of kidney transplant candidates. Introduction :
HLA matching is considered first and most important step in kidney transplant work up. the increased mismatch at HLA A-B – DR. associated with increased risk of graft rejection and impact the outcome base on many studies which revealed that multiple mismatches translated into a 64% higher risk, while the risk dropped to 13% with just one HLA mismatch.
Different methods are available for HLA typing.
-Serological HLA typing:
Old method complement dependent by adding the recipient lymphocytes to a tray that contain sera of antibodies to all known HLA antigens,( usually from multiparous women after certain incubation will add the complement and coloring dye Once Ag-AB reaction occurs complement activation occur, lead to cell lysis , then examine under the microscope the percentage of dead cells which take the dye (red ).The serologic typing are important in deceased donor renal transplantation as the results are available in a short period.
– Molecular DNA-based HLA typing techniques
It includes different types: –
· SSOP sequence specific oligonucleotide probe hybridization
· SSP sequence-specific primer amplification
· Direct DNA sequencing determines the precise order of nucleotides in the gene of interest, allowing HLA type comparison, through the amino acid level, to provide insight to the risk monitoring
Molecular based HLA typing are more sensitive and accurate than serological typing. soluble or recombinant HLA molecules based not targeting lymphocytes as lymphocytes can express HLA and non-HLA antigens.
–Enzyme-linked immunosorbent assay platform when purified HLA molecules are applied to enzyme-linked immunosorbent assay (ELISA) platforms and will bind to HLA antibody after the addition of recipient serum enzyme conjugated antibodies to IgG (human) is then added to detect the presence of HLA antibody antigen and interpretation by optical density of AB-Ag reaction.
–Solid phase assay Luminex single bead antigen:
Estimate PRA by the proportion of positive beads, SAB assays permit
identification of anti-HLA antibodies for all common and many rare antigens and alleles. Its range of identification is up to eleven HLA loci.
SAB assays are rapid the results available in 3-4 h. The assay is also quite efficient in a single reaction chamber up to one hundred unique antigen beads can be tested or multiplex testing can allow testing many patients at one time .
Results from SAB enable virtual crossmatching (VXM) to identify DSA pre-transplant, thereby enabling organ allocation and risk stratification by calculating the percentage of unacceptable Ags(cPRA %) its more specific and helpful for allocation of donors for highly sensitized patients.
Limitation with Luminex SAB, can give false positive due to denatured Abs, Drugs like monoclonal AB
Prozone effect can give false negative results (can overcome by more dilution)
Epitope sharing
Drugs like IVIG
Being too sensitive they can detect antibody that is below the threshold associated with apositive crossmatch. The detected antibody may not always have clinical implications and can preclude a potential donor.
2 – Screening of antibodies
1-Complement dependent or antibody-dependent cell-mediated cytotoxicity (ADCC) or CDCXM:
Similar to the cytotoxic assay with low sensitivity its used limited by false positive results due to the presence of autoantibodies, IgM, non-HLA and non-complement fixing ABS, using AHG and DTT may improve the sensitivity for auto-antibodies, also positive B CDCXM indicate presence of autoimmune disease or treatment with rituximab. CDCXM also less specific test and associated with false negative result that can occur due to non-complement fixing AB or low level of DSAs or technical errors.
2– Flow cytometry crossmatch (FXCM):
The technique based on the incubation of donor’s lymphocytes with recipient sera, then add second antibody with florescence dye (green) that will bind ABS (DSA) attached to donor lymphocytes Flow FCXM detects DSA independent of complement fixation.in flow cytometry method usually measure fluorescence intensity and compare it to control (median channel shift- MCS) With the use of flow cytometry, both B and T lymphocytes can be easily identified and have their HLA related DSA usually IgG class1.11 abs I not IgM. more sensitive but less specific for non-HLA abs and can give false positive B FXCM in case of treatment with monoclonal ABS or non-HLA abs.
3 – Virtual cross-match(VXM):
In virtual cross-matching (VXM), computer-based analysis of donor HLA typing and solid phase antibody screening are used together It is correlated well with FCM cross match and graft survival even in sensitized patients Define unacceptable antigens by CPRA % so donors can be excluded, and allow identification of suitable donor in short times, useful for DD program with the advantage of shorting cold ischemia time and reduce the waiting time for allocation of donors for highly sensitized recipients.
False positive VXM due to Denatured HLA antigens on the SAB or due to the presence of null alleles.
False negative VXM either due to incomplete crossmatch or absence of certain alleles
4 – Immunological risk stratification:
1- High immunological risk
high titer of circulating HLA-DSAs
2-Intermediate immunological risk
low DSA titer and the historical DSA is not detectable.
3- Standard immunological risk
no evidence of targeted donor sensitization to HLA.
Conclusion :
understanding the concept of basic transplant immunology is the key factor that would help the practicing clinicians and guide them for better immunological risk stratification, better organ allocation for sensitized patients and improve the transplant outcome especially with recent advancing in molecular methods for HLA typing and antibodies quantification techniques .
Asmaa Khudhur
3 years ago
HLA typing is a crucial step in renal transplantation ,HLA laboratories currently perform serological and molecular typing methods.
1-)Serological typing :her a tray containing sera with Abs to a many known types of HLA alleles to which recipient lymphocytes are introduced in edition to complement and dye .
Cons:
1- the results are available in short period especially in deceased donor renal transplantation which mean less cold ischemia times.
2-the other benefit is that it offer the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigens expression which is called NULL HLA alleles with less immunological significance.
Prons:
1-) is the lake of sera with Ab specificities that are capable of identifying the ever-growing number of HLA alleles.
2-)do not readily detect differences in HLA proteins small amino acids which may be antigenic enough to trigger potent immunological responses.
Serological typing has fallen into disuse with more advanced methods of typing currently available.
2-) molecular typing
– sequence-specific oligonucleatide probes.
– Sequence -specific primer
– Direct DNA sequencing
Advantages:
1-) regardless of the method, molecular typing can identify differences in HLA Ag between donor and recipient with details to the amino acid level.
2-) HLA typing by PCR is highly specific with no cross-reactivity.
Disadvantages:
New alleles not currently on the HLA sequence databank will fail to be identified.
HLA AB screening:
The usual route of sensitization toward HLA Ag accurs by pregnancy,post blood transfusion and prior transplantation.
There are different methods used for HLA antibody screening
1. Cytotoxic (cell-based) Ab screening
False positive result due to non-HLA antibody ,autoantibodies and non -specific Igm antibodies.
False negative results because it complement dependent that requires high Ab titer to be activated.
2.Solid phase antibody screening.
a-Enzyme-linked immunosorbent assay platform
b-micro bead platform /single-Antigen beads.
CROSSMATCHING (XM)
Complement-dependent cytotoxicity crossmatch
Complement-dependent cytotoxicity crossmatch (CDC-XM) can be done for B and T lymphocytes. Sensitivity is limited if the relevant antibody is in low titres,
Similar to cytotoxic PRA this method could miss low titre antibody resulting in false negatives. CDC-XM can also give false positives by detecting autoantibody, IgM/IgG HLA or non-HLA.
Flow cytometry crossmatch
Flow cytometry crossmatch (FCXM) detects DSA independent of complement fixation.
Compared to complement-dependent cytotoxicity crossmatch this offers greater sensitivity[9]. Different laboratories use different methods, and this can result in a difference in the results between them[50]
Virtual crossmatching
In virtual crossmatch (VXM), both donor HLA typing and solid phase antibody screening are utilised together.
False positive results of VXM may arise where there are significantly low titre and/or non-complement binding antibodies, thereby, resulting in the wrong exclusion of potential donors.
The VXM can also give false negative results due to the fact that the list of all potential HLA donor antigens have been classed differently and, therefore, can not be correctly represented.
Ibrahim Omar
3 years ago
Please summarise this article with emphasis on the different techniques of tissue typing and crossmatching :
cross matching techniques are of utmost importance before doing solid organs transplantation. they are done to check if there are any preformed antibodies in the patient serum against graft antigens. if these antibodies are found, variable antigen antibody reactions and graft rejection will occur.
there are 2 main techniques for detection of these antibodies, cell-phase and solid phase. the 1st includes complement mediated cytotoxicity and flow cytometry and the 2nd includes ELISA and Bead techniques. the followings are some related details.
1- Complement dependent cytotoxicity (CDC) :
Recipient serum will be mixed with donor T and B lymphocytes then a complement is added. if reaction occurs, the complement will be activated and cause a degree of cell lysis. 20 % was taken as the minimum cut-off for a +ve result. the sensitivity of this test can be increased by adding anti-human globulin to this reaction as multiple AHGs can bind to a single DSA.
Limitations of this test are due to either being false -ve or false +ve. false -ve occurs if DSA are of very low titre or of non-complement fixing types. false +ve occurs due to presence of auto-antibodies as these abs are of IgM and can mediated complement activation. However this can be prevented by adding Dithiothreitol (DTT).
As B cells epress both class I and II HLA antigens and T cells express only class I antigens, therefore +ve T-cell crossmatch alone means a technical error and +ve B-cell crossmatch alone means DSA to class II antigens and not consistently associated with rejection. rejection will definitly occur with +ve both cell types cros match.
2- Flow cytometry: using impedence flow cytometer
it is more sensitive than CDC as it detects low levels of abs.
also, it detects 2 extra types of abs namely, non-complement fixing and non- HLA abs.
however, the clinical significance of such detection of these abs, is a matter of debate in managing transplantation.
3- Solid phase immunoassay :
it is more specific for HLA antibodies, so it eliminates false +ve antibodies detected by CDC and FC.
it includes the following :
A- ELISA :
it depends on using HLA glycoproteins fixed into microtitre wells. serum will be added to these wells then washing to finally get an accurate estmate of DSA.
it is more sensitive than CDC.
B- Luminex technology :
it has replaced ELISA.
it is more sensitive than CDC and FC
C- C1q assay :
it is a new modification of Luminex.
it detects both complement dependent and non-complement dependant antibodies.
D- Epitope matching :
it is more specfic to certain epitopes on HLA molecule.
it is more predictive of cross match results.
4- Virtual cross match :
depends on comparing anti-HLA antibodies, detected by Luminx assay, to HLA profile of the donor.
it will define unacceptable HLA antigens so some donors can be excluded.
amiri elaf
3 years ago
* Renal transplantation is the best solution for patients with end stage renal disease (ESRD).
* Immunological methods like human leukocyte antigen (HLA) typing ,crossmatch and antibodies screening play important role in improving the outcome of kidney transplantion.
* HLA TYPING
HLA typing is recognition of foreign HLA antigen by recipient T lymphocytes and trigger of immune response.
*methods:
*Serological typing
A tray containing sera with antibodies
to a multitude of known HLA alleles is used, recipient lymphocytes are then introduced into the tray wells, the antibodies can bind to the antigens on the surface of lymphocytes, complement is activated when it added, and this result in cell death, allowing the dye to enter the cell that can be visualized microscopy.
Through a process of comparison and elimination of positive wells the HLA type is assigned.
* Advantage of serologic typing :
-Short period and less cold ischemia time this is important in deceased dononation.
-Ability to differentiate HLA alleles
that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression“null” HLA alleles are of less immunological significance.
*Disadvantage
-Lack of sera with antibody specificities
that are capable of identifying the ever-growing number of HLA alleles ( HLA-Cw, DQ, and DP) that may have clinically significant effects on the outcomes of allografts.
-Not detect differences in HLA protein small amino acids.
* Molecular typing :
– Sequence-specific primer polymerase chain reaction:
Extracted DNA from the subject is amplified in several wells , each well has primers that are complementary to specific HLA alleles, this is then instilled into an agarose gel and undergoes electrophoresis where they appear as a band, then matching of amplificated primers to the DNA sequences of several alleles.
-Sequence specific oligonucleotid probes:
Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles, unique HLA alleles are then identified using fluorescent tags.
– Direct DNA sequencing:
This method determines the precise order of nucleotides in the gene of interest, using published HLA allele sequences by comparison.
* Advantage:
– Identify differences in HLA antigen between donor and recipient with detail of amino acid level and epitopes
– HLA typing based on polymerase chain reaction (PCR) is highly specific, with no cross-reactivity.
*( Cross-reactivity is the identification of an allele which is essentially similar to the allele of interest).
* Disadvantage:
New alleles not currently on the HLA sequence databank will fail to be identified.
* HLA ANTIBODY SCREENING
– The usual route for sensitisation towards HLA antigens occurs in three instances:
pregnancy, post blood transfusion and prior transplantation.
_ Preformed antibodies increase the chances of immunological failure even low titers of DSA by causing positive crossmatches.
-Successful transplantation is possible
by employing strategies such as desensitisation, paired exchange and acceptable mismatching.
– Different methods used for HLA antibody screening:
* Cytotoxic (cell-based) antibody screening:
A set of cell donors are randomly selected to be representative of a population ,each panel consists of around 30 to 40 different donor lymphocytes.
The method is similar to that of serologic typing, recipient serum is mixed with cell donor lymphocytes with complement and dye.
If the serum antibodies bind to the cell surface receptors and activate complement pathways, this leads to cell death and subsequent uptake of dye.
The degree of cell death is quantified using as percentage PRA (panel reactive antibody).
*Disadvantage:
– The PRA percent can be different numerically with out corresponding change in the type or amount of antibody.
– False positive results can be produced due to nonHLA antibodies, autoanti- bodies and non specific IgM antibodies.
– False negative results are possible
as this is purely complement dependent that requires higher antibody titres to be activated.
– Antibody specificities and unacceptable antigens cannot be identified using this method as there are several antigens in each well.
* Solid phase antibody screening:
– This method employs soluble or recombinant HLA molecule instead of lymphocytes targets
– The variants of these methods are:
* Enzyme-linked immunosorbent assay platform:
– Purified HLA molecules are applied to
(ELISA) platforms and will bind individually to HLA antibody after the
addition of recipient serum, enzyme conjugated antibodies to IgG is added to detect the presence of HLA antibody in the serum that bound to the antigen. Detection is performed by optical density reading.
* Microbead platform/single-antigen beads:
– Pooled panel beads with several different class Ⅰ or Ⅱ HLA antigens on a bead give a positive or negative result. -The phenotype or also known as ID beads are individually coated with class
Ⅰ or Ⅱ HLA antigens of an individual patient-derived cell line.
– Microbead that is fluorescent dye conjugated is then added to detect the presence of HLA antibod using a flow
cytometer or single-antigen beads (SAB) Luminex® platform.
These estimate PRA by the proportion of positive beads. SAB are individuall coated with a single HLA antigen and yield alist of distinct antibody specificitied Specificities are subsequently compared with HLA frequencies in the donor .
-To determine the calculate panel reactive antibody (cPRA), this give estimate of the of a positive crossmatch/donor specific antibody to a randomly selected donor.
– cPRA is based on unacceptable HLA antigens that patient has been sensitized to give high probability of a positive
crossmatch.
* CROSSMATCHING (XM)
– The cytotoxic assay was implemented as the requisite test prior to transplantation when it was shown that
recipients with DSA had significantly higher rates of allograft failure due to hyperacute rejection as well as
primary failure.
* Complement-dependent cytotoxicity crossmatch:
-Similar to cytotoxic assay the complement dependent cytotoxicity crossmatch is interpreted as positive if a
considerable number of lymphocytes are destroyed after the incorporation of complement.
(CDC-XM) can be done for B and T lymphocytes.
– Sensitivity is limited if the relevant antibody is in low titres, but this can be overcome by increasing the incubation
time, the use of the AHG-enhanced method as well as additional wash step.
-False negative due to low titre antibody. -False positives by detecting autoantibody IgM/IgG HLA or non-HLA.
* Flow cytometry crossmatch( FCXM): Detects DSA independent of complement – fixation.
– Recipient serum is mixed with donor
lymphocytes and then tagged with a fluoro chrome conjugated anti-IgG antibody.
– Several antibodies with separate fluorochromes particular to B and T lymphocyte surface proteins can be added with the use of flow-cytometry, B and T lymphocytes can be readily
identified and have their DSA individually interrogated.
– Compared to complement dependent cytotoxicity crossmatch this offers greater sensitivity.
* Virtual crossmatching (VXM):
– In(VXM) both donor HLA typing and solid phase antibody screening are utilised together .
– VXM lead to shorter wait times and improved outcomes for sensitised transplant recipients.
– VXM permits transplant physicians
to consider a potentially positive
crossmatch a risk factor for donor selection.
– Titres, specificities, and presence or absence of antibodies could significantly vary over time.
– False positive results of VXM may arise where there are significantly low titre and/or non complement binding antibodies resulting in the wrong exclusion of donors.
– Give false negative results due to the list of all HLA donor antigens have been classed differently and can not be correctly represented.
– Does not identify the HLA “Null” alleles.
* DEFINING RISK
– High immunological risk
At the time of transplantation there are high titres of DSA and this can lead to hyperacute rejection.
– Intermediate immunological risk
At the time of transplantation, there is a low titer of DSA and historic DSA is not detectable.
– Standard immunological risk
Where there is no evidence of donor directed sensitisation to HLA.
mai shawky
3 years ago
· Immunological risk stratification in kidney transplantation is crucial to determine risk of rejection, tailor immunosuppressive regimens and expect the graft and patient survival.
· This includes 3 steps:
o HLA typing of recipient.
o HLA antibodies screening (DSA detection)
o Cross matching.
· HLA typing either by:
o Serological method: adding recipient lymphocytes to sera containing multiple wells with addition of complement, then detection of complement dependent cell lysis. It is rapid and useful in deceased donor, but does not detect HLA type Cw, DP or DQ which may affect transplant outcome.
o Molecular technique: more accurate and use DNA probes and PCR reactions. It includes:
Sequence specific primer PCR.
Sequence specific oligonucleotide probs.
Direct DNA sequencing.
o Mainly we stress on HLA A, B and DR.
· Screening for Anti HLA antibodies (DSA) done by 2 methods either:
o Complement dependent cytotoxicity (CDC);
Similar to serological method of HLA typing.
Adding recipient serum to donor lymphocytes, with addition of complement and detect lymphocytes lysis.
Determine PRA (reaction of recipient serum to many tested donor lymphocytes, about 50-100 variable lymphocytes).
o Solid phase antibody screening: it is more sensitive.
It utilizes HLA molecules common in donor population rather than donor lymphocytes
. It incudes:
– ELISA.
-Flowcytometry.
-Luminex SAB (use beads coated with HLA type I and II antigens)
§ It determines cPRA which represents the unaccepted antigens.
o MIF: mean fluorescent intensity:
-represent semiquantitative assay of DSA strength.
-Correlates with risk of AR, but no absolute CT to transplantation.
– MIF accepted if > 2000 for class I and > 5000 in class II HLA.
· Cross matching (XM):
o It detects preformed DSA in recipient serum against donor lymphocytes.
o It means either Mixing both donor lymphocytes and recipient serum (real XM) or by matching results of donor HLA typing and solid phase assay of DSA to expect actual invitro cross match (virtual XM).
o Positive cross match against T lymphocytes is an absolute contraindication to transplantation unless desensitization protocols as (plasmapheresis, IvIg or rituximab is used and the cross match turn negative together with close monitoring of DSA after transplantation.
o While positive crossmatch against B cell may be relative CI. It is considered mainly in retransplantation or negative auto cross match.
o Real cross match either:
-CDC: detect lysis of donor lymphocytes on adding recipient serum and complement. It is done for both B and T lymphocytes. False negative results if low titer of DSA or non-complement dependent antibody fixation. In addition, false positive results if patient has auto antibodies as in SLE (avoided by autocross match test) or IgM antibodies (avoided by adding dithiothreitol (DTT).
– Flow cytometry: more sensitive, it detects non complement depend fixing antibodies by using fluorescenated anti IgG antibodies attach to antibodies reacting against B or T lymphocytes.
o Virtual XM: It is not 100 % accurate.
· Based on above provided information, transplantation immunological risk can be classified into;
o High risk: high titer of DSA, better avoid transplantation as hyperacute rejection and graft loss is inevitable.
o Intermediate: low titer of DSA, need induction therapy and strong immunosuppressive protocol.
o Standard risk: no DSA and need standard immunosuppressive protocol, not essentially induction therapy.
MOHAMMED GAFAR medi913911@gmail.com
3 years ago
RIVEW OF HLA TPING METHODS AND DIFFERENT CROSS MATCH METHODS
FIRST TO DISCUSS HLA TYPING METHODS,
A-Serologic Typing: lymphocytes are taken from the person who has HLA antigen on its surface mixed many serum and all incubated with complement, and if the complement activated due to presence of antibody in the serum that leads to formation of complement complexes and cell lysis and death and dye enters the cell which can be read by special microscpe.
Advantages; rapid results, less immunological relevance
Limitation; unable to find high quality serum with increasing the importance of HLA subtypes like HLA DQ, HLA CW, which in many studies started to show some immunological responses and affect the graft survival by rejection episodes down the road.
B-Molecular Typing: sequence specific primer polymerase chain reaction (SSP-PCR), sequence specific oligonucleotide probes (SSOP).
Molecular typing gives us detailed idea about the difference between the donor and serum HLA, to the extent of suballels. Which in turn give us detailed information about the mismatches between them which affects the graft down the road and help us expecting the rejection episodes and may help us choosing the immunosuppression protocol?
SECOND TO DISCUSS DIFFERENT CROSS MATCH TECNIQUE;
Different cross match techniques are available in many centers around the world , pros and cons of each one will be discussed briefly in the section below,
1- Complement-Dependent Cytotoxicity Crossmatch Methods: recipient Serum antibodies will be mixed with donor lymphocytes (b cell and t cell), if the reaction occurs with help of complement, then cell lysis will occur and cross match will be positive. and important note should be mentioned here HLA CLASS 1,2 are present on b lymphocytes and only class 1 present on t lymphocyte.
Cons: Miss low titer antibody.
Pros: detect non-HLA IgG antibody, autoantibody, or IgM HLA/non-HLA antibody.
2-Flow Cytometry Crossmatch Methods: recipient serum which has different types of antibodies are mixed with donors lymphocytes , and conjugated ig antibodies colored with special dye and if the recipient serum has specific antibodies to the donors ,conjugation with ig happened and there is special flow cytometer which has baseline number can detect the number above the limit which is considered positive for cross match.
PROS: detects non complement antibodies, and sensitive for low titre antibody.
CONS: may be false positive for patient, who had received before any monloclonalantibdies like rituximab.
3-Applications of Antibody Screening/PRA Testing: pra once introduced in clinical practice in kidney transplantation made a big difference in the descion of physician’s regarding each clinical scenario they face.
What is pra ? it a collection of about 10000 serum HLA antibodies from different donors which mostly hade all the known HLA discovered or known till know ,
Each recipient serum will be mixed with these serum HLA antibodies presented as beads in machine which represent in numbers that give us an idea about the recipient antibodies in his blood, as he may be exposed to any procedure before that provoked his immune system to perform antibodies like blood transfusion or transplantation before and for females’ pregnancy also.
The more percentage that the recipient has in his report the less likely transplantation , for example the patient has 80% pra that means that he has 80% antibodies to the donors that he will be offered.
For this kind of patient, with high pra , the paired exchange program are offered if they have positive cross match with any living donors and also the will be on the top of the list of deceased list .
4-Virtual Cross matching: (VXM) is not a true crossmatch. it is just an application comparing the recipient HLA PRA screening with the donors HLA , as predictors of the actual crossmatch
Limitations: antibodies are dynamic in the body, they are changing from time to time may be cleared by the immune system and may return back .so updated samples of the patient or the recipient serum may be needed every three month not to miss any new antibody .
Pros and cons of vxm
False positive readings: in non-complement antibodies
False negative readings: due to variable HLA typing alleles, detailed HLA of the donor serum should be carefully read as HLA pra are not fully presented as mentioned before in the pra method, which may give us false positive readings.
Last edited 3 years ago by MOHAMMED GAFAR medi913911@gmail.com
Amit Sharma
3 years ago
I. Human leukocyte antigen typing and crossmatch: A comprehensive review
Please summarise this article with emphasis on the different techniques of tissue typing and crossmatching.
This review article deals with reviewing the Human Leukocyte Antigen (HLA) typing and crossmatch techniques with respect to an index case of End Stage Renal Disease (ESRD) with lupus nephritis as the basic disease having a positive B cell complement dependent cytotoxicity (CDC) crossmatch and a negative flow cytometry crossmatch (FCXM) in absence of Donor Specific Antibodies (DSA). This false positive result was due to the autoimmune disease, i.e. SLE.
HLA typing techniques include serological assay and DNA based molecular methods.
a) Serological assay: This method utilizes antibody-dependent cell mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Recipient lymphocyte is added to tray with wells containing serum with antibodies, complement and a dye leading to cell lysis which is observed under a phase contrast microscope. Results are rapidly available leading to a decreased cold ischemia time. They do not identify ‘null’ alleles (having low immunological significance). These methods are not used nowadays due to limitations like lack of commercially available serum containing specific antibodies against different HLA alleles, esp. HLA Cw, DQ and DP. They are not able to detect difference in HLA protein small amino acids.
b) Molecular methods: These are DNA based methods which are more sensitive, more accurate and have higher resolution, ultimately helping in better HLA typing. These methods include: i. SSP (Sequence Specific Primer): Extracted DNA from patient is mixed with primers complementary to specific alleles in wells forming an amplification product (using PCR) which, on an agarose gel electrophoresis, forms a band helping in HLA typing. ii. SSOP (Sequence Specific Oligonucleotide Probe hybridization): Amplified DNA is mixed with oligonucleotide probe complementary to specific DNA segment of different alleles which are identified using fluorescent markers leading to HLA typing.
iii. Direct DNA sequencing: With his method, the exact order of nucelotides in the gene can be understood and the HLA allele sequences can be comared to assign the HLA type.
Molecular methods are more specific but time consuming. Another drawback is with respect to absence of newly identified alleles in the HLA sequence databank.
HLA antibody screening: This is especially important in patients with history of sensitization, like prior pregnancy, blood transfusion of transplantation.
Methods for HLA antibody screening include cytotoxic (cell-based) and solid phase antibody screening.
A) Cell based (cytotoxic) method: It involves mixing recipient serum in a well containing 30-40 different ‘donor’ lymphocytes serum with complement and a dye leading to cell lysis (if antibodies are present in the recipient serum) which is observed under a phase contrast microscope. The degree of cell lysis is expressed as panel reactive antibody (PRA). Disadvantages with this method include lack of adequate representation of all the HLA types present in the population; presence of false postive values due to autoantibodies, IgM, and non-HLA antibodies. False negative results can be seen in conditions with low antibody titres. Prices documentation of unacceptable antigens cannot be done with this method.
B) Solid phase antibody screening: It involves using HLA molecules in place of lymphocytes.
1) Enzyme linked immunosorbent assay (ELISA): It is a 3 step procedure in which recipient serum is added to HLA glycoprotein labelled microtiter wells and after giving a wash, anti IgG with a passenger reporter molecule (alkaline phosphatase) is added followed by a wash. The third step involves addition of a substrate which undergoes a color change (due to dephosphorylation by the reporter molecule) if antibodies are present.
2) Microbead/ single-antigen bead technology: It is a very sensitive method in which recipient serum is added to beads labelled with fluorescein (reporter dye) and incorporated with HLA molecules. After giving a wash, Anti IgG lebelled with phycoerythron (detector antibody) is added and the result can be visualized using 2 laser beams, each detecting the reporter dye and specific bead. The results can be interpreted as either channel shift associated with the antibody binding (flowcytometry) or degree of fluorescence (mean fluorescence intensity, MFI) using Luminex method. It is useful in conditions with low level of DSA, non-HLA antibodies and non-complement binding antibodies, seen in a situation whereby CDC crossmatch comes out negative. The drawback with this method is that the bead kits available might not be representative of the HLA antigens in the community. They are specific, rapid, efficient, have increased range of identification (upto 11 HLA loci) and enable virtual crossmatch. But these methods are too sensitive, the antibodies detected might not have a clinical relevance. A false negative value can be seen due to high titre antibodies (prozone phenomenon), presence of IgM, IVIG use and due to epitope sharing (leading to reduction in MFI).
Microbead assays are more specific than ELISA based assays, which, in turn, are more specific than the cell based assays.
Crossmatches are used to identify the risk of graft rejection in a prospective transplant recipient.
Crossmatch techniques can be divided into cytotoxic (cell based) assays and virtual crossmatch.
A. Cell-based assays: These are of 2 types.
1) CDC technique: In this, complement is added to a mixture of recipient serum and donor lymphocytes (T and B cells separately). If donor-specific antibody (DSA) is present, it will bind with the lymphocytes, complement will get activated and cell lysis will take place, giving a positive result. To increase its sensitivity, anti human immunoglobulin (AHG) can be added to the mix. A positive result can be given either on the basis of a cut-off value of >20% cell lysis (semi-quantitative), or a titred value of dilution required to get a negative result (for quantification). Presence of autoantibodies give a false positive result, addition of DTT (ditheothreitol) inhibits IgM mediated complement activation. A false negative result is seen if the DSA level is low, or in presence of non-HLA activating antibodies.
A positive T and positive B cell CDC crossmatch denotes DSA to HLA type I and II antigen. A negative T with positive B cell CDC crossmatch denotes DSA to HLA type II or low level DSA to HLA type I antigen. A positive T with negative B cell CDC crossmatch denotes a technical error.
2) Flowcytometry Crossmatch (FCXM): This is a test with higher sensitivity.In this, a fluorescein labelled anti IgG antibody is added to a mixture of recipient serum and donor (T and B) lymphocytes. A flowcytometer is used to detect the lymphocytes bound to the anti IgG labelled DSA. It is useful in conditions with low level of DSA, non-HLA antibodies and non-complement binding antibodies, seen in a situation whereby CDC crossmatch is negative, while FCXM is positive. So, it is useful in sensitized individuals. The result is expressed as either a measure of fluorescence intensity as a ratio of control (channel shift), or a titred value of dilution required to get a negative result (for quantification).
3) Virtual crossmatch (VXM): It involves utilizing both donor HLA typing and the recipient solid phase assay antibody screening together. The HLA of the donor is compared with the antibodies present in the historical recipient sera. The advantage with VXM is that it is a rapid, on paper, crossmatch and can be done while the donor is still being evaluated. The drawback includes false positivity in patients with low antibody titre and non-complement binding antibodies.
Last edited 3 years ago by Amit Sharma
Tahani Ashmaig
3 years ago
HLA typing:
HLA typing is a crucial step in renal transplantation.
Methods of HLA typing includes serological typing & molecular typing
A.Serological typing:
In this method recipient lymphocytes are introduced into a tray wells which contain anti HLA antibodies. Then complement is added. When the antibody bind to the antigens on the surface of lymphocytes; the complement is activated. This results in cell lysis and death which allow the dye to enter the cell. Tray wells are then identified under phase contrast microscopy.
Pros:
1. Results are available in a short period so it’s important in deceased donor renal transplantation.
2. Offers the ability to differentiate ” Null” HLA alleles.
Cons:
1. Aren’t capable of identifying the ever-growing number of HLA alleles due to lack of sera (eg: HLA-Cw, DQ, and DP antigens)
2. Can’t detect differences in HLA protein small amino which may trigger potent immunological responses.
B.Molecular typing:
These are many but all of them depend on DNA extraction.
Types:
1) Sequence-specific primer polymerase chain reaction (SSP-PCR):
Steps:
i. DNA extraction
ii. PCR to acquire amplified DNA of specific primers.
iii. Gel electrophoresis by installing the amplified allels into an agarose gel where they appear as bands.
iv. Matching primers of the amplification product to DNA sequences of several candidate alleles.
2) Sequence specific oligonucleotide probes(SSOP):
Steps:
i. DNA extraction
ii. DNA amplification.
iii. Mixing of the amplified DNA with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles.
iv. HLA alleles identification using fluorescent tags.
v. HLA type is then assigned using available HLA allele sequences.
3) Direct DNA sequencing:
This method determines the precise order of nucleotides in the gene of interest.
Using published HLA allele sequences, HLA type is subsequently assigned by comparison.
Pros and cons of HLA typing based on PCR
Pros:
1. Can identify differences in HLA antigen between donor and recipient with detail to the amino acid level.
2. Highly specific where specific alleles are identified with no cross-reactivity.
Cons:
Not all Molecular techniques can identify new alleles (eg: SSP & SSOP).
Notes:
▪︎HLA-A, HLA-B and HLA-DR are considered as the most important ones in kidney transplantation.
▪︎Eurotransplant and old United Kingdom transplant data suggest that HLA.DR matching has a far greater effect than HLA-A or HLA-B.
▪︎ Data from the United Network for Organ Sharing (UNOS) registry highlighted the significance of paying attention to having the least number of mismatches.
HLA antibody screening:
Causes of HLA antibodies formation:
i. Pregnancy
ii. Post blood transfusion.
iii.Prior transplantation.
▪︎Methods used for HLA antibody screening:
A. Cell based (Complement Dependent Cytotoxicity):
In which recipient serum is mixed with donor cell’s lymphocytes and adequate complement. If there’s DSA complement is activated which result in cell death and uptake of dye. The degree of cytotoxicity is expressed as percentage PRA.
▪︎Limitations:
1. PRA percent can be different numerically without a corresponding change in the type or amount of antibody which largely depends on the cell panel used.
2.False positive results can be produced due to non HLA antibodies, autoantibodies and nonspecific IgM antibodies.
3. False negative results are possible due to low antibody titres.
4. Complete lists of antibody specificities and unacceptable antigens cannot be identified as there are several antigens in each well.
B. Solid phase antibody screening:
This depends on recombinant HLA molecules instead of lymphocytes
(to avoid false positivity due to non- HLA antigens in the lymphocytes).
▪︎Methods:
1) Enzyme-linked immunosorbent assay platform. Steps:
· Purified HLA molecules are applied to ELISA platforms.
· Then the recipient serum and enzyme conjugated antibodies to IgG are added.
· Detect the presence of HLA antibody by optical density reading.
2) Microbead platform/single-antigen beads: Types:
2.1)Screening test:
· A panel beads with several different class Ⅰ or Ⅱ HLA antigens on a bead are used.
· It gives a positive or negative result.
2.2)The phenotype (ID beads):
· Beads are individually coated with class Ⅰ or Ⅱ HLA antigens of an individual patient-derived cell line.
· Microbead which is conjugated with fluorescent dye is then added to detect the presence of HLA antibody in the serum.
· Fluorescence detection can be done traditionally using a flow cytometer (Flow PRA®) or via single-antigen beads (SAB) Luminex® platform.
· These estimate PRA by the proportion of positive beads.
Notes:
· SAB are individually coated with a single HLA antigen and yield a list of distinct antibody specificities which are subsequently compared with HLA frequencies in the donor population to determine the calculated panel-reactive antibody (cPRA).
Pros:
1. Can discriminate between immunologically relevant positive crossmatches from false-positive results.
2. Microbead assays (both Flow PRA® and Luminex®) are more sensitive for lower titre antibody than ELISA( which is ten percent more sensitive compared to anti-human globulin enhanced cytotoxicity based assays).
3. Specific for anti-HLA antibodies.
4. SAB assays are rapid with results available in 3-4 h.
5. Quite efficient in a single reaction. (chamber up to one hundred unique antigen beads can be tested).
6. Permits testing many patients simultaneously.
7. Results from SAB enable virtual crossmatching (VXM) to identify DSA pre-transplant.
8. SAB assays permit identification of anti-HLA antibodies for all common
and numerous rare antigens and alleles.
Cons:
1. They detect both complement and non-complement dependent Abs.
2. Can detect antibody that is below the threshold associated with a positive crossmatch which may not always have clinical implications.
3. Can not detect non- HLA antibodies.
4. Give false results in these conditions:
– Prozon phenomenon whih can affect
SAB – Luminex® assays
– Binding of IgM antibodies or other
serum factors to the beads.
– Nonspecific binding by serum proteins
and drugs such as IVIG.
– Epitopes sharing
3)CROSSMATCHING (XM)
· The presence of DSA depicted as a positive crossmatch was a contraindication to transplantation.
· Crossmatch tests will identify if a recipient had antibodies to a specific donor cells.
· T cell cytotoxic crossmatch had a twenty percent false positive rate and a four percent false negative rate. Therefore, it is insufficient to identify all relevant antibodies.
· The solid-phase antibody test should be used together with crossmatch results to identify those that are immunologically relevant.
3.1) Complement-dependent cytotoxicity crossmatch
Similar to cytotoxic assay the complement-dependent cytotoxicity crossmatch is interpreted as positive if a considerable number of lymphocytes are destroyed after the incorporation of complement (Figure 2A in the paper) .
– Can be done for B and T lymphocytes.
Limitation:
1. Sensitivity is limited if the relevant
antibody is in low titres.
2.Can give false positives by detecting
autoantibody, IgM/IgG HLA or non-HLA.
3.2)Flow cytometry crossmatch (FCXM):
· Detects DSA independent of complement fixation.
· In this method, recipient serum is mixed with donor lymphocytes and then tagged with a fluorochrome conjugated anti-IgG antibody.
· Several antibodies with separate fluorochromes particular to B and T lymphocyte surface proteins can be added.
· With the use of flow-cytometry, B and T lymphocytes can be identified and have their DSA individually interrogated
Advantages: It is more sensitive than CDC-crossmatch.
3.3)Virtual crossmatching (VXM):
· Both donor HLA typing and solid phase antibody screening data are utilised together.
Pros:
1. Shorter wait times.
2. Improved outcomes for sensitised transplant recipients.
3. Permits transplant physicians to consider a potentially positive crossmatch (a risk factor for donor selection).
Cons:
1.Titres, specificities, and presence or absence of antibodies could significantly vary over time. Thus, the use of antibody specificity from historical serum sample (earlier than six months) could not predict a crossmatch with certainty.
2. False positive results of VXM may arise where there are significantly low titre and/or non-complement binding antibodies, thereby, resulting in the wrong exclusion of potential donors.
3. Give false negative results due to the fact that the list of all potential HLA donor antigens have been classed differently and, therefore, can not be correctly represented.
4. Does not identify the HLA “Null” alleles.
Ben Lomatayo
3 years ago
Introduction; The knowledge of cross-match and HLA typing is important to have a good transplant outcome. Explaining cross-match results can very difficult for those clinicians without basic course in applied transplant immunology.
HLA typing ; This is important because donor HLA is recognised as a foreign by recipient T lymphocytes. Methods are ;
Serological typing ; A tray full of sera with antibodies to a large numbers of HLA alleles is the basic of this test. It uses complement which causes cell death and uptake of the dye by dead cells as seen by microscopy.
Advantages ;a) the result is fast which means minimization of cold ischemia time b) identification of null alleles (10) Disadvantage ; a) Absence of sera with antibodies specificities against increasing numbers of HLA alleles(10) b) they are rare serological assays for HLA-cw,DQ, and DP loci b) inability to identify differences in HLA small amino acids proteins. 2 Molecular typing ;
Sequence-Specific Primer Polymerase Chain Reaction ; Here DNA from the sample is amplified in many wells. Every well contain primers complementary to the specific HLA alleles
Sequence Specific Oligonucleotide Probes ; Amplified DNA is added to oligonucleotide probes complementary to specific parts of DNA of different alleles
Direct DNA Sequencing ; It establish the precise order of nucleotide in the gene of interest
Advantages ; Identifications of differences in HLA antigen between donor & recipient
HLA antibody Screening ; this is important to avoid immunological failure and positive cross-.match test. Methods are ;
Cyto-toxic antibody screening (cell- based); The principle is the same as serologic typing but recipient serum is mixed with donor lymphocytes. Complement causes cell death and the dye enters the cells. This complement dependent cyto-toxicity is measured as panel reactive antibodies( PRA).
Disadvantages ;a) Variations in the value which affecting type or amount of antibodies b) Uses commerial kids which may not give the true picture of the population c) False positives e.g. IgM (26-29) d) False negative if low antibody titre(29) 2 Solid phase assay ; It utilises soluble or recombinant HLA molecules and not lymphocytes. Methods are ;
ELISA ; based on ELISA plate forms
Single Antigen Beads(SAB) : Binding of antibodies to a recombinant HLA beads. Fluorescent tag antibodies is added. It helps to determine cPRA. cPRA indicates the % of donors to whom the recipients are likely to be incompatible.
Advantages ; a) quick test with results in 4 hours b) large numbers of patients can be tested at once c) provide the bases for virtual cross-match (VXM) (37) Disadvantage ; a) it may pick up antibodies with no clinical relevants = high sensitivity b) Cannot detect non-HLA antibodies which are equally important as well(42,43) c) Not all HLA alleles are avialable on the beads e) Artefacts e.g. prozone = false negative due C1q binding. The test can turn positive after dilution (45-46) f) epitope sharing = single antibodie binds to many antigen beads
Cross-matching; posivitive cross-match is generally a contraindications to transplantation.
Complement-dependent Cyto-totoxicity ; The same principle of cell based assay. It has low sensitivity but this can be improve by increasing incubation time, adding Anti-human IgG, and additional wash out step. False positives are seen in autoimmune disease due to IgM or non-HLA Ab
Flow cytometry ; Serum from the recipient is mixed with donor lyphmocytes. This then tagged with fluorochrome conjugated anti-IgG antibodies. This is subjected to flow cytometry and the read out is in form of median channel shift. There is a lots of variations between laboratories and the test is existing in many places around the world (50)
Virtual cross-match ; it is comparing recipients antibodies specificities to the donors HLA antigens. The donor antigens to which the recipients has prefomred antibodies is called unacceptable antigens. Avoid historical serum sample > 6 months use in this test.
Disadvantages are ; a) false negative b) failure to detect null alleles
Abdul Rahim Khan
3 years ago
HLA typing is an essential component of renal transplantation. HLA interpretations can be difficult and various modalities can be used for this purpose with different pros and cons.
HLA typing methds include:
1- Serological
In this method the recipient lymphocytes are mixed with sera containing different know HLA alleles . Null alleles can be exclude easily. These methods do not recognise HLA Cw ,DQ and DP
2- Molecular
These include –
Sequence specific PCR– In this method the DNA from subject is amplified in several wells containing Primes which are complimentary to HLA alleles
Sequence specific oligonucleotide Probes; Precise order of nucleotides of gene of interest can be determined.
Direct DNA sequencing- It can detect significant alleles and also specific alleles without cross reactivity
HLA NTIBODY SCREENING
Cytotoxic antibody screening
In this recipient serum is mixed with 30-40 donor lymphocytes , dye and Compliment . Results are expressed as PRA
Solid phase antibody screening
Uses recombinant HLA molecules instead of lymphocytes
ELISA platform
Microbead platform
Estimates PRA and more sensitive than ELISA. cPRA identifies unacceptable antigens
CROSS MATCHING
Compliment dependent Cytotoxic cross match-It can be done for B and T lymphocytes
Flow cytometry– In this recipient serum is added to donor lymphocytes and Fluorescent tags are used. It can detect DSA independent of compliment fixation .
Virtual cross match-Its combination of Solid phase antibody screening and HLA Typing
Immunological Risk Groups
High immunological risk groups-
Have high level of DSA and can lead to hyper acute Rejection
Intermediate Risk Groups-
Low titres of DSA and may require extensive immuno suppression
Standard Risk group-
No evidence of donor directed HLA sensitization
Innocent lule segamwenge
3 years ago
This paper is an excellent clinical review of HLA typing and crossmatch methods.
HLA Typing
HLA mismatches between recipient and donor represents an immunological burden that the donor graft is likely to be exposed to once transplantation takes place.
The more HLA mismatches between donor and recipient the higher the likelihood for reduced graft survival. For example, 6 mismatches are likely could lead to 64% higher risk of graft failure compared to 13% when there is one mismatch.
There two methods employed in HLA typing; Serological method and Molecular methods.
Serological method; Has the advantage of quicker turnaround of results, which can be useful in deceased donor transplant as it reduced cold ischaemic times.
It is helpful in excluding null alleles which are detectable by molecular techniques but not expressed on the cell surface. The disadvantage is that there may not be commercially available sera to identify newly discovered HLA alleles and also for HLA-C, DQ and DP. Molecular methods; There are 3 different methods of molecular typing.
Sequence specific primer pcr; Extracted DNA is amplified with primers. HLA type is determined by matching primers to HLA sequence.
Sequence specific oligonucleotide probe; HLA is determined by mixing amplified DNA with oligonucleotide probes of different alleles.
Direct DNA sequencing; specific nucleotides are determined and HLA is assigned based on known HLA allele sequences.
HLA antibody screening
This can be done using Cell based and Solid phase methods. Cytotoxic antibody screening;
Recipient serum is tested against a set of 30 to 40 different donor lymphocytes, dye and complement are added. Cells that bind antibodies in the recipient’s serum die and take up die. The frequency of this reaction happening with the different cells is expressed as the Panel reactive antibody (PRA). Solid phase antibody screening
This done either by ELISA or Single antigen beads
The single antigen bead method involves commercially prepared beads which are coated with Class I or II HLA molecules. When mixed with the donor serum, specific antigen reactions are determined.
The antigen specificities are then compared with HLA common in the donor population to determine the calculated panel reactive antibody(cPRA).
Unlike PRA, cPRA is actually based on antigens the donor is likely to be sensitized to (unacceptable antigens).
Crossmatching Complement-dependent cytotoxicity crossmatch (CDC) Similar to cytotoxic assay, done on B and T lymphocytes and indicated presence of complement dependent cytotoxicity of lymphocytes to which the donor may be sensitized to and has antibodies directed against them.
False negative results can be obtained if the antibody titres are low or if the recipient has antibodies that do not fix complement.
False positive results can be obtained in the presence of autoimmune antibodies or IgM antibodies. This can be corrected for by doing auto crossmatch test and addition of dithiothreitol (DTT) respectively. Flow cytometry crossmatch
Detects donor specific antibodies independent of complement fixation.
Utilises fluorochrome labelled anti-IgG antibody which attaches to antibodies that have reacted to B or T lymphocytes. This makes it more sensitive than CDC crossmatch. Virtual crossmatch
Uses both HLA typing and Solid phase antibody detection. This information identifies the donor HLA type and the DSA that he possesses. This information is then used to work out what the real crossmatch would turn out to be if performed as the donor unacceptable antigens are known and the associated DSA is determined. In conclusion, this is a useful summary of the different HLA typing methods, screening for preformed DSA and Crossmatch methods.
Theepa Mariamutu
3 years ago
Human leukocyte antigen typing and cross match: A comprehensive review
Crosshatch and HLA typing plays a crucial role in improving outcome of organ transplantation and afford better matches to recipient.
HLA typing
Serological typing
• Recipient lymphocytes are introduced into the tray well contacting sera, complement and dye
• Antibodies in the tray will bind to the antigens on the surface of lymphocytes and complement activated which will induce cell death.
• Benefit
o able to produce results quickly and important in DCD transplant where the cold ischemia time can be reduced
o Able to differentiate HLA alleles with no cell surface antigen expression -null HLA
• Disadvantages – lack of sera with antibody specificities that’s are capable of identifying the ever growing number of HLA alleles
• HLA Cw,DQ and DP antigen may have clinical benefit but are scarce for these loci.
Molecular Typing
Sequence -specific primer polymerase chain reaction
• Extracted DNA is amplified in several wells in which each well has specific HLA alleles
• In wells, amplification product is formed when DNA probes are complementary to the specific sequence of HLA molecule
• Band will be formed when it is instilled into an agarose gel and undergoes electrophoresis
• Highly specific where specific alleles are identified with no cross reactivity
• Cross reactivity – identification of alleles that are similar to alleles of interest
Sequence specific oligonucleotide probes
• Amplified DNA is mixed with oligonucleotide probes that compatible to specific DNA of different alleles
• unique HLA alleles will be identified by fluorescent labels
Direct DNA sequencing
• identify the exact order if nucleotides in the interested genes.
• According to HLA alleles sequencing, HLA type is assigned by comparison
HLA mismatches
• CTS- HLA DR and HLA B antigens offer the most alloimmune burden with less from HLA A
• Eurotransplant and UK transplant data (old)- HLA DR mismatches has far greater effect than HLA A or HLA B
• Owen WF et al showed HLA DR mismatching had most effect during first 6/12 post TX while maximal effect of HLA B mismatching occurred 2 years post TX
• UNOS – 6 mismatches -64% higher risk then 1 mismatches 13% only
• Taboo mismatches – 81% one year survival and 50% 5 year survival
HLA antibody screening
Cytotoxic antibody screening
• Representative of the population of potential deceased donors – consists of 30-40 different donor lymphocytes
• The degree of cytotoxicity is shown as PRA %
• Limitations
o depend on the cell panel used
o HLA frequencies and racial differences need to be factored
o False positive – non-HLA antibodies, autoantibodies non-specific IgM antibodies
o False negative- requires higher antibody titres to be activated
Solid phase antibody
• Uses soluble or recombinant HLA molecules instead of lymphocytes targets
ELISA
• Purified HLA molecules – applied to ELISA and will bind individually to HLA antibodies and detected by optical density reading
Micro bead platform/ SAB
• Fluorescence detection thru flow cytometer ( FLOW PRA) or via the single antigen beads (SAB) luminex platform
• SAB- rapid and results available in 3-4h
• Enable virtual XM
• Allow identification of anti-HLA antibodies for all common and numerous rare antigens and alleles up to 11 HLA loci
PRA vs cPRA
• PRA- translate into high probability of a positive crossmatch
• cPRA- based on unacceptable antigens -that those the patient is sensitised to
Crossmatching (XM)
• presence of DS cytotoxic antibody – positive XM -contraindication to TX
• 20% false positive rate and 4% false negative rate
• So used with SAB
CDCXM
• Positive – large amount of lymphocytes are destroyed – suggest significant amount of DSA found
• Done for B cell and T cell
• Sensitivity is limited – can improve by increasing the time of incubation and use of AHG-enhanced method
• Negative – 18% graft loss in 1 year, positive 36% graft loss
FCXM
• Detect DSA independent of complement
• Accurately detect the presence or lack of IgM/IgG on donor lymphocytes
• Greater sensitivity than CDCXM
Virtual XM
• The use of data to XM and forecast the actual in vitro crossmatch
• Lead to shorter time for waiting and improved outcomes for sensitised transplant recipient
• Does not identify null HLA
The clinical scenario
Positive CDC, negative FXCM ands SAB showed standard immunological risk, so can proceed with transplant
Heba Wagdy
3 years ago
HLA typing:
HLA matching has a significant effect on graft survival. Serologic techniques: Pros: Quick results which decrease ischemia time in deceased donor transplant determine “null” HLA alleles (of low immunological significance as has identifiable DNA sequence with no cell surface antigen expression) Cons: The available sera can’t identify all HLA alleles can’t identify HLA-cw, DQ and DP antigens which are clinically significant can’t detect small amino acids in HLA protiens which may be antigenic Not used now Molecular techniques:
Sequence-specific primer PCR
Sequence specific oligonucleotide probes
Direct DNA sequencing
Pros: detect significant alleles and differentiate between donor and recipient at level of antigens, epitopes and amino acids. Detect specific alleles without crossreactivity. Cons: Can’t identify new alleles that are not on primers used in HLA-typing Studies showed that: HLA-DR and B antigen mismatches have higher risk than HLA-A mismatch. HLA-DR mismatch has the highest immunological risk. The effect of HLA-DR mismatch occurs post transplant in the first 6 months while the effect of HLA-B mismatch appears 2 years post transplant Data from UNOS registry showed that increased number of mismatches increases the risk of transplant failure.
HLA antibody screening
Detection of preformed anti-HLA antibodies is important as they increase the immunological risk of graft failure. Low titer of DSA may cause early or late AMR even in presence of negative crossmatch. Cytotoxic antibody screening: Expressed as percentage PRA, used to estimate risk of recipient having positive crossmatch Cons: Depends on commercially available cell panel which may not represent the population HLA frequency and racial differences can’t be considered Can’t determine antibody specificity and unacceptable antigens. False positive results due to non-HLA antibodies, autoantibodies or non specific IgM antibodies. False negative results due to low titer antibodies that fail to activate complement. Solid phase antibody screening:
Use recombinant HLA molecules instead of lymphocytes and identify antibody specificity of recipient serum ELISA Microbead platform/single antigen beads:
More sensitive than ELISA
Identify list of distinct antibody specificity
Estimate PRA which identifies several antibodies to cluster of donors when PRA increase, it indicates high probability of positive crossmatch
Determine cPRA by comparing antibody specificities with HLA frequencies in donor population cPRA: determine unacceptable HLA antigens that a recipient is sensitized to and represent actual proportion of donors who have unacceptable antigens and the recipient is compatible with. Pros:
Specific for HLA antibodies and identify anti-HLA antibodies up to 11 HLA loci
Rapid and tests many patients simultaneously
Enables virtual crossmatch and identify DSA pre transplant. Cons:
detect both complement and non complement binding antibodies simultaneously
detects antibody which is below threshold and may be clinically insignificant leading to exclusion of potential donor unnecessarily
can’t detect non HLA antibodies
can’t fully represent the ever-growing HLA alleles False negative results due to prozone phenomenon, complement component C1, IgM antibodies, serum proteins and IVIG and epitope sharing which may decrease MFI on single bead
Crossmatching:
Identifies if a recipient has antibodies to specific donor. CDC-XM
Done for B and T lymphocytes
False negative results due to low titer antibodies not detected
False positive results due to detection of autoantibodies, IgM/IgG HLA or non HLA antibodies. FCXM
Recipient serum mixed with donor lymphocytes
Identify T and B lymphocytes with no need for complement fixation
more sensitive than CDC and detect IgG DSA on donor lymphocytes. Virtual crossmatch:
Donor HLA typing and solid phase antibody screening are used together to predict the actual crossmatch in vitro
Leads to shorter waiting time, better outcome in sensitized recipients and better donor selection as predict potentially positive crossmatch.
should include all serum results with recent sample as antibodies presence, titer and specificity vary with time especially with sensitizing events. Cons:
False positive results due to low titer antibodies (clinically insignificant but detected by SAB) and/or non complement binding antibodies.
False negative results as all potential HLA donor antigens may be not correctly represented.
not 100% accurate and actual crossmatch is mandatory.
Defining immunological risk
Based on donor crossmatch and antibody screening at time of transplantation
Detailed study of DSA by molecular techniques prevent wrongly exclusion of donors, allow improved organ allocation and shorter waiting time. High risk:
High titer of circulating DSA
may lead to hyperacute rejection and preclude transplant
desensitization decreases the risk Intermediate risk:
low titer of DSA
Historic DSA not detectable
Acceptable transplant with intensified immunosuppression and close monitoring post transplant Standard risk:
No evidence of sensitization to HLA of the donor
Mohamed Fouad
3 years ago
Dear professors,
Regarding HLA antigen mismatches, is there any clinical significance for HLA matching at the epitope level? in regards of de novo DSA,long term graft survival and allocation system from clinical point of view.
Zahid Nabi
3 years ago
HUMAN LEUCOCYTE ANTIGEN TYPING AND CROSS MATCH:A COMPREHENSIVE REVIEW
This article by Althaf and colleagues is an excellent review emphasizing the value of interpretation of cross match results in a given clinical scenario . The fact that kidney transplantation is the best option for patients suffering from end stage renal disease(ESRD), all efforts should be made to ensure that appropriate patients are given this opportunity and misinterpreting a cross match result can deny any patient getting a transplant
The case scenario reflects the same that a positive CDC cross match in a patient of lupus is not because of a DSA rather it is result of immune complex binding to Fc receptors giving a false positive B cell cross match.
The authors have thoroughly explained different techniques of HLA typing mentioning there pros and cons as well like
SEROLOGICAL TYPING
Lymphocytes of the recipient are added to a tray containing sera, complement and dye, and if there is complement activation, this will lead to cell death, where the membrane integrity of the cells are compromised, leading to the dye being able to enter into the cells. These can be identified using phase contrast microscopy.
The key benefit of serological typing is that results are available in short period and is particularly important in deceased donor transplant.
As more advanced method of typing are available currently available serological typing has fallen into disuse.
MOLECULAR TYPING
These are of three types :
Sequence specific primer polymerase chain reaction : The principle of DNA amplification is applied with electrophoresis in order to identify DNA sequences for HLA typing.
Sequence specific oligonucleotide probes : DNA amplification is done with oligonucleotide probes and unique fluorescent tags are used to identify HLA alleles.
Direct DNA sequencing : HLA type identification is done by comparing with established allele sequences.
HLA ANTIBODY SCREENING
The authors reported that almost third of patients on waiting list for kidney transplant have some degree of anti HLA antibodies so it is crucial to detect these antibodies to ensure a safe kidney transplant .
Different methods for detecting these antibodies are
CYTOTOXIC ( cell based) ANTIBODY SCREENING
SOLID PHASE ANTIBODY SCREENING
ENZYME LINKED IMMUNOSORBANT ASSAY PLATFORM
MICROBEAD PLATFORM/ SINGLE ANTIGEN BEAD
The authors have further elaborated different cross matching techniques and finally given the concept of immunological categorization after all these testings
High immunological risk
At the time of transplantation there are high timers of circulating antibodies HLA DSA
This can lead to hyper acute rejection
Intermediate immunological risk
Historic DSA is not detectable and at the time of transplantation there are low titers of DSA.
Standard immunological risk
No DSA
I think the concept of this paper is to ensure that treating Nephrologist should have adequate knowledge of transplant immunology and any positive cross match result should be read in that given clinical scenario
CARLOS TADEU LEONIDIO
3 years ago
This article provides a review of the main methods used to assess the immunobiological risk of a transplant.
HLA typing to evaluate HLA alleles: HLA A-B – DR. Studies revealed that mismatches translated into a 64% higher risk, while the risk dropped to 13% with just one HLA mismatch. Thus, HLA Recognition is the first and most important step in this process. Possible methods are:
1 – HLA TYPING
A-Serological: in this case sera with antibodies to a multitude of known HLA alleles is used, juntamente com recipient lymphocytes and complement. The comparasion of cell death indentified in microscopy proporcionara o resultado a ser avaliado.
The key benefit of serologic typing is that results are available in a short period. This is particularly important in deceased donor renal transplantation.
B- Molecular:
B.1 – Sequence-specific primer polymerase chain reaction: extracted DNA from amplified in several wells are submetidos a eletroforese para formação de bandas a serem estudadas
B.2 – Sequence specific oligonucleotide probes: amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles, so HLA alleles are then identified using fluorescent tags.
B.3 – Direct DNA sequencing: determines the precise order of nucleotides in the gene of interest, allowing HLA type comparison, through the amino acid level, to provide insight to the risk monitoring mismatched donor-recipient antigens, epitopes and amino acid.
2 – SCREENING OF HLA ANTIBODIES:
After evaluation of the direct HLA alleles, the evaluation of HLA is the second step. Here, comparative panel studies (PRA) with population controls of specific antibodies compared the risk of replication through specific donors (DAS). They are used: cell-based or solids (divided in ELISA or fluorescence microspheres (Flow PRA or SAB Luminex))
There is the possibility of calculating panel reactive antibodies (cPRA), which provide as best estimates of the probability of a positive donor-specific antibody/crossmatch for a randomly selected donor. cPRA is based on unacceptable HLA antigens – those to which the patient has been sensitized, an offer to a recipient with a high cPRA is a high probability of a positive crossmatch.
3 – CROSSING
With cytotoxic time it was necessary to test that the T cell cross-matching was insufficient to identify all relevant antibodies and, moreover, could exclude transplant patients unnecessarily. Therefore, it should be used in conjunction with cross-matching results to identify those that are immunologically relevant.
A – Crossmatch of complement-dependent cytotoxicity:
Similar to the cytotoxic assay, complement-dependent cytotoxicity cross-matching is interpreted as positive if a considerable number of lymphocytes are destroyed after complement incorporation. Use B and T lymphocytes.
B – Flow cytometry crossmatch
Flow cytometry crossmatch (FCXM) detects DSA independent of complement fixation. With the use of flow cytometry, B and T lymphocytes can be easily identified and have their DSA interrogated individually. Compared with complement-dependent cytotoxicity cross-matching, this offers greater sensitivity.
C – Virtual cross-match
In virtual cross-matching (VXM), donor HLA typing and solid phase antibody screening are used together
4 – RISK CLASSIFICATION:
A – High immunological risk
High titers of circulating HLA-specific antibodies from mismatched donor
B – Intermediate immunological risk
there is a low DSA titer and the historical DSA is not detectable.
C – Standard immunological risk
there is no evidence of targeted donor sensitization to HLA
This excellent article is a review titled HLA typing, focussing on the different types of tissue typing and crossmatch involved in kidney transplant. It also expresses the application of said crossmatch results and how to interpret the same to an extent in clinical scenarios. The given article takes the example of a lupus ESRD patient. The entire article is based on the significant impact that HLA matching has on survival of the allograft.
The article also defines immunological risk by performing a risk assessment and gives principles for three categories of high, intermediate, and standard immunological risk.
High immunological risk is classified as high titers of DSA, which can lead to hyper acute rejection.
Intermediate immunological risk is classified as low DSA titer, where intense immunosuppressive regimens and careful monitoring post transplant can potentially give a good outcome for the recipient.
Standard immunological risk is classified as a situation wherein there is no donor directed sensitization to HLA.
HLA typing is of two kinds :
A. Serological typing
Lymphocytes of the recipient are added to a tray containing sera, complement and dye, and if there is complement activation, this will lead to cell death, where the membrane integrity of the cells are compromised, leading to the dye being able to enter into the cells. These can be identified using phase contrast microscopy.
B. Molecular typing
These are of three types :
Sequence specific primer polymerase chain reaction : The principle of DNA amplification is applied with electrophoresis in order to identify DNA sequences for HLA typing.
Sequence specific oligonucleotide probes : DNA amplification is done with oligonucleotide probes and unique fluorescent tags are used to identify HLA alleles.
Direct DNA sequencing : HLA type identification is done by comparing with established allele sequences.
All methods of molecular typing can differentiate between donor and recipient HLA antigen.
HLA ANTIBODY SCREENING
This is essential to identify previous sensitisation, which is generally seen due to pregnancy, prior transplantation, or following blood transfusion due to any cause. Once the patient has been sensitized, there are several methods that can be used in order to treat the patient such as desensitization, acceptable mismatching, and paired exchange schemes in order to bring about the best possible transplant outcome.
There are several methods of HLA screening, which are listed below along with their advantages and disadvantages.
CYTOTOXIC ANTIBODY SCREENING :
In this method, lymphocytes from 30-40 donors are selected as a representation of the population and the recipient serum is mixed with this panel along with complement and dye. If the serum antibodies bind to the cell surface receptors and activate complement pathways, this leads to cell death and subsequent uptake of dye. The degree of cell death is quantified using PRA of cancel reactive antibody. This is the risk of the recipient in having a positive crossmatch, lowering the survival chances for the allograft.
Disadvantages :
A. The PRA percent taken may not be exactly representative of the cell population because of racial differences and HLA frequencies. This means we do not get an accurate outcome, which can lead us to reject viable donors. This will significantly limit the already scarce options for transplant for the recipient and place unnecessary burden on waiting lists.
B. False positive results due to non HLA antibodies, non specific IgM antibodies and autoantibodies.
C. False negative results because this method is dependent purely on complement activation and if there are low tires then this can prevent complement activation, causing the presence of a true antibody to be hidden, leading to a bad outcome post kidney transplant.
D. Complete and accurate list of antibody specificities cannot be identified due to the multiple antigens in each well.
SOLID PHASE ANTIBODY SCREENING :
Recombinant HLA molecules are employed in this method instead of lymphocytes. This is because lymphocytes present both HLA and non-HLA molecules.
There are different methods under this category, listed below.
ELISA platform
Optical density reading helps to identify HLA antibody in serum of the recipient. Enzyme conjugated antibodies to IgG detects the HLA antibody.
This process is done by adding HLA molecules to ELISA platforms which then bind to HLA antibody from the recipient serum.
Advantages :
A. More sensitive than AHG enhanced cytotoxicity based assays.
MICROBEAD PLATFORM
Different panel beads with class I or class II HLA antigens are used to obtain positive or negative result. Proportion of positive beads are calculated using flow cytometer or Luminex. This will produce the PRA result. Antibody specificities are used to estimate cPRA or calculated panel reactive antibody percentage. cPRA is based on unacceptable HLA antigens, which the patient has already been sensitized to previously. This will estimate the chances of a positive cross match.
Advantages :
A. More sensitive for low tire antibody detection.
B. More rapid results.
C. Multiple patients can be tested simultaneously.
D. DSA can be detected pre transplant, allowing space for organ allocation and risk assessment.
E. Anti HLA antibodies for common and rare antigens and alleles can be identified.
Disadvantages :
A. Both complement and non-complement binding detected in parallel. Antibodies below threshold level are detected, which can lead to preclusion of a potential donor.
B. SAB Luminex is affected by prozone phenomenon, wherein high titer anti-hula antibodies are not detected when tested in a concentrated form, but are recognized only when diluted 1:10.
C. False negative reports Can arise because of epitope sharing. Low MFI is seen.
CROSSMATCHING :
The methods of cross matching have been detailed below :
COMPLEMENT DEPENDENT CYTOTOXICITY CROSSMATCH (CDC -XM)
This method gives a positive result if significant DSA binds to cell surface, leading to complement activation and destruction of lymphocytes.
Advantages :
A. Can be done for B and T lymphocytes.
Disadvantages :
A. Limited sensitivity in low titers. Solution is to increase incubation time.
B. Negative test result has been associated with 18% graft loss in one year.
C. Gives false negatives by detecting IgM/IgG HLA or non HLA.
FCXM :
Detects DSA independent of complement fixation. Recipient serum is added to donor lymphocytes and fluorescent tags are used for identification of DSA.
Advantages :
A. Does not depend on complement pathway for results hence false negatives due to low titers are generally reduced or avoided.
B. Can accurately identify absence of presence of DSA IgG on donor lymphocytes.
C. B and T lymphocytes immediately identified and their DSA individually sorted.
D. More sensitive than CDC XM.
Disadvantages :
A. No standardization of results threshold because of different methods used by different labs.
B. Unclear impact on assessment of immunological risk.
VIRTUAL CROSSMATCH :
Both donor HLA typing and solid phase antibody screening done together.
Advantages :
A. Shorter wait time
B. Better outcome for sensitized recipients
C. Sensitive and specific tech
D. Can be done at the time of donor identification
E. Allow physician to consider donor organs that otherwise might not be available for donor selection.
Disadvantages :
A. Significantly low antibody titers, non complement binding antibodies can lead to false positive, which results in excluding a potential donor.
B. False negative results seen when the potential HLA donor antigens are incorrectly represented because of being classed differently.
C. Not fully accurate and thus requires an actual crossmatch to be done along with it to ensure correct results.
D. Does not recognize HLA null alleles.
There are tow different techniques of tissue typing
1.serological typing :Microcytotoxicity assay
Principle: complement dependant cytotoxicity
After lymphocyte separation well be added in tray wells which contain sera which contain antibodies then complement added and dye.
When these antibodies bind to lymphocytes surface antigens complement activation occurs leading to cell lysis .The percentage of lysed cells are counted under phase contrast microscopy then HLA type is i assigned.
2. Molecular methods
A.Sequence-specific primer polymerase chain reaction:Extracted DNA is added in the wells which contains oligonuclotide primers complementary to small segment of one’s HLA allele then amplified and runs in agarose gel and undergo electrophoresis and appear as a band then typing can be assigned.
B.Sequence specific oligonucleotide probes:
HLA type assigned by mixing amplified DNA with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles.
C. Direct DNA sequencing:
Determines the precise order of nucleotides in the gene of interest.
CROSSMATCHING (XM) . There are tow methods
1.Complement-dependent cytotoxicity crossmatch(CDC-XM) the principle of the test as above. Its specific and less sensitive.
2.Flowcytometry (FCXM) Sensitive and specific
Donor lymphocyte added to the patient serum
then tagged with a fluoro-chrome conjugated anti-Ig G antibody.
3.virtual cross match: acomparative one we comper the PRA result with the donor HLA typing.
The paper takes advantage of a specific situation of autoimmunity induced by an immune complex secondary to SLE to review the definitions of immunity and its impact on different laboratory tests.
Serological methods are the most used commercially. Molecular methods have been used in research with the need to have earlier and more specific results, reducing cold ischemia time. The lack of a comprehensive database of these alleles is still a limiting factor for the method, which may result in false negatives.
HLA-Antibodies screening
It can occur by the cytotoxic method, using a reactive panel of antibodies with donor lymphocytes and patient serum. The most specific methods are solid phase, mainly ELISA, flow cytometry, and single antigen beads. These can calculate a PRA (cPRA) with higher specificity of detecting the Crossmatch and is based on unacceptable HLA antigens.
Crossmatch (XM)
There are similar methods, but with some peculiarities that differ from previous tests.
1. Complement-dependent Cytotoxicity Crossmatch – CDC-XM
Here we add the complement and a complex organic molecule to the cytotoxic method, which can expose both B and T lymphocytes. Adding an anti-human IgG immunoglobulin increases the sensitivity of the method. Remember the possibility of false positives with high levels of IgM, autoantibodies, or non-HLA antigens.
2. Flow cytometry – FCXM
This method detects DSA independent of complement fixation using recipient serum, donor lymphocytes and using a fluor-conjugated IgG antibody.
3. Virtual Crossmatch – VXM
It is a combination of previous methods, reducing the waiting time for cold ischemia and with the possibility of comparisons with previous and current exposures between donor and recipient. False positives can occur in low antibody titers or absence of complement, erroneously removing the donor from the list. False negatives occur when antigens are not expressed on the donor HLA.
Based on these tests, it can generate enough data for the classification of immunological risk, which can be high, intermediate or standard.
Conclusion
The patient has lupus nephritis
Deceased donor kidney with a 1-0-0 mismatch (low risk)
(CDC) crossmatch reported positive for B lymphocyte, probably based on humoral disease as SLE
Flow cytometry crossmatch (FCXM) negative for both B and T lymphocytes
Luminex-SAB (single antigen bead) did not identify any DSA
He never had a blood transfusion
HLA typing and crossmatch
HLA typing is a cornerstone in renal transplantation. donor HLA recognized by the recipient T lymphocytes as a foreign antigen; ultimately activate the immune response against the allograft.
HLA typing made by serologic and molecular methods Serological typing:
Recipient lymphocytes are added to a tray containing sera with antibodies to many known HLA alleles commercially available. Complement and dye are added thereafter. If the antibody can bind to the antigen on the surface of the lymphocyte complement is activated forming a membrane attack complex causing cell lyses so dye enters it. Tray wells with significant cell death are then identified under a microscope. By comparison and elimination of positive wells the HLA type is assigned. Advantage:
1- Results are given in a short time, which is important in deceased donors as it decreases the cold ischemia time.
2- It offers the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression. These alleles called null HLA alleles, they are of less immunological significance. Disadvantages:
1- Lack of sera with Abs specificities that are capable of identifying the ever-growing number of HLA alleles.
2- HLA- CW, DQ, DP Ag may have clinically significant effects on the outcomes of allografts. However, serological assays are scant for these loci.
3- Serological methods do not readily detect differences in HLA protein-small amino acids that may be antigenic enough to trigger a potent immunologic reaction.
Molecular typing:
1- Sequence-specific primer polymerase chain reaction:
DNA from the subject is amplified in several wells. Each well has primers that are complementary to specific HLA alleles. Where DNA probes are complementary to the specific sequence of the HLA molecule, an amplification product is formed which is then implanted into agarose gel and undergoes electrophoresis where they appear as bands. HLA typing is then allocated by matching the primers of the amplification product to the DNA sequence of several candidate alleles.
2- Sequence-specific oligonucleotide probes: Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles which are then identified using fluorescent tags.
3- Direct DNA sequencing: it determines the precise order of nucleotides in the gene of interest. HLA type is assigned by comparison with published HLA allele sequences. Advantages of molecular typing:
1- Can clearly identify differences in HLA Ag between donor and recipient with detail to the epitope, and amino acid level allows better risk stratification. HLA typing using PCR is highly specific where specific alleles are identified with no cross-reactivity. Cross-reactivity is the identification of an allele that is essentially similar to the allele of interest.
Disadvantage: new alleles not currently on the HLA sequence databank will fail to be identified. Why is HLA typing needed?
The dissimilarity in the HLA Ag reflects the alloimmune burden that a donor’s kidney presents to the recipient. Ultimately affecting the outcome.
HLA alleles do not have equal weight. HLA-DR, then –B, then –A which has the less alloimmune burden. The influence of HLA-DR mismatching had the most effect during the first six months post transplantation, while the maximal effect of HLA-B mismatching occurs 2 years posttransplantation.
UNOS looked at quantifying the risk of transplant failure with HLA mismatch in deceased allografts. Patient with 6 HLA mismatches translated to 64% higher risk while the risk was 13% with just one HLA mismatch, this is independent of locus. Study identify seven specific HLA mismatch combinations that were associated with decreased allograft survival. These called taboo mismatches translated to 81% one-year survival and 50% five-year survival. HLA antibody screen:
Sensitization towards HLA-Ag (DSA formation) occurs in pregnancy, post blood transfusion and prior transplantation.
Why DSA screen important?
1- They Contribute to immunological failure
2- Cause positive CXMm thereby; resulting in the exclusion of donor.
So; both sensitive and specific detection of these Abs is important. Because this influence the decision of transplantation either with desensitization strategy, or paired exchange, and acceptable mismatching with potent immunosuppression.
Methods for DSA screen:
1- Cytotoxic (cell-based) way: a group of donor cells selected randomly from the population of potential deceased donors. 30 to 40 different donors lymphocytes mixed with recipient serum in individual walls along with complement and dye, if the recipient serum contains Abs that can bind to the cell surface Ag, the complement-mediated reaction will occur, ends with cell lyses and the dye enters it. Coloured cells identified by polarised microscopy. The degree of cell death is expressed as a percentage of PRA (panel reactive Ab).
PRA tell us about the possibility of the recipient to have positive crossmatch, and so the likely to have a donor from that population. Disadvantages:
1- PRA% can change without a corresponding change in the type or amount of antibodies.
2- The cell panels are commercially produced and may not truly represent the population.
3- False positive which can be in the presence of non-HLA antibodies, auto-Abs, and non-specific IgM Abs
4- False-negative result due to low titre antibodies.
5- Each well contains several Ags so, precise complete lists of antibody specificities and unacceptable Ag cannot be identified by this method.
2- Solid-phase antibody screen:
Soluble or recombinant HLA assay
a- Enzyme-Linked immunosorbent assay platform: ELISA recipient serum will add to ELISA platform containing purified HLA molecules, enzyme-conjugated Ab IgG is then added to detect the presence of HLA antibody which bound to Ag by optical density reading. b- Microbead platform/ single-antigen beads: pools of beads coated with different class I or II HLA antigens used for screening.
Fluorescence detection based either on cytometer (Flow PRA) or by the single antigen bead (SAB) Luminex platform.
The proportion of positive beads estimates PRA. SAB yields antibodies specificity. Specificities are subsequently compared with HLA frequencies in the donor population to determine the cPRA .
cPRA represents actual donors who express unacceptable HLA antigens to which the recipient has been sensitized. It is used in organ allocation.
The combination of the traditional cell-based assay with solid-phase assays help in better determining relevant positive CXM from false-positive results.
Limitations of solid-phase assays;
1- Detect both complement and non-complement fixing Abs
2- Can detect very low titer Abs or Abs with no clinical significance, but can preclude the potential donor. Also, detect non- HLA antibodies
3- SAB-Luminex may have an artefact known as prozone phenomenon. This occurs if sera containing high titer Abs give negative results when tested dry, and rendered strongly positive after dilution or addition of DTT which prevent IgM or any protein competing to Ab binding site.
4- Epitops can give negative results, different Ags on different beads share common (same) epitopes to the binding of Ab to more than one bead. Leads to the reduction of MFI on a single bead. Crossmatching: is required pretransplant
1- complement-dependent cytotoxicity CXM: can be done for B and T lymphocytes. It has same principles of the cytotoxic assay. In presence of low titre Abs the test sensitivity is low that can be increased by increasing the incubation period, use of AHG-enhanced method or wash steps.
False negative results due to low titer Abs are clinically significant. False-positive CDC-XM can be due to IgM/IgG or non-HLA.
Flow cytometry crossmatch: detects the presence or lack of IgG DSA on donor lymphocytes, independent of complement fixation. It is more sensitive than CDCXM. Virtual crossmatching: The data is used for prediction of the actual in vitro crossmatch results by mixing identified antibody specificities of recipient serum with donor HLA antigens. The use of VXM can lead to shorter wait times and improved outcomes for sensitized recipients. The use of a historical sera could not predect Ab specificities with certainty so, recent sera should be used. In current practice an actual CXM should be performed as well. VXM does not identify Null HLA alleles which carry significant risk. Recipient risk stratification: High immunological risk: High titres of DSA, that preclude transplantation. pretransplant desensitization regimens can reduce the risk. Intermediate immunological risk: low titer of DSA, and historic DSA is not detectable. In them use intensified immunosuppression, in addition to immunological monitoring posttransplantation. Standard risk: no DSA
WEEK 1 JOURNAL CLUB I.Human leukocyte antigen typing and crossmatch: A comprehensive review Mohammed Mahdi Althaf, Mohsen El Kossi, Jon Kim Jin, Ajay Sharma, Ahmed Mostafa Halawa Mohammed Mahdi Althaf, Jack Pryor Renal Unit, Norfolk and Norwich University Hospital – NHS Foundation Trust, Norwich NR4 7UY, United Kingdom
Background and Introduction:
Kidney transplantation is the best renal replacement therapy for patients with end stage renal disease (ESRD). Human leukocyte antigen (HLA) typing & cross-matching are done before any transplantation so as to improve organ allocation & allow better matches to recipients. Nephrologists can be challenged by interpretation of the results of these immunological studies, as they have no formal training in immunology. In this review article the author presents one of these challenging scenarios. CLINICAL SCENARIO:
The author reports a case of a 30-year-old man with ESRD secondary to Lupus nephritis. He was offered a deceased donor kidney with a 1-0-0 mismatch. Other immunological workup was as follow:
-Complement dependent cytotoxicity (CDC) cross-match positive for B lymphocyte
-Flow cytometrycross-match (FCXM) negative for both B & T lymphocytes.
-Luminex- single antigen bead (SAB): no any donor specific antibodies (DSA).
The positive CDC cross-match result is not concordant with DSA status. The patient had no history of blood transfusion and this is his first transplantation. Autoimmune diseases such as systemic lupus erythematosus (SLE) are known to give false positive CDC cross-match result. Failure to consider such points may unnecessarily lead to exclusion of adequate kidney grafts. The application of molecular techniques to study DSA would prevent wrong exclusion of such donors.
The author aimed to discuss an immunological risk stratification by using an example of an index case & the following review.
HLA TYPING METHODS: A. Serological typing
Recipient lymphocytes are instilled into a tray wells containing sera, complement & dye.
The sera contains antibodies that can react to a multiple of known HLA alleles. When these antibodies bind to lymphocytes surface antigens complement dependent cell lysis occurs.The percentage of lysed cells are counted under phase contrast microscopy.
This method is quick & thus less cold ischemia time that suits the deceased donor renal transplantation.
However, serological typing fails to identify HLA-Cw, DQ, & DP antigen which may have significant impact on graft outcomes.
B. Molecular typing (i) Sequence-specific primer polymerase chain reaction:Extracted DNA is amplified in several wells containing primers that are complementary to specific HLA alleles.HLA typing is defined by matching the primers of the amplification product to DNA sequences
of several candidate alleles. (ii) Sequence specific oligonucleotide probes:
HLA type assigned by mixing amplified DNA with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles. (iii) Direct DNA sequencing:
Determines the precise order of nucleotides in the gene of interest.
Molecular typing methods give details to amino acid level & clearly identify differences
in HLA antigen between donor & recipient providing insight to the risk accompanying mismatched donor-recipient antigens, epitopes & amino acid[1,2].
HLA ANTIBODY SCREENING METHODS Cytotoxic (cell-based) antibody screening
A panel of donor lymphocytes representative of the population is used.
Similar to that of serologic typing however here recipient serum is mixed with “cell donor” lymphocytes in individual wells along with complement & dye.
Limitations of this method include:
-may not be truly representative of the population.
-HLA frequencies & racial differences cannot be done.
-false positive results due to non-HLA antibodies, autoantibodies & nonspecific IgM
antibodies.
-false negative results are possible if low titers fail to activate complment [3-5]. Solid phase antibody screening
Soluble or recombinant HLA molecules are used instead of lymphocytes targets (i)Enzyme-linked immunosorbent assay (ELISA) platform:
-purified HLA molecules are applied to ELISA platforms to bind individually to HLA antibody after the addition of recipient serum [6,7]. (ii)Microbead platform/single-antigen beads: –Pooled panel beads with several different class I or II HLA antigens on a bead yield a positive or negative result and are utilised for screening [8].
-Microbead that is fluorescent dye conjugated is then added to detect the presence of HLA antibody in the serum which is bound to the antigen.
-Fluorescence detection can be done by a flow cytometer (Flow PRA®) or usingthe single-antigen beads (SAB) Luminex® platform. Advantages: -SAB gives rapid results (3-4 h)
-Flow PRA® & Luminex® are 10% more sensitive for lower titre antibody than ELISA.
-ELISA is ten 10% more sensitive than AHG) enhanced cytotoxicity based assays
-SAB is efficient (up to one hundred unique antigen beads can be tested in a single reaction
chamber.
-SAB enables virtual crossmatching (VXM) to identify DSA pre-transplant
-SAB identifies anti-HLA antibodies for all common & many rare antigens & alleles [9]. Limitations of SAB:-Being too sensitive they can detect antibody that is below the threshold
associated with a positive cross-match(clinically irrelevant antibody
detected may preclude a potential donor).
-susceptible to an artifact(prozone phenomenon) [11].
CROSSMATCHING (XM) A.Complement-dependent cytotoxicity crossmatch(CDC-XM)
-Positive if a considerable number of lymphocytes are destroyed after the incorporation of complement (similar to cytotoxic assay).
-CDC-XM can be done for B and T lymphocytes
-limited sensivity if the relevant antibody is in low titers(this can be overcome by increasing incubation time, use of AHG-enhanced method as well as additional wash steps)[3,10].
-20% false positives( by detecting autoantibody, IgM/IgG HLA or non-HLA).
– 4% false negative rate
B.Flow cytometry crossmatch (FCXM)
-recipient serum is mixed with donor lymphocytes & then tagged with a fluoro-
chrome conjugated anti-Ig G antibody.
-detects DSA independent of complement fixation.
-precisely detects the presence or lack of IgG DSA on donor lymphocytes.
-B & T lymphocytes can be readily identified & have their DSA individually examined.
-greater sensitivity compared to CDC-XM[12].
-not widely available & its role in assessing immunological risk is still unclear. C.Virtual crossmatching (VXM)
-not precisely a crossmatch as there is no mixing of serum & lymphocytes.
-both donor HLA typing & solid phase antibody screening are used (“mixed” to identify
actualin vitrocross-match) [12].
-shorter wait times (performed at the time of donor identification)
-improved outcomes for sensitized transplant recipients.
-permits acceptance of donor organs that would otherwise be rejected(allows to consider a
potentially positive crossmatch a risk factor for donor selection) [13,14].
– use of antibody specificity from historical serum sample (earlier than 6 months) could not
predict a crossmatch with certainty.
-factors that can influence antibody specificities(pregnancies, transplants & blood
transfusions) should be considered.
-VXM results not a hundred percent accurate & current practice mandates an actual
crossmatch be performed as well [15].
– wrong exclusion of potential donors due to false positive results if significantly low titer
&/or non-complement binding antibodies
Risk categories:
Gebel et al suggested the following risk stratification categories in prospective renal transplant patients according to immunological risk: High immunological risk
These patients have high titers of circulating DSA specific for mismatched donor HLA. Such patients are usually excluded from transplantation as hyper-acute rejection is inevitable in the presence of DSA. Intermediate immunological risk
In this category there is a low titer of DSA at the time of transplantation, and no detectable historic DSA. Intensified immunosuppression as well as immunological monitoring in the post-transplant period are required in these patients. Standard immunological risk
Here there is no evidence of DSA; & the pre-transplant immunological risk assessment is based on donor cross-match and antibody screening results. Risk Assessment Of The Index Case
This patient had positive CDC-XM for B &T lymphocytes but negative FCXM forboth B & T lymphocytes. No DSA detected by Luminex-SAB. According to these results, this patient can be consideredas “standard immunological risk”, and he can proceed with transplantation. But the positive CDC-XM result is not inharmony with DSA status. These discordant resultsare caused by the underlying SLE which leads tofalse-positive B- lymphocyte cross-matches as a result ofbinding immune complexes to Fc-receptors.
Conclusion
HLA typing of the index pair was 1-0-0-mismatch which corresponds to the pair of alleles mismatched at HLA-A, HLA-B & HLA-DR, respectively. These 3 antigens are usually the most important ones in kidney transplantation & the fewer the mismatches, the better the match between donor & recipient leading to a successful outcome. In this case, there is one mismatch at HLA-A; this poses less immunological burden compared to that at HLA-B & HLA-DR. Euro-transplant & old United Kingdom transplant data suggest that HLA-DR matching has a far greater effect than HLA-A or HLA-B
This case illustrates a common clinical scenario in transplant practice; and a detailed immunological testing & cautious interpretation of the results is crucial before deciding to preclude or accept a case for transplantation. Simply relying on the CDC-XM result would have unnecessarily excluded this patient from transplantation. The false positive CDC-XM result in the case presented was due SLE. The false-positive B-lymphocyte cross-match results from immune complexes binding to Fc-receptors [16,17].
False positive
CDC-XM could also be due to medications such as Isoniazid &
Hydralazine [18,19]. Detailed study of DSA by molecular technique would
improve organ allocation, shorten waiting time, & prevent unnecessary
exclusion of such donors. References
1 Duquesnoy RJ, Claas FH. Is the application of HLAMatchmaker relevant in kidney transplantation? Transplantation 2005; 79: 250-251 [PMID: 15665779 DOI: 10.1097/01.TP.0000144327.92898.A6]
2 Claas FH, Roelen DL, Oudshoorn M, Doxiadis II. Future HLA matching strategies in clinical transplantation. Dev Ophthalmol 2003; 36: 62-73 [PMID: 12494682 DOI: 10.1159/000067657]
3 Amos DB, Cohen I, Klein WJ Jr. Mechanisms of immunologic enhancement. Transplant Proc 1970; 2: 68-75 [PMID: 4107294]
4 Ful l e r TC, F u l l e r A A , G o l d e n M , R o d e y G E . H L A alloantibodies and the mechanism of the antiglobulin-augmented lymphocytotoxicity procedure. Hum Immunol 1997; 56: 94-105 [PMID: 9455498 DOI: 10.1016/S0198-8859(97)00174-2]
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Human leukocyte antigen typing and crossmatch: A comprehensive review
Complement dependent cytotoxicity (CDC) cross match results could be false positive as a result of underlying autoimmune disease such as SLE, which may lead to inadvertent refusal of adequate kidney grafts. Using molecular technique to study DSA can prevent inadvertent refusal of such donors. We are going to discuss HLA typing, HLA screening and cross match methods.
HLA Typing.
HLA typing is very important in renal transplantation. In the case of recipient T lymphocyte recognize foreign HLA that will result in T lymphocyte activation which will lead to immune response against the allograft. There are two ways to perform HLA typing which are serologic and molecular typing methods.
Serologic method where the recipient lymphocytes are introduced to a tray wells containing sera with antibodies to a multitude of known HLA alleles then complement and dye will be added. If antibodies bind to the antigen a complement activation will occur and finally the dye will enter the cell. Phase contrast microscopy is then used to identify wells with significant cell death.
Advantages of serologic methods are they are fast with results in short time which is important in deceased donor transplantation and the ability to identify non HLA alleles which have identifiable DNA sequences with molecular typing but no cell surface antigen expression.
Disadvantaged of serologic methods are the inability to detect the increasing number of HLA alleles because of the lake of sera which contains specific antibodies. In addition that serologic method is not able to detect differences in HLA protein small amino acids that may be antigenic enough to induce potent immune response.
There are three molecular HLA typing methods. First, sequence-specific primer PCR where extracted DNA is amplified in several wells where DNA probes are complementary to the specific sequence of HLA molecule, then is instilled into an agarose gel and undergo electrophoresis and appear as a band. Second, Sequence-specific oligonucleotide probes where amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles and unique HLA alleles identified with fluorescent tags. Finally, Direct DNA sequencing where HLA type is assigned by comparison to published HLA allele sequences.
There are many pros of molecular methods of HLA typing, they can clearly identify the differences in HLA antigen between the donor and the recipient regardless of the method up to the amino acids level which make them highly specific methods. PCR typing can identify specific alleles with no cross reactivity – identification of allele which is essentially similar to the allele of interest – although a gene may occur in two or more forms (alleles). While this is highly specific it has the disadvantage that new alleles not on HLA databank will not be identified.
HLA antibody screening.
Screening for HLA antibodies is crucial in renal transplantation because of the increased chances of immunological graft failure in sensitized patients especially that there are strategies such as desensitization, paired exchange and acceptable mismatching which can lead to successful transplantation. There are three main ways for sensitization towards HLA antigen which are Pregnancy, post blood transfusion and prior transplantation. There are two methods for HLA antibody screening which are cytotoxic (cell-based) antibody and solid-phase antibody screening.
In cytotoxic HLA antibody screening the recipient serum is mixed with a set of cell donors where each panel consists of about 30 to 40 different donor lymphocyte to represent the potential deceased donor population. It’s similar to serologic typing as if the recipient serum contains antibodies against donor lymphocytes complement pathway will be activated followed by cell death and dye uptake. The degree of cytotoxicity is expressed as percentage PRA.
There are many limitations to cytotoxic HLA antibody screening as PRA percentage may be different numerically without a corresponding change in the type or amount of antibody. In addition, HLA frequencies and racial differences need to be factored in but can’t be done. False-positive results can be produced by autoantibodies, IgM and non-HLA antibodies. As well as, false-negative results can occur due to non-complement activation due to low titers of true antibodies.
The other method for HLA antibody screening is solid-phase essay which uses soluble HLA molecules instead of lymphocyte targets. It can be done through three ways: ELISA, Flow cytometry and single antigen beads SAB. In ELISA purified HLA molecules will bind individually to HLA antibody after adding the recipient serum and then detected by optical density reading. The other two ways include a panel of beads loaded with either class I or II HLA antigens where patient serum added to each bead then a fluorescent dye is used to detect antibody-antigen complex by flow-cytometry or SAB. These estimate PRA by the proportion of positive beads.
SAB are individually coated with single HLA antigen and yield a list of distinct antibody specifities – Highly specific – and then compared with HLA frequencies in donor population to determine the calculated PRA which is the best estimate of positive crossmatch/ donor specific antibody to a randomly selected donor. It has many advantages as its more sensitive compared to ELISA by 10%, rapid with results available within 3-4 hours, permits testing many patients simultaneously and enables virtual cross match which help in risk stratification. Additionally, SAB can identify anti-HLA antibodies for all common and numerous rare antigens and alleles up to 11 HLA loci.
SAB has its own limitation with false negative results called as the prozone phenomenon which occurs when sera with high titer anti-HLA antibodies give negative results when tested neat which can be overcome by dilution. Similar scenario can occur with the binding of IgM antibodies or other serum factors to the beads. In general, solid phase antibody screening have limitations as they detect both complement and non-complement binding simultaneously. In addition, they are too sensitive to detect antibody levels below the threshold with a positive cross match and can result in precluding a potential donor.
Cross Matching:
Crossmatch will identify if a recipient had antibodies to a specific donor of interest where PRA identifies antibodies to a potential cluster of donors. Cytotoxic cross matching is insufficient to identify all relevant antibodies and it may exclude patients from transplant. Therefore, it should be used together with solid-phase antibody tests to identify those that are immunologically relevant. There are three methods of cross matching.
First, complement-dependent cytotoxicity which considered positive if a considerable number of lymphocytes are destroyed after adding the complement and can be done for B and T lymphocytes. False negative results can occur because its sensitivity is limited in low titer antibodies but can overcome by increasing the incubation time. False positive results can occur in the presence of autoantibodies, IgM, non-HLA antibodies.
Second, flow-cytometry crossmatch which detect DSA independent of complement fixation. In this method, the recipient serum is mixed with donor lymphocytes and then tagged with fluorochrome-conjugated anti-IgG antibody. It can detect the presence or lack of IgG DSA on donor lymphocytes. It has greater sensitivity than CDC.
Finally, virtual crossmatching (VXM) where donor HLA typing and solid-phase antibody screening are utilized together. It’s not accurate as cross match in term of mixing serum and lymphocytes but it can lead to shorter waiting times. The use of antibody specificity from historical serum sample (earlier than six months) could not predict a crossmatch because it vary over time. Therefore, serum results including at least one recent within less than 3-6 months should be used. Because the results of virtual cross matching are not accurate an actual crossmatch should be performed. Lastly VXM does not identify the HLA null alleles.
In Conclusion, understanding of crossmatch results and the limitations of each individual test is crucial for successful outcome. We can’t rely only on CDC-XM as false positive results can occur due autoimmune disease or immune complexes binding to Fc-receptors in the case of B-cell cross match. Detailed study of DSA by molecular technique would prevent erroneous exclusion of such donors.
HLA TYPING
HLA typing is a cornerstone in renal transplantation. It can be done either by serologic method or molecular typing method. Serological typing :
Recipient lymphocytes are mixed into the tray wells contacting sera,
complement and dye. In-tray wells where antibodies can bind to the antigens on the surface of lymphocytes; complement is activated which results in cell death, and the dye enters the cell. Tray wells with significant cell death are then identified under microscopy. Advantages :
1. Results are available in a short period so less cold ischemia times.
2..Differentiate HLA alleles that have identifiable DNA sequences with molecular
typing but with no cell surface antigen expression(null HLA alleles). Disadvantages:
1. It is a lack of sera with antibody specificities that are capable of identifying HLA-Cw, DQ, and DP antigen which may have clinically significant effects on the outcomes of allografts.
2. It does not detect differences in HLA protein-small amino acids which may trigger potent immunological responses. Molecular typing :
It can identify differences in HLA antigen between donor and
recipient. It includes:
1. Sequence-specific primer polymerase chain reaction.
2. Sequence-specific oligonucleotide probes.
3. Direct DNA sequencing. HLA antibody screening
Cytotoxic (cell-based) antibody screening
The patient, s serum is mixed with donor lymphocytes in individual wells along with complement and dye.
If the serum contains antibodies will activate complement pathways that result in cell death and uptake of the dye. The degree of cytotoxicity is expressed
as PRA %.
False-positive results can be produced due to non- HLA antibodies, autoantibodies, and nonspecific IgM antibodies. False-negative results are possible because it requires high antibody titers to activate complement. Solid-phase antibody screening
Enzyme-linked immunosorbent assay platform (ELISA):
Purified HLA molecules are applied to ELISA platforms and will bind individually to HLA antibodies.
Microbead platform/single-antigen beads( SAB): Pooled panel beads with several different classⅠ or Ⅱ HLA antigens on a bead yield a positive or negative result
and are utilized for screening. Cross-matching: Complement-dependent cytotoxicity crossmatch
Similar to cytotoxic assay the complement-dependent cytotoxicity crossmatch.CDC-XM is representative of what would happen in vivo. Low titer antibodies detected by this method were associated with 36% 1-year allograft loss compared with 18% loss in those with a negative test.
CDC-XM may be applied to both B and T cells. CDC-XM for T cell reflects the presence of HLA-I antibodies while for B cell reflects HLA I and II antibodies. B cells express a higher amount of class I antigens so appositive B cell CDC-XM associated with a negative Tcell CDC-XM indicate a low level of class I antibodies.
CDC-XM has a false positive rate of 20% that may be to antibodies which are generally of IgM and non-HLA IgG type. There have been reports of a false positive B –cell CDC-XM following treatment with rituximab and basiliximab.
One disadvantage of CDC-XM is that it only detects complement-fixing antibodies, while non-complement fixing antibodies may still affect graft function. Flow cytometry crossmatch
Recipient serum is mixed with donor lymphocytes and then tagged with a fluorochrome-conjugated anti-IgG antibody.
It is the most sensitive test to detect donor-specific antibodies that were being missed by the CDC-XM. Virtual crossmatching
In virtual crossmatch (VXM), both donor HLA typing and solid phase antibody screening are utilized together. It is compared specific anti-HLA antibodies in the recipient with the HLA profile of the donor.
It can lead to shorter wait times and improved outcomes for sensitized transplant recipients.
The results from VXM are not precise and current practice mandates an actual
crossmatch be performed as well. It does not identify the HLA “Null” alleles. Renal recipients can be classified into : High immunological risk
They have high titers of DSA which can lead to hyperacute rejection. Intermediate immunological risk
They have a low titer of DSA, and historic DSA is not detectable. Standard immunological risk
Where there is no evidence of DSA.
Renal transplantation is the best modality of treatment for patients with ESRD. It improve survival and quality of life. So accurate & careful cross-match & tissue typing are crucial for successful transplantation.
There are several methods of tissue typing:
Seriological: it is widely available, the results available in short time, so less cold ischemia time. But this method can not identify HLA-Cw, -DQ &- DP Ag.
molecular methods: (a): Sequences-specific primer PCR.
(b) Sequence-specific oligonucleotide probe
(c) Direct DNA sequence.
Molecular typing can detect the differences in HLA Ag at amino acid & epitopes level. But it had a disadvantage that it can’t detect new allelfsthat not found in databank.
About half of patients on waiting list are sensitized due pregnancy, blood transfusion or previous organ transplantation. The presence of antibodies increase the risk of rejection, so antibodies detection is very important which can be done by:
cytotoxic (cell bead) Ab screening: this method had several limitation as HLA frequency & racial difference can’t be applied , false +ve results due to non HLA Ab or autoAb &false neg results may be due to low titer Ab that can’t activate the complement
Solid phase Ab: (a) ELIZA platform
(b) Micro-bead platform/ single Ag bead
SAB result can be obtained through 3-4 h. It can detect both common & rare HLA-Ab.
SPA can detect complement & non complement binding Abs even with very low level. But non HLA Abs can’t be measured solely by this method.
Cross-match techniques:
CDCXM: can be done for B & T cells. it had low sensitivity in low titer of Abs, but the sensitivity can be increased by increasing incubation time or adding AHG.
FCXM: independent of complement fixation in detection of DSA. Can detect Abs against B & T cells . It not widely used.
VXM: used for both donor HLA typing & SPA screening. Useful for sensitized transplant recipients with less waiting time. Serum sample better to be 3-6 months before transplantation because Abs titer is differ with time. False +ve occurs with low HLA titer Abs & non complement binding Abs. It can’t identify null alleles ( identified DNA sequence with out expression of HLA product on cell surface).
According to tissue typing & cross match the immunological risk can be classified into :
High risk ( high DSA Abs for mismatched donor)
intermediate risk ( low titer DSA or previous DSA that disappear at time of transplantation)
standard risk ( no evidence of previous or recent DSA)
Drtalib Salman
3 years ago
HLA TYPING
serology:
used in deceased donor (quick method),serum with Ab in tray mix with donor lymphocyte and see reaction rarely used now .
Molecular typing :
PCR amplification process word wise use.
Direct DNA sequence precise of nucleotide .
HLA mismatch carry high risk of rejection
HLA class 1 (A,B,C) .
HLA class 2 ( DP,DQ,DR).
A,B.DR mismatch carry high risk of rejection du to more allogenicity.
DQ mismatch risk for denovo DSA.
DR mismatch may cause early rejection (first 6 month ).
risk of rejection increase with increase in mismatch may reach up to 60 percent in case of 6 mismatch .
HLA Ab screening :
1-cell based Ab screening .
2-Solid phase Ab screening
-Luminex (single antigen bead ).
quick method 3-4 H
commonly used in allocating system .
disadvantage
miss non HLA.
canot differentiate complement from non complement.
used in virtual cross match .
false negative from prozone phenomena either due to high titer or complement (c1q)
or IgM and drug (IV Ig).
Cross Match:
CDC (complement dependent cytotoxicity )
complement dependent reaction.
false negative may be due to low titer .
false positive due to auto Ab, IgM, IgM reaction.
T cell positive absolute contra indication.
B cell positive relative contraindication.
Flow cytometry cross match :
highly sensitive used in highly sensitized patient .
non complement reaction.
Virtual cross match:
not true cross match.
commonly used in deceased donor.
according to lab result patient divide to 3 group:
-high immunological risk.
-intermediate immunological risk .
-standard immunological risk.
Please elaborate more in classification of Risk as this is the value you get for clinical practice
Huda Al-Taee
3 years ago
HLA Typing:
serological typing
molecular typing
Serological Typing:
In this method, the recipient lymphocytes are introduced into a tray wells contacting sera, complement, and dye. when antibodies bind to the antigen, complements are activated resulting in cell death, so the dye will enter into the cells. Wells with significant cell death are identified under a microscope.
The benefit of this method:
results are available in a short time and this is important in the deceased donation.
the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but no cell surface antigen expression( null HLA alleles).
Disadvantages:
lack of sera with antibody specificities that can identify other HLA alleles such as HLA-Cw, DP, & DQ which have an impact on graft outcomes.
Do not detect differences in HLA protein-small amino acids that may be immunogenic enough to trigger an immune response.
Molecular Typing:
Sequence-specific primer polymerase chain: extracted DNA from the subject is amplified in several wells.
Sequence-specific oligonucleotide probes: amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles.
Direct DNA sequencing: using published HLA allele sequences, HLA type is subsequently assigned by comparison.
Advantages:
clearly identify the differences in HLA antigen between the donor and the recipient.
give details till the amino acid level
highly specific
there is cross-reactivity( identification of an allele that is essentially similar to the allele of interest).
Disadvantages:
new alleles that are not on the HLA sequence databank will fail to be identified.
HLA antibody screening:
cytotoxic (cell-based) antibody screening.
solid-phase antibody screening.
Cytotoxic antibody screening:
recipient serum is mixed with donor lymphocytes in a well with complement and dye.
if the serum contains antibodies against the donor, the binding will happen and complement system activation resulting in cell death and uptake of the dye.
the degree of cytotoxicity is expressed as percentage PRA.
Limitations:
PRA percent can be different numerically without a corresponding change in the type or amount of antibody.
HLA frequencies and racial differences need to be factored in.
false-positive results can be produced by autoantibodies, IgM, non-HLA antibodies.
false-negative results due to non-complement fixing antibodies.
Solid-phase antibody screening:
enzyme-linked immunosorbent assay platform: HLA molecules applied to ELISA platform and will bind to HLA antibody after the addition of recipient serum.
microbead platform/single-antigen beads: a panel of beads loaded with either class I or II HLA antigens, patient serum added to each bead, a fluorescent dye is added to detect the presence of antibody which is bound to the antigen, detection is done by flow-cytometer or through the SAB Luminex platform.
Advantage of SAB Luminex testing:
more sensitive for low titer antibody than ELISA.
specific for HLA antibodies.
rapid test( results are available within 3-4 hrs).
enable virtual CXM
identify anti-HLA antibodies for all common and numerous rare antigens and alleles.
Limitations:
susceptible to prozone phenomenon.
IgM binding
Crossmatching:
CDC CXM
Flow-cytometry ( FCXM)
Virtual CXM
Complement-dependent cytotoxicity:
considered positive if a considerable number of lymphocytes are destroyed after adding the complement.
can be done for B & T lymphocytes.
sensitivity is limited in low titer antibodies.
false-positive results in the presence of autoantibodies, IgM, non-HLA antibodies.
Flow-cytometry crossmatch:
recipient serum is mixed with donor lymphocytes and then tagged with fluorochrome-conjugated anti-IgG antibody.
independent of complement-fixing.
precisely detects the presence of IgG DSA.
has greater sensitivity than CDC
Virtual crossmatching;
donor HLA typing and solid-phase antibody screening are utilized together.
leads to shorter wait times.
titers, specificities, and the presence or absence of antibodies vary over time.
false-positive results since the list of all potential HLA donor antigens has been classed differently.
the results from VXM are not 100% accurate and wet CXM is mandatory.
1- Serologic method using either antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)
· In this method recipient lymphocytes are introduced to a tray that contain sera of antibodies to all known HLA antigens, complement and coloring dye
· Once Ag-AB reaction occurs complement activation occur, MAC is generated, cell lysis , then cells take the dye which appear red under microscope
· Although it is rapid method but its use dropped due to growing numbers of new HLA alleles with which can not be covered by the sera
2- DNA-based HLA typing methods which is more sensitive and accurate than serological technique and it includes the following:
· SSOP sequence specific oligonucleotide probe hybridization
· SSP sequence-specific primer amplification
· Direct DNA sequencing CROSS MATCH ASSAYS I– CDC cross match
– It involves incubation of donors lymphocytes in recipient serum, washing to remove unbound antibodies, then adding complement and after incubation period, coloring dye is added.
– If antibodies to donor HLA present complement activation occur, MAC is generated, cell lysis , then cells take the dye which appear red under microscope
Uses
– CDC is used either in determining compatibility between donor and recipient or in determining sensitization in case of PRA
– A positive CDCXM indicating the presence of complement fixing antibodies IgG or IgM, against HLA or non HLA antigens or autoantibodies, but it does not detect non complement fixing AB against HLA Ag which may be significant (IgG2, IgG4) and low level DSA.
– CDC has low sensitivity with false positive result that can occur due to the presence of clinically non-significant non HLA abs or autoimmune IgM antibodies which can be eliminated either by heating or adding reducing agent also Rituximab can cause false positive B cell cross match or may be due to technichal error.
– CDC has low specificity with false negative result that can occur due to non complement fixing AB or low level of HLA antibodies or technical errors, also HLA Ag expression may be low compared to flow cytometry II- Flowcytometry
– It involves incubating donors lymphocytes with recipient serum , then add second antibody with fleouricin dye (green) that will bind to bound ABS (DSA) attached to donor lymphocytes, then adding 2 other ABs one bind to CD19 in B cells (red)and the other bind to CD3 in T cells (yellow) to know the type of cell affected and this is detected by laser
– So seeing green and yellow dye means T cell + cross match, green and red dye means B cell+ cross match and seeing all 3 dyes means T and B cell + cross match
Uses
-FCM is used either in determining compatibility between donor and recipient or in determining sensitization in case of cPRA
Significance
– A positive cross match using FCM indicating the presence of complement fixing or non fixing antibodies IgG only (not IgM), againest HLA or non HLA antigens or autoantibodies and low level DSA, So CDC – FCM + cross match indicates the presence of non complement fixing AB or low level DSA.
– More sensitive than CDC as it use lymphocytes which express massive HLA antigens and detect only IgG and not IgM
– False positive results can occur due to binding of non HLA IgG or auto antibody to B cell, also rituximab was found to cause false positive results
– Flow cytometry method, that measure fluorescence intensity and compare it to control (median channel shift- MCS)
III- Solid phase assays (ELISA or beed assay- luminex)
– Beed assay (luminex) replace ELISA
– In beed assay the patient serum is tested against HLA antigens attached to solid beeds labeled with fluorescein, each bead either have single (SAB) or multiple HLA molecule then anti human globulin labeled with phycoirythrin is added. Antigen-antibody reaction is detected via laser based fuorescent imaging
– Used to detect DSA
– Can detect complement and non complement fixing antibodies, low level DSA
Sensitivity
– Very sensitive, as it detect only Abs against HLA antigens so avoid false positive results due to non HLA antibodies or auto ABS
– False positive result can occur due to denatured antigens attached to beeds, leading to alteration in protein configuration that can be overcomed by treating beeds with acid that fully denature the beads protein , also 2 kits may be used
– False positive result can occur due to detection of low level DSA which may not be significant leading to positive luminex negative cross match, thus another tes is added to increase the sensitivity of luminex which is C1q assay that identify only complement fixing antibodies that are clinically significant, but the test is expensive so its use is limited
– Very specific, but false negative result can occur if patient has high levels of IgM or Complement factors (C1) (such as those on long term HD) that can bind to antigen beeds and prevent binding to actual IgG-DSA, or may occur due to very high level of DSA that agglutinate so fail to bind to the beed.
– Calculates the degree of fluorescence (median fluorescence intensity -MFI) through the use of 2 lasers to excite the florochrome of the beed and the phycoerythrin bound to the antibody. IV- Virtual cross match
– By comparing anti HLA antibodies of recipient detected by luminex SAB to HLA profile of the donor.
– It is correlated well with FCM cross match and graft survival even in sensitized patients
– Define unacceptable antigens so donors can be excluded, and allow identification of suitable donor
– Wet cross match should be performed in only sensitized patients as there may be a new DSA that develop, this should be detected by wet cross match (positive FCXM, negative VXM), in non-sensitized recipient we can rely on VXM in proceeding in transplantation
– A positive VXM with a negative FCXM can occur due to non clinically significant low titre DSA that will not affect transplantation, so it is safe to proceed to transplantation
Well read ,very well understood Thankyou for referring to MFI ,MCS though you skipped risk stratification which is your main goal as a clinician.
The table supplied is useful but could be much simpler.
MICHAEL Farag
3 years ago
HLA TYPING
HLA typing is a crucial step in renal transplantation, as recognition of foreign HLA by recipient T lymphocytes would trigger an immune response. T lymphocyte activation initiates a cascade of mediators that direct the immune system against the allograft
There are 2 types of HLA typing
I) Serological typing Idea: a tray containing sera with antibodies to a multitude of known HLA alleles is used. These are commercially available. For typing, recipient lymphocytes are introduced into the tray wells contacting sera, complement and dye. In tray wells where antibodies can bind to the antigens on the surface of lymphocytes; complement is activated. This results in complement pathways triggered resulting in cell death, ultimately allowing the dye to enter the cell. Tray wells with significant cell death are then identified under phase contrast microscopy.
Advantage: – the results are available in a short period. This is particularly important in deceased donor renal transplantation. Quick results mean less cold ischemia times. – This method also offers the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression. These alleles termed “null” HLA alleles are of less immunological significance. Disadvantage:
– the lack of sera with antibody specificities that are capable of identifying the ever-growing number of HLA alleles. The HLA-Cw, DQ, and DP antigen may have clinically significant effects on the outcomes of allografts. However, serologic assays are scarce for these loci.
– serologic methods do not readily detect differences in HLA protein small aminoacids. These may be antigenic enough to trigger potent immunological responses. With more advanced methods of typing currently available serological typing has fallen into disuse
II) Molecular typing There are some techniques to do that:
– Sequence-specific primer polymerase chain reaction
– Sequence specific oligonucleotide probes – Direct DNA sequencing
Molecular typing regardless of the method can clearly identify differences in HLA antigen between donor and recipient. Often with detail to the amino acid level that can provide insight to the risk accompanying mismatched donor-recipient antigens, epitopes and amino acid
HLA ANTIBODY SCREENING
Preformed antibodies increase the chances of immunological failure of the allograft.
Both sensitive and specific detection of anti-HLA antibodies is crucial. Where crossmatch is negative, even low titres of DSA can lead to early as well as late antibody mediated rejection.
There are different methods used for HLA antibody screening as shown below.
I) Cytotoxic (cell-based) antibody screening Idea: therecipient serum is mixed with “cell donor” lymphocytes in individual wells along with complement and dye. Where the serum contains antibodies that bind to the cell surface with adequate density complement pathways are activated which results in cell death and uptake of the dye. The degree of cytotoxicity is expressed as percentage PRA (panel reactive antibody)
Disadvantages:
– PRA percent can be different numerically without a corresponding change in the type or amount of antibody. This largely depends on the cell panel used which are commercially produced and may not truly represent the population.
– Significant false positive results can be produced due to non-HLA antibodies, autoantibodies and nonspecific IgM antibodies.
– false negative results are possible as this is purely complement dependent that requires higher antibody titres to be activated
– The lack of a complement activation simply due to low titres allows a true antibody to be hidden
– Precise, complete lists of antibody specificities and unacceptable antigens cannot be identified using this method as there are several antigens in each well II) Solid phase antibody screening
This method employs soluble or recombinant HLA molecules instead of lymphocytes targets – as lymphocytes present both HLA as well as non-HLA molecules. The variants of these methods are: –Enzyme-linked immunosorbent assay platform -Microbead platform/single-antigen beads III) CROSSMATCHING (XM)
With PRA (HLA antibody screening) that identifies several antibodies to a potential cluster of donors, the crossmatch will identify if a recipient had antibodies to a specific donor of interest.
The solid-phase antibody test should be used together with crossmatch results to identify those that are immunologically relevant
The techniques of cross match are: – Complement-dependent cytotoxicity crossmatch – Flow cytometry crossmatch – Virtual cross match
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Sahar elkharraz
3 years ago
renal transportation is the best option in patient with ESRD even with autoimmune disease.
this article focus on risk of immunological stratification in renal transplants.
careful testing of HLA cross match help to good outcome of graft and avoid of allograft rejection.
HLA typing is the first and important step in renal transplantation. it helps for recognition of foreign HLA T. lymphocytes by recipient against allograft.
2 main study to recognise T lymphocyte activation by HLA. (serology/ molecular studying).
serology typing: identifying lymphocytes activation by uptake dye. it’s limitation not identifying specific type of HLA proteins.
molecular typing: identifying specific type of HLA alleles.
the most important HLA typing are HLA A, HLA B, HLA DR.
HLA DR matching has great effects than HLA A,B
HLA antibody screening: most of wait listed patients have anti HLA Ab detected.
most highly sensitised patients had exposure to blood transfusion/ pregnancy/ previous transplant. Presence of these antibodies increases chance of organ failure. But sensitised patients successfully transplantation possible by using method of desensitisation (paired exchanges & acceptable mismatching.
cytotoxic antibody screening: it’s same as serological typing (if positive uptake dye indicates positive cross match & degree of cytotoxicity expressed by percent of PRA. this method has limitations because it depend on amount of antibody in panel and it may hidden the true antibody. so this method difficult to identify specific AB & unacceptable Ag.
Solid phase Ab: this method to detect HLA molecule & non HLA molecule by ELISA // micro bead platform (SAB)
ELSA : to detect HLA antibody bind to Ag.
micro bead platform/ single antigen beads to detect HLA antibody Antigen by flow cytometry or by single antigen luminex platform.
this method detect specific HLA Ab-Ag and calculated panel reactive Ab the best to detect positive cross match donor specific Ab.
there’s difference between PRA & c-PRA.
PRA value is reflecting high probability of positive cross match
c-PRA better than PRA in detect unacceptable HLA Ag especially in high sensitized patients.
C-PRA is useful in allocation of kidney & pancreas transplantation.
high titer of c-PRA indicated incompatible donor & it’s called positive cross match.
micro bead assays (flow PRA & Luminex are 10% more sensitive for lower titer antibody than ELSA.
ELSA is 10% more sensitive compared to anti-human globulin.
SAB are rapid test & result available in 3 to 4 hr ; it help to identify DSA pre transplant & organ allocation. SAB help to identification of anti- HLA Ab for all common & rare Ag & alleles. Also help to identify complement & non complement binding.
SAB luminex assay: this method need dilution of sera to avoid prozone phenomena which can give false negative. this artifact results from several factors like complement compound 1 which prevent bind HLA Ab to Ag. it can also arise from IgM Ab and use of intravenous immunoglobulin.
Cross Matching:
presence of DSA indicate higher rates of organ failure.
T cell cytotoxicity cross match may had false positive & false negative. So it insufficient to identify all relevant AB.
Solid phase antibody test should be used together with cross match to identify immunological relevant.
CDC cross match become positive if high number of lymphocytes destroyed after incorporated of complement. this indicates significant DSA has been found in surface of cell.
CDC cross match can done for B & T lymphocytes.
it limited if titer is low with relevant Ab.
Flow cytometry cross match: detect DSA independent of complement fixation. it is detect presence Or lack of IgG DSA on donor lymphocytes. this method is not widely used because different laboratories method & different results; also it’s immunological risk unclear.
Vitiral cross match: used in high sensitized patients.
Thankyou for trying but you need to try harder.
Read ,understand then write short ,simple sentences.
Avoid spelling mistakes which could be serious eg.ELISA notElSA
Comp. component not compound.
Keep trying .
Mohamed Saad
3 years ago
Transplantation is the best modality of Renal replacement therapy.
Understanding of transplant immunology is decisive and effective in transplant outcome. There are two methods for HLA typing:
-Serological.
– Molecular typing methods.
================================
1-Serological HLA typing:
-Depending on reaction between recipient lymphocyte (ag)+serum (anti bodies) +dye, reaction led to trigger of complement pathway which led to cell death. then identified under phase contrast microscopy.
Advantages of this method:
Results are available in a short period so it’s important in deceased donor renal transplantation because it decreases cold ischemia times.
Detection of “null” HLA alleles which are of less immunological significance.
Disadvantages:
Lack of sera with antibody specificities that are capable of identifying the ever-growing number of HLA alleles
e.g.: The HLA-Cw, DQ, and DP antigen
Do not detect differences in HLA protein small amino which may be antigenic enough to trigger potent immunological responses.
2- Molecular typing:
Molecular typing regardless of the method can clearly identify differences in HLA antigen between donor and recipient.
=> Sequence-specific primer polymerase chain reaction.
-In which HLA typing is allocated by matching the primers of the amplification product to DNA sequences of several candidate alleles.
=> Sequence specific oligonucleotide probes.
In which amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles.
=> Direct DNA sequencing.
Initial Collaborative Transplant Study (CTS) analysis that HLA-DR and HLA-B antigens offer the most alloimmune burden with less so from HLA-A.
Euro transplant and old United Kingdom transplant data suggest that HLA-DR matching has a far greater effect than HLA-A or HLA-B.
====================================== HLA antibody screening:
The usual route for sensitization towards HLA antigens occurs in three instances; pregnancy, post blood transfusion and prior transplantation.
successful transplantation is possible by employing strategies such as desensitization, paired exchange and acceptable mismatching. Methods used for HLA antibody screening:
= Cytotoxic (cell-based) antibody screening.
Recipient serum is mixed with “cell donor” lymphocytes with complement and dye, similar to that of serologic typing.
False positive results can be produced due to non-HLA antibodies, autoantibodies and nonspecific IgM antibodies.
False negative results are possible as this is complement dependent which requires higher antibody titers to be activated.
= Solid phase antibody screening.
It depends on recombinant HLA molecules instead of lymphocytes (to avoid non-HLA antibodies).
Many methods of solid phase antibody screening
1- Enzyme-linked immunosorbent assay platform.
Enzyme conjugated antibodies to IgG (human) is then added to detect the presence of HLA antibody in the serum which is bound to the antigen.
2-Microbead platform/single-antigen beads:
Microbead that is fluorescent dye conjugated is then added to detect the presence of HLA antibody in the serum which is bound to the antigen.
Fluorescence detection can be done traditionally using a flow cytometer or by the single-antigen beads (SAB) Luminex platform.
These estimate PRA by the proportion of positive beads. SAB are individually coated with a single HLA antigen and yield a list of distinct antibody specificities.
cPRA is useful in the allocation of kidney and pancreas transplants. cPRA estimates the proportion of donors with whom a particular recipient would be incompatible.
N.B: The SAB – Luminex assay has been shown to be susceptible to an artefact known as the prozone phenomenon which give false negative which might be due to many causes (C1q which prevents HLA antibody binding to the HLA antigen on the bead,IgM abs, intravenous immunoglobulin and epitope sharing). CROSSMATCHING (XM)
==Complement-dependent cytotoxicity crossmatch (CDC-XM) can be done for B and T lymphocytes.
CDC-XM can also give false positives by detecting autoantibody, IgM/IgG HLA or non-HLA.
== Flow cytometry crossmatch:
Here recipient serum is mixed with donor lymphocytes and then tagged with a fluorochrome conjugated anti-IgG antibody which is more sensitive than CDC-XM.
=Virtual crossmatching:
In virtual crossmatch (VXM), both donor HLA typing and solid phase antibody screening are utilized together.
Professor Ahmed Halawa
Admin
3 years ago
Thank you All I’m still waiting for all colleagues to complete their logging
Riham Marzouk
3 years ago
Cross match can be done using several methods like CDC (complement dependent cytotoxicity crossmatch) and FCXM (flow cytometry crossmatch) , the problem of CDC; it can not be recognize autoantibody it could be positive due to presence of autoantibodies as in autoimmune diseases, but the patient is positive by FCXM, also CDC can detect complement dependent antibody.
There are several methods to detect antiHLA antibody :
1- Serological methods which depend on the recipient lymphocytes which will react with different antibodies against different HLA alleles in tray containing wells, it is rapid method can be used in deceased donor transplant to decrease cold ischemic time , also can detect NULL alleles which has no antigen expression but this has less immunological significance , it has some disadvantages like being less specific or limited can not be used to detect more specific growing alleles.
2- Molecular methods: a- Sequence-specific primer polymerase chain reaction
b-Sequence specific oligonucleotide probes
c-Direct DNA sequencing
PCR based HLA typing is more specific to detect more alleles and detect cross reactivity with the allele of interest but if alleles not present in database of alleles bank , will not be detected and this is its disadvantage.
HLA mismatch has important impact on graft survival especially DR and B more than A , the more mismatch especially in DR and B the more negative impact on graft survival.
HLA-DR mismatch affects graft in the first six months but HLA-B mismatch affects graft in the first two years.
Screening of HLA antibodies can be done as the following :
1- CDC complement dependent cytotoxicity lymphocyte of recipient mixed with donor cells of 30-40 donors…if antibody found and detected by complement reaction and cell lysis, the result will be positive test , this test has limitation as it cannot differentiate autoantibodies and also cannot detect non complement dependent antibody, also the degree of positivity can be presented as panel reactive antibody which depends on the quality and quantity of antibodies used in the test which may differ and not truly represent the population as a whole, also cannot differentiate between HLA and non-HLA antibodies.
2- Solid phase antibody screening use soluble or recombinant HLA molecule as lymphocytes carry HLA and non-HLA antibodies:
Can be done by : Enzyme-linked immunosorbent assay platform Microbead platform/single-antigen beads : Fluorescence detection by using a flow cytometery or single-antigen beads (SAB) Luminex.
Calculated PRA can be used from that point which use a pool of many donors reach more than 10000 donors to get more specificities and depend on the nationality and race and the place, also it yields the list of unacceptable genes which help in the program of kidney transplant.
3- Flow cytometry CM
It detect non complement IgG DSA, it is more sensitive than CDC
4- Virtual CM
It is not an actual cross match but it is in vitro one , HLA typing of recipient is compared to a pool of donors HLA typing and the match will be seen , this to find a donor for sensitized patient and to improve their transplant outcome.
Based on presence or absence of DSA at the time of transplantation, will stratify the patients as regard their risk; high risk who has high titer of DSA at the time of transplant and need pretransplant desensitization protocol and of course potent(high risk) induction and maintance protocol with close monitoring post transplant, intermediate risk who has low titer of DSA or history of its presence so need potent (high risk) induction and close immunological monitoring post transplant, low risk patients has no DSA at the time of transplant and need standard risk protocol of induction.
HLA typing:
Identification of a foreign HLA by recipient T cell can trigger an immune response against the allograft.
Types :
1- Serological typing: sera with antibodies to multiple Known HLA alleles are used, to this T lymphocytes of the recipient are added with complement and dye. It gives rapid results. Which leads to less cold ischemia times.
2- Molecular typing: Sequence-specific primer PCR: PCR HLA typing is highly specific with identification of specific allele with no cross-reactivity.
HLA antibody screen
Preformed anti-HLA antibodies (DSA) may occur as a result of previous sensitization as in ; blood transfusion, and prior transplantation. they can cause positive crossmatch, so refuse this donor. DSA low titres can make ABMR.
1- Cytotoxic antibody screening
A group of donors cells . Recipient serum is mixed with donor lymphocyte along with complement and dye. If the serum contains antibody, it will bind cell surface antigen causing complement activation results in cell death and dye enter the cell. The percentage of cell death represents as PRA. false positive results can occur as in non-HLA antibodies, autoantibodies.false -ve if low AB titres.
2- Solid phase antibody screening:
I. ELISA platform contains purified HLA molecules that bind to individual HLA antibody after the addition of recipient serum.
II- single antigen beads: (SAB)
Beads coated with varible class I or II HLA antigens are added to fluorescent dye conjugated microbead to detect the HLA antibody in the patient serum. The fluorescence detected by flow cytometer or via the single antigen beads (SAB) Luminex platform. Positive beads represent PRA percent which ; represents a probability of a positive crossmatch.
cPRA is based on unacceptable HLA antigens- to which the patient has been sensitized to. High c PRA carries high probability of positive crossmatch.
SAB can identify anti-HLA antibodies for all common and numerous rare alleles.
Solid phase assays can detect both complement and non-complement binding antibodies. They are so sensitive.
But ; false negative results can occur in: Sera with high anti-HLA antibodies titer give negative results when tested neat, while react positive after 1:10 dilution. This called prozone phenomenon, Presence of IgM antibodies, Drugs like IVIG , Epitope sharing.
Crossmatching (XM)
Presence of DSAs represented as a positive crossmatch was contraindicated to transplantation. it is insufficient to identify all relevant antibodies, and may exclude potential donor unnecessarily, to avoid this; solid phase test should be used together with crossmatch.
1- Complement dependent cytotoxicity crossmatch
Positive test as determined by number of destroyed cells, points to the presence of DSA. It is done for both B and T lymphocytes. In the presence of low titre DSA the sensitivity of the test is low. False negative can occur in low titre antibodies. While false positive can occur with autoantibodies, nonHLA antibodies.
2- Flow cytometry crossmatch:
It detects DSA whether complement fixing or non-complement fixing. Recipient serum is mixed with donor lymphocytes . With the use of flow-cytometry, B and T lymphocytes can be determined and their DSA individually determined. It is highly sensitive but not available widely.
Virtual crossmatching (VXM): both donor HLA typing and solid phase screening are utilized together to predict true in vitro crossmatch results. Its result changes over time, so it should be done within 3 – 6 months. It does not identify Null HLA alleles
Immunological recipient risk :due to prior transplantation, recipient age, DSA, sex, original disease:
High immunological risk:
High titres of DSA at time of transplantation . Treatment with desensitization protocols.
Intermediate risk:
Low titre of DSA. Treatment : intensification of the immunosuppressive drugs and post transplant monitoring of DSA
Standard risk: No DSA , for standard conventional immunosuppression therapy.
Human leukocyte antigen typing and crossmatch: A comprehensive review
there are two main methods for HLA typing
1. Serology method
2. Molecular method.
1)Serology method
In which Recipient’s lymphocytes (which carries unknown antigens) are mixed with antibodies to known HLA antigens, dye and complement in a tray.
If the antigen reacted with the specific corresponding antibody, cell lysis occurs mediated by complement system. Dead cells are stained by the dye and identified by phase contrast microscope then HLA typing is diagnosed.
Advantages:
· rapid HLA typing which reflect in reducing cold ischemia time in cadaveric transplantation.
· Able to diagnose “null” HLA alleles
Disadvantages:
· Unable to diagnose all HLA alleles.
· Cross reactivity with CREG specially in using nonspecific broad antibodies.
· Can’t be use in diagnosis epitopic analysis.
That’s why most of HLA labs now using molecular methods.
2)Molecular method.
HLA typing on nuclear level which translated by m-RNA to HLA antigens on cell surface which done by
1. Sequence specific primer PCR.
-HLA related DNA sequences on chromosome 6 is diagnosed by specific manufactured primer.
2. Sequence specific oligo-nucleotide probes.
-HLA related DNA sequencing diagnosed by oligo-nucleotide probes tagged by specific fluorescent dye.
3. Direct DNA sequencing.
-direct diagnosis of the DNA sequencing.
Advantages:
· Specific
· Can be used in diagnosis of epitopic matching specially in high resolution typing.
Disadvantages:
· Unable to identify new alleles
· It takes time which means prolonged ischemia time
Crossmatch for antibodies screening.
2 main methods for antibodies screening:
Wet crossmatch which means actual mixing donor’s lymphocytes which carries HLA typing with recipient’s serum in which Ab is present.
1. Wet crossmatch includes (CDCXM and FCXM)
2. Solid phase crossmatch which means mixing recipient’s serum with beads carrying different HLA typing.
1)Wet crossmatch:
A) Cytotoxic antibody screening:
It’s one of the wet crossmatch modalities in which recipient’s serum (containing Ab) is mixed with donor’s lymphocytes (carrying HLA) along with complement and dye in a tray. Positive results are considered if cell death occurred and uptake of the dye. The result is express as percentage against both B cell (which carries both class I, II) and T cell (which carries only class I)
Advantages:
Specific in detection in complement fixing antibodies.
Disadvantages:
· less sensitive give you false negative due to low antibody titer.
· False positive due to non-HLA antibodies, autoantibodies, some drugs like Rituximab (can be avoided by adding pronase) and nonspecific Ig M antibodies (which can be avoided by adding DTT).
· Can’t identify antibody specificities and unacceptable antigens.
b) Flow cytometry crossmatch. (FCXM)
it’s wet crossmatch in which recipient serum (containing antibodies) mixed with donor’s lymphocytes then anti-anti HLA antibodies are added if some positive reactions are presents it will be diagnosed by florescent tagged antibodies. Results are diagnosed by flowcytometry and expressed by MCS.
Advantages:
-very sensitive can diagnose all antibodies even non-HLA antibodies and non-complement fixing Ab which may not carry harmful effect on the graft.
Disadvantages:
-non specific
2)Solid phase antibody screening.
I. ELISA.
It’s type of solid phase crossmatch in which purified HLA antigen mixed with recipient serum. Enzyme conjugated antibodies to IgG is added. Antigen antibody positive reaction is identified by optical density reading.
II. mixed microbead platform/ single -antigen beads.
it’s type of solid phase crossmatch in which latex beads coated with class I or II HLA antigens are mixed with recipient serum and used for antibody screening. Fluorescent dye is added and AB are detected via flowcytometry.
Advantage:
-it gives specific list of antibodies and unacceptable antigens.
Disadvantage:
-false positive cause by denaturated proteins
-false negative cause by prozone phenomenon.
Risk assessment in renal transplant patients:
Depend on multiple factors like age of recipient, sex ,1ry kidney disease, HLA mismatch and DSA
Specific definition and risk stratifications differs from transplant center to another
The more HLA mismatch and the Prescence of DSA consider immunological risk and associated with increase risk of rejection, poor long term and short-term graft survival.
This article is displaying the different methods performed to outline the immunologic risk pertaining to kidney transplantation elucidating on the HLA typing and anti-HLA antibody detection methods .It started by showcasing a clinical vignette of SLE case with end stage kidney disease tested positive with CDC and negative with FCMX to both T and B cells. and SAB Luminex did not reveal any antibodies. He don’t have a positive history of blood transfusion. This contradicting result was stemmed from the presence of non HLA antibodies binding to the FC region. The HLA A, B and DR are the major determinants of Allo-sensitization, with DR antigen has the major impact than HLA A and B, One study suggested that the DR mismatch affect the the outcome in the first 6 months. And the HLA B effect is usually after 2 years.
6 antigens mismatch is reflecting 65% higher risk of rejection in comparison to 13% with 1 antigen mismatch.
Taboo mismatches:
Seven specific HLA mismatch combinations were associated with reduced renal allograft survival.
one third of waitlisted patients are having anti-HLA antibodies. They are usually stemming from previous transplantation, blood transfusion or pregnancy.
quantification and specification of DSAs are important to be stratify further management.
Notwithstanding negative cross match, even low DSAs titer might portend a deleterious consequences.
Methods to overcome the DSAs barrier entail desensitization, paired kidney donation and acceptable mismatches.
Human leukocyte antigen typing and crossmatch: A comprehensive review
Understanding crossmatch and HLA typing for kidney transplantation and applying it in clinical practice is an important step to achieving a good outcome
HLA typing:
Identification of a foreign HLA by the recipient T cell can trigger an immune response with activation of complement cascade directed against the allograft causing its rejection. Therefore; HLA typing is a key step in transplantation.
There are serologic and molecular typing.
1- Serological typing
Tray wells containing sera with antibodies to multiple Known HLA alleles are used, to this T lymphocytes of the recipient are added with complement and dye. If antigen on T-cell reacts to antibody in the well, complement activates and cell lysis occurs and dye enters the cells. coloured cells identified by polarized microscope. By elimination and comparison of positive wells, the HLA type is assigned.
Advantage:
· It gives rapid results. Which leads to less cold ischemia times.
· Differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression. This allele called null HLA are of less immunological significance.
Disadvantages:
1- As the HLA is polymorphic, sera with antibody specificities that are capable of identifying the ever-growing number of HLA alleles are lacking. For example; the HLA-Cw, DQ and DP antigen that may have clinically significant effects on the transplantation, their serologic assays are scare.
2- Serologic methods do not exactly detect differences in HLA protein small amino acids that may be antigenic and trigger potent immunological responses.
2- Molecular typing: done by:
· Sequence-specific primer polymerase chain reaction: in this method, recipient DNA is amplified in several wells. Each has a primer that is integral to specific HLA alleles. Amplification of the DNA occurs if meets the complementary DNA probe. Which then implanted into an agarose gel and undergoes electrophoresis, ultimately appearing as a band. By matching the primer that cause amplification to DNA sequences of several candidate alleles, the HLA typing is assigned.
· Sequence specific oligonucleotide probes:
Probes with oligonucleotide complementary to specific DNA segments of different alleles is mixed with amplified DNA. Then by fluorescent tags, unique HLA alleles are identified. HLA type is then assigned using available sequences.
· Direct DNA sequencing:
This method allows for a precise gene sequence. HLA typing is determined by comparison.
PCR HLA typing is highly specific with identification of specific allele with no cross-reactivity. Cross-reactivity is the identification of an allele similar to the allele of concern.
The new alleles that not on the HLA sequence databank will fail to be identified by this method.
The dissimilarity in the HLA antigen contributes to the alloimmune burden.
Recently, with the use of potent immunosuppression, the HLA mismatching in deceased donor kidney transplants become less significant.
HLA antibody screen
Preformed anti-HLA antibodies (DSA) may occur as a result of previous sensitization during blood transfusion, pregnancy, and prior transplantation. they can cause positive crossmatch, leading to exclusion of the donor. DSA even in low titres can lead to antibody mediated rejection. Still transplantation in sensitized patients is possible by using strategies such as desensitization, paired exchange and acceptable mismatching.
1- Cytotoxic antibody screening
A group of donors cells that represent the population of potential deceased donors are selected. Recipient serum is mixed with donor lymphocyte along with complement and dye. If the serum contains antibody, it will bind cell surface antigen causing complement activation results in cell death and dye enter the cell. The percentage of cell death represents as PRA, which is a tool that can estimate the risk of recipient to have a positive crossmatch to a likely donor organ taken from a similar population.
Limitations of cytotoxic crossmatch:
The cell panels which are commercially produced, may not represent the population truly, causing PRA percent numercally is not correlate with the type or amount of antibody.
Significant false positive results can occur as a result to non-HLA antibodies, autoantibodies, and nonspecific IgM antibodies.
False negative results can be if there is low antibody titres leading to miss of significant antibody.
Because; the well contains several antigens, the specificity of these antibodies cannot be determined as well the unacceptable antigens
2- Solid phase antibody screening:
Soluble or recombinant HLA molecules are used.
I. Enzyme-linked immunosorbent assay platform:
ELISA platform contains purified HLA molecules that bind to individual HLA antibody after the addition of recipient serum, enzyme conjugated antibodies to IgG is then added to detect the presence of HLA antibody in the serum which is bound to the antigen.
II- Microbead platform/single antigen beads: (SAB)
Beads coated with varible class I or II HLA antigens are added to fluorescent dye conjugated microbead to detect the HLA antibody in the patient serum. The fluorescence detected by flow cytometer or via the single antigen beads (SAB) Luminex platform. Positive beads represent PRA percent.
SAB specificities are compared with HLA frequencies in the donor population to determine the cPRA which estimate the possibility of a positive crossmatch/DSA to a randomly selected donor.
PRA represents a probability of a positive crossmatch.
cPRA is based on unacceptable HLA antigens- to which the patient has been sensitized to. It is calculated from HLA antigen frequencies among about twelve thousand donors and used for allocation of kidney offers. High c PRA carries high probability of positive crossmatch.
SAB can identify anti-HLA antibodies for all common and numerous rare alleles.
Solid phase assays can detect both complement and non-complement binding antibodies. They are so sensitive, can detect antibody with low titre which may exclude a potential donor.
As the list of HLA alleles is always growing, complete spectrum of the specific HLA antigens cannot be fully presented on the solid phase assays.
False negative results can occur in:
1- Sera with high anti-HLA antibodies titer give negative results when tested neat, while react positive after 1:10 dilution. This called prozone phenomenon. Which occurs due to C1 effect that compete with the antibodies binding to HLA molecules.
2- Presence of IgM antibodies. Resolved with DTT and serum dilution.
3- Drugs like IVIG
4- Epitope sharing. The HLA antigens on the beads share mutual antibody binding epitopes causing same antibodies bind to more than one bead. resulting in reduction in the MFI on a single bead.
Crossmatching (XM)
Presence of DSAs represented as a positive crossmatch was contraindicated to transplantation. it is insufficient to identify all relevant antibodies, and may exclude potential donor unnecessarily, to avoid this; solid phase test should be used together with crossmatch results to identify those that are immunologically relevant.
1- Complement dependent cytotoxicity crossmatch
Positive test as determined by number of destroyed cells, points to the presence of DSA. It is done for both B and T lymphocytes. In the presence of low titre DSA the sensitivity of the test is low. That can be increased by increasing the incubation time, addition of AHG-enhanced method or use of additional wash steps. False negative can occur with low titre antibodies. While false positive can occur with autoantibodies, IgM/IgG HLA or nonHLA antibodies.
2- Flow cytometry crossmatch:
It detects DSA whether complement fixing or non-complement fixing. Recipient serum is mixed with donor lymphocytes and then tagged with fluorochrome conjugated anti-IgG antibody. With the use of flow-cytometry, B and T lymphocytes can be determined and their DSA individually determined. It is highly sensitive but not available widely, so its role in assessing the immunological risk is still unclear.
Virtual crossmatching (VXM):
In this, both donor HLA typing and solid phase screening are utilized together to predict the true in vitro crossmatch results.
The data is used to predict the true in vitro crossmatch results by “mixing” identified antibody specificities of recipient serum with donor HLA antigens. It gives rapid result so can shorten the wait times. VXM allows physicians to consider donor organs that would not otherwise be available. Its result changes over time, therefore it should be done considering all available serum results including at least one recent within 3 – 6 months.
False positive results that may lead to wrong exclusion of potential donors, can occur in presence of:
1- Low titer DSA
2- Non-complement fixing antibodies
False negative results:
1- HLA donor antigens cannot be correctly represented, because they have been classed differently.
Disadvantages:
· Its result is not fully accurate and actual crossmatch must be done.
· It does not identify Null HLA alleles that can carry a significant risk if misidentified.
Defining recipient risk based on pre-transplant donor crossmatch and antibody screening:
High immunological risk:
High titres of DSA at time of transplantation for mismatched donor HLA. Treatment with desensitization
Intermediate risk
Low titre of DSA, and historic DSA is not detectable. Treatment option; intensification of the immunosuppressive drugs and post –transplant monitoring of DSA
Standard risk:
No evidence of DSA
· This article discuses about an interesting case of SLE. Recurrence rate of lupus in transplant kidneys is low. A 30 y/o man with SLE has been doing hemodialysis since 5 year ago, candidate for receiving kidney from a deceased donor 100 mismatch, with B cell CDC XM positive while FCXM and Luminex-SAB negative. To interpret this case, different method of HLA-typing and XM is necessary.
· HLA typing methods:
Serological: Donors lymphocytes are added to Trays well with special HLA Abs. If donor’s HLA is the same with lymphocytes will lysis by complement and dye enters cells. This is a quick method and so suitable for deceased donors.
Limitation: serologic assay is limited for HLA-CW and DQ, DP. Molecular method uses PCR-SSP and SSO probes and DNA sequencing.
HLA-DR, HLA-B and HLA-A are important in alloimmune burden, respectively. Higher number of HLA mismatches are associated with higher risk of graft failure. Six HLA mismatches causes 64%risk while for one HLA mismatch risk is 13%.
· Screening for HLA:
One third of in waiting list have anti-HLA Abs which if it is DSA, will be important.
· Two type methods:
· Cell based or cytotoxic method: There are thirty to forty potential donor’s lymphocytes in each panel. After adding recipient’s serum and washing, complement and dye is added. If patient has antibody, uptake of dye due to cell death will occur. Its grade is expressed as PRA and indicates risk for positive XM.
False positive: Non-HLA Abs, Auto antibodies, nonspecific IgM antibodies.
False negative: Non-complement fixing antibodies and low titer antibodies.
· Solid phase assays: ELISA and Microbeads platform or single-antigen beads.
· Luminex-SAB: In this method, there are different class I or II HLA antigens on a bead. If added recipient’s serum contains anti-HLA Abs, then by adding fluorescent dye, anti-HLA Abs can be detected by Flow cytometer (Flow PRA) or Luminex –SAB. This method can determine a complete list of specific anti-HLA antibodies. The HLA frequency in the donor population is compared to determine CPRA which is the best estimate of having a positive cross match with a random donor. It is calculated among twelve thousand kidney donors in the USA and indicates the proportion of donors who express these unacceptable antigens. Sensitized recipients would be higher on the list. SAB assays could be available in 3-4 h and virtual XM could be done to determine pre-transplant DSA.
· Limitation for SAB:
High sensitivity for detection of anti-HLA Abs even if they are insufficient to cause positive XM. Both complement fixing and non-complement fixing Abs are detected but non-HLA Abs won’t be detected by this method. Increasing list of HLA alleles is another limitation.
Prozone phenomenon: False negative results are seen would be positive results due to effect of C1 or IgM Abs. IgM Abs could be removed by adding DTT. Some serum proteins and IVIg and epitope sharing would cause false negative results.
· Cross match (XM): it is used to determine if recipient has antibodies against donor’s Ag.
1-T cell cytotoxic XM: 20% False positive and 4% False negative result.
CDC-XM could be done for both B cell and T cells.
If antibody is low titer, its sensitivity would be low which increases by adding AHG or high incubation time and more washing.
False positive CDC-XM: Autoantibodies, IgM HLA or non-HLA Abs.
False negative CDC-XM: Low titer antibodies
2-FCXM: This method detects DSA without correlation with complement –fixing. Recipient’s serum is mixed with donor’s lymphocytes then tagged with a flour-chrome conjugated with anti-IgG Abs. This method is more sensitive than CDC-XM.
3-VXM: This method predicts result of a real XM by using donors HLA typing and recipients Luminex-SAB. Luminex should be repeated after pregnancy, Tx or blood transfusion and every 3-6 months to interpret correctly, all previous Luminex results should be considered. False positive VXM: Low titer Abs or non-complement fixing Abs and null-alleles.
False negative results: Because of different classification for donors HLA. VXM should be confirmed by a real XM before transplantation.
· Risk defining:
High immunological risk: High titers of DSA are present at the time of TX which leads to hyper acute rejection.
Intermediate risk: There are low titer DSA at the time of TX but no historic DSA is detected and needs intensified immunosuppression and then immunological monitoring after TX.
Standard risk: There are no DSA at the time of TX.
Renal transplantation is the best for ESKD patients .
Lupus nephritis can recur in quarter of tge pts after Tx .
* HLA typing methods :
Serological typing (and remember the null alleles)
Molecular methods
A.PCR using primers
B. Oligonucleotid probes
C. Direct DNA sequencing
* HLA antibody Screening
Sensitization due to Pregnancy , blood Tx , prior Tx
A. CDC
B . Solid phase screening using ELISA or SAB
N.B SAB : artefact is improved by dilution method , DDT
and also need to be mentioned the prozone phenomenon and The IVIG effect on SAB results
*Cross matching
A. CDC
B. Flow cytometry
*Virtual Cross match not 100% accurate can give false positive or false negative
Defining risk
A. HIGH Risk
B. Intermediate Risk
C. Standard Risk
Conclusion
Tx immunology is very important in successful Tx outcome
Renal transplantation remains the best modalities of RRT for ESRD patients.
HLA TYPING:
HLA typing is essential step in renal transplant as reorganization of foreign HLA by recipient T lymphocyte will trigger an immune response
HLA typing perform serologic or molecular typing methods
Serological typing:
Recipient lymphocytes are introduce into the tray wells contracting sera ,complement and dye, then the cell death take place as a result of complement activation .finally the cell death identify under phase contrast microscopy
Advantage:
Ø the results are available in short time
Ø decrease cold ischemia time
Ø able to identify null HLA alleles
Disadvantage:
Molecular typing:
there are different methods to apply molecular typing:
HLA typing is perform by matching the amplification product of subject DNA to the DNA sequences of other patients alleles.
Subject amplified DNA is mixed with oligonucloetide probes. HLA alleles recognized by fluorescent tags. Precise nucloetide order identified via sequencing.
Disadvantage:
1-not identify new alleles on currently in HLA sequence
HLA-A, HLA-B,and HLA-DR are most important antigens in kidney transplantation.so it has significant impact on allograft survival
HLA mismatching in deceased donor kidney transplantation is of lesser significance due to use of potent immunosuppression
HLA ANTIBODY SCREENING:
Recipient serum is mixed with cell donor lymphocytes along with complement and dye,
In a tray complement activation result in cell death and uptake of the dye so the result is express as percentage panel reactive(PRA)
Advantage:
risk of positive cross matching of a given recipient
Disadvantage:
Solid phase antibody screening
use soluble or recombinant HLA molecule in this method instead of lymphocyte target:
a) Enzyme-linked immunosorbent assay platform:
depend on using HLA glycoproteins fixed into microtiter well then add serum to wells and wash it to estimate DSA
b) single antigen beads(SAB):
It is more sensitive than CDC and FC
the SAB Luminex assay can suspect to some artifact although can be overcome by delusion or add DTT
reduction in MFI can occur due to HLA antigens bounding
prevent hyperacute rejection
detect DSA
positive result is contraindicated to transplantation
identify specific recipient antibody to the donor
it has false negative and positive results
need solid phase antibody test to identify those that are immunological relevant
CROSSMATCHING (XM)
Complement-dependent cytotoxicity crossmatch
Ø positive test is considerable number of lymphocytes are destroyed after the incorporation of complement
Ø less sensitive than the other test
Ø false negative result and false positive result
Ø CDC -XM can be done for B &T lymphocytes
Flow cytometry crossmatch:
In this method recipient serum is mixed with donor lymphocytes and then tagged with a fluorochrome conjugated anti IgG antibody.
Advantage:
Ø More sensitive test
Disadvantage:
Ø Different methods used between different laboratories can result in a different results
Ø Assessing immunological risk still not clear.
Virtual cross matching:
Advantage:
Disadvantage:
Defining risk:
High immunological risk:
Intermediate immunological risk:
Standard immunological risk:
1) Donor crossmatching
2) Antibody screening outcome
Conclusion:
HLA typing is an essential part of renal transplantation.
HLA typing include meth0ds :
Serological
Recipient lymphocytes are mixed with sera with different known HLA alleles . HLA Cw ,DQ and DP are not recognized by that method.
Molecular
Sequence specific PCR: DNA from subject is amplified in several wells with primes which are complimentary to HLA alleles
Sequence specific oligonucleotide Probes; certain order of nucleotides of gene of concern is determined.
Direct DNA sequencing-: detects significant alleles and specific alleles without cross reactivity
HLA NTIBODY SCREENING
Cytotoxic antibody screening
Recipient serum is mixed with 30-40 donor lymphocytes , dye and Complement . Results are shown as PRA
Solid phase antibody screening
Using recombinant HLA molecules instead of lymphocytes
ELISA platform
Microbead platform
Estimating PRA ; more sensitive than ELISA. c PRA determines unacceptable antigens
CROSS MATCHING
Complement dependent Cytotoxic cross match: It can be processed for B and T lymphocytes
Flow cytometry: recipient serum is added to donor lymphocytes and Fluorescence for tagging is used. DSA detected independent of compliment fixation .
Virtual cross match-:combination of Solid phase antibody screening and HLA Typing; predicting suitable donors.
Immunological Risk Groups are high,intermediate and low groups according to DSA levels and history of sensitization.
HLA TYPING :
HLA typing play essential role in renal transplantation, as recognition of foreign HLA by recipient T lymphocytes will stimulate an immune response.
1. Serology method
a tray containing sera with antibodies to a multitude of known HLA alleles is used recipient lymphocytes are introduced into the tray wells contacting sera, complement and dye. In tray wells where antibodies can bind to the antigens on the surface of lymphocytes;
complement is activated. results in complement pathways triggered resulting in cell death, ultimately allowing the dye to enter the cell. Tray wells with significant cell death are then identified under phase contrast microscopy.
This method also offers the ability to differentiate HLA alleles
Disadvantages he HLA-Cw, DQ, and DP antigen have clinically significant effects on the outcomes of allografts. However, serologic assays are scarce for these loci.
2. Molecular method
( Sequence-specific primer polymerase chain reaction )
extracted DNA from the subject is amplified in several wells. Each well has primers that are complementary to specific HLA alleles.DNA probes are complementary to the specific sequence of the HLA molecule, an amplification product is formed. This is then instilled into an agarose gel and undergoes electrophoresis where they appear as a band.
( equence specific oligonucleotide probes )
Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles. Unique HLA alleles are then identifiedusing fluorescent tags.
( Direct DNA sequencing)
determines the precise order of nucleotides in the gene of interest.
Using published HLA allele sequences, HLA type is subsequently assigned by comparison.
.
HLA ANTIBODY SCREENING
methods used for HLA antibody screening
Cytotoxic (cell-based) antibody screening
set of cell donors are randomly selected to be representative of a population. This should be representative of the population of potential deceased donors. Each panel consists of around 30 to 40 different donor lymphocytes.
false positive results can be produced due to nonHLA antibodies, autoantibodies and nonspecific IgM antibodies. false negative results complement dependent method requires higher antibody titres to be activated.
Solid phase antibody screening
recombinant HLA molecules instead of lymphocytes targets – as lymphocytes present
both HLA as well as non-HLA molecule.
Enzyme-linked immunosorbent assay platform
purified HLA molecules are applied toenzyme-linked immunosorbent assay (ELISA) platforms and will bind individually to HLA antibody after the addition of recipient serum nzyme conjugated antibodies to IgG (human) is then added to detect the presence of HLA antibody in the serum which is bound to the antigen. Detection is performed by optical density reading.
Microbead platform/single-antigen beads
Pooled panel beads with several different class Ⅰ or Ⅱ HLA antigens on a bead
Microbead that is fluorescent dye conjugated detect the presence of HLA antibody in the serum which is bound to the antigen. solid phase antibody screening addresses detect both complement and non-complement binding simultaneously. More sensitive detecting antibody that is below the threshold associated with a positive crossmatch.
CROSSMATCHING (XM)
Complement-dependent cytotoxicity crossmatch
positive if a considerable number of lymphocytes are destroyed after the incorporation of complement when significant DSA has been bound to the cell surface.done for B and T lymphocytes. Sensitivity is limited.
Flow cytometry crossmatch
detects DSA independent of complement fixation
detects the presence or lack of IgG DSA on donor lymphocytes.recipient serum is mixed with donor lymphocytes and then tagged with a fluorochromeconjugated anti-IgG antibody.
more sensitive
Virtual crossmatch
both donor HLA typing and solid phase antibody screening are utilised together. It is not precisely a crossmatch in the sense of mixing serum and lymphocytes. The data is used to forecast the actual in vitro crossmatch results by “mixing” identified antibody specificities of recipient serum with donor HLA antigens.
VXM can lead to shorter wait times and improved outcomes for sensitised transplant recipients.
permits transplant physiciansto consider donor organs that would not otherwise be
available by means of a prospective crossmatch strategy,and thereby, allows to consider a potentially positive crossmatch a risk factor for donor selection.
Comprehensive review for HLA typing and crossmatch.
Proper HLA typing and crossmatch ensure good clinical outcome in renal transplant patient.
HLA typing method
1. Serology method
2. Molecular method.
1) Serology method
Main concept :
Recipient blood lymphoctes containing antigen mix with control sera with antibodies of know HLA alleles , dye and complement in a tray. Complement activation through antigen antibody reaction resulted cell death which stained by the dye. The death cell is identified by phase contrast microscope.
Advantages :
I. Short process time for HLA typing.
II. Reduce cold ischemia time.
III. Able to idenitfy “null” HLA alleles.
Disadvantages:
I. Not able to identify all HLA alleles, especially HLA- Cw , DQ, DP antigen.
II. Unable to detect different HLA small amino acids.
Outcome : Not popular due to its limitation and more advance HLA typing method.
2) Molecular method.
Main concept :
A) Sequence specific primer PCR.
-HLA typing is perform by matching the amplification product of subject DNA to the DNA sequences of other candidate alleles.
B) Sequence specific oligonycleotide probes.
-Subject amplified DNA is mixed with oligonucloetide probes. HLA alleles recognized by fluorescent tags. Precise nucloetide order identified via sequencing.
C) Direct DNA sequencing.
-Determine precise order of nucleotides in a gene directly.
Advantages :
I. Highly specific.
II. Provide clear indentification of HLA antigens between donor and recipient.
III. Provide insight to future mismatch by assesing donor recipient antigens, epitopes and amino acids sequence of HLA typing.
Disadvantages:
I. Unable to identify new alleles not currently available on the HLA sequence databank.
Outcome : Prefer method for HLA typing due to high accuracy.
Key notes
1) Mismatch for different HLA antigens provide different clinical outcomes. According to Eurotransplant and old UK transplant data the clinical impact of HLA mismatch is greatest for HLA-DR followed by HLA-A or HLA-B.
2) Taboo mismatch indicate 7 specific HLA mismatch which result in poor renal allograft survival.
HLA Antibody screening.
A) Cytotoxic antibody screening.
Concept :
Recipient serum is mixed with “cell donor” lymphocytes along with complement and dye in a tray. Complement activation resulted in cell death and uptake of the dye. The result is express as percentage panel reactive body ( PRB )
Advantages :
I. Approximation the risk of positive corssmatch of a given recipient.
Disadvantages:
I. Cell panel used may not represent the true population.
II. False positive due to non HLA antibodies , autoantibodies and nonspecific Ig M antibodies.
III. False negative due to low antibody titre.
IV. Unable to identify complete lists of antibody specificities and unacceptable antigens.
B) Solid phase antibody screening.
I. ELISA .
Concept :
Adding purified HLA antigen bind to HLA antibody after adding recipient serum. Enzyme conjugated antibodies to Ig G is added. Antigen antibody reaction is identify by optical density reading.
II. Microbead platform/ single -antigen beads.
Concept:
Pooled panel beads coated with class I or II HLA antigens are used for antibody screening. Fluorescent dye is added and can be detetcted via FLOW PRA or SAB. C PRA is obtained by comparing the HLA frequencies in the donor population. Best estimate positive crossmatch.
Key notes
1. C PRA : Detect antibody in recipeint via following methods. Serological, Sold phase assay ( ELISA, FLOW Cytometry, SAB )
2. PRA : Recipient’s serum is examined in a pool of local donar antigens via complement dependent cytotoxicity .
3. Microbead assays (PRA , SAB Luminex ) 10% more sensitive for lower antibody then ELISA.
4. ELISA 10% more sensitive then AHG.
5. SAB assays results available in 3-4 hours.
6. SAB enable VXM to identify DSA before transplant. Provide extra benefit for organ allocation and risk stratification.
7. SAB allow identification of rare antigens and alleles beside the common one.
8. SAB -Luminex susceptible for prozone phenomenon.
Crossmatching.
Crossmatch via PRA able to identify specific recipient antibody to donor.
3 methods
1) Complement dependent cytotoxicity corssmatch.
Concept : Activated complement causing B or T lyphocytes lysis which indicate that significant DSA boubd to the cell surface. Sensitivity dependant on antibody titre, but this can be overcome by increasing incubation duration, AHG enhanced method and additional wash steps.
2) Flow cytometry crossmatch. (FCXM)
Concept: Accurately detect Ig G DSA on donor lymphocytes idependent of complement fixation. DSA towards B and T lymphoctes can be identified seperately. Provide huge variation of results among different laboratories. Not widely available.
3) Virtual crossmatching. (VXM)
Concept : Applying both donor HLA typing and solid phase antibody screening. Recipient serum which contain identified antibody is mix with donor HLA antigens. Reduce the waiting times and improve outcomes for sensitised transplant recipients. Prosspective crossmatch allows to consider probable positive crossmatch a risk for donor selection. However, actual crossmatch is needed as VXM is not highly accurate.
Risk assessment in renal transplant patients
1) High Immunological Risk
-High antibodies specific for mismatch donor HLA upon transplantation.
-risk of hyperacute rejection.
-Generally presence of DSA precludes transplantation. However, role of pre transplant desensitisation regimens being reported.
2) Intermediate Immunological Risk
-Low DSA titre and historic DSA not detected upon transplantation.
-May proceed with transplant with intensified immunosuppression.
3) Standard Immunological risk
-No evidence of donor HLA sensitisation.
HLA matching has a significant effect on graft survival.
Serologic techniques:
Quick results which decrease ischemia time in deceased donor transplant
determine “null” HLA alleles (of low immunological significance as has identifiable DNA sequence with no cell surface antigen expression) but the available sera can’t identify all HLA alleles , can’t identify HLA-cw, DQ and DP antigens which are clinically significant, can’t detect small amino acids in HLA proteins which may be antigenic so not used now.
Molecular techniques:
Sequence-specific primer PCR
Sequence specific oligonucleotide probes-Direct DNA sequencing
detect significant alleles and differentiate between donor and recipient at level of antigens, epitopes and amino acids but can’t identify new alleles that are not on primers used in HLA-typing.
HLA antibody screening
Detection of preformed anti-HLA antibodies is important as they increase the immunological risk of graft failure.
Cytotoxic antibody screening:
Expressed as percentage PRA, used to estimate risk of recipient having positive cross match
but depends on commercially available cell panel which may not represent the population
HLA frequency and racial differences can’t be considered
Can’t determine antibody specificity and unacceptable antigens.
Solid phase antibody screening:
Use recombinant HLA molecules instead of lymphocytes and identify antibody specificity of recipient serum
Cross-matching:
Identifies if a recipient has antibodies to specific donor.
CDC-XM
Done for B and T lymphocytes
False negative results due to low titer antibodies not detected
False positive results due to detection of autoantibodies, IgM/IgG HLA or non HLA antibodies.
FCXM
Recipient serum mixed with donor lymphocytes
Identify T and B lymphocytes with no need for complement fixation
more sensitive than CDC and detect IgG DSA on donor lymphocytes
Virtual crossmatch:
Donor HLA typing and solid phase antibody screening are used together to predict the actual crossmatch in vitro
Leads to shorter waiting time, better outcome in sensitized recipients and better donor selection as predict potentially positive crossmatch.
should include all serum results with recent sample as antibodies presence, titer and specificity vary with time especially with sensitizing events but false positive results due to low titer antibodies (clinically insignificant but detected by SAB) and/or non-complement binding antibodies.
False negative results as all potential HLA donor antigens may be not correctly represented.
Defining immunological risk:
High
High titer of circulating DSA
Intermediate
low titer of DSA
Historic DSA not detectable
Acceptable transplant with intensified immunosuppression and close monitoring post-transplant
Standard
No evidence of sensitization to HLA of the donor
Crossmatch methods and human leukocyte antigen (HLA) typing play a pivotal role in
improving organ allocation and afford better matches to recipients.
HLA TYPING:
HLA typing is a crucial step in renal transplantation, as recognition of foreign HLA by
recipient T lymphocytes would trigger an immune response. HLA laboratories currently
perform serologic as well as molecular typing methods.
Serological typing:
Recipient lymphocytes are introduced into the tray wells contacting sera, complement
and dye.
In tray wells where antibodies can bind to the antigens on the surface of lymphocytes;
complement is activated. This results in complement pathways triggered resulting in cell
death, ultimately allowing the dye to enter the cell. Tray wells with significant cell death
are then identified under phase contrast microscopy.
Advantage
Short time.
Disadvantage:
Lack of sera with antibody specificities that are capable of identifying the ever-growing
number of HLA not readily detect differences in HLA protein small amino.
Molecular typing:
Sequence-specific primer polymerase chain reaction.
Direct DNA sequencing: This method determines the precise order of nucleotides in the
gene of interest.
Sequence specific oligonucleotide probes.
Advantage:
Identify differences in HLA antigen between donor and recipient. Often with detail to the
amino acid level that can provide insight to the risk accompanying mismatched donor-
recipient antigens, epitopes and amino acid.
HLA typing based on polymerase chain reaction (PCR) is highly specific where specific
allele Where specific alleles are identified with no cross-reactivity.
Disadvantage: it poses is that new alleles not currently on the HLA sequence databank
will fail to be identified.
HLA ANTIBODY SCREENING:
Third of patients who are waitlisted for transplantation may have a degree of anti-HLA
antibodies detected. Preformed antibodies increase the chances of immunological
failure of the allograft e chances of immunological failure of the allograft by causing
positive crosshatches and, thereby, result in the exclusion of donors.
Methods used for HLA antibody screening:
Cytotoxic (cell-based) antibody screening:
A set of cell donors are randomly selected to be representative of a population.
Each panel consists of around 30 to 40 different donor lymphocytes.
Recipient serum is mixed with “cell donor” lymphocytes in individual wells along with
complement and dye. The serum contains antibodies that bind to the cell surface with
adequate density complement pathways are activated which results in cell death and
uptake of the dye.
Degree of cytotoxicity is expressed as percentage PRA (panel reactive antibody). It is a
tool that can be employed to approximate the risk of a given recipient of having a
positive crossmatch.
Disadvantage:
PRA percent can be different numerically without a corresponding change in the type or
amount of antibody.
This largely depends on the cell panel used which are commercially produced and may
not truly represent the population.
HLA frequencies and racial differences need to be factored in but cannot be done.
False positive results can be produced due to non HLA antibodies, autoantibodies and
nonspecific IgM antibodies.
False negative results are possible as this is purely complement dependent that
requires higher antibody titers to be activated, the lack of a complement activation
simply due to low titers allows a true antibody to be hidden.
Solid phase antibody screening:
Use soluble or recombinant HLA molecules: it’s either
Enzyme-linked immunosorbent assay platform:
HLA molecules are applied to ELISA platforms and will bind individually to HLA antibody
after the addition of recipient serum. Enzyme conjugated antibodies to IgG (human) is
then added to detect the presence of HLA antibody in the serum which is bound to the
antigen. Detection is performed by optical density
Microbeads platform/single-antigen beads:
Pooled panel beads with several different class Ⅰ or Ⅱ HLA antigens on a bead yield a
positive or negative result and are utilized for screening. Microbeads that is fluorescent
dye conjugated is then added to detect the presence of HLA antibody in the serum
which is bound to the antigen.
Fluorescence detection can be done traditionally using:
A flow cytometer (Flow PRA®) or
Via the single-antigen beads (SAB) Luminex® platform.
These estimate PRA by the proportion of positive beads.
Advantage:
Better discriminate immunologically relevant positive crossmatches from false-positive
results when traditional cell based methods are complemented with solid phase assays
Microbeads assays (both Flow PRA® and Luminex®) are ten percent more sensitive for
lower titer antibody than ELISA.
Assays are specific for anti-HLA antibodies.
SAB assays are rapid with results available 3-4hrs.
SAB assays permit identification of anti-HLA antibodies for all common and numerous
rare antigens and alleles. to eleven HLA loci.
Disadvantage:
Being too sensitive they can detect antibody that is below the threshold associated with
a positive crossmatch. The detected antibody may preclude a potential donor.
With growing list of HLA alleles, the complete spectrum of unique HLA antigens cannot
be fully presented on solid phase assays.
CROSSMATCHING:
Either:
Complement-dependent cytotoxicity crossmatch:
The complement-dependent cytotoxicity crossmatch is interpreted as positive if a
considerable number of lymphocytes are destroyed after the incorporation of
complement. This suggests that a significant DSA has been bound to the cell surface.
Complement-dependent cytotoxicity crossmatch (CDC-XM) can be done for B and T
lymphocytes.
Sensitivity is limited if the relevant antibody is in low titres, but this can be overcome by
increasing the incubation time, the use of the AHG-enhanced method as well as
additional wash steps.
Low titre antibody resulting in false negatives. CDC-XM can also give false positives by
detecting autoantibody, IgM/IgG HLA or non-HLA.
Flow cytometry crossmatch:
FCXM detects DSA independent of complement fixation:
Recipient serum is mixed with donor lymphocytes and then tagged with a fuorochromes
conjugated anti-IgG antibody.
Virtual cross matching:
In VXM, both donor HLA typing and solid phase antibody screening are utilized together.
By “mixing” identified antibody specificities of recipient serum with donor HLA antigens.
DEFINING RISK:
High immunological risk:
High titres of circulating antibodies specific for mismatched donor HLA (DSA).
Can lead to hperacute rejection. The presence of DSA precludes transplantation.
However, there are reports of innovative pre-transplant desensitization regimens to
reduce this risk.
Intermediate immunological risk:
There is a low titer of DSA, and historic DSA is not detectable.
It may be acceptable to consider intensified immunosuppression as well as
immunological monitoring in the post-transplant period.
Standard immunological risk:
There is no evidence of donor directed sensitization.
Renal transplantation Remains the best option for patients with end-stage renal disease, however, careful selection and preparation must be carried out to improve the success of these of this treatment modality.
The important steps to ensure maximal matching between the donor and recipient are :
First. HLA typing
Second. HLA antibody screening. this is mainly to look for donor specific antibodies. DSA.
Third. Cross-matching.
Regarding th first step HLA typing. This can be done by either serologic methods, or molecular typing methods.
for the serological typing, It includes the use of commercially available sera containing antibodies to a multitude of known HLA alleles , along with complement use .Recipient lymphocytes are introduced into the tray which contain the serum And when antibodies bind to antigens on the surface of lymphocytes, then the complement is is activated ultimately leading to cell death and this can be identified on the phase contrast microscopy.
The key benefit of serologic typing is that the results are available in a short period, which is particularly important in case of deceased donor kidney transplantation. Which means less cold ischemia time. This method. Also. Helps to differentiate HLA alleles. That have identifiable, DNA sequences. But no cell surface antigen expression. Termed as the null HLA alleles.
The drawback of this method is the lack of Sera with antibody specificity that are capable of identifying the ever-growing number of HLA alleles. Another drawback of this method. Is that the HLA Cw , DQ. DP. Antigens which May have clinically significant effects on the outcomes of allograft. However, these assays are scars for these loci. The third drawback is that serologic methods Do not readily detect differences in HLA protein small amino acids,these may be antigenic enough to trigger potent immunological response.
The other method is molecular typing. And here we have three ways first. Sequence-specific primer, polymerase chain reaction. Second sequence specific oligo nucleotide probes ,Third is direct DNA sequencing.
It can readily and clearly identified differences in HLA antigens between donor and recipient. HLA typing based on PCR is highly specific
The Three Most important HLA antigens Considered in kidney Transplantation are HLA-A, HLA-B and HLA DR. Where the fewer of the mismatches, the better the match between the donor and the recipient. And the better Transplant outcome.
HLA antibody screening.
Almost a third of patients who are on the waiting list for kidney transplantation. Art sensitized. Which means having HLA antibodies which usually occur in three instances :pregnancy. Post blood, transfusion. And in case of Prior transplantation.
The presence of preformed antibodies, increases the chances of immunological failure of The graft. And By causing positive cross-match , thereby leading to exclusion of the donor.
Methods used for HLA. Antigens. Screening are the following first cytotoxic antibody screening. 2nd is solid phase antibody screening.
Where in the first the Recipient,s Serum is mixed with donor lymphocytes. And the degree of cytotoxicity is expressed as percentage panel reactive antibody (PRA)
The limitations of this of this method are that PRA percent can be different numerically without a corresponding change in the type or amount of antibody.
False positive results. Can be due to non HLA antibodies autoantibodies or nonspecific IGM antibodies. Whereas false negative results Can occur cases of low antibody titers Where are the complement Is not activated.
Solid phase screening
Here it includes the use of recombinant HLA molecules. Instead of using lymphocytes targets, variants of this method.
1)Enzyme-linked immunosorbent assay platform.
2)microbead platform. Or called single antigen Beads. :the beads are individually coated with class 1 or 2 HLA, antigens of an individual patient. And as the fluorescent dye is used in this method , So fluorescence detection can be done traditionally using a flow cytometerOr with single antigen beads luminex. Platform.
The importance of single antigen bead Is thatIt can Detect A list of distinct antibody specificities that are subsequently compared with the HLA frequencies in the donor population, to determine the calculated panel reactive antibody cPRA ,This yields, the best estimate of the likelihood of a positive cross-match.
Regarding the difference between pra and cpra.
in traditional PRA a high value translated to a high probability of a positive cross-match. Where in cPRA is based on unacceptable HLA, antigens that The patient has been sensitized to , cPRA estimate the proportion of donors with whom a particular recipient would be incompatible.
Despite the benefits of the solid phase antibodies. Some limitations are there ; Being too sensitive, they can detect antibod That is below the threshold associated with the positive cross-match. Also that non HLA antibodies Cannot be accounted for utilizing this method solely. Moreover, this single antigen bead luminex essay has been shown to be susceptible to an artifact known as the protozoan phenomenal. Where sera with high anyi HLA antibody titers give negative results when tested neat, but become strongly positive after dilution , also a false positive result can occur with igM antibodies binding to the beads as it can also occur with the use of IVIG therapy.
, cross-matching
We have three ways. First complement dependent cytotoxicity cross-match
Complement dependent cytotoxicity, cross-match can be done for B and Tlymphocytes. It’s sensitivity is limited if the relevant antibody Is in low titer, But this can be overcome by increasing the incubation time and with the use of anti-human globulin enhanced method. The CDC cross-match also can give false positive results by detecting autoantibodies, IgM. Antibody or non HLA and antibodies.
The second way is flow cytometry cross-match.
This method is more sensitive than the complement dependent cytotoxicity Cross-match.
Third is virtual cross-match.
In which both donor HLA typing and solid phase antibody screening are utilized together.
The data is used to forecast the actual in vitro cross match results by mixing identified antibody specificities of recipients Serum with donor HLA, antigens, the use of virtual cross-match can lead to short-term, wait time and improved outcomes for sensitized transplant Recipients.
It’s important to notice that titres, specificities and presence or absence of antibodies could significantly vary over time ; Thus.The use of antibody specificity. From serum samples earlier than six months, could not predict a cross match with certainty. However, results from virtual cross-match are not 100% accurate and current practice mandates The actual cross-match to be performed as well.
virtual cross-match does not identify HLA. Null allele is not a jelly and leads. Our ones have identifiable, DNA sequences with more connect typing, but not express HLA products on sale surface.
Defining the risk.
So, we have three categories of the immunological risk in the kidney transplantation.
First high immunological risk ; here at the type of transplantation, there are high titers of circulating. Antibodies specific for mismatched donor DSA. This can lead to hyperacute rejection.
Intermediate risk : low DSA titres , need intensified immunosuppression with close monitoring
Standard immunological risk. Where there is no evidence of donor directed sensitization to HLA .
in conclusion :Interpretation and clinical application of transplant Immunology are crucial steps to successful outcome.
Renal transplantation remains the best option for patients suffering from end stage renal
disease (ESRD). Given the worldwide shortage of organs and growing population of patients with ESRD
HLA TYPING
is a important step in renal transplantation, as recognition of foreign HLA by recipient T lymphocytes would trigger an immune response.
HLA laboratories currently perform serologic as well as molecular typing methods.
HLA laboratories currently perform serologic as well as molecular typing methods.
recipient lymphocytes are introduced into the tray wells contacting sera, complement and dye.
When antibodies bind to the antigens on the surface of lymphocytes the complement is activated resulting in cell death and dye to enter the cell. Tray wells with significant cell death are then identified under phase contrast microscopy. Through a process of comparison and elimination of positive wells the HLA type is assigned.
Advantages
results are available in a short period.
This is important in deceased donor renal transplantation because decrease cold ischemia times.
This method also offers the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression. These alleles termed “null” HLA alleles are of less immunological significance
Disadvantages
lack of sera with antibody specificities that are capable of identifying the ever-growing number of HLA alleles
The HLA-Cw, DQ, and DP antigen have clinically significant effects on the outcomes of allografts. However, serologic assays are scarce for these loci.
serologic methods do not readily detect differences in HLA protein small amino acids. These may be antigenic enough to trigger potent immunological responses
Molecular typing
Sequence-specific primer polymerase chain reaction
In this approach extracted DNA from the subject is amplified in several wells. Each well has primers that are complementary to specific HLA alleles.
Sequence specific oligonucleotide probes
Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles. Unique HLA alleles are then identified using fluorescent tags.
Direct DNA sequencing
This method determines the precise order of nucleotides in the gene of interest. Using published HLA allele sequences, HLA type is subsequently assigned by comparison.
Molecular typing regardless of the method can identify differences in HLA antigen between donor and recipient. Often with detail to the amino acid level that can provide insight to the risk accompanying mismatched donor-recipient antigens, epitopes and amino acid.
HLA typing based on polymerase chain reaction (PCR) is highly specific where specific alleles are identified with no cross-reactivity.
disadvantage
it poses is that new alleles not currently on the HLA sequence databank will fail to be identified.
HLA ANTIBODY SCREENING
detection of anti-HLA antibodies is important , when the crossmatch is negative, even low titres of DSA can lead to early as well as late antibody mediated rejection
For sensitised patients, successful transplantation is possible by using strategies such as desensitisation, paired exchange and acceptable mismatching
Cytotoxic (cell-based) antibody screening
recipient serum is mixed with “cell donor” lymphocytes in individual wells along with complement and dye. Where the serum contains antibodies that bind to the cell surface with adequate density complement pathways are activated which results in cell death and uptake of the dye
false positive results
can be produced due to nonHLA antibodies, autoantibodies and nonspecific IgM antibodies.
false negative results
complement dependent method requires higher antibody titres to be activated
Solid phase antibody screening
Enzyme-linked immunosorbent assay platform
purified HLA molecules are applied to enzyme-linked immunosorbent assay (ELISA) platforms and will bind individually to HLA antibody after the addition of recipient serum
Enzyme conjugated antibodies to IgG (human) is then added to detect the presence of HLA antibody in the serum which is bound to the antigen. Detection is performed by optical density reading.
Microbead platform/single-antigen beads
Pooled panel beads with several different class Ⅰ or Ⅱ HLA antigens on a bead
Microbead that is fluorescent dye conjugated detect the presence of HLA antibody in the serum which is bound to the antigen.
solid phase antibody screening addresses detect both complement and non-complement binding simultaneously. More sensitive detecting antibody that is below the threshold associated with a positive crossmatch.
CROSSMATCHING (XM)
Complement-dependent cytotoxicity crossmatch
the complement-dependent cytotoxicity crossmatch is considered as positive if a considerable number of lymphocytes are destroyed after the incorporation of complement when significant DSA has been bound to the cell surface.
Complement-dependent cytotoxicity crossmatch (CDC-XM) done for B and T lymphocytes. Sensitivity is limited when there is in low antibodies titres, this can be overcome by increasing the incubation
Time and use of the AHG-enhanced method as well as additional wash steps
this method could miss low titre antibody resulting in false negatives. CDC-XM can also give false positives by detecting autoantibody, IgM/IgG HLA or non-HLA.
Flow cytometry crossmatch
Flow cytometry crossmatch (FCXM) detects DSA independent of complement fixation
detects the presence or lack of IgG DSA on donor lymphocytes.
The recipient serum is mixed with donor lymphocytes and then tagged with a fluorochromeconjugated anti-IgG antibody.
Compared to complement-dependent cytotoxicity crossmatch FCXM give greater sensitivity
Virtual crossmatch
in vitro crossmatch results by “mixing” identified antibody specificities of recipient serum with donor HLA antigens
The use of VXM can lead to shorter wait times and improved outcomes for sensitised transplant recipients
VXM should, be done on all available serum results including at least one recent within less than 3-6 mo for a given patient because the titers , specificities, and presence or absence of antibodies could significantly vary over time.
False positive results of VXM may arise where there are significantly low titre and/or non-complement binding antibodies, thereby, resulting in the wrong exclusion of potential donors
Conclusion
Interpretation and clinical application of transplant immunology are crucial steps to a successful outcome.
Hla typing
Three important steps for successful transplantation are
1- HLA TYPING
2- DSA SCREENING
3- CROSSMATING
1- HLA typing is crucial step in successful kidney transplantation .
There is two main method of HLA typing
A-Serological typing
a tray containing sera with antibodies ( commercially available )
to a multitude of known HLA alleles is used. For typing, recipient lymphocytes are introduced into the tray wells contacting sera, complement and dye. In tray wells where antibodies can bind to the antigens on the surface of lymphocytes; complement is activated. This results in complement pathways triggered resulting in cell death, ultimately allowing the dye to enter the cell. Tray wells with significant cell death are then identified under phase contrast microscopy .
advantages : – the result is available in short time , which means less ischemia in deseased kidney transplantation .
disadvantages :- un availability of ab against overgrowing number of HLA allels .
B -Molecular typing
a. Sequence-specific primer polymerase chain reaction .
b. Sequence specific oligonucleotide probes.
c. Direct DNA sequencing.
2- HLA ANTIBODY SCREENING
– About 1/3 of of waitlist patients are DSA positive .
– Sensitization occur by (blood transfusion , pregnancy ,previous transplan )
– Even low titre of DSA may lead to early as well as to late rejection .
– For sensitized patients transplantation is possible by
Ø Desensitization
Ø Paired exchanged
Ø Acceptable mismatching
2-1 Cytotoxic (cell-based) antibody screening
The method is similar to that of serologic typing howeverhere recipient serum (instead of commercially prepared ANT HLA ab ) is mixed with “cell donor” lymphocytes
in individual wells along with complement and dye.
It is a tool by which we can approximate a risk of positive CM .
false positive results can be produced due to
Ø non-HLA antibodies,
Ø autoantibodies
Ø nonspecific IgM antibodies.
false negative results are possible simply due to
Ø low antibody
Ø complete lists of antibody specificities and unacceptable antigens cannot be identified as there are several antigens in each well
2-2 Solid phase antibody screening
This method employs soluble or recombinant HLA molecules instead of lymphocytes.
The variants of these methods are
A-Enzyme-linked immunosorbent assay platform:
In this method, purified HLA molecules are applied to enzyme-linked immunosorbent assay (ELISA) platforms and will bind individually to HLA antibody after the addition of recipient serum[. Enzyme conjugated antibodies to IgG (human) is then added to detect the presence of HLA antibody in the serum which is bound to the antigen. Detection is performed by optical density reading.
B- Microbead platform/single-antigen beads:
Pooled panel beads with several different class Ⅰ or Ⅱ HLA antigens on a bead yield a positive or negative result and are utilised for screening .
Fluorescence detection can be done traditionally using
a flow cytometer (Flow PRA) or
via the single-antigen beads (SAB) Luminex platform.
These estimate PRA by the proportion of positive beads.
SAB are individually coated with a single HLA antigen and yield a list of distinct antibody specificities.
PRA estimates the probability of positive XM by using small no of antigens .
cPRA is the same as PRA , but using large no of antigens ( 10,000- 12,000) by using computer .it is useful in allocation of kidney and pancrease .
microbeads assay is more sensitive than ELISA method which is more sensitive than cell based cytotoxic method .
SAB is specific and sensitive and available within 3-4 hours .
Solid phase diadvantages
1. They detect both complement and non-complement binding simultaneously.
2. they can detect antibody that is below the threshold associated with a positive crossmatch which can preclude a potential donor.
3. Cannot detect Non-HLA antibodies .
In the SAB – Luminex® assay the very high titre AB may give negative result( Prozone phenomenon) which needs to be diluted after (1:10 dilution) to give the correct result .
Also to avoid a false positive result from IgM AB the sample needs to be treated by dithiothreitol (DTT) which removes IgM from sample .
3- CROSSMATCHING (XM)
3-1 Complement-dependent cytotoxicity crossmatch
Positive XM is contraindication to transplantation .
Similar to cytotoxic assay the complement-dependent cytotoxicity crossmatch is interpreted as positive if a considerable number of lymphocytes are destroyed after the incorporation of complement . This suggests that a significant DSA has been bound to the cell surface. Complement-dependent cytotoxicity crossmatch (CDC-XM) can be done for B and T lymphocytes.
Sensitivity is limited if the relevant antibody is in low titres, but this can be overcome by
Ø increasing the incubation time .
Ø the use of the AHG-enhanced method .
this method has low sensitivity which may miss the low titre antibody which is clinically significant .
3-2 Flow cytometry crossmatch
Flow cytometry crossmatch (FCXM) detects DSA independent of complement fixation. It precisely detects the presence or lack of IgG DSA on donor lymphocytes.
In this method, recipient serum is mixed with donor lymphocytes and then tagged with a fluorochromeconjugated anti-IgG antibody.
Several antibodies with separate fluorochromes particular to B and T lymphocyte
surface proteins can be added .
With the useof flow-cytometry, B and T lymphocytes can be readily identified and have their DSA individually interrogated.
It has high sensitivity .
3-3 – Virtual crossmatching
In virtual crossmatch (VXM), both donor HLA typing and solid phase antibody screening are utilised together.
It is not precisely a crossmatch in the sense of mixing serum and lymphocytes .
The data is used to forecast the actual in vitro crossmatch results by “mixing” identified antibody
specificities of recipient serum with donor HLA antigens .
The use of VXM can lead to shorter wait times and improved outcomes for sensitised transplant recipients .
The results from VXM are not a hundred percent accurate and current practice mandates an actual
crossmatch be performed as well .
DEFINING RISK
High immunological risk
there are high titres of circulating antibodies specific for mismatched donor HLA (DSA).
This can lead to hyperacute rejection.
Thepresence of DSA precludes transplantation. But this risk can be reduced by desensitization .
Intermediate immunological risk
There is a low titer of DSA, and historic DSA is not detectable. It may be acceptable
to consider intensified immunosuppression as well as immunological monitoring in the post-transplant period.
Standard immunological risk
Where there is no evidence of donor directed sensitisation
to HLA. Transplantation can be done even without induction therap in low risk patients .
*HLA typing is a crucial step in renal
transplantation, as recognition of foreign HLA by recipient T lymphocytes would trigger an immune response.
*** Serological typing:
-a tray containing sera with antibodies to a multitude of known HLA alleles is used.
-For typing, recipient lymphocytes are introduced into the tray wells contacting sera, complement and dye. In tray wells where antibodies can bind to the antigens on the surface of lymphocytes; complement is activated. This results in complement pathways triggered resulting in cell death, ultimately allowing the dye to enter the cell.
-The key benefit of serologic typing is that results are available in a short period. This is particularly important in deceased donor renal transplantation. Quick results mean less cold ischemia times.
-This method also offers the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression.
-serologic methods do not readily detect differences in HLA protein small amino acids .
***Molecular typing :
-Sequence- specific primer polymerase chain reaction.
– Sequence specific oligonucleotide probes.
-Direct DNA sequencing.
*** HLA antibodies screening:
-Sensitised patients who are pregnant, post blood transfusions and prior transplantation which causes positive crossmatch and increase chance of immunological failure .
-Low titres DSA even with negative crossmatch cause early and late antibody mediated rejection.
For successful transplantation ; desensitisation , paired exchange and acceptable mismatching must be planed .
>>>Screening by :
-Cytotoxic ( cell-based ) antibody screening.
-Solid phase antibody screening.
-Enzyme-linked immunosorbent assay platform.
-Microbead platform /single-antigen beads.
***CROSSMATCHING (XM):
-The presence of donor-specific cytotoxic antibodies depicted as a positive crossmatch was a contraindication to transplantation. With PRA that identifies several antibodies to a potential cluster of donors, the crossmatch will identify if a recipient had antibodies to a specific donor of interest.
-The solid-phase antibody test should be used together with crossmatch results to identify those that are immunologically relevant.
••• Complement-dependent cytotoxicity crossmatch :
-It is interpreted as positive if a considerable number of lymphocytes are destroyed after the incorporation of complement.This suggests that a significant DSA has been bound to the cell surface. Complement-dependent cytotoxicity crossmatch (CDC-XM) can be done for B and T lymphocytes. Sensitivity is limited if the relevant antibody is in low titres, but this can be overcome by increasing the incubation time, the use of the AHG-enhanced method as well as additional wash steps. The complement fixing antibody to anti-human immunoglobulin (AHG) will bind to any DSA present on lymphocytes.
••• Flow cytometry crossmatch :
It detects DSA independent of complement fixation. It precisely detects the presence or lack of IgG DSA on donor lymphocytes. In this method, recipient serum is mixed with donor lymphocytes and then tagged with a fluorochrome- conjugated anti-IgG antibody.
••• Virtual crossmatching :
– In virtual crossmatch (VXM), both donor HLA typing and solid phase antibody screening are utilised together. It is not precisely a crossmatch in the sense of mixing serum and lymphocytes.
– The use of VXM can lead to shorter wait times and improved outcomes for sensitised transplant recipients. The speed of results generated allows a VXM to be performed at the time of donor identification owing to the fact that there is progressively sensitive and specific flow cytometry technology.
*** DEFINING RISK :
•••High immunological risk :
At the time of transplantation, there are high titres of circulating antibodies specific for mismatched donor HLA (DSA). This can lead to hyperacute rejection. The presence of DSA precludes transplantation. However, there are reports of innovative pre-transplant desensitisation regimens to reduce this risk.
•••Intermediate immunological risk :
At the time of transplantation, there is a low titer of DSA, and historic DSA is not detectable. It may be acceptable to consider intensified immunosuppression as well as immunological monitoring in the post-transplant period.
••• Standard immunological risk
Where there is no evidence of donor directed sensitisation to HLA.
REFERENCES :
1.Mojcik CF, Klippel JH. End-stage renal disease and systemic lupus erythematosus. Am J Med 1996; 101: 100-107 [PMID: 8686702 DOI: 10.1016/S0002-9343(96)00074-5]
Nossent HC, Swaak TJ, Berden JH. Systemic lupus erythematosus after renal transplantation: patient and graft survival and disease activity. The Dutch Working Party on Systemic Lupus Erythematosus. Ann Intern Med 1991; 114: 183-188 [PMID: 1984742 DOI: 10.7326/0003-4819-114-3-183]
Ponticelli C, Moroni G. Renal transplantation in lupus nephritis. Lupus 2005; 14: 95-98 [PMID: 15732296 DOI: 10.1191/09612033 05lu2067oa]
Contreras G, Mattiazzi A, Guerra G, Ortega LM, Tozman EC, Li H, Tamariz L, Carvalho C, Kupin W, Ladino M, LeClercq B, Jaraba I, Carvalho D, Carles E, Roth D. Recurrence of lupus nephritis after kidney transplantation. J Am Soc Nephrol 2010; 21: 1200-1207 [PMID: 20488956 DOI: 10.1681/asn.2009101093] Stone JH, Amend WJ, Criswell LA. Outcome of renal transplantation in systemic lupus erythematosus. Semin Arthritis Rheum 1997; 27: 17-26 [PMID: 9287386 DOI: 10.1016/ S0049-0172(97)80033-9]
Costenbader KH, Feskanich D, Stampfer MJ, Karlson EW. Reproductive and menopausal factors and risk of systemic lupus erythematosus in women. Arthritis Rheum 2007; 56: 1251-1262 [PMID: 17393454 DOI: 10.1002/art.22510]
Gilbert EL, Ryan MJ. Estrogen in cardiovascular disease during systemic lupus erythematosus. Clin Ther 2014; 36: 1901-1912 [PMID: 25194860 DOI: 10.1016/j.clinthera.2014.07.021]
von Andrian UH, Mackay CR. T-cell function and migration. Two sides of the same coin. N Engl J Med 2000; 343: 1020-1034 [PMID: 11018170 DOI: 10.1056/nejm200010053431407]
Tinckam KJ. Basic histocompatibility testing methods. In: Chandraker A, editor. Core concepts in renal transplantation. New York: Springer Science + Business Media, LLC, 2012: 21-42 Robinson J, Waller MJ, Parham P, de Groot N, Bontrop R, Kennedy LJ, Stoehr P, Marsh SG. IMGT/HLA and IMGT/MHC: sequence databases for the study of the major histocompatibility complex. Nucleic Acids Res 2003; 31: 311-314 [PMID: 12520010 DOI: 10.1093/nar/gkg070]
Claas FH, Witvliet MD, Duquesnoy RJ, Persijn GG, Doxiadis II. The acceptable mismatch program as a fast tool for highly sensitized patients awaiting a cadaveric kidney transplantation: short waiting time and excellent graft outcome. Transplantation 2004; 78: 190-193 [PMID: 15280676 DOI: 10.1097/01. TP.0000129260.86766.67].
When it comes to kidney transplantation, HLA typing is a critical component of the process. HLA interpretations may be tricky, and a variety of modalities can be utilized for this aim, each with its own set of advantages and disadvantages.
The following are examples of HLA typing methods:
1- Serological research
In this procedure, the recipient cells are mixed with sera carrying various HLA alleles that have been identified before. Null alleles may be readily excluded from a population. These approaches do not distinguish between HLA Cw, DQ, and DP.
2- The molecular level
These are some examples:
In this procedure, DNA from the individual is amplified in many wells containing Primes that are complementary to HLA alleles, and the result is a single amplicon of DNA.
The use of sequence-specific oligonucleotide probes allows for the determination of the precise order of nucleotides in a gene of interest.
Direct DNA sequencing has the advantage of being able to identify both significant alleles and particular alleles without the need for cross-reactivity.
SCREENING OF THE HLA ANTIBODY
Antibody screening for cytotoxic activity
This recipient serum is combined with 30-40 donor lymphocytes, dye, and Compliment to create a unique mixture. The results are provided in the form of PRA.
Antibody screening using solid-phase technology
Instead of lymphocytes, it makes use of recombinant HLA molecules.
ELISA is a platform for research.
Platform made of microbeads
PRA is estimated using this method, which is more sensitive than ELISA. The cPRA detects antigens that are deemed undesirable.
MATCHING CROSSWORDS
Dependent on the compliment It is possible to do a cytotoxic crossmatch on both B and T cells.
Transfection with recipient serum and the addition of fluorescent tags to donor cells is performed using flow cytometry. It is capable of detecting DSA in the absence of complement fixation.
The virtual crossmatch is a mix of solid-phase antibody screening and HLA typing that provides a virtual cross match.
Risk stratification categories: According to on immunological risk, Gebel et al. proposed the following risk stratification categories in prospective renal transplant patients:
High level of immunological hazard
These individuals exhibit high titers of circulating DSA that is specific for mismatched donor HLA, which indicates that they are infected. Due to the high risk of acute rejection in the presence of DSA, such individuals are often barred from receiving a transplant in most cases.
Immunological hazard of intermediate severity
In this group, the DSA titer at the time of transplantation is low, and there is no identifiable historical DSA. In these individuals, intensified immunosuppression, as well as immunological surveillance throughout the post-transplant period, are essential.
Immunological hazard as defined by standard practice
There is no indication of DSA in this case, and the pre-transplant immunological risk assessment is based on the findings of the donor cross-match and antibody screening tests.
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Summarize the article
====================
This article provides a case-based review of the different methods used for HLA -typing and quantification of the DSAs ,also facilitate the assessment of the patient immunological status. and help in risk stratification of kidney transplant candidates.
Introduction :
HLA matching is considered first and most important step in kidney transplant work up. the increased mismatch at HLA A-B – DR. associated with increased risk of graft rejection and impact the outcome base on many studies which revealed that multiple mismatches translated into a 64% higher risk, while the risk dropped to 13% with just one HLA mismatch.
Different methods are available for HLA typing.
-Serological HLA typing:
Old method complement dependent by adding the recipient lymphocytes to a tray that contain sera of antibodies to all known HLA antigens,( usually from multiparous women after certain incubation will add the complement and coloring dye Once Ag-AB reaction occurs complement activation occur, lead to cell lysis , then examine under the microscope the percentage of dead cells which take the dye (red ).The serologic typing are important in deceased donor renal transplantation as the results are available in a short period.
– Molecular DNA-based HLA typing techniques
It includes different types: –
· SSOP sequence specific oligonucleotide probe hybridization
· SSP sequence-specific primer amplification
· Direct DNA sequencing determines the precise order of nucleotides in the gene of interest, allowing HLA type comparison, through the amino acid level, to provide insight to the risk monitoring
Molecular based HLA typing are more sensitive and accurate than serological typing. soluble or recombinant HLA molecules based not targeting lymphocytes as lymphocytes can express HLA and non-HLA antigens.
–Enzyme-linked immunosorbent assay platform
when purified HLA molecules are applied to enzyme-linked immunosorbent assay (ELISA) platforms and will bind to HLA antibody after the addition of recipient serum enzyme conjugated antibodies to IgG (human) is then added to detect the presence of HLA antibody antigen and interpretation by optical density of AB-Ag reaction.
–Solid phase assay Luminex single bead antigen:
Estimate PRA by the proportion of positive beads, SAB assays permit
identification of anti-HLA antibodies for all common and many rare antigens and alleles. Its range of identification is up to eleven HLA loci.
SAB assays are rapid the results available in 3-4 h. The assay is also quite efficient in a single reaction chamber up to one hundred unique antigen beads can be tested or multiplex testing can allow testing many patients at one time .
Results from SAB enable virtual crossmatching (VXM) to identify DSA pre-transplant, thereby enabling organ allocation and risk stratification by calculating the percentage of unacceptable Ags(cPRA %) its more specific and helpful for allocation of donors for highly sensitized patients.
Limitation with Luminex SAB, can give false positive due to denatured Abs, Drugs like monoclonal AB
Prozone effect can give false negative results (can overcome by more dilution)
Epitope sharing
Drugs like IVIG
Being too sensitive they can detect antibody that is below the threshold associated with apositive crossmatch. The detected antibody may not always have clinical implications and can preclude a potential donor.
2 – Screening of antibodies
1-Complement dependent or antibody-dependent cell-mediated cytotoxicity (ADCC) or CDCXM:
Similar to the cytotoxic assay with low sensitivity its used limited by false positive results due to the presence of autoantibodies, IgM, non-HLA and non-complement fixing ABS, using AHG and DTT may improve the sensitivity for auto-antibodies, also positive B CDCXM indicate presence of autoimmune disease or treatment with rituximab. CDCXM also less specific test and associated with false negative result that can occur due to non-complement fixing AB or low level of DSAs or technical errors.
2– Flow cytometry crossmatch (FXCM):
The technique based on the incubation of donor’s lymphocytes with recipient sera, then add second antibody with florescence dye (green) that will bind ABS (DSA) attached to donor lymphocytes Flow FCXM detects DSA independent of complement fixation.in flow cytometry method usually measure fluorescence intensity and compare it to control (median channel shift- MCS) With the use of flow cytometry, both B and T lymphocytes can be easily identified and have their HLA related DSA usually IgG class1.11 abs I not IgM. more sensitive but less specific for non-HLA abs and can give false positive B FXCM in case of treatment with monoclonal ABS or non-HLA abs.
3 – Virtual cross-match(VXM):
In virtual cross-matching (VXM), computer-based analysis of donor HLA typing and solid phase antibody screening are used together It is correlated well with FCM cross match and graft survival even in sensitized patients Define unacceptable antigens by CPRA % so donors can be excluded, and allow identification of suitable donor in short times, useful for DD program with the advantage of shorting cold ischemia time and reduce the waiting time for allocation of donors for highly sensitized recipients.
False positive VXM due to Denatured HLA antigens on the SAB or due to the presence of null alleles.
False negative VXM either due to incomplete crossmatch or absence of certain alleles
4 – Immunological risk stratification:
1- High immunological risk
high titer of circulating HLA-DSAs
2-Intermediate immunological risk
low DSA titer and the historical DSA is not detectable.
3- Standard immunological risk
no evidence of targeted donor sensitization to HLA.
Conclusion :
understanding the concept of basic transplant immunology is the key factor that would help the practicing clinicians and guide them for better immunological risk stratification, better organ allocation for sensitized patients and improve the transplant outcome especially with recent advancing in molecular methods for HLA typing and antibodies quantification techniques .
HLA typing is a crucial step in renal transplantation ,HLA laboratories currently perform serological and molecular typing methods.
1-)Serological typing :her a tray containing sera with Abs to a many known types of HLA alleles to which recipient lymphocytes are introduced in edition to complement and dye .
Cons:
1- the results are available in short period especially in deceased donor renal transplantation which mean less cold ischemia times.
2-the other benefit is that it offer the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigens expression which is called NULL HLA alleles with less immunological significance.
Prons:
1-) is the lake of sera with Ab specificities that are capable of identifying the ever-growing number of HLA alleles.
2-)do not readily detect differences in HLA proteins small amino acids which may be antigenic enough to trigger potent immunological responses.
Serological typing has fallen into disuse with more advanced methods of typing currently available.
2-) molecular typing
– sequence-specific oligonucleatide probes.
– Sequence -specific primer
– Direct DNA sequencing
Advantages:
1-) regardless of the method, molecular typing can identify differences in HLA Ag between donor and recipient with details to the amino acid level.
2-) HLA typing by PCR is highly specific with no cross-reactivity.
Disadvantages:
New alleles not currently on the HLA sequence databank will fail to be identified.
HLA AB screening:
The usual route of sensitization toward HLA Ag accurs by pregnancy,post blood transfusion and prior transplantation.
There are different methods used for HLA antibody screening
1. Cytotoxic (cell-based) Ab screening
False positive result due to non-HLA antibody ,autoantibodies and non -specific Igm antibodies.
False negative results because it complement dependent that requires high Ab titer to be activated.
2.Solid phase antibody screening.
a-Enzyme-linked immunosorbent assay platform
b-micro bead platform /single-Antigen beads.
CROSSMATCHING (XM)
Complement-dependent cytotoxicity crossmatch
Complement-dependent cytotoxicity crossmatch (CDC-XM) can be done for B and T lymphocytes. Sensitivity is limited if the relevant antibody is in low titres,
Similar to cytotoxic PRA this method could miss low titre antibody resulting in false negatives. CDC-XM can also give false positives by detecting autoantibody, IgM/IgG HLA or non-HLA.
Flow cytometry crossmatch
Flow cytometry crossmatch (FCXM) detects DSA independent of complement fixation.
Compared to complement-dependent cytotoxicity crossmatch this offers greater sensitivity[9]. Different laboratories use different methods, and this can result in a difference in the results between them[50]
Virtual crossmatching
In virtual crossmatch (VXM), both donor HLA typing and solid phase antibody screening are utilised together.
False positive results of VXM may arise where there are significantly low titre and/or non-complement binding antibodies, thereby, resulting in the wrong exclusion of potential donors.
The VXM can also give false negative results due to the fact that the list of all potential HLA donor antigens have been classed differently and, therefore, can not be correctly represented.
Please summarise this article with emphasis on the different techniques of tissue typing and crossmatching :
1- Complement dependent cytotoxicity (CDC) :
2- Flow cytometry: using impedence flow cytometer
3- Solid phase immunoassay :
A- ELISA :
B- Luminex technology :
C- C1q assay :
D- Epitope matching :
4- Virtual cross match :
* Renal transplantation is the best solution for patients with end stage renal disease (ESRD).
* Immunological methods like human leukocyte antigen (HLA) typing ,crossmatch and antibodies screening play important role in improving the outcome of kidney transplantion.
* HLA TYPING
HLA typing is recognition of foreign HLA antigen by recipient T lymphocytes and trigger of immune response.
*methods:
*Serological typing
A tray containing sera with antibodies
to a multitude of known HLA alleles is used, recipient lymphocytes are then introduced into the tray wells, the antibodies can bind to the antigens on the surface of lymphocytes, complement is activated when it added, and this result in cell death, allowing the dye to enter the cell that can be visualized microscopy.
Through a process of comparison and elimination of positive wells the HLA type is assigned.
* Advantage of serologic typing :
-Short period and less cold ischemia time this is important in deceased dononation.
-Ability to differentiate HLA alleles
that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression“null” HLA alleles are of less immunological significance.
*Disadvantage
-Lack of sera with antibody specificities
that are capable of identifying the ever-growing number of HLA alleles ( HLA-Cw, DQ, and DP) that may have clinically significant effects on the outcomes of allografts.
-Not detect differences in HLA protein small amino acids.
* Molecular typing :
– Sequence-specific primer polymerase chain reaction:
Extracted DNA from the subject is amplified in several wells , each well has primers that are complementary to specific HLA alleles, this is then instilled into an agarose gel and undergoes electrophoresis where they appear as a band, then matching of amplificated primers to the DNA sequences of several alleles.
-Sequence specific oligonucleotid probes:
Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles, unique HLA alleles are then identified using fluorescent tags.
– Direct DNA sequencing:
This method determines the precise order of nucleotides in the gene of interest, using published HLA allele sequences by comparison.
* Advantage:
– Identify differences in HLA antigen between donor and recipient with detail of amino acid level and epitopes
– HLA typing based on polymerase chain reaction (PCR) is highly specific, with no cross-reactivity.
*( Cross-reactivity is the identification of an allele which is essentially similar to the allele of interest).
* Disadvantage:
New alleles not currently on the HLA sequence databank will fail to be identified.
* HLA ANTIBODY SCREENING
– The usual route for sensitisation towards HLA antigens occurs in three instances:
pregnancy, post blood transfusion and prior transplantation.
_ Preformed antibodies increase the chances of immunological failure even low titers of DSA by causing positive crossmatches.
-Successful transplantation is possible
by employing strategies such as desensitisation, paired exchange and acceptable mismatching.
– Different methods used for HLA antibody screening:
* Cytotoxic (cell-based) antibody screening:
A set of cell donors are randomly selected to be representative of a population ,each panel consists of around 30 to 40 different donor lymphocytes.
The method is similar to that of serologic typing, recipient serum is mixed with cell donor lymphocytes with complement and dye.
If the serum antibodies bind to the cell surface receptors and activate complement pathways, this leads to cell death and subsequent uptake of dye.
The degree of cell death is quantified using as percentage PRA (panel reactive antibody).
*Disadvantage:
– The PRA percent can be different numerically with out corresponding change in the type or amount of antibody.
– False positive results can be produced due to nonHLA antibodies, autoanti- bodies and non specific IgM antibodies.
– False negative results are possible
as this is purely complement dependent that requires higher antibody titres to be activated.
– Antibody specificities and unacceptable antigens cannot be identified using this method as there are several antigens in each well.
* Solid phase antibody screening:
– This method employs soluble or recombinant HLA molecule instead of lymphocytes targets
– The variants of these methods are:
* Enzyme-linked immunosorbent assay platform:
– Purified HLA molecules are applied to
(ELISA) platforms and will bind individually to HLA antibody after the
addition of recipient serum, enzyme conjugated antibodies to IgG is added to detect the presence of HLA antibody in the serum that bound to the antigen. Detection is performed by optical density reading.
* Microbead platform/single-antigen beads:
– Pooled panel beads with several different class Ⅰ or Ⅱ HLA antigens on a bead give a positive or negative result. -The phenotype or also known as ID beads are individually coated with class
Ⅰ or Ⅱ HLA antigens of an individual patient-derived cell line.
– Microbead that is fluorescent dye conjugated is then added to detect the presence of HLA antibod using a flow
cytometer or single-antigen beads (SAB) Luminex® platform.
These estimate PRA by the proportion of positive beads. SAB are individuall coated with a single HLA antigen and yield alist of distinct antibody specificitied Specificities are subsequently compared with HLA frequencies in the donor .
-To determine the calculate panel reactive antibody (cPRA), this give estimate of the of a positive crossmatch/donor specific antibody to a randomly selected donor.
– cPRA is based on unacceptable HLA antigens that patient has been sensitized to give high probability of a positive
crossmatch.
* CROSSMATCHING (XM)
– The cytotoxic assay was implemented as the requisite test prior to transplantation when it was shown that
recipients with DSA had significantly higher rates of allograft failure due to hyperacute rejection as well as
primary failure.
* Complement-dependent cytotoxicity crossmatch:
-Similar to cytotoxic assay the complement dependent cytotoxicity crossmatch is interpreted as positive if a
considerable number of lymphocytes are destroyed after the incorporation of complement.
(CDC-XM) can be done for B and T lymphocytes.
– Sensitivity is limited if the relevant antibody is in low titres, but this can be overcome by increasing the incubation
time, the use of the AHG-enhanced method as well as additional wash step.
-False negative due to low titre antibody. -False positives by detecting autoantibody IgM/IgG HLA or non-HLA.
* Flow cytometry crossmatch( FCXM): Detects DSA independent of complement – fixation.
– Recipient serum is mixed with donor
lymphocytes and then tagged with a fluoro chrome conjugated anti-IgG antibody.
– Several antibodies with separate fluorochromes particular to B and T lymphocyte surface proteins can be added with the use of flow-cytometry, B and T lymphocytes can be readily
identified and have their DSA individually interrogated.
– Compared to complement dependent cytotoxicity crossmatch this offers greater sensitivity.
* Virtual crossmatching (VXM):
– In(VXM) both donor HLA typing and solid phase antibody screening are utilised together .
– VXM lead to shorter wait times and improved outcomes for sensitised transplant recipients.
– VXM permits transplant physicians
to consider a potentially positive
crossmatch a risk factor for donor selection.
– Titres, specificities, and presence or absence of antibodies could significantly vary over time.
– False positive results of VXM may arise where there are significantly low titre and/or non complement binding antibodies resulting in the wrong exclusion of donors.
– Give false negative results due to the list of all HLA donor antigens have been classed differently and can not be correctly represented.
– Does not identify the HLA “Null” alleles.
* DEFINING RISK
– High immunological risk
At the time of transplantation there are high titres of DSA and this can lead to hyperacute rejection.
– Intermediate immunological risk
At the time of transplantation, there is a low titer of DSA and historic DSA is not detectable.
– Standard immunological risk
Where there is no evidence of donor directed sensitisation to HLA.
· Immunological risk stratification in kidney transplantation is crucial to determine risk of rejection, tailor immunosuppressive regimens and expect the graft and patient survival.
· This includes 3 steps:
o HLA typing of recipient.
o HLA antibodies screening (DSA detection)
o Cross matching.
· HLA typing either by:
o Serological method: adding recipient lymphocytes to sera containing multiple wells with addition of complement, then detection of complement dependent cell lysis. It is rapid and useful in deceased donor, but does not detect HLA type Cw, DP or DQ which may affect transplant outcome.
o Molecular technique: more accurate and use DNA probes and PCR reactions. It includes:
o Mainly we stress on HLA A, B and DR.
· Screening for Anti HLA antibodies (DSA) done by 2 methods either:
o Complement dependent cytotoxicity (CDC);
o Solid phase antibody screening: it is more sensitive.
– ELISA.
-Flowcytometry.
-Luminex SAB (use beads coated with HLA type I and II antigens)
§ It determines cPRA which represents the unaccepted antigens.
o MIF: mean fluorescent intensity:
-represent semiquantitative assay of DSA strength.
-Correlates with risk of AR, but no absolute CT to transplantation.
– MIF accepted if > 2000 for class I and > 5000 in class II HLA.
· Cross matching (XM):
o It detects preformed DSA in recipient serum against donor lymphocytes.
o It means either Mixing both donor lymphocytes and recipient serum (real XM) or by matching results of donor HLA typing and solid phase assay of DSA to expect actual invitro cross match (virtual XM).
o Positive cross match against T lymphocytes is an absolute contraindication to transplantation unless desensitization protocols as (plasmapheresis, IvIg or rituximab is used and the cross match turn negative together with close monitoring of DSA after transplantation.
o While positive crossmatch against B cell may be relative CI. It is considered mainly in retransplantation or negative auto cross match.
o Real cross match either:
-CDC: detect lysis of donor lymphocytes on adding recipient serum and complement. It is done for both B and T lymphocytes. False negative results if low titer of DSA or non-complement dependent antibody fixation. In addition, false positive results if patient has auto antibodies as in SLE (avoided by autocross match test) or IgM antibodies (avoided by adding dithiothreitol (DTT).
– Flow cytometry: more sensitive, it detects non complement depend fixing antibodies by using fluorescenated anti IgG antibodies attach to antibodies reacting against B or T lymphocytes.
o Virtual XM: It is not 100 % accurate.
· Based on above provided information, transplantation immunological risk can be classified into;
o High risk: high titer of DSA, better avoid transplantation as hyperacute rejection and graft loss is inevitable.
o Intermediate: low titer of DSA, need induction therapy and strong immunosuppressive protocol.
o Standard risk: no DSA and need standard immunosuppressive protocol, not essentially induction therapy.
RIVEW OF HLA TPING METHODS AND DIFFERENT CROSS MATCH METHODS
FIRST TO DISCUSS HLA TYPING METHODS,
A-Serologic Typing: lymphocytes are taken from the person who has HLA antigen on its surface mixed many serum and all incubated with complement, and if the complement activated due to presence of antibody in the serum that leads to formation of complement complexes and cell lysis and death and dye enters the cell which can be read by special microscpe.
Advantages; rapid results, less immunological relevance
Limitation; unable to find high quality serum with increasing the importance of HLA subtypes like HLA DQ, HLA CW, which in many studies started to show some immunological responses and affect the graft survival by rejection episodes down the road.
B-Molecular Typing: sequence specific primer polymerase chain reaction (SSP-PCR), sequence specific oligonucleotide probes (SSOP).
Molecular typing gives us detailed idea about the difference between the donor and serum HLA, to the extent of suballels. Which in turn give us detailed information about the mismatches between them which affects the graft down the road and help us expecting the rejection episodes and may help us choosing the immunosuppression protocol?
SECOND TO DISCUSS DIFFERENT CROSS MATCH TECNIQUE;
Different cross match techniques are available in many centers around the world , pros and cons of each one will be discussed briefly in the section below,
1- Complement-Dependent Cytotoxicity Crossmatch Methods: recipient Serum antibodies will be mixed with donor lymphocytes (b cell and t cell), if the reaction occurs with help of complement, then cell lysis will occur and cross match will be positive. and important note should be mentioned here HLA CLASS 1,2 are present on b lymphocytes and only class 1 present on t lymphocyte.
Cons: Miss low titer antibody.
Pros: detect non-HLA IgG antibody, autoantibody, or IgM HLA/non-HLA antibody.
2-Flow Cytometry Crossmatch Methods: recipient serum which has different types of antibodies are mixed with donors lymphocytes , and conjugated ig antibodies colored with special dye and if the recipient serum has specific antibodies to the donors ,conjugation with ig happened and there is special flow cytometer which has baseline number can detect the number above the limit which is considered positive for cross match.
PROS: detects non complement antibodies, and sensitive for low titre antibody.
CONS: may be false positive for patient, who had received before any monloclonalantibdies like rituximab.
3-Applications of Antibody Screening/PRA Testing: pra once introduced in clinical practice in kidney transplantation made a big difference in the descion of physician’s regarding each clinical scenario they face.
What is pra ? it a collection of about 10000 serum HLA antibodies from different donors which mostly hade all the known HLA discovered or known till know ,
Each recipient serum will be mixed with these serum HLA antibodies presented as beads in machine which represent in numbers that give us an idea about the recipient antibodies in his blood, as he may be exposed to any procedure before that provoked his immune system to perform antibodies like blood transfusion or transplantation before and for females’ pregnancy also.
The more percentage that the recipient has in his report the less likely transplantation , for example the patient has 80% pra that means that he has 80% antibodies to the donors that he will be offered.
For this kind of patient, with high pra , the paired exchange program are offered if they have positive cross match with any living donors and also the will be on the top of the list of deceased list .
4-Virtual Cross matching: (VXM) is not a true crossmatch. it is just an application comparing the recipient HLA PRA screening with the donors HLA , as predictors of the actual crossmatch
Limitations: antibodies are dynamic in the body, they are changing from time to time may be cleared by the immune system and may return back .so updated samples of the patient or the recipient serum may be needed every three month not to miss any new antibody .
Pros and cons of vxm
False positive readings: in non-complement antibodies
False negative readings: due to variable HLA typing alleles, detailed HLA of the donor serum should be carefully read as HLA pra are not fully presented as mentioned before in the pra method, which may give us false positive readings.
I. Human leukocyte antigen typing and crossmatch: A comprehensive review
This review article deals with reviewing the Human Leukocyte Antigen (HLA) typing and crossmatch techniques with respect to an index case of End Stage Renal Disease (ESRD) with lupus nephritis as the basic disease having a positive B cell complement dependent cytotoxicity (CDC) crossmatch and a negative flow cytometry crossmatch (FCXM) in absence of Donor Specific Antibodies (DSA). This false positive result was due to the autoimmune disease, i.e. SLE.
HLA typing techniques include serological assay and DNA based molecular methods.
a) Serological assay: This method utilizes antibody-dependent cell mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Recipient lymphocyte is added to tray with wells containing serum with antibodies, complement and a dye leading to cell lysis which is observed under a phase contrast microscope. Results are rapidly available leading to a decreased cold ischemia time. They do not identify ‘null’ alleles (having low immunological significance). These methods are not used nowadays due to limitations like lack of commercially available serum containing specific antibodies against different HLA alleles, esp. HLA Cw, DQ and DP. They are not able to detect difference in HLA protein small amino acids.
b) Molecular methods: These are DNA based methods which are more sensitive, more accurate and have higher resolution, ultimately helping in better HLA typing. These methods include:
i. SSP (Sequence Specific Primer): Extracted DNA from patient is mixed with primers complementary to specific alleles in wells forming an amplification product (using PCR) which, on an agarose gel electrophoresis, forms a band helping in HLA typing.
ii. SSOP (Sequence Specific Oligonucleotide Probe hybridization): Amplified DNA is mixed with oligonucleotide probe complementary to specific DNA segment of different alleles which are identified using fluorescent markers leading to HLA typing.
iii. Direct DNA sequencing: With his method, the exact order of nucelotides in the gene can be understood and the HLA allele sequences can be comared to assign the HLA type.
Molecular methods are more specific but time consuming. Another drawback is with respect to absence of newly identified alleles in the HLA sequence databank.
HLA antibody screening: This is especially important in patients with history of sensitization, like prior pregnancy, blood transfusion of transplantation.
Methods for HLA antibody screening include cytotoxic (cell-based) and solid phase antibody screening.
A) Cell based (cytotoxic) method: It involves mixing recipient serum in a well containing 30-40 different ‘donor’ lymphocytes serum with complement and a dye leading to cell lysis (if antibodies are present in the recipient serum) which is observed under a phase contrast microscope. The degree of cell lysis is expressed as panel reactive antibody (PRA). Disadvantages with this method include lack of adequate representation of all the HLA types present in the population; presence of false postive values due to autoantibodies, IgM, and non-HLA antibodies. False negative results can be seen in conditions with low antibody titres. Prices documentation of unacceptable antigens cannot be done with this method.
B) Solid phase antibody screening: It involves using HLA molecules in place of lymphocytes.
1) Enzyme linked immunosorbent assay (ELISA): It is a 3 step procedure in which recipient serum is added to HLA glycoprotein labelled microtiter wells and after giving a wash, anti IgG with a passenger reporter molecule (alkaline phosphatase) is added followed by a wash. The third step involves addition of a substrate which undergoes a color change (due to dephosphorylation by the reporter molecule) if antibodies are present.
2) Microbead/ single-antigen bead technology: It is a very sensitive method in which recipient serum is added to beads labelled with fluorescein (reporter dye) and incorporated with HLA molecules. After giving a wash, Anti IgG lebelled with phycoerythron (detector antibody) is added and the result can be visualized using 2 laser beams, each detecting the reporter dye and specific bead. The results can be interpreted as either channel shift associated with the antibody binding (flowcytometry) or degree of fluorescence (mean fluorescence intensity, MFI) using Luminex method. It is useful in conditions with low level of DSA, non-HLA antibodies and non-complement binding antibodies, seen in a situation whereby CDC crossmatch comes out negative. The drawback with this method is that the bead kits available might not be representative of the HLA antigens in the community. They are specific, rapid, efficient, have increased range of identification (upto 11 HLA loci) and enable virtual crossmatch. But these methods are too sensitive, the antibodies detected might not have a clinical relevance. A false negative value can be seen due to high titre antibodies (prozone phenomenon), presence of IgM, IVIG use and due to epitope sharing (leading to reduction in MFI).
Microbead assays are more specific than ELISA based assays, which, in turn, are more specific than the cell based assays.
Crossmatches are used to identify the risk of graft rejection in a prospective transplant recipient.
Crossmatch techniques can be divided into cytotoxic (cell based) assays and virtual crossmatch.
A. Cell-based assays: These are of 2 types.
1) CDC technique: In this, complement is added to a mixture of recipient serum and donor lymphocytes (T and B cells separately). If donor-specific antibody (DSA) is present, it will bind with the lymphocytes, complement will get activated and cell lysis will take place, giving a positive result. To increase its sensitivity, anti human immunoglobulin (AHG) can be added to the mix. A positive result can be given either on the basis of a cut-off value of >20% cell lysis (semi-quantitative), or a titred value of dilution required to get a negative result (for quantification). Presence of autoantibodies give a false positive result, addition of DTT (ditheothreitol) inhibits IgM mediated complement activation. A false negative result is seen if the DSA level is low, or in presence of non-HLA activating antibodies.
A positive T and positive B cell CDC crossmatch denotes DSA to HLA type I and II antigen. A negative T with positive B cell CDC crossmatch denotes DSA to HLA type II or low level DSA to HLA type I antigen. A positive T with negative B cell CDC crossmatch denotes a technical error.
2) Flowcytometry Crossmatch (FCXM): This is a test with higher sensitivity. In this, a fluorescein labelled anti IgG antibody is added to a mixture of recipient serum and donor (T and B) lymphocytes. A flowcytometer is used to detect the lymphocytes bound to the anti IgG labelled DSA. It is useful in conditions with low level of DSA, non-HLA antibodies and non-complement binding antibodies, seen in a situation whereby CDC crossmatch is negative, while FCXM is positive. So, it is useful in sensitized individuals. The result is expressed as either a measure of fluorescence intensity as a ratio of control (channel shift), or a titred value of dilution required to get a negative result (for quantification).
3) Virtual crossmatch (VXM): It involves utilizing both donor HLA typing and the recipient solid phase assay antibody screening together. The HLA of the donor is compared with the antibodies present in the historical recipient sera. The advantage with VXM is that it is a rapid, on paper, crossmatch and can be done while the donor is still being evaluated. The drawback includes false positivity in patients with low antibody titre and non-complement binding antibodies.
HLA typing:
HLA typing is a crucial step in renal transplantation.
Methods of HLA typing includes serological typing & molecular typing
A.Serological typing:
In this method recipient lymphocytes are introduced into a tray wells which contain anti HLA antibodies. Then complement is added. When the antibody bind to the antigens on the surface of lymphocytes; the complement is activated. This results in cell lysis and death which allow the dye to enter the cell. Tray wells are then identified under phase contrast microscopy.
Pros:
1. Results are available in a short period so it’s important in deceased donor renal transplantation.
2. Offers the ability to differentiate ” Null” HLA alleles.
Cons:
1. Aren’t capable of identifying the ever-growing number of HLA alleles due to lack of sera (eg: HLA-Cw, DQ, and DP antigens)
2. Can’t detect differences in HLA protein small amino which may trigger potent immunological responses.
B.Molecular typing:
These are many but all of them depend on DNA extraction.
Types:
1) Sequence-specific primer polymerase chain reaction (SSP-PCR):
Steps:
i. DNA extraction
ii. PCR to acquire amplified DNA of specific primers.
iii. Gel electrophoresis by installing the amplified allels into an agarose gel where they appear as bands.
iv. Matching primers of the amplification product to DNA sequences of several candidate alleles.
2) Sequence specific oligonucleotide probes(SSOP):
Steps:
i. DNA extraction
ii. DNA amplification.
iii. Mixing of the amplified DNA with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles.
iv. HLA alleles identification using fluorescent tags.
v. HLA type is then assigned using available HLA allele sequences.
3) Direct DNA sequencing:
This method determines the precise order of nucleotides in the gene of interest.
Using published HLA allele sequences, HLA type is subsequently assigned by comparison.
Pros and cons of HLA typing based on PCR
Pros:
1. Can identify differences in HLA antigen between donor and recipient with detail to the amino acid level.
2. Highly specific where specific alleles are identified with no cross-reactivity.
Cons:
Not all Molecular techniques can identify new alleles (eg: SSP & SSOP).
Notes:
▪︎HLA-A, HLA-B and HLA-DR are considered as the most important ones in kidney transplantation.
▪︎Eurotransplant and old United Kingdom transplant data suggest that HLA.DR matching has a far greater effect than HLA-A or HLA-B.
▪︎ Data from the United Network for Organ Sharing (UNOS) registry highlighted the significance of paying attention to having the least number of mismatches.
HLA antibody screening:
Causes of HLA antibodies formation:
i. Pregnancy
ii. Post blood transfusion.
iii.Prior transplantation.
▪︎Methods used for HLA antibody screening:
A. Cell based (Complement Dependent Cytotoxicity):
In which recipient serum is mixed with donor cell’s lymphocytes and adequate complement. If there’s DSA complement is activated which result in cell death and uptake of dye. The degree of cytotoxicity is expressed as percentage PRA.
▪︎Limitations:
1. PRA percent can be different numerically without a corresponding change in the type or amount of antibody which largely depends on the cell panel used.
2.False positive results can be produced due to non HLA antibodies, autoantibodies and nonspecific IgM antibodies.
3. False negative results are possible due to low antibody titres.
4. Complete lists of antibody specificities and unacceptable antigens cannot be identified as there are several antigens in each well.
B. Solid phase antibody screening:
This depends on recombinant HLA molecules instead of lymphocytes
(to avoid false positivity due to non- HLA antigens in the lymphocytes).
▪︎Methods:
1) Enzyme-linked immunosorbent assay platform. Steps:
· Purified HLA molecules are applied to ELISA platforms.
· Then the recipient serum and enzyme conjugated antibodies to IgG are added.
· Detect the presence of HLA antibody by optical density reading.
2) Microbead platform/single-antigen beads: Types:
2.1)Screening test:
· A panel beads with several different class Ⅰ or Ⅱ HLA antigens on a bead are used.
· It gives a positive or negative result.
2.2)The phenotype (ID beads):
· Beads are individually coated with class Ⅰ or Ⅱ HLA antigens of an individual patient-derived cell line.
· Microbead which is conjugated with fluorescent dye is then added to detect the presence of HLA antibody in the serum.
· Fluorescence detection can be done traditionally using a flow cytometer (Flow PRA®) or via single-antigen beads (SAB) Luminex® platform.
· These estimate PRA by the proportion of positive beads.
Notes:
· SAB are individually coated with a single HLA antigen and yield a list of distinct antibody specificities which are subsequently compared with HLA frequencies in the donor population to determine the calculated panel-reactive antibody (cPRA).
Pros:
1. Can discriminate between immunologically relevant positive crossmatches from false-positive results.
2. Microbead assays (both Flow PRA® and Luminex®) are more sensitive for lower titre antibody than ELISA( which is ten percent more sensitive compared to anti-human globulin enhanced cytotoxicity based assays).
3. Specific for anti-HLA antibodies.
4. SAB assays are rapid with results available in 3-4 h.
5. Quite efficient in a single reaction. (chamber up to one hundred unique antigen beads can be tested).
6. Permits testing many patients simultaneously.
7. Results from SAB enable virtual crossmatching (VXM) to identify DSA pre-transplant.
8. SAB assays permit identification of anti-HLA antibodies for all common
and numerous rare antigens and alleles.
Cons:
1. They detect both complement and non-complement dependent Abs.
2. Can detect antibody that is below the threshold associated with a positive crossmatch which may not always have clinical implications.
3. Can not detect non- HLA antibodies.
4. Give false results in these conditions:
– Prozon phenomenon whih can affect
SAB – Luminex® assays
– Binding of IgM antibodies or other
serum factors to the beads.
– Nonspecific binding by serum proteins
and drugs such as IVIG.
– Epitopes sharing
3)CROSSMATCHING (XM)
· The presence of DSA depicted as a positive crossmatch was a contraindication to transplantation.
· Crossmatch tests will identify if a recipient had antibodies to a specific donor cells.
· T cell cytotoxic crossmatch had a twenty percent false positive rate and a four percent false negative rate. Therefore, it is insufficient to identify all relevant antibodies.
· The solid-phase antibody test should be used together with crossmatch results to identify those that are immunologically relevant.
3.1) Complement-dependent cytotoxicity crossmatch
Similar to cytotoxic assay the complement-dependent cytotoxicity crossmatch is interpreted as positive if a considerable number of lymphocytes are destroyed after the incorporation of complement (Figure 2A in the paper) .
– Can be done for B and T lymphocytes.
Limitation:
1. Sensitivity is limited if the relevant
antibody is in low titres.
2.Can give false positives by detecting
autoantibody, IgM/IgG HLA or non-HLA.
3.2)Flow cytometry crossmatch (FCXM):
· Detects DSA independent of complement fixation.
· In this method, recipient serum is mixed with donor lymphocytes and then tagged with a fluorochrome conjugated anti-IgG antibody.
· Several antibodies with separate fluorochromes particular to B and T lymphocyte surface proteins can be added.
· With the use of flow-cytometry, B and T lymphocytes can be identified and have their DSA individually interrogated
Advantages: It is more sensitive than CDC-crossmatch.
3.3)Virtual crossmatching (VXM):
· Both donor HLA typing and solid phase antibody screening data are utilised together.
Pros:
1. Shorter wait times.
2. Improved outcomes for sensitised transplant recipients.
3. Permits transplant physicians to consider a potentially positive crossmatch (a risk factor for donor selection).
Cons:
1.Titres, specificities, and presence or absence of antibodies could significantly vary over time. Thus, the use of antibody specificity from historical serum sample (earlier than six months) could not predict a crossmatch with certainty.
2. False positive results of VXM may arise where there are significantly low titre and/or non-complement binding antibodies, thereby, resulting in the wrong exclusion of potential donors.
3. Give false negative results due to the fact that the list of all potential HLA donor antigens have been classed differently and, therefore, can not be correctly represented.
4. Does not identify the HLA “Null” alleles.
Advantages ;a) the result is fast which means minimization of cold ischemia time b) identification of null alleles (10)
Disadvantage ; a) Absence of sera with antibodies specificities against increasing numbers of HLA alleles(10) b) they are rare serological assays for HLA-cw,DQ, and DP loci b) inability to identify differences in HLA small amino acids proteins.
2 Molecular typing ;
Advantages ; Identifications of differences in HLA antigen between donor & recipient
Disadvantages ;a) Variations in the value which affecting type or amount of antibodies b) Uses commerial kids which may not give the true picture of the population c) False positives e.g. IgM (26-29) d) False negative if low antibody titre(29)
2 Solid phase assay ; It utilises soluble or recombinant HLA molecules and not lymphocytes. Methods are ;
Advantages ; a) quick test with results in 4 hours b) large numbers of patients can be tested at once c) provide the bases for virtual cross-match (VXM) (37)
Disadvantage ; a) it may pick up antibodies with no clinical relevants = high sensitivity b) Cannot detect non-HLA antibodies which are equally important as well(42,43)
c) Not all HLA alleles are avialable on the beads e) Artefacts e.g. prozone = false negative due C1q binding. The test can turn positive after dilution (45-46) f) epitope sharing = single antibodie binds to many antigen beads
Disadvantages are ; a) false negative b) failure to detect null alleles
HLA typing is an essential component of renal transplantation. HLA interpretations can be difficult and various modalities can be used for this purpose with different pros and cons.
HLA typing methds include:
1- Serological
In this method the recipient lymphocytes are mixed with sera containing different know HLA alleles . Null alleles can be exclude easily. These methods do not recognise HLA Cw ,DQ and DP
2- Molecular
These include –
Sequence specific PCR– In this method the DNA from subject is amplified in several wells containing Primes which are complimentary to HLA alleles
Sequence specific oligonucleotide Probes; Precise order of nucleotides of gene of interest can be determined.
Direct DNA sequencing- It can detect significant alleles and also specific alleles without cross reactivity
HLA NTIBODY SCREENING
Cytotoxic antibody screening
In this recipient serum is mixed with 30-40 donor lymphocytes , dye and Compliment . Results are expressed as PRA
Solid phase antibody screening
Uses recombinant HLA molecules instead of lymphocytes
ELISA platform
Microbead platform
Estimates PRA and more sensitive than ELISA. cPRA identifies unacceptable antigens
CROSS MATCHING
Compliment dependent Cytotoxic cross match- It can be done for B and T lymphocytes
Flow cytometry– In this recipient serum is added to donor lymphocytes and Fluorescent tags are used. It can detect DSA independent of compliment fixation .
Virtual cross match- Its combination of Solid phase antibody screening and HLA Typing
Immunological Risk Groups
High immunological risk groups-
Have high level of DSA and can lead to hyper acute Rejection
Intermediate Risk Groups-
Low titres of DSA and may require extensive immuno suppression
Standard Risk group-
No evidence of donor directed HLA sensitization
This paper is an excellent clinical review of HLA typing and crossmatch methods.
HLA Typing
HLA mismatches between recipient and donor represents an immunological burden that the donor graft is likely to be exposed to once transplantation takes place.
The more HLA mismatches between donor and recipient the higher the likelihood for reduced graft survival. For example, 6 mismatches are likely could lead to 64% higher risk of graft failure compared to 13% when there is one mismatch.
There two methods employed in HLA typing; Serological method and Molecular methods.
Serological method; Has the advantage of quicker turnaround of results, which can be useful in deceased donor transplant as it reduced cold ischaemic times.
It is helpful in excluding null alleles which are detectable by molecular techniques but not expressed on the cell surface. The disadvantage is that there may not be commercially available sera to identify newly discovered HLA alleles and also for HLA-C, DQ and DP.
Molecular methods; There are 3 different methods of molecular typing.
Sequence specific primer pcr; Extracted DNA is amplified with primers. HLA type is determined by matching primers to HLA sequence.
Sequence specific oligonucleotide probe; HLA is determined by mixing amplified DNA with oligonucleotide probes of different alleles.
Direct DNA sequencing; specific nucleotides are determined and HLA is assigned based on known HLA allele sequences.
HLA antibody screening
This can be done using Cell based and Solid phase methods.
Cytotoxic antibody screening;
Recipient serum is tested against a set of 30 to 40 different donor lymphocytes, dye and complement are added. Cells that bind antibodies in the recipient’s serum die and take up die. The frequency of this reaction happening with the different cells is expressed as the Panel reactive antibody (PRA).
Solid phase antibody screening
This done either by ELISA or Single antigen beads
The single antigen bead method involves commercially prepared beads which are coated with Class I or II HLA molecules. When mixed with the donor serum, specific antigen reactions are determined.
The antigen specificities are then compared with HLA common in the donor population to determine the calculated panel reactive antibody(cPRA).
Unlike PRA, cPRA is actually based on antigens the donor is likely to be sensitized to (unacceptable antigens).
Crossmatching
Complement-dependent cytotoxicity crossmatch (CDC)
Similar to cytotoxic assay, done on B and T lymphocytes and indicated presence of complement dependent cytotoxicity of lymphocytes to which the donor may be sensitized to and has antibodies directed against them.
False negative results can be obtained if the antibody titres are low or if the recipient has antibodies that do not fix complement.
False positive results can be obtained in the presence of autoimmune antibodies or IgM antibodies. This can be corrected for by doing auto crossmatch test and addition of dithiothreitol (DTT) respectively.
Flow cytometry crossmatch
Detects donor specific antibodies independent of complement fixation.
Utilises fluorochrome labelled anti-IgG antibody which attaches to antibodies that have reacted to B or T lymphocytes. This makes it more sensitive than CDC crossmatch.
Virtual crossmatch
Uses both HLA typing and Solid phase antibody detection.
This information identifies the donor HLA type and the DSA that he possesses. This information is then used to work out what the real crossmatch would turn out to be if performed as the donor unacceptable antigens are known and the associated DSA is determined.
In conclusion, this is a useful summary of the different HLA typing methods, screening for preformed DSA and Crossmatch methods.
Human leukocyte antigen typing and cross match: A comprehensive review
Crosshatch and HLA typing plays a crucial role in improving outcome of organ transplantation and afford better matches to recipient.
HLA typing
Serological typing
• Recipient lymphocytes are introduced into the tray well contacting sera, complement and dye
• Antibodies in the tray will bind to the antigens on the surface of lymphocytes and complement activated which will induce cell death.
• Benefit
o able to produce results quickly and important in DCD transplant where the cold ischemia time can be reduced
o Able to differentiate HLA alleles with no cell surface antigen expression -null HLA
• Disadvantages – lack of sera with antibody specificities that’s are capable of identifying the ever growing number of HLA alleles
• HLA Cw,DQ and DP antigen may have clinical benefit but are scarce for these loci.
Molecular Typing
Sequence -specific primer polymerase chain reaction
• Extracted DNA is amplified in several wells in which each well has specific HLA alleles
• In wells, amplification product is formed when DNA probes are complementary to the specific sequence of HLA molecule
• Band will be formed when it is instilled into an agarose gel and undergoes electrophoresis
• Highly specific where specific alleles are identified with no cross reactivity
• Cross reactivity – identification of alleles that are similar to alleles of interest
Sequence specific oligonucleotide probes
• Amplified DNA is mixed with oligonucleotide probes that compatible to specific DNA of different alleles
• unique HLA alleles will be identified by fluorescent labels
Direct DNA sequencing
• identify the exact order if nucleotides in the interested genes.
• According to HLA alleles sequencing, HLA type is assigned by comparison
HLA mismatches
• CTS- HLA DR and HLA B antigens offer the most alloimmune burden with less from HLA A
• Eurotransplant and UK transplant data (old)- HLA DR mismatches has far greater effect than HLA A or HLA B
• Owen WF et al showed HLA DR mismatching had most effect during first 6/12 post TX while maximal effect of HLA B mismatching occurred 2 years post TX
• UNOS – 6 mismatches -64% higher risk then 1 mismatches 13% only
• Taboo mismatches – 81% one year survival and 50% 5 year survival
HLA antibody screening
Cytotoxic antibody screening
• Representative of the population of potential deceased donors – consists of 30-40 different donor lymphocytes
• The degree of cytotoxicity is shown as PRA %
• Limitations
o depend on the cell panel used
o HLA frequencies and racial differences need to be factored
o False positive – non-HLA antibodies, autoantibodies non-specific IgM antibodies
o False negative- requires higher antibody titres to be activated
Solid phase antibody
• Uses soluble or recombinant HLA molecules instead of lymphocytes targets
ELISA
• Purified HLA molecules – applied to ELISA and will bind individually to HLA antibodies and detected by optical density reading
Micro bead platform/ SAB
• Fluorescence detection thru flow cytometer ( FLOW PRA) or via the single antigen beads (SAB) luminex platform
• SAB- rapid and results available in 3-4h
• Enable virtual XM
• Allow identification of anti-HLA antibodies for all common and numerous rare antigens and alleles up to 11 HLA loci
PRA vs cPRA
• PRA- translate into high probability of a positive crossmatch
• cPRA- based on unacceptable antigens -that those the patient is sensitised to
Crossmatching (XM)
• presence of DS cytotoxic antibody – positive XM -contraindication to TX
• 20% false positive rate and 4% false negative rate
• So used with SAB
CDCXM
• Positive – large amount of lymphocytes are destroyed – suggest significant amount of DSA found
• Done for B cell and T cell
• Sensitivity is limited – can improve by increasing the time of incubation and use of AHG-enhanced method
• Negative – 18% graft loss in 1 year, positive 36% graft loss
FCXM
• Detect DSA independent of complement
• Accurately detect the presence or lack of IgM/IgG on donor lymphocytes
• Greater sensitivity than CDCXM
Virtual XM
• The use of data to XM and forecast the actual in vitro crossmatch
• Lead to shorter time for waiting and improved outcomes for sensitised transplant recipient
• Does not identify null HLA
The clinical scenario
Positive CDC, negative FXCM ands SAB showed standard immunological risk, so can proceed with transplant
HLA matching has a significant effect on graft survival.
Serologic techniques:
Pros:
Quick results which decrease ischemia time in deceased donor transplant
determine “null” HLA alleles (of low immunological significance as has identifiable DNA sequence with no cell surface antigen expression)
Cons:
The available sera can’t identify all HLA alleles
can’t identify HLA-cw, DQ and DP antigens which are clinically significant
can’t detect small amino acids in HLA protiens which may be antigenic
Not used now
Molecular techniques:
Pros:
detect significant alleles and differentiate between donor and recipient at level of antigens, epitopes and amino acids.
Detect specific alleles without crossreactivity.
Cons:
Can’t identify new alleles that are not on primers used in HLA-typing
Studies showed that:
HLA-DR and B antigen mismatches have higher risk than HLA-A mismatch.
HLA-DR mismatch has the highest immunological risk.
The effect of HLA-DR mismatch occurs post transplant in the first 6 months while the effect of HLA-B mismatch appears 2 years post transplant
Data from UNOS registry showed that increased number of mismatches increases the risk of transplant failure.
Detection of preformed anti-HLA antibodies is important as they increase the immunological risk of graft failure.
Low titer of DSA may cause early or late AMR even in presence of negative crossmatch.
Cytotoxic antibody screening:
Expressed as percentage PRA, used to estimate risk of recipient having positive crossmatch
Cons:
Depends on commercially available cell panel which may not represent the population
HLA frequency and racial differences can’t be considered
Can’t determine antibody specificity and unacceptable antigens.
False positive results due to non-HLA antibodies, autoantibodies or non specific IgM antibodies.
False negative results due to low titer antibodies that fail to activate complement.
Solid phase antibody screening:
Use recombinant HLA molecules instead of lymphocytes and identify antibody specificity of recipient serum
ELISA
Microbead platform/single antigen beads:
More sensitive than ELISA
Identify list of distinct antibody specificity
Estimate PRA which identifies several antibodies to cluster of donors when PRA increase, it indicates high probability of positive crossmatch
Determine cPRA by comparing antibody specificities with HLA frequencies in donor population
cPRA: determine unacceptable HLA antigens that a recipient is sensitized to and represent actual proportion of donors who have unacceptable antigens and the recipient is compatible with.
Pros:
Specific for HLA antibodies and identify anti-HLA antibodies up to 11 HLA loci
Rapid and tests many patients simultaneously
Enables virtual crossmatch and identify DSA pre transplant.
Cons:
detect both complement and non complement binding antibodies simultaneously
detects antibody which is below threshold and may be clinically insignificant leading to exclusion of potential donor unnecessarily
can’t detect non HLA antibodies
can’t fully represent the ever-growing HLA alleles
False negative results due to prozone phenomenon, complement component C1, IgM antibodies, serum proteins and IVIG and epitope sharing which may decrease MFI on single bead
Identifies if a recipient has antibodies to specific donor.
CDC-XM
Done for B and T lymphocytes
False negative results due to low titer antibodies not detected
False positive results due to detection of autoantibodies, IgM/IgG HLA or non HLA antibodies.
FCXM
Recipient serum mixed with donor lymphocytes
Identify T and B lymphocytes with no need for complement fixation
more sensitive than CDC and detect IgG DSA on donor lymphocytes.
Virtual crossmatch:
Donor HLA typing and solid phase antibody screening are used together to predict the actual crossmatch in vitro
Leads to shorter waiting time, better outcome in sensitized recipients and better donor selection as predict potentially positive crossmatch.
should include all serum results with recent sample as antibodies presence, titer and specificity vary with time especially with sensitizing events.
Cons:
False positive results due to low titer antibodies (clinically insignificant but detected by SAB) and/or non complement binding antibodies.
False negative results as all potential HLA donor antigens may be not correctly represented.
not 100% accurate and actual crossmatch is mandatory.
Based on donor crossmatch and antibody screening at time of transplantation
Detailed study of DSA by molecular techniques prevent wrongly exclusion of donors, allow improved organ allocation and shorter waiting time.
High risk:
High titer of circulating DSA
may lead to hyperacute rejection and preclude transplant
desensitization decreases the risk
Intermediate risk:
low titer of DSA
Historic DSA not detectable
Acceptable transplant with intensified immunosuppression and close monitoring post transplant
Standard risk:
No evidence of sensitization to HLA of the donor
Dear professors,
Regarding HLA antigen mismatches, is there any clinical significance for HLA matching at the epitope level? in regards of de novo DSA,long term graft survival and allocation system from clinical point of view.
HUMAN LEUCOCYTE ANTIGEN TYPING AND CROSS MATCH:A COMPREHENSIVE REVIEW
This article by Althaf and colleagues is an excellent review emphasizing the value of interpretation of cross match results in a given clinical scenario . The fact that kidney transplantation is the best option for patients suffering from end stage renal disease(ESRD), all efforts should be made to ensure that appropriate patients are given this opportunity and misinterpreting a cross match result can deny any patient getting a transplant
The case scenario reflects the same that a positive CDC cross match in a patient of lupus is not because of a DSA rather it is result of immune complex binding to Fc receptors giving a false positive B cell cross match.
The authors have thoroughly explained different techniques of HLA typing mentioning there pros and cons as well like
SEROLOGICAL TYPING
Lymphocytes of the recipient are added to a tray containing sera, complement and dye, and if there is complement activation, this will lead to cell death, where the membrane integrity of the cells are compromised, leading to the dye being able to enter into the cells. These can be identified using phase contrast microscopy.
The key benefit of serological typing is that results are available in short period and is particularly important in deceased donor transplant.
As more advanced method of typing are available currently available serological typing has fallen into disuse.
MOLECULAR TYPING
These are of three types :
Sequence specific primer polymerase chain reaction : The principle of DNA amplification is applied with electrophoresis in order to identify DNA sequences for HLA typing.
Sequence specific oligonucleotide probes : DNA amplification is done with oligonucleotide probes and unique fluorescent tags are used to identify HLA alleles.
Direct DNA sequencing : HLA type identification is done by comparing with established allele sequences.
HLA ANTIBODY SCREENING
The authors reported that almost third of patients on waiting list for kidney transplant have some degree of anti HLA antibodies so it is crucial to detect these antibodies to ensure a safe kidney transplant .
Different methods for detecting these antibodies are
CYTOTOXIC ( cell based) ANTIBODY SCREENING
SOLID PHASE ANTIBODY SCREENING
ENZYME LINKED IMMUNOSORBANT ASSAY PLATFORM
MICROBEAD PLATFORM/ SINGLE ANTIGEN BEAD
The authors have further elaborated different cross matching techniques and finally given the concept of immunological categorization after all these testings
High immunological risk
At the time of transplantation there are high timers of circulating antibodies HLA DSA
This can lead to hyper acute rejection
Intermediate immunological risk
Historic DSA is not detectable and at the time of transplantation there are low titers of DSA.
Standard immunological risk
No DSA
I think the concept of this paper is to ensure that treating Nephrologist should have adequate knowledge of transplant immunology and any positive cross match result should be read in that given clinical scenario
This article provides a review of the main methods used to assess the immunobiological risk of a transplant.
HLA typing to evaluate HLA alleles: HLA A-B – DR. Studies revealed that mismatches translated into a 64% higher risk, while the risk dropped to 13% with just one HLA mismatch. Thus, HLA Recognition is the first and most important step in this process. Possible methods are:
1 – HLA TYPING
A-Serological: in this case sera with antibodies to a multitude of known HLA alleles is used, juntamente com recipient lymphocytes and complement. The comparasion of cell death indentified in microscopy proporcionara o resultado a ser avaliado.
The key benefit of serologic typing is that results are available in a short period. This is particularly important in deceased donor renal transplantation.
B- Molecular:
B.1 – Sequence-specific primer polymerase chain reaction: extracted DNA from amplified in several wells are submetidos a eletroforese para formação de bandas a serem estudadas
B.2 – Sequence specific oligonucleotide probes: amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles, so HLA alleles are then identified using fluorescent tags.
B.3 – Direct DNA sequencing: determines the precise order of nucleotides in the gene of interest, allowing HLA type comparison, through the amino acid level, to provide insight to the risk monitoring mismatched donor-recipient antigens, epitopes and amino acid.
2 – SCREENING OF HLA ANTIBODIES:
After evaluation of the direct HLA alleles, the evaluation of HLA is the second step. Here, comparative panel studies (PRA) with population controls of specific antibodies compared the risk of replication through specific donors (DAS). They are used: cell-based or solids (divided in ELISA or fluorescence microspheres (Flow PRA or SAB Luminex))
There is the possibility of calculating panel reactive antibodies (cPRA), which provide as best estimates of the probability of a positive donor-specific antibody/crossmatch for a randomly selected donor. cPRA is based on unacceptable HLA antigens – those to which the patient has been sensitized, an offer to a recipient with a high cPRA is a high probability of a positive crossmatch.
3 – CROSSING
With cytotoxic time it was necessary to test that the T cell cross-matching was insufficient to identify all relevant antibodies and, moreover, could exclude transplant patients unnecessarily. Therefore, it should be used in conjunction with cross-matching results to identify those that are immunologically relevant.
A – Crossmatch of complement-dependent cytotoxicity:
Similar to the cytotoxic assay, complement-dependent cytotoxicity cross-matching is interpreted as positive if a considerable number of lymphocytes are destroyed after complement incorporation. Use B and T lymphocytes.
B – Flow cytometry crossmatch
Flow cytometry crossmatch (FCXM) detects DSA independent of complement fixation. With the use of flow cytometry, B and T lymphocytes can be easily identified and have their DSA interrogated individually. Compared with complement-dependent cytotoxicity cross-matching, this offers greater sensitivity.
C – Virtual cross-match
In virtual cross-matching (VXM), donor HLA typing and solid phase antibody screening are used together
4 – RISK CLASSIFICATION:
A – High immunological risk
High titers of circulating HLA-specific antibodies from mismatched donor
B – Intermediate immunological risk
there is a low DSA titer and the historical DSA is not detectable.
C – Standard immunological risk
there is no evidence of targeted donor sensitization to HLA
Excellent summary
Well done Carlos
This excellent article is a review titled HLA typing, focussing on the different types of tissue typing and crossmatch involved in kidney transplant. It also expresses the application of said crossmatch results and how to interpret the same to an extent in clinical scenarios. The given article takes the example of a lupus ESRD patient. The entire article is based on the significant impact that HLA matching has on survival of the allograft.
The article also defines immunological risk by performing a risk assessment and gives principles for three categories of high, intermediate, and standard immunological risk.
High immunological risk is classified as high titers of DSA, which can lead to hyper acute rejection.
Intermediate immunological risk is classified as low DSA titer, where intense immunosuppressive regimens and careful monitoring post transplant can potentially give a good outcome for the recipient.
Standard immunological risk is classified as a situation wherein there is no donor directed sensitization to HLA.
HLA typing is of two kinds :
A. Serological typing
Lymphocytes of the recipient are added to a tray containing sera, complement and dye, and if there is complement activation, this will lead to cell death, where the membrane integrity of the cells are compromised, leading to the dye being able to enter into the cells. These can be identified using phase contrast microscopy.
B. Molecular typing
These are of three types :
All methods of molecular typing can differentiate between donor and recipient HLA antigen.
HLA ANTIBODY SCREENING
This is essential to identify previous sensitisation, which is generally seen due to pregnancy, prior transplantation, or following blood transfusion due to any cause. Once the patient has been sensitized, there are several methods that can be used in order to treat the patient such as desensitization, acceptable mismatching, and paired exchange schemes in order to bring about the best possible transplant outcome.
There are several methods of HLA screening, which are listed below along with their advantages and disadvantages.
CYTOTOXIC ANTIBODY SCREENING :
In this method, lymphocytes from 30-40 donors are selected as a representation of the population and the recipient serum is mixed with this panel along with complement and dye. If the serum antibodies bind to the cell surface receptors and activate complement pathways, this leads to cell death and subsequent uptake of dye. The degree of cell death is quantified using PRA of cancel reactive antibody. This is the risk of the recipient in having a positive crossmatch, lowering the survival chances for the allograft.
Disadvantages :
A. The PRA percent taken may not be exactly representative of the cell population because of racial differences and HLA frequencies. This means we do not get an accurate outcome, which can lead us to reject viable donors. This will significantly limit the already scarce options for transplant for the recipient and place unnecessary burden on waiting lists.
B. False positive results due to non HLA antibodies, non specific IgM antibodies and autoantibodies.
C. False negative results because this method is dependent purely on complement activation and if there are low tires then this can prevent complement activation, causing the presence of a true antibody to be hidden, leading to a bad outcome post kidney transplant.
D. Complete and accurate list of antibody specificities cannot be identified due to the multiple antigens in each well.
SOLID PHASE ANTIBODY SCREENING :
Recombinant HLA molecules are employed in this method instead of lymphocytes. This is because lymphocytes present both HLA and non-HLA molecules.
There are different methods under this category, listed below.
ELISA platform
Optical density reading helps to identify HLA antibody in serum of the recipient. Enzyme conjugated antibodies to IgG detects the HLA antibody.
This process is done by adding HLA molecules to ELISA platforms which then bind to HLA antibody from the recipient serum.
Advantages :
A. More sensitive than AHG enhanced cytotoxicity based assays.
MICROBEAD PLATFORM
Different panel beads with class I or class II HLA antigens are used to obtain positive or negative result. Proportion of positive beads are calculated using flow cytometer or Luminex. This will produce the PRA result. Antibody specificities are used to estimate cPRA or calculated panel reactive antibody percentage. cPRA is based on unacceptable HLA antigens, which the patient has already been sensitized to previously. This will estimate the chances of a positive cross match.
Advantages :
A. More sensitive for low tire antibody detection.
B. More rapid results.
C. Multiple patients can be tested simultaneously.
D. DSA can be detected pre transplant, allowing space for organ allocation and risk assessment.
E. Anti HLA antibodies for common and rare antigens and alleles can be identified.
Disadvantages :
A. Both complement and non-complement binding detected in parallel. Antibodies below threshold level are detected, which can lead to preclusion of a potential donor.
B. SAB Luminex is affected by prozone phenomenon, wherein high titer anti-hula antibodies are not detected when tested in a concentrated form, but are recognized only when diluted 1:10.
C. False negative reports Can arise because of epitope sharing. Low MFI is seen.
CROSSMATCHING :
The methods of cross matching have been detailed below :
COMPLEMENT DEPENDENT CYTOTOXICITY CROSSMATCH (CDC -XM)
This method gives a positive result if significant DSA binds to cell surface, leading to complement activation and destruction of lymphocytes.
Advantages :
A. Can be done for B and T lymphocytes.
Disadvantages :
A. Limited sensitivity in low titers. Solution is to increase incubation time.
B. Negative test result has been associated with 18% graft loss in one year.
C. Gives false negatives by detecting IgM/IgG HLA or non HLA.
FCXM :
Detects DSA independent of complement fixation. Recipient serum is added to donor lymphocytes and fluorescent tags are used for identification of DSA.
Advantages :
A. Does not depend on complement pathway for results hence false negatives due to low titers are generally reduced or avoided.
B. Can accurately identify absence of presence of DSA IgG on donor lymphocytes.
C. B and T lymphocytes immediately identified and their DSA individually sorted.
D. More sensitive than CDC XM.
Disadvantages :
A. No standardization of results threshold because of different methods used by different labs.
B. Unclear impact on assessment of immunological risk.
VIRTUAL CROSSMATCH :
Both donor HLA typing and solid phase antibody screening done together.
Advantages :
A. Shorter wait time
B. Better outcome for sensitized recipients
C. Sensitive and specific tech
D. Can be done at the time of donor identification
E. Allow physician to consider donor organs that otherwise might not be available for donor selection.
Disadvantages :
A. Significantly low antibody titers, non complement binding antibodies can lead to false positive, which results in excluding a potential donor.
B. False negative results seen when the potential HLA donor antigens are incorrectly represented because of being classed differently.
C. Not fully accurate and thus requires an actual crossmatch to be done along with it to ensure correct results.
D. Does not recognize HLA null alleles.
Reference :
Given article.
Thanks, Well done
There are tow different techniques of tissue typing
1.serological typing :Microcytotoxicity assay
Principle: complement dependant cytotoxicity
After lymphocyte separation well be added in tray wells which contain sera which contain antibodies then complement added and dye.
When these antibodies bind to lymphocytes surface antigens complement activation occurs leading to cell lysis .The percentage of lysed cells are counted under phase contrast microscopy then HLA type is i assigned.
2. Molecular methods
A.Sequence-specific primer polymerase chain reaction:Extracted DNA is added in the wells which contains oligonuclotide primers complementary to small segment of one’s HLA allele then amplified and runs in agarose gel and undergo electrophoresis and appear as a band then typing can be assigned.
B.Sequence specific oligonucleotide probes:
HLA type assigned by mixing amplified DNA with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles.
C. Direct DNA sequencing:
Determines the precise order of nucleotides in the gene of interest.
CROSSMATCHING (XM) . There are tow methods
1.Complement-dependent cytotoxicity crossmatch(CDC-XM) the principle of the test as above. Its specific and less sensitive.
2.Flowcytometry (FCXM) Sensitive and specific
Donor lymphocyte added to the patient serum
then tagged with a fluoro-chrome conjugated anti-Ig G antibody.
3.virtual cross match: acomparative one we comper the PRA result with the donor HLA typing.
Thanks Manal
The paper takes advantage of a specific situation of autoimmunity induced by an immune complex secondary to SLE to review the definitions of immunity and its impact on different laboratory tests.
Serological methods are the most used commercially. Molecular methods have been used in research with the need to have earlier and more specific results, reducing cold ischemia time. The lack of a comprehensive database of these alleles is still a limiting factor for the method, which may result in false negatives.
HLA-Antibodies screening
It can occur by the cytotoxic method, using a reactive panel of antibodies with donor lymphocytes and patient serum. The most specific methods are solid phase, mainly ELISA, flow cytometry, and single antigen beads. These can calculate a PRA (cPRA) with higher specificity of detecting the Crossmatch and is based on unacceptable HLA antigens.
Crossmatch (XM)
There are similar methods, but with some peculiarities that differ from previous tests.
1. Complement-dependent Cytotoxicity Crossmatch – CDC-XM
Here we add the complement and a complex organic molecule to the cytotoxic method, which can expose both B and T lymphocytes. Adding an anti-human IgG immunoglobulin increases the sensitivity of the method. Remember the possibility of false positives with high levels of IgM, autoantibodies, or non-HLA antigens.
2. Flow cytometry – FCXM
This method detects DSA independent of complement fixation using recipient serum, donor lymphocytes and using a fluor-conjugated IgG antibody.
3. Virtual Crossmatch – VXM
It is a combination of previous methods, reducing the waiting time for cold ischemia and with the possibility of comparisons with previous and current exposures between donor and recipient. False positives can occur in low antibody titers or absence of complement, erroneously removing the donor from the list. False negatives occur when antigens are not expressed on the donor HLA.
Based on these tests, it can generate enough data for the classification of immunological risk, which can be high, intermediate or standard.
Conclusion
The patient has lupus nephritis
Deceased donor kidney with a 1-0-0 mismatch (low risk)
(CDC) crossmatch reported positive for B lymphocyte, probably based on humoral disease as SLE
Flow cytometry crossmatch (FCXM) negative for both B and T lymphocytes
Luminex-SAB (single antigen bead) did not identify any DSA
He never had a blood transfusion
Well done
HLA typing and crossmatch
HLA typing is a cornerstone in renal transplantation. donor HLA recognized by the recipient T lymphocytes as a foreign antigen; ultimately activate the immune response against the allograft.
HLA typing made by serologic and molecular methods
Serological typing:
Recipient lymphocytes are added to a tray containing sera with antibodies to many known HLA alleles commercially available. Complement and dye are added thereafter. If the antibody can bind to the antigen on the surface of the lymphocyte complement is activated forming a membrane attack complex causing cell lyses so dye enters it. Tray wells with significant cell death are then identified under a microscope. By comparison and elimination of positive wells the HLA type is assigned.
Advantage:
1- Results are given in a short time, which is important in deceased donors as it decreases the cold ischemia time.
2- It offers the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression. These alleles called null HLA alleles, they are of less immunological significance.
Disadvantages:
1- Lack of sera with Abs specificities that are capable of identifying the ever-growing number of HLA alleles.
2- HLA- CW, DQ, DP Ag may have clinically significant effects on the outcomes of allografts. However, serological assays are scant for these loci.
3- Serological methods do not readily detect differences in HLA protein-small amino acids that may be antigenic enough to trigger a potent immunologic reaction.
Molecular typing:
1- Sequence-specific primer polymerase chain reaction:
DNA from the subject is amplified in several wells. Each well has primers that are complementary to specific HLA alleles. Where DNA probes are complementary to the specific sequence of the HLA molecule, an amplification product is formed which is then implanted into agarose gel and undergoes electrophoresis where they appear as bands. HLA typing is then allocated by matching the primers of the amplification product to the DNA sequence of several candidate alleles.
2- Sequence-specific oligonucleotide probes: Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles which are then identified using fluorescent tags.
3- Direct DNA sequencing: it determines the precise order of nucleotides in the gene of interest. HLA type is assigned by comparison with published HLA allele sequences.
Advantages of molecular typing:
1- Can clearly identify differences in HLA Ag between donor and recipient with detail to the epitope, and amino acid level allows better risk stratification. HLA typing using PCR is highly specific where specific alleles are identified with no cross-reactivity. Cross-reactivity is the identification of an allele that is essentially similar to the allele of interest.
Disadvantage: new alleles not currently on the HLA sequence databank will fail to be identified.
Why is HLA typing needed?
The dissimilarity in the HLA Ag reflects the alloimmune burden that a donor’s kidney presents to the recipient. Ultimately affecting the outcome.
HLA alleles do not have equal weight. HLA-DR, then –B, then –A which has the less alloimmune burden. The influence of HLA-DR mismatching had the most effect during the first six months post transplantation, while the maximal effect of HLA-B mismatching occurs 2 years posttransplantation.
UNOS looked at quantifying the risk of transplant failure with HLA mismatch in deceased allografts. Patient with 6 HLA mismatches translated to 64% higher risk while the risk was 13% with just one HLA mismatch, this is independent of locus. Study identify seven specific HLA mismatch combinations that were associated with decreased allograft survival. These called taboo mismatches translated to 81% one-year survival and 50% five-year survival.
HLA antibody screen:
Sensitization towards HLA-Ag (DSA formation) occurs in pregnancy, post blood transfusion and prior transplantation.
Why DSA screen important?
1- They Contribute to immunological failure
2- Cause positive CXMm thereby; resulting in the exclusion of donor.
So; both sensitive and specific detection of these Abs is important. Because this influence the decision of transplantation either with desensitization strategy, or paired exchange, and acceptable mismatching with potent immunosuppression.
Methods for DSA screen:
1- Cytotoxic (cell-based) way: a group of donor cells selected randomly from the population of potential deceased donors. 30 to 40 different donors lymphocytes mixed with recipient serum in individual walls along with complement and dye, if the recipient serum contains Abs that can bind to the cell surface Ag, the complement-mediated reaction will occur, ends with cell lyses and the dye enters it. Coloured cells identified by polarised microscopy. The degree of cell death is expressed as a percentage of PRA (panel reactive Ab).
PRA tell us about the possibility of the recipient to have positive crossmatch, and so the likely to have a donor from that population.
Disadvantages:
1- PRA% can change without a corresponding change in the type or amount of antibodies.
2- The cell panels are commercially produced and may not truly represent the population.
3- False positive which can be in the presence of non-HLA antibodies, auto-Abs, and non-specific IgM Abs
4- False-negative result due to low titre antibodies.
5- Each well contains several Ags so, precise complete lists of antibody specificities and unacceptable Ag cannot be identified by this method.
2- Solid-phase antibody screen:
Soluble or recombinant HLA assay
a- Enzyme-Linked immunosorbent assay platform: ELISA recipient serum will add to ELISA platform containing purified HLA molecules, enzyme-conjugated Ab IgG is then added to detect the presence of HLA antibody which bound to Ag by optical density reading.
b- Microbead platform/ single-antigen beads: pools of beads coated with different class I or II HLA antigens used for screening.
Fluorescence detection based either on cytometer (Flow PRA) or by the single antigen bead (SAB) Luminex platform.
The proportion of positive beads estimates PRA. SAB yields antibodies specificity. Specificities are subsequently compared with HLA frequencies in the donor population to determine the cPRA .
cPRA represents actual donors who express unacceptable HLA antigens to which the recipient has been sensitized. It is used in organ allocation.
The combination of the traditional cell-based assay with solid-phase assays help in better determining relevant positive CXM from false-positive results.
Limitations of solid-phase assays;
1- Detect both complement and non-complement fixing Abs
2- Can detect very low titer Abs or Abs with no clinical significance, but can preclude the potential donor. Also, detect non- HLA antibodies
3- SAB-Luminex may have an artefact known as prozone phenomenon. This occurs if sera containing high titer Abs give negative results when tested dry, and rendered strongly positive after dilution or addition of DTT which prevent IgM or any protein competing to Ab binding site.
4- Epitops can give negative results, different Ags on different beads share common (same) epitopes to the binding of Ab to more than one bead. Leads to the reduction of MFI on a single bead.
Crossmatching: is required pretransplant
1- complement-dependent cytotoxicity CXM: can be done for B and T lymphocytes. It has same principles of the cytotoxic assay. In presence of low titre Abs the test sensitivity is low that can be increased by increasing the incubation period, use of AHG-enhanced method or wash steps.
False negative results due to low titer Abs are clinically significant. False-positive CDC-XM can be due to IgM/IgG or non-HLA.
Flow cytometry crossmatch: detects the presence or lack of IgG DSA on donor lymphocytes, independent of complement fixation. It is more sensitive than CDCXM.
Virtual crossmatching: The data is used for prediction of the actual in vitro crossmatch results by mixing identified antibody specificities of recipient serum with donor HLA antigens. The use of VXM can lead to shorter wait times and improved outcomes for sensitized recipients. The use of a historical sera could not predect Ab specificities with certainty so, recent sera should be used. In current practice an actual CXM should be performed as well. VXM does not identify Null HLA alleles which carry significant risk.
Recipient risk stratification:
High immunological risk: High titres of DSA, that preclude transplantation. pretransplant desensitization regimens can reduce the risk.
Intermediate immunological risk: low titer of DSA, and historic DSA is not detectable. In them use intensified immunosuppression, in addition to immunological monitoring posttransplantation.
Standard risk: no DSA
Well done
WEEK 1
JOURNAL CLUB
I.Human leukocyte antigen typing and crossmatch: A
comprehensive review
Mohammed Mahdi Althaf, Mohsen El Kossi, Jon Kim Jin, Ajay Sharma, Ahmed Mostafa Halawa
Mohammed Mahdi Althaf, Jack Pryor Renal Unit, Norfolk and Norwich University Hospital – NHS Foundation Trust, Norwich NR4 7UY, United Kingdom
Background and Introduction:
Kidney transplantation is the best renal replacement therapy for patients with end stage renal disease (ESRD). Human leukocyte antigen (HLA) typing & cross-matching are done before any transplantation so as to improve organ allocation & allow better matches to recipients. Nephrologists can be challenged by interpretation of the results of these immunological studies, as they have no formal training in immunology. In this review article the author presents one of these challenging scenarios.
CLINICAL SCENARIO:
The author reports a case of a 30-year-old man with ESRD secondary to Lupus nephritis. He was offered a deceased donor kidney with a 1-0-0 mismatch. Other immunological workup was as follow:
-Complement dependent cytotoxicity (CDC) cross-match positive for B lymphocyte
-Flow cytometry cross-match (FCXM) negative for both B & T lymphocytes.
-Luminex- single antigen bead (SAB): no any donor specific antibodies (DSA).
The positive CDC cross-match result is not concordant with DSA status. The patient had no history of blood transfusion and this is his first transplantation. Autoimmune diseases such as systemic lupus erythematosus (SLE) are known to give false positive CDC cross-match result. Failure to consider such points may unnecessarily lead to exclusion of adequate kidney grafts. The application of molecular techniques to study DSA would prevent wrong exclusion of such donors.
The author aimed to discuss an immunological risk stratification by using an example of an index case & the following review.
HLA TYPING METHODS:
A. Serological typing
Recipient lymphocytes are instilled into a tray wells containing sera, complement & dye.
The sera contains antibodies that can react to a multiple of known HLA alleles. When these antibodies bind to lymphocytes surface antigens complement dependent cell lysis occurs.The percentage of lysed cells are counted under phase contrast microscopy.
This method is quick & thus less cold ischemia time that suits the deceased donor renal transplantation.
However, serological typing fails to identify HLA-Cw, DQ, & DP antigen which may have significant impact on graft outcomes.
B. Molecular typing
(i) Sequence-specific primer polymerase chain reaction:Extracted DNA is amplified in several wells containing primers that are complementary to specific HLA alleles.HLA typing is defined by matching the primers of the amplification product to DNA sequences
of several candidate alleles.
(ii) Sequence specific oligonucleotide probes:
HLA type assigned by mixing amplified DNA with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles.
(iii) Direct DNA sequencing:
Determines the precise order of nucleotides in the gene of interest.
Molecular typing methods give details to amino acid level & clearly identify differences
in HLA antigen between donor & recipient providing insight to the risk accompanying mismatched donor-recipient antigens, epitopes & amino acid[1,2].
HLA ANTIBODY SCREENING METHODS
Cytotoxic (cell-based) antibody screening
A panel of donor lymphocytes representative of the population is used.
Similar to that of serologic typing however here recipient serum is mixed with “cell donor” lymphocytes in individual wells along with complement & dye.
Limitations of this method include:
-may not be truly representative of the population.
-HLA frequencies & racial differences cannot be done.
-false positive results due to non-HLA antibodies, autoantibodies & nonspecific IgM
antibodies.
-false negative results are possible if low titers fail to activate complment [3-5].
Solid phase antibody screening
Soluble or recombinant HLA molecules are used instead of lymphocytes targets
(i)Enzyme-linked immunosorbent assay (ELISA) platform:
-purified HLA molecules are applied to ELISA platforms to bind individually to HLA antibody after the addition of recipient serum [6,7].
(ii)Microbead platform/single-antigen beads:
–Pooled panel beads with several different class I or II HLA antigens on a bead yield a positive or negative result and are utilised for screening [8].
-Microbead that is fluorescent dye conjugated is then added to detect the presence of HLA antibody in the serum which is bound to the antigen.
-Fluorescence detection can be done by a flow cytometer (Flow PRA®) or using the single-antigen beads (SAB) Luminex® platform.
Advantages: -SAB gives rapid results (3-4 h)
-Flow PRA® & Luminex® are 10% more sensitive for lower titre antibody than ELISA.
-ELISA is ten 10% more sensitive than AHG) enhanced cytotoxicity based assays
-SAB is efficient (up to one hundred unique antigen beads can be tested in a single reaction
chamber.
-SAB enables virtual crossmatching (VXM) to identify DSA pre-transplant
-SAB identifies anti-HLA antibodies for all common & many rare antigens & alleles [9].
Limitations of SAB:-Being too sensitive they can detect antibody that is below the threshold
associated with a positive cross-match(clinically irrelevant antibody
detected may preclude a potential donor).
-susceptible to an artifact(prozone phenomenon) [11].
CROSSMATCHING (XM)
A.Complement-dependent cytotoxicity crossmatch(CDC-XM)
-Positive if a considerable number of lymphocytes are destroyed after the incorporation of complement (similar to cytotoxic assay).
-CDC-XM can be done for B and T lymphocytes
-limited sensivity if the relevant antibody is in low titers(this can be overcome by increasing incubation time, use of AHG-enhanced method as well as additional wash steps)[3,10].
-20% false positives( by detecting autoantibody, IgM/IgG HLA or non-HLA).
– 4% false negative rate
B.Flow cytometry crossmatch (FCXM)
-recipient serum is mixed with donor lymphocytes & then tagged with a fluoro-
chrome conjugated anti-Ig G antibody.
-detects DSA independent of complement fixation.
-precisely detects the presence or lack of IgG DSA on donor lymphocytes.
-B & T lymphocytes can be readily identified & have their DSA individually examined.
-greater sensitivity compared to CDC-XM[12].
-not widely available & its role in assessing immunological risk is still unclear.
C.Virtual crossmatching (VXM)
-not precisely a crossmatch as there is no mixing of serum & lymphocytes.
-both donor HLA typing & solid phase antibody screening are used (“mixed” to identify
actual in vitro cross-match) [12].
-shorter wait times (performed at the time of donor identification)
-improved outcomes for sensitized transplant recipients.
-permits acceptance of donor organs that would otherwise be rejected(allows to consider a
potentially positive crossmatch a risk factor for donor selection) [13,14].
– use of antibody specificity from historical serum sample (earlier than 6 months) could not
predict a crossmatch with certainty.
-factors that can influence antibody specificities(pregnancies, transplants & blood
transfusions) should be considered.
-VXM results not a hundred percent accurate & current practice mandates an actual
crossmatch be performed as well [15].
– wrong exclusion of potential donors due to false positive results if significantly low titer
&/or non-complement binding antibodies
Risk categories:
Gebel et al suggested the following risk stratification categories in prospective renal transplant patients according to immunological risk:
High immunological risk
These patients have high titers of circulating DSA specific for mismatched donor HLA. Such patients are usually excluded from transplantation as hyper-acute rejection is inevitable in the presence of DSA.
Intermediate immunological risk
In this category there is a low titer of DSA at the time of transplantation, and no detectable historic DSA. Intensified immunosuppression as well as immunological monitoring in the post-transplant period are required in these patients.
Standard immunological risk
Here there is no evidence of DSA; & the pre-transplant immunological risk assessment is based on donor cross-match and antibody screening results.
Risk Assessment Of The Index Case
This patient had positive CDC-XM for B & T lymphocytes but negative FCXM for both B & T lymphocytes. No DSA detected by Luminex-SAB. According to these results, this patient can be considered as “standard immunological risk”, and he can proceed with transplantation. But the positive CDC-XM result is not in harmony with DSA status. These discordant results are caused by the underlying SLE which leads to false-positive B- lymphocyte cross-matches as a result of binding immune complexes to Fc-receptors.
Conclusion
HLA typing of the index pair was 1-0-0-mismatch which corresponds to the pair of alleles mismatched at HLA-A, HLA-B & HLA-DR, respectively. These 3 antigens are usually the most important ones in kidney transplantation & the fewer the mismatches, the better the match between donor & recipient leading to a successful outcome. In this case, there is one mismatch at HLA-A; this poses less immunological burden compared to that at HLA-B & HLA-DR. Euro-transplant & old United Kingdom transplant data suggest that HLA-DR matching has a far greater effect than HLA-A or HLA-B
This case illustrates a common clinical scenario in transplant practice; and a detailed immunological testing & cautious interpretation of the results is crucial before deciding to preclude or accept a case for transplantation. Simply relying on the CDC-XM result would have unnecessarily excluded this patient from transplantation. The false positive CDC-XM result in the case presented was due SLE. The false-positive B-lymphocyte cross-match results from immune complexes binding to Fc-receptors [16,17].
False positive
CDC-XM could also be due to medications such as Isoniazid &
Hydralazine [18,19]. Detailed study of DSA by molecular technique would
improve organ allocation, shorten waiting time, & prevent unnecessary
exclusion of such donors.
References
1 Duquesnoy RJ, Claas FH. Is the application of HLAMatchmaker relevant in kidney transplantation? Transplantation 2005; 79: 250-251 [PMID: 15665779 DOI: 10.1097/01.TP.0000144327.92898.A6]
2 Claas FH, Roelen DL, Oudshoorn M, Doxiadis II. Future HLA matching strategies in clinical transplantation. Dev Ophthalmol 2003; 36: 62-73 [PMID: 12494682 DOI: 10.1159/000067657]
3 Amos DB, Cohen I, Klein WJ Jr. Mechanisms of immunologic enhancement. Transplant Proc 1970; 2: 68-75 [PMID: 4107294]
4 Ful l e r TC, F u l l e r A A , G o l d e n M , R o d e y G E . H L A alloantibodies and the mechanism of the antiglobulin-augmented lymphocytotoxicity procedure. Hum Immunol 1997; 56: 94-105 [PMID: 9455498 DOI: 10.1016/S0198-8859(97)00174-2]
5 Kerman RH, Kimball PM, Van Buren CT, Lewis RM, DeVera V, Baghdahsarian V, Heydari A, Kahan BD. AHG and DTE/AHG procedure identification of crossmatch-appropriate donor-recipient pairings that result in improved graft survival. Transplantation 1991; 51: 316-320 [PMID: 1994522 DOI: 10.1097/00007890-1991 02000-00008]
6 Zachary AA, Ratner LE, Graziani JA, Lucas DP, Delaney NL, Leffell MS. Characterization of HLA class I specific antibodies by ELISA using solubilized antigen targets: II. Clinical relevance. Hum Immunol 2001; 62: 236-246 [PMID: 11250041 DOI: 10.1016/ S0198-8859(00)00253-6]
7 Zachary AA, Delaney NL, Lucas DP, Leffell MS. Characterization of HLA class I specific antibodies by ELISA using solubilized antigen targets: I. Evaluation of the GTI QuikID assay and analysis of antibody patterns. Hum Immunol 2001; 62: 228-235 [PMID:
11250040 DOI: 10.1016/S0198-8859(00)00254-8]
8 Pei R, Wang G, Tarsitani C, Rojo S, Chen T, Takemura S, Liu A, Lee J. Simultaneous HLA Class I and Class II antibodies screening with flow cytometry. Hum Immunol 1998; 59: 313-322 [PMID: 9619770 DOI: 10.1
016/S0198-8859(98)00020-2]
9 Pei R, Lee JH, Shih NJ, Chen M, Terasaki PI. Single human leukocyte antigen flow cytometry beads for accurate identification of human leukocyte antigen antibody specificities. Transplantation 2003; 75: 43-49 [PMID: 12544869 DOI: 10.1097/01.tp.0000040431.80510.98]
10 Ful l e r TC, F u l l e r A A , G o l d e n M , R o d e y G E . H L A alloantibodies and the mechanism of the antiglobulin-augmented lymphocytotoxicity procedure. Hum Immunol 1997; 56: 94-105 [PMID: 9455498 DOI: 10.1016/S0198-8859(97)00174-2]
11 Weinstock C, Schnaidt M. The complement-mediated prozone effect in the Luminex single-antigen bead assay and its impact on HLA antibody determination in patient sera. Int J Immunogenet 2013; 40: 171-177 [PMID: 22913810 DOI: 10.1111/j.1744-313X.2012.01147.x]
12 Tinckam KJ. Basic histocompatibility testing methods. In: Chandraker A, editor. Core concepts in renal transplantation. New York: Springer Science + Business Media, LLC, 2012: 21-42
13 Pescovitz MD. B cells: a rational target in alloantibody-mediated solid organ transplantation rejection. Clin Transplant 2006; 20: 48-54 [PMID: 16556153 DOI: 10.1111/j.1399-0012.2005.00439.x]
14 Zangwill S, Ellis T, Stendahl G, Zahn A, Berger S, Tweddell J. Practical application of the virtual crossmatch. Pediatr Transplant 2007; 11: 650-654 [PMID: 17663689 DOI: 10.1111/ j.1399-3046.2007.00746.x]
15 Bray RA, Nolen JD, Larsen C, Pearson T, Newell KA, Kokko K, Guasch A, Tso P, Mendel JB, Gebel HM. Transplanting the highly sensitized patient: The emory algorithm. Am J Transplant 2006; 6:2307-2315 [PMID: 16939516 DOI: 10.1111/j.1600-6143.2006.01521.x]
16 Schlaf G, Mauz-Körholz C, Ott U, Leike S, Altermann W. General insufficiency of the classical CDC-based crossmatch to detect donor-specific anti-HLA antibodies leading to invalid results under recipients’ medical treatment or underlying diseases. Histol Histopathol 2012; 27: 31-38 [PMID: 22127594 DOI: 10.14670/ hh-27.31]
17 Eggleton P. Hypersensitivity: Immune Complex Mediated (Type III). John Wiley Sons, Ltd, 2001: 1-14 [DOI: 10.1038/npg. els.0001138]
18 Poli F, Innocente A, Cagni N, Brambilla C, Crespiatico L, Colombo MB, Scalamogna M. Isoniazid in patient plasma may cause a false-positive result on the complement-dependent cytotoxicity test. Hum Immunol 2009; 70: 758-759 [PMID: 19539003 DOI: 10.1016/j.humimm.2009.06.012]
19 Kucharz EJ, Goodwin JS. Hydralazine causes nonspecific binding of antibodies to human lymphocytes in vitro. Immunopharmacology 1990; 19: 1-4 [PMID: 2307578 DOI: 10.10 16/0162-3109(90)90020-F]
Well done
Human leukocyte antigen typing and crossmatch: A comprehensive review
Complement dependent cytotoxicity (CDC) cross match results could be false positive as a result of underlying autoimmune disease such as SLE, which may lead to inadvertent refusal of adequate kidney grafts. Using molecular technique to study DSA can prevent inadvertent refusal of such donors. We are going to discuss HLA typing, HLA screening and cross match methods.
HLA Typing.
HLA typing is very important in renal transplantation. In the case of recipient T lymphocyte recognize foreign HLA that will result in T lymphocyte activation which will lead to immune response against the allograft. There are two ways to perform HLA typing which are serologic and molecular typing methods.
Serologic method where the recipient lymphocytes are introduced to a tray wells containing sera with antibodies to a multitude of known HLA alleles then complement and dye will be added. If antibodies bind to the antigen a complement activation will occur and finally the dye will enter the cell. Phase contrast microscopy is then used to identify wells with significant cell death.
Advantages of serologic methods are they are fast with results in short time which is important in deceased donor transplantation and the ability to identify non HLA alleles which have identifiable DNA sequences with molecular typing but no cell surface antigen expression.
Disadvantaged of serologic methods are the inability to detect the increasing number of HLA alleles because of the lake of sera which contains specific antibodies. In addition that serologic method is not able to detect differences in HLA protein small amino acids that may be antigenic enough to induce potent immune response.
There are three molecular HLA typing methods. First, sequence-specific primer PCR where extracted DNA is amplified in several wells where DNA probes are complementary to the specific sequence of HLA molecule, then is instilled into an agarose gel and undergo electrophoresis and appear as a band. Second, Sequence-specific oligonucleotide probes where amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles and unique HLA alleles identified with fluorescent tags. Finally, Direct DNA sequencing where HLA type is assigned by comparison to published HLA allele sequences.
There are many pros of molecular methods of HLA typing, they can clearly identify the differences in HLA antigen between the donor and the recipient regardless of the method up to the amino acids level which make them highly specific methods. PCR typing can identify specific alleles with no cross reactivity – identification of allele which is essentially similar to the allele of interest – although a gene may occur in two or more forms (alleles). While this is highly specific it has the disadvantage that new alleles not on HLA databank will not be identified.
HLA antibody screening.
Screening for HLA antibodies is crucial in renal transplantation because of the increased chances of immunological graft failure in sensitized patients especially that there are strategies such as desensitization, paired exchange and acceptable mismatching which can lead to successful transplantation. There are three main ways for sensitization towards HLA antigen which are Pregnancy, post blood transfusion and prior transplantation. There are two methods for HLA antibody screening which are cytotoxic (cell-based) antibody and solid-phase antibody screening.
In cytotoxic HLA antibody screening the recipient serum is mixed with a set of cell donors where each panel consists of about 30 to 40 different donor lymphocyte to represent the potential deceased donor population. It’s similar to serologic typing as if the recipient serum contains antibodies against donor lymphocytes complement pathway will be activated followed by cell death and dye uptake. The degree of cytotoxicity is expressed as percentage PRA.
There are many limitations to cytotoxic HLA antibody screening as PRA percentage may be different numerically without a corresponding change in the type or amount of antibody. In addition, HLA frequencies and racial differences need to be factored in but can’t be done. False-positive results can be produced by autoantibodies, IgM and non-HLA antibodies. As well as, false-negative results can occur due to non-complement activation due to low titers of true antibodies.
The other method for HLA antibody screening is solid-phase essay which uses soluble HLA molecules instead of lymphocyte targets. It can be done through three ways: ELISA, Flow cytometry and single antigen beads SAB. In ELISA purified HLA molecules will bind individually to HLA antibody after adding the recipient serum and then detected by optical density reading. The other two ways include a panel of beads loaded with either class I or II HLA antigens where patient serum added to each bead then a fluorescent dye is used to detect antibody-antigen complex by flow-cytometry or SAB. These estimate PRA by the proportion of positive beads.
SAB are individually coated with single HLA antigen and yield a list of distinct antibody specifities – Highly specific – and then compared with HLA frequencies in donor population to determine the calculated PRA which is the best estimate of positive crossmatch/ donor specific antibody to a randomly selected donor. It has many advantages as its more sensitive compared to ELISA by 10%, rapid with results available within 3-4 hours, permits testing many patients simultaneously and enables virtual cross match which help in risk stratification. Additionally, SAB can identify anti-HLA antibodies for all common and numerous rare antigens and alleles up to 11 HLA loci.
SAB has its own limitation with false negative results called as the prozone phenomenon which occurs when sera with high titer anti-HLA antibodies give negative results when tested neat which can be overcome by dilution. Similar scenario can occur with the binding of IgM antibodies or other serum factors to the beads. In general, solid phase antibody screening have limitations as they detect both complement and non-complement binding simultaneously. In addition, they are too sensitive to detect antibody levels below the threshold with a positive cross match and can result in precluding a potential donor.
Cross Matching:
Crossmatch will identify if a recipient had antibodies to a specific donor of interest where PRA identifies antibodies to a potential cluster of donors. Cytotoxic cross matching is insufficient to identify all relevant antibodies and it may exclude patients from transplant. Therefore, it should be used together with solid-phase antibody tests to identify those that are immunologically relevant. There are three methods of cross matching.
First, complement-dependent cytotoxicity which considered positive if a considerable number of lymphocytes are destroyed after adding the complement and can be done for B and T lymphocytes. False negative results can occur because its sensitivity is limited in low titer antibodies but can overcome by increasing the incubation time. False positive results can occur in the presence of autoantibodies, IgM, non-HLA antibodies.
Second, flow-cytometry crossmatch which detect DSA independent of complement fixation. In this method, the recipient serum is mixed with donor lymphocytes and then tagged with fluorochrome-conjugated anti-IgG antibody. It can detect the presence or lack of IgG DSA on donor lymphocytes. It has greater sensitivity than CDC.
Finally, virtual crossmatching (VXM) where donor HLA typing and solid-phase antibody screening are utilized together. It’s not accurate as cross match in term of mixing serum and lymphocytes but it can lead to shorter waiting times. The use of antibody specificity from historical serum sample (earlier than six months) could not predict a crossmatch because it vary over time. Therefore, serum results including at least one recent within less than 3-6 months should be used. Because the results of virtual cross matching are not accurate an actual crossmatch should be performed. Lastly VXM does not identify the HLA null alleles.
In Conclusion, understanding of crossmatch results and the limitations of each individual test is crucial for successful outcome. We can’t rely only on CDC-XM as false positive results can occur due autoimmune disease or immune complexes binding to Fc-receptors in the case of B-cell cross match. Detailed study of DSA by molecular technique would prevent erroneous exclusion of such donors.
Thanks Murad
HLA TYPING
HLA typing is a cornerstone in renal transplantation. It can be done either by serologic method or molecular typing method.
Serological typing :
Recipient lymphocytes are mixed into the tray wells contacting sera,
complement and dye. In-tray wells where antibodies can bind to the antigens on the surface of lymphocytes; complement is activated which results in cell death, and the dye enters the cell. Tray wells with significant cell death are then identified under microscopy.
Advantages :
1. Results are available in a short period so less cold ischemia times.
2..Differentiate HLA alleles that have identifiable DNA sequences with molecular
typing but with no cell surface antigen expression(null HLA alleles).
Disadvantages:
1. It is a lack of sera with antibody specificities that are capable of identifying HLA-Cw, DQ, and DP antigen which may have clinically significant effects on the outcomes of allografts.
2. It does not detect differences in HLA protein-small amino acids which may trigger potent immunological responses.
Molecular typing :
It can identify differences in HLA antigen between donor and
recipient. It includes:
1. Sequence-specific primer polymerase chain reaction.
2. Sequence-specific oligonucleotide probes.
3. Direct DNA sequencing.
HLA antibody screening
Cytotoxic (cell-based) antibody screening
The patient, s serum is mixed with donor lymphocytes in individual wells along with complement and dye.
If the serum contains antibodies will activate complement pathways that result in cell death and uptake of the dye. The degree of cytotoxicity is expressed
as PRA %.
False-positive results can be produced due to non- HLA antibodies, autoantibodies, and nonspecific IgM antibodies. False-negative results are possible because it requires high antibody titers to activate complement.
Solid-phase antibody screening
Enzyme-linked immunosorbent assay platform (ELISA):
Purified HLA molecules are applied to ELISA platforms and will bind individually to HLA antibodies.
Microbead platform/single-antigen beads( SAB): Pooled panel beads with several different classⅠ or Ⅱ HLA antigens on a bead yield a positive or negative result
and are utilized for screening.
Cross-matching:
Complement-dependent cytotoxicity crossmatch
Similar to cytotoxic assay the complement-dependent cytotoxicity crossmatch.CDC-XM is representative of what would happen in vivo. Low titer antibodies detected by this method were associated with 36% 1-year allograft loss compared with 18% loss in those with a negative test.
CDC-XM may be applied to both B and T cells. CDC-XM for T cell reflects the presence of HLA-I antibodies while for B cell reflects HLA I and II antibodies. B cells express a higher amount of class I antigens so appositive B cell CDC-XM associated with a negative Tcell CDC-XM indicate a low level of class I antibodies.
CDC-XM has a false positive rate of 20% that may be to antibodies which are generally of IgM and non-HLA IgG type. There have been reports of a false positive B –cell CDC-XM following treatment with rituximab and basiliximab.
One disadvantage of CDC-XM is that it only detects complement-fixing antibodies, while non-complement fixing antibodies may still affect graft function.
Flow cytometry crossmatch
Recipient serum is mixed with donor lymphocytes and then tagged with a fluorochrome-conjugated anti-IgG antibody.
It is the most sensitive test to detect donor-specific antibodies that were being missed by the CDC-XM.
Virtual crossmatching
In virtual crossmatch (VXM), both donor HLA typing and solid phase antibody screening are utilized together. It is compared specific anti-HLA antibodies in the recipient with the HLA profile of the donor.
It can lead to shorter wait times and improved outcomes for sensitized transplant recipients.
The results from VXM are not precise and current practice mandates an actual
crossmatch be performed as well. It does not identify the HLA “Null” alleles.
Renal recipients can be classified into :
High immunological risk
They have high titers of DSA which can lead to hyperacute rejection.
Intermediate immunological risk
They have a low titer of DSA, and historic DSA is not detectable.
Standard immunological risk
Where there is no evidence of DSA.
Well done
Renal transplantation is the best modality of treatment for patients with ESRD. It improve survival and quality of life. So accurate & careful cross-match & tissue typing are crucial for successful transplantation.
There are several methods of tissue typing:
(b) Sequence-specific oligonucleotide probe
(c) Direct DNA sequence.
Molecular typing can detect the differences in HLA Ag at amino acid & epitopes level. But it had a disadvantage that it can’t detect new allelfsthat not found in databank.
About half of patients on waiting list are sensitized due pregnancy, blood transfusion or previous organ transplantation. The presence of antibodies increase the risk of rejection, so antibodies detection is very important which can be done by:
(b) Micro-bead platform/ single Ag bead
SAB result can be obtained through 3-4 h. It can detect both common & rare HLA-Ab.
SPA can detect complement & non complement binding Abs even with very low level. But non HLA Abs can’t be measured solely by this method.
Cross-match techniques:
According to tissue typing & cross match the immunological risk can be classified into :
HLA TYPING
serology:
used in deceased donor (quick method),serum with Ab in tray mix with donor lymphocyte and see reaction rarely used now .
Molecular typing :
PCR amplification process word wise use.
Direct DNA sequence precise of nucleotide .
HLA mismatch carry high risk of rejection
HLA class 1 (A,B,C) .
HLA class 2 ( DP,DQ,DR).
A,B.DR mismatch carry high risk of rejection du to more allogenicity.
DQ mismatch risk for denovo DSA.
DR mismatch may cause early rejection (first 6 month ).
risk of rejection increase with increase in mismatch may reach up to 60 percent in case of 6 mismatch .
HLA Ab screening :
1-cell based Ab screening .
2-Solid phase Ab screening
-Luminex (single antigen bead ).
quick method 3-4 H
commonly used in allocating system .
disadvantage
miss non HLA.
canot differentiate complement from non complement.
used in virtual cross match .
false negative from prozone phenomena either due to high titer or complement (c1q)
or IgM and drug (IV Ig).
Cross Match:
CDC (complement dependent cytotoxicity )
complement dependent reaction.
false negative may be due to low titer .
false positive due to auto Ab, IgM, IgM reaction.
T cell positive absolute contra indication.
B cell positive relative contraindication.
Flow cytometry cross match :
highly sensitive used in highly sensitized patient .
non complement reaction.
Virtual cross match:
not true cross match.
commonly used in deceased donor.
according to lab result patient divide to 3 group:
-high immunological risk.
-intermediate immunological risk .
-standard immunological risk.
Please elaborate more in classification of Risk as this is the value you get for clinical practice
HLA Typing:
Serological Typing:
In this method, the recipient lymphocytes are introduced into a tray wells contacting sera, complement, and dye. when antibodies bind to the antigen, complements are activated resulting in cell death, so the dye will enter into the cells. Wells with significant cell death are identified under a microscope.
The benefit of this method:
Disadvantages:
Molecular Typing:
Advantages:
Disadvantages:
HLA antibody screening:
Cytotoxic antibody screening:
recipient serum is mixed with donor lymphocytes in a well with complement and dye.
if the serum contains antibodies against the donor, the binding will happen and complement system activation resulting in cell death and uptake of the dye.
the degree of cytotoxicity is expressed as percentage PRA.
Limitations:
Solid-phase antibody screening:
Advantage of SAB Luminex testing:
Limitations:
Crossmatching:
Complement-dependent cytotoxicity:
Flow-cytometry crossmatch:
Virtual crossmatching;
Well done
HLA typing
1- Serologic method using either antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)
· In this method recipient lymphocytes are introduced to a tray that contain sera of antibodies to all known HLA antigens, complement and coloring dye
· Once Ag-AB reaction occurs complement activation occur, MAC is generated, cell lysis , then cells take the dye which appear red under microscope
· Although it is rapid method but its use dropped due to growing numbers of new HLA alleles with which can not be covered by the sera
2- DNA-based HLA typing methods which is more sensitive and accurate than serological technique and it includes the following:
· SSOP sequence specific oligonucleotide probe hybridization
· SSP sequence-specific primer amplification
· Direct DNA sequencing
CROSS MATCH ASSAYS
I– CDC cross match
– It involves incubation of donors lymphocytes in recipient serum, washing to remove unbound antibodies, then adding complement and after incubation period, coloring dye is added.
– If antibodies to donor HLA present complement activation occur, MAC is generated, cell lysis , then cells take the dye which appear red under microscope
Uses
– CDC is used either in determining compatibility between donor and recipient or in determining sensitization in case of PRA
– A positive CDCXM indicating the presence of complement fixing antibodies IgG or IgM, against HLA or non HLA antigens or autoantibodies, but it does not detect non complement fixing AB against HLA Ag which may be significant (IgG2, IgG4) and low level DSA.
– CDC has low sensitivity with false positive result that can occur due to the presence of clinically non-significant non HLA abs or autoimmune IgM antibodies which can be eliminated either by heating or adding reducing agent also Rituximab can cause false positive B cell cross match or may be due to technichal error.
– CDC has low specificity with false negative result that can occur due to non complement fixing AB or low level of HLA antibodies or technical errors, also HLA Ag expression may be low compared to flow cytometry
II- Flowcytometry
– It involves incubating donors lymphocytes with recipient serum , then add second antibody with fleouricin dye (green) that will bind to bound ABS (DSA) attached to donor lymphocytes, then adding 2 other ABs one bind to CD19 in B cells (red)and the other bind to CD3 in T cells (yellow) to know the type of cell affected and this is detected by laser
– So seeing green and yellow dye means T cell + cross match, green and red dye means B cell+ cross match and seeing all 3 dyes means T and B cell + cross match
Uses
-FCM is used either in determining compatibility between donor and recipient or in determining sensitization in case of cPRA
Significance
– A positive cross match using FCM indicating the presence of complement fixing or non fixing antibodies IgG only (not IgM), againest HLA or non HLA antigens or autoantibodies and low level DSA, So CDC – FCM + cross match indicates the presence of non complement fixing AB or low level DSA.
– More sensitive than CDC as it use lymphocytes which express massive HLA antigens and detect only IgG and not IgM
– False positive results can occur due to binding of non HLA IgG or auto antibody to B cell, also rituximab was found to cause false positive results
– Flow cytometry method, that measure fluorescence intensity and compare it to control (median channel shift- MCS)
III- Solid phase assays (ELISA or beed assay- luminex)
– Beed assay (luminex) replace ELISA
– In beed assay the patient serum is tested against HLA antigens attached to solid beeds labeled with fluorescein, each bead either have single (SAB) or multiple HLA molecule then anti human globulin labeled with phycoirythrin is added. Antigen-antibody reaction is detected via laser based fuorescent imaging
– Used to detect DSA
– Can detect complement and non complement fixing antibodies, low level DSA
Sensitivity
– Very sensitive, as it detect only Abs against HLA antigens so avoid false positive results due to non HLA antibodies or auto ABS
– False positive result can occur due to denatured antigens attached to beeds, leading to alteration in protein configuration that can be overcomed by treating beeds with acid that fully denature the beads protein , also 2 kits may be used
– False positive result can occur due to detection of low level DSA which may not be significant leading to positive luminex negative cross match, thus another tes is added to increase the sensitivity of luminex which is C1q assay that identify only complement fixing antibodies that are clinically significant, but the test is expensive so its use is limited
– Very specific, but false negative result can occur if patient has high levels of IgM or Complement factors (C1) (such as those on long term HD) that can bind to antigen beeds and prevent binding to actual IgG-DSA, or may occur due to very high level of DSA that agglutinate so fail to bind to the beed.
– Calculates the degree of fluorescence (median fluorescence intensity -MFI) through the use of 2 lasers to excite the florochrome of the beed and the phycoerythrin bound to the antibody.
IV- Virtual cross match
– By comparing anti HLA antibodies of recipient detected by luminex SAB to HLA profile of the donor.
– It is correlated well with FCM cross match and graft survival even in sensitized patients
– Define unacceptable antigens so donors can be excluded, and allow identification of suitable donor
– Wet cross match should be performed in only sensitized patients as there may be a new DSA that develop, this should be detected by wet cross match (positive FCXM, negative VXM), in non-sensitized recipient we can rely on VXM in proceeding in transplantation
– A positive VXM with a negative FCXM can occur due to non clinically significant low titre DSA that will not affect transplantation, so it is safe to proceed to transplantation
Well read ,very well understood Thankyou for referring to MFI ,MCS though you skipped risk stratification which is your main goal as a clinician.
The table supplied is useful but could be much simpler.
HLA TYPING
HLA typing is a crucial step in renal transplantation, as recognition of foreign HLA by recipient T lymphocytes would trigger an immune response. T lymphocyte activation initiates a cascade of mediators that direct the immune system against the allograft
There are 2 types of HLA typing
I) Serological typing
Idea: a tray containing sera with antibodies to a multitude of known HLA alleles is used. These are
commercially available. For typing, recipient lymphocytes are introduced into the tray wells contacting sera, complement and dye. In tray wells where antibodies can bind to the antigens on the surface of lymphocytes; complement is activated. This results in complement pathways triggered resulting in cell death, ultimately allowing the dye to enter the cell. Tray wells with significant cell death are then identified under phase contrast microscopy.
Advantage:
– the results are available in a short period. This is particularly important in deceased donor renal transplantation. Quick results mean less cold ischemia times.
– This method also offers the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression. These alleles termed “null” HLA alleles are of less immunological significance.
Disadvantage:
– the lack of sera with antibody specificities that are capable of identifying the ever-growing number of HLA alleles. The HLA-Cw, DQ, and DP antigen may have clinically significant effects on the outcomes of allografts. However, serologic assays are scarce for these loci.
– serologic methods do not readily detect differences in HLA protein small aminoacids. These may be antigenic enough to trigger potent immunological responses. With more advanced
methods of typing currently available serological typing has fallen into disuse
II) Molecular typing
There are some techniques to do that:
– Sequence-specific primer polymerase chain reaction
– Sequence specific oligonucleotide probes
– Direct DNA sequencing
Molecular typing regardless of the method can clearly identify differences in HLA antigen between donor and recipient. Often with detail to the amino acid level that can provide insight to the risk accompanying mismatched donor-recipient antigens, epitopes and amino acid
HLA ANTIBODY SCREENING
Preformed antibodies increase the chances of immunological failure of the allograft.
Both sensitive and specific detection of anti-HLA antibodies is crucial. Where crossmatch is negative, even low titres of DSA can lead to early as well as late antibody mediated rejection.
There are different methods used for HLA antibody screening as shown below.
I) Cytotoxic (cell-based) antibody screening
Idea: the recipient serum is mixed with “cell donor” lymphocytes in individual wells along with complement and dye.
Where the serum contains antibodies that bind to the cell surface with adequate density complement pathways are activated which results in cell death and uptake of the dye. The degree of cytotoxicity is expressed as percentage PRA (panel reactive antibody)
Disadvantages:
– PRA percent can be different numerically without a corresponding change in the type or amount of antibody. This largely depends on the cell panel used which are commercially
produced and may not truly represent the population.
– Significant false positive results can be produced due to non-HLA antibodies, autoantibodies and nonspecific IgM antibodies.
– false negative results are possible as this is purely complement dependent that requires
higher antibody titres to be activated
– The lack of a complement activation simply due to low titres allows a true antibody to be hidden
– Precise, complete lists of antibody specificities and unacceptable antigens cannot be identified using this method as there are several antigens in each well
II) Solid phase antibody screening
This method employs soluble or recombinant HLA molecules instead of lymphocytes targets – as lymphocytes present both HLA as well as non-HLA molecules. The variants of these methods are:
–Enzyme-linked immunosorbent assay platform
-Microbead platform/single-antigen beads
III) CROSSMATCHING (XM)
With PRA (HLA antibody screening) that identifies several antibodies to a potential cluster
of donors, the crossmatch will identify if a recipient had antibodies to a specific donor of interest.
The solid-phase antibody test should be used together with crossmatch results to identify those that are immunologically relevant
The techniques of cross match are:
– Complement-dependent cytotoxicity crossmatch
– Flow cytometry crossmatch
– Virtual cross match
Need to finish your homework
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renal transportation is the best option in patient with ESRD even with autoimmune disease.
this article focus on risk of immunological stratification in renal transplants.
careful testing of HLA cross match help to good outcome of graft and avoid of allograft rejection.
HLA typing is the first and important step in renal transplantation. it helps for recognition of foreign HLA T. lymphocytes by recipient against allograft.
2 main study to recognise T lymphocyte activation by HLA. (serology/ molecular studying).
serology typing: identifying lymphocytes activation by uptake dye. it’s limitation not identifying specific type of HLA proteins.
molecular typing: identifying specific type of HLA alleles.
the most important HLA typing are HLA A, HLA B, HLA DR.
HLA DR matching has great effects than HLA A,B
HLA antibody screening: most of wait listed patients have anti HLA Ab detected.
most highly sensitised patients had exposure to blood transfusion/ pregnancy/ previous transplant. Presence of these antibodies increases chance of organ failure. But sensitised patients successfully transplantation possible by using method of desensitisation (paired exchanges & acceptable mismatching.
cytotoxic antibody screening: it’s same as serological typing (if positive uptake dye indicates positive cross match & degree of cytotoxicity expressed by percent of PRA. this method has limitations because it depend on amount of antibody in panel and it may hidden the true antibody. so this method difficult to identify specific AB & unacceptable Ag.
Solid phase Ab: this method to detect HLA molecule & non HLA molecule by ELISA // micro bead platform (SAB)
ELSA : to detect HLA antibody bind to Ag.
micro bead platform/ single antigen beads to detect HLA antibody Antigen by flow cytometry or by single antigen luminex platform.
this method detect specific HLA Ab-Ag and calculated panel reactive Ab the best to detect positive cross match donor specific Ab.
there’s difference between PRA & c-PRA.
PRA value is reflecting high probability of positive cross match
c-PRA better than PRA in detect unacceptable HLA Ag especially in high sensitized patients.
C-PRA is useful in allocation of kidney & pancreas transplantation.
high titer of c-PRA indicated incompatible donor & it’s called positive cross match.
micro bead assays (flow PRA & Luminex are 10% more sensitive for lower titer antibody than ELSA.
ELSA is 10% more sensitive compared to anti-human globulin.
SAB are rapid test & result available in 3 to 4 hr ; it help to identify DSA pre transplant & organ allocation. SAB help to identification of anti- HLA Ab for all common & rare Ag & alleles. Also help to identify complement & non complement binding.
SAB luminex assay: this method need dilution of sera to avoid prozone phenomena which can give false negative. this artifact results from several factors like complement compound 1 which prevent bind HLA Ab to Ag. it can also arise from IgM Ab and use of intravenous immunoglobulin.
Cross Matching:
presence of DSA indicate higher rates of organ failure.
T cell cytotoxicity cross match may had false positive & false negative. So it insufficient to identify all relevant AB.
Solid phase antibody test should be used together with cross match to identify immunological relevant.
CDC cross match become positive if high number of lymphocytes destroyed after incorporated of complement. this indicates significant DSA has been found in surface of cell.
CDC cross match can done for B & T lymphocytes.
it limited if titer is low with relevant Ab.
Flow cytometry cross match: detect DSA independent of complement fixation. it is detect presence Or lack of IgG DSA on donor lymphocytes. this method is not widely used because different laboratories method & different results; also it’s immunological risk unclear.
Vitiral cross match: used in high sensitized patients.
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Read ,understand then write short ,simple sentences.
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Comp. component not compound.
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Transplantation is the best modality of Renal replacement therapy.
Understanding of transplant immunology is decisive and effective in transplant outcome.
There are two methods for HLA typing:
-Serological.
– Molecular typing methods.
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1-Serological HLA typing:
-Depending on reaction between recipient lymphocyte (ag)+serum (anti bodies) +dye, reaction led to trigger of complement pathway which led to cell death. then identified under phase contrast microscopy.
Advantages of this method:
Results are available in a short period so it’s important in deceased donor renal transplantation because it decreases cold ischemia times.
Detection of “null” HLA alleles which are of less immunological significance.
Disadvantages:
Lack of sera with antibody specificities that are capable of identifying the ever-growing number of HLA alleles
e.g.: The HLA-Cw, DQ, and DP antigen
Do not detect differences in HLA protein small amino which may be antigenic enough to trigger potent immunological responses.
2- Molecular typing:
Molecular typing regardless of the method can clearly identify differences in HLA antigen between donor and recipient.
=> Sequence-specific primer polymerase chain reaction.
-In which HLA typing is allocated by matching the primers of the amplification product to DNA sequences of several candidate alleles.
=> Sequence specific oligonucleotide probes.
In which amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles.
=> Direct DNA sequencing.
Initial Collaborative Transplant Study (CTS) analysis that HLA-DR and HLA-B antigens offer the most alloimmune burden with less so from HLA-A.
Euro transplant and old United Kingdom transplant data suggest that HLA-DR matching has a far greater effect than HLA-A or HLA-B.
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HLA antibody screening:
The usual route for sensitization towards HLA antigens occurs in three instances; pregnancy, post blood transfusion and prior transplantation.
successful transplantation is possible by employing strategies such as desensitization, paired exchange and acceptable mismatching.
Methods used for HLA antibody screening:
= Cytotoxic (cell-based) antibody screening.
Recipient serum is mixed with “cell donor” lymphocytes with complement and dye, similar to that of serologic typing.
False positive results can be produced due to non-HLA antibodies, autoantibodies and nonspecific IgM antibodies.
False negative results are possible as this is complement dependent which requires higher antibody titers to be activated.
= Solid phase antibody screening.
It depends on recombinant HLA molecules instead of lymphocytes (to avoid non-HLA antibodies).
Many methods of solid phase antibody screening
1- Enzyme-linked immunosorbent assay platform.
Enzyme conjugated antibodies to IgG (human) is then added to detect the presence of HLA antibody in the serum which is bound to the antigen.
2-Microbead platform/single-antigen beads:
Microbead that is fluorescent dye conjugated is then added to detect the presence of HLA antibody in the serum which is bound to the antigen.
Fluorescence detection can be done traditionally using a flow cytometer or by the single-antigen beads (SAB) Luminex platform.
These estimate PRA by the proportion of positive beads. SAB are individually coated with a single HLA antigen and yield a list of distinct antibody specificities.
cPRA is useful in the allocation of kidney and pancreas transplants. cPRA estimates the proportion of donors with whom a particular recipient would be incompatible.
N.B: The SAB – Luminex assay has been shown to be susceptible to an artefact known as the prozone phenomenon which give false negative which might be due to many causes (C1q which prevents HLA antibody binding to the HLA antigen on the bead,IgM abs, intravenous immunoglobulin and epitope sharing).
CROSSMATCHING (XM)
==Complement-dependent cytotoxicity crossmatch (CDC-XM) can be done for B and T lymphocytes.
CDC-XM can also give false positives by detecting autoantibody, IgM/IgG HLA or non-HLA.
== Flow cytometry crossmatch:
Here recipient serum is mixed with donor lymphocytes and then tagged with a fluorochrome conjugated anti-IgG antibody which is more sensitive than CDC-XM.
=Virtual crossmatching:
In virtual crossmatch (VXM), both donor HLA typing and solid phase antibody screening are utilized together.
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Cross match can be done using several methods like CDC (complement dependent cytotoxicity crossmatch) and FCXM (flow cytometry crossmatch) , the problem of CDC; it can not be recognize autoantibody it could be positive due to presence of autoantibodies as in autoimmune diseases, but the patient is positive by FCXM, also CDC can detect complement dependent antibody.
There are several methods to detect antiHLA antibody :
1- Serological methods which depend on the recipient lymphocytes which will react with different antibodies against different HLA alleles in tray containing wells, it is rapid method can be used in deceased donor transplant to decrease cold ischemic time , also can detect NULL alleles which has no antigen expression but this has less immunological significance , it has some disadvantages like being less specific or limited can not be used to detect more specific growing alleles.
2- Molecular methods: a- Sequence-specific primer polymerase chain reaction
b-Sequence specific oligonucleotide probes
c-Direct DNA sequencing
PCR based HLA typing is more specific to detect more alleles and detect cross reactivity with the allele of interest but if alleles not present in database of alleles bank , will not be detected and this is its disadvantage.
HLA mismatch has important impact on graft survival especially DR and B more than A , the more mismatch especially in DR and B the more negative impact on graft survival.
HLA-DR mismatch affects graft in the first six months but HLA-B mismatch affects graft in the first two years.
Screening of HLA antibodies can be done as the following :
1- CDC complement dependent cytotoxicity lymphocyte of recipient mixed with donor cells of 30-40 donors…if antibody found and detected by complement reaction and cell lysis, the result will be positive test , this test has limitation as it cannot differentiate autoantibodies and also cannot detect non complement dependent antibody, also the degree of positivity can be presented as panel reactive antibody which depends on the quality and quantity of antibodies used in the test which may differ and not truly represent the population as a whole, also cannot differentiate between HLA and non-HLA antibodies.
2- Solid phase antibody screening use soluble or recombinant HLA molecule as lymphocytes carry HLA and non-HLA antibodies:
Can be done by : Enzyme-linked immunosorbent assay platform
Microbead platform/single-antigen beads : Fluorescence detection by using a flow cytometery or single-antigen beads (SAB) Luminex.
Calculated PRA can be used from that point which use a pool of many donors reach more than 10000 donors to get more specificities and depend on the nationality and race and the place, also it yields the list of unacceptable genes which help in the program of kidney transplant.
3- Flow cytometry CM
It detect non complement IgG DSA, it is more sensitive than CDC
4- Virtual CM
It is not an actual cross match but it is in vitro one , HLA typing of recipient is compared to a pool of donors HLA typing and the match will be seen , this to find a donor for sensitized patient and to improve their transplant outcome.
Based on presence or absence of DSA at the time of transplantation, will stratify the patients as regard their risk; high risk who has high titer of DSA at the time of transplant and need pretransplant desensitization protocol and of course potent(high risk) induction and maintance protocol with close monitoring post transplant, intermediate risk who has low titer of DSA or history of its presence so need potent (high risk) induction and close immunological monitoring post transplant, low risk patients has no DSA at the time of transplant and need standard risk protocol of induction.
Well done.