Dear All
How would you explain the following result Positive cross match (both T and B cells are positive), but no DSA.
There is a present for the correct answers. I will announce it On Saturday morning
It is Recent Advances and Clinical Outcomes of Kidney Transplantation Volume 1 and 2 2020 EDITION, Original copy.
presence of non-HLA antibodies like angiotensin II type 1 receptor antibody (IgG) in the recipient.
can be positive crossmatch in case of donor treated by rituximab recently.
history of prior desensitization of the patient.
very low level of DSA not detected by SAB single antigen bead because of complement interference.
B cell positive alone is considered false in more than 50 % of cases, and can not be relied on, so if no DSA ….consider it false.
WILLIAM R MULLEY, JOHN KANELLIS. Understanding crossmatch testing in organ transplantation: A case-based guide for the general nephrologist. 19 October 2010
Judith Desoutter,1Marie-Joëlle Apithy,1 Ségolène Bartczak,1 and Nicolas Guillaume. False Positive B-Cells Crossmatch after Prior Rituximab Exposure of the Kidney Donor. Hindawi. Case reports in transplantation.2016.
Non-HLA antibodies This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
How would you explain the following result Positive cross match (both T and B cells are positive), but no DSA.
we should be very carful in interpreting this results as still could be false postive results if done by the CDC assay either due to techincal erro so better to repeat by using DTT assayby fruther dilutions to cleave out the auto-AB by reduces the disulfide bonds in IgM ,also we can do auto-crossmatch test in which the recipient serum is crossmatched against self- lymphocytes not the donor thereby preventing IgM antibodies from generating a positive result. IgM antibodies are generally regarded as having no pathological significance in transplantation.
if this crossmatch results done by flowcytomtery assay
so still could be false postive due to the presence of following :
1- non- HLA AB
2- non-complement fixing AB
3- low level of the DSA which calculated by MFI with the variable cutoff value for postive results between different laberotories usualy postive flow cytometry in senesitzed recipient more senesitive and specific and some centres even with low values will go for desensitazation or allocated for pair donation program .
Non-HLA antibodies This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
A positive cross match in the absence of DSA needs to be evaluated properly.
History:
Any history of immunological disease like SLE
Any history of injection rituximab (but only B cell cross match should be positive)
Any history of desensitization.
Method of crossmatch:
False positive CDC cross match can be seen in presence of autoantibodies. Hence DTT should be used to remove autoantibody related false positives.
The test kit used for DSA detection, if is not representative of the HLA prevalent in the community, it is possible that the single antigen bead test might miss the antibodies present in the recipient serum due to lack of corresponding HLA antigen on the beads, giving a false negative DSA report.
Positive cross match (both T and B cells are positive), but no DSA.
This result can be seen in lupuspatients, usually crossmatching by CDC method will be positive( false positive) due to the presence of IgM antibodies, but when we do flow crossmatch the result will be negative and DSA testing by solid phase assay will be negative. Another explanation is the presence of non-HLA antibodies.
Reference:
Chughtai Sh. A.,Sharma A.K., Halawa A. Interpretation of Crossmatch reports in a patient with Lupus Nephritis. Arch Organ Transplant 2(1): 023-029.
Non-HLA antibodies This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
The result should be interpreted with caution.
This can be due to autoantibodies or due to non HLA antibodies .
Or due to incomplete typing of the HLA of the donor HLA C ,
or HLA antibody to rare HLA antigen that not expressed in the Luminex SAB panel
Reference :
Carrie A. Schinstock, MD, Manish J. Gandhi, MD, and Mark D. Stegall, MD
Interpreting Anti-HLA Antibody Testing Data: A Practical Guide for Physicians
Transplantation 2016;100: 1619–1628DOI: 10.1097/TP.0000000000001203
Non-HLA antibodies This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Positive crossmatch (both T and B cells are positive), but no DSA.”
Possible causes include autoantibodies, non- HLA antibodies, IgM antibodies or history of ATG or Alemtuzumab use (1).
I found a very comprehensive and organized table that collected causes of the positive crossmatch and their clinical interpretation, and I attached a screenshot of it in the attached photo (1). The table will guide us to differentiate each of the above mentioned possible causes of the proposed scenario.
Non-HLA antibodies This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
As crossmatch may have false-positive results due to autoantibodşes and those of IgM type.
DDT will resolve the problem of IgM. Despite all measures some patients still have false-positive CDC crossmatch and or Flowcytometry. This is mainly due to autoantibodies. Some therapies like rituximab will induce complement-dependent cytotoxicity thereby interfering with CDC. Also, it may be recognized by anti-human antibodies used in the flow cytometry technique.
You are right. Non-HLA antibodies such as drugs (Rituximab) or MICA MICB, anti-endothelial antibodies, Angiotensin II receptors antibodies.
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
There is a reported case of positive flow cytometry crossmatch (FCXM) in a renal transplant candidate following a recent COVID-19 vaccination. Interestingly, the donor-specific antibody (DSA) was not detectablewith the sensitive solid-phase single-antigen beads (SAB) assay. Infection and vaccination have been reported to be associated with allo-sensitization either in healthy population or in solid organ transplant recipients.
Positive flow cytometry crossmatch with discrepant antibody testing results following COVID-19 vaccinationQingyong Xu,Puneet Sood,Dennis Helmick,Jon S. Lomago,Amit D. Tevar,Adriana Zeevi First published: 09 July 2021https://doi.org/10.1111/ajt.16753
Non-HLA antibodies You looked for unusual cases. What you described is non-HLA antibodies such as drugs (Rituximab) for MICA MICB and anti-endothelial antibodies, Angiotensin II receptors antibodies.
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
I will considered you a winner as you thought outside the box, but remember we are looking for common events. Do not think of the rarities first.
This could be explained by presence of non-IgG Ab like IgM (rarely encountered)
the IgM-antibodies were responsible for the positive AHG-CDC-XM result. The patient with positive CDC XM has a good outcome in the absence of DSA. Further work is needed to determine under what circumstances CDCXM positive transplants can be performed with FCXM and DSA negative.
a positive T cell and B cells cross match would indicate presence of antibodies to HLA type I and II antigens while a positive B cell cross match could indicate either:
(i) DSAs to type II antigens alone or
(ii) Low levels of DSAs to type 1 antigens.
A positive T cell crossmatch alone is usually due to technical error.
persistent positive T cell crossmatch result post desensitization is according to current guidelines, an absolute contraindication to renal transplant.
a positive T cell and B cells cross match would indicate presence of antibodies to HLA type I and II antigens while a positive B cell cross match could indicate either:
(i) DSAs to type II antigens alone or
(ii) Low levels of DSAs to type 1 antigens.
A positive T cell crossmatch alone is usually due to technical error.
persistent positive T cell crossmatch result post desensitization is according to current guidelines, an absolute contraindication to renal transplant.
Non-HLA antibodies This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Donor specific antibodies are against HLA antibodies and the absence of DSA doesn’t mean that other non-HLA antibodies that can lead to positive cross match are absent, such as MICA, angiotensin II type 1 receptor, H-Y antigen, antibodies against graft endothelium, antibodies against nsSNPs, and antibodies against LIMS1 proteins.
In addition, false positive results may be an explanation for positive crossmatch and negative DSA results such as presence of IgM antibody in the context of autoimmune disease or using rituximab in the CDC test.
Another reason may be the absence of rare antigen among the HLA antigen profile of bead assays and so DSA will not be detected.
You made it very complicated, but well done. Yes, this is a typical presentation of non-HLA antibodies. Unlikely due to IgM. It is a standard practice now that the HLA lap will run the autocross match to exclude autoantibodies.
Very impressed with your knowledge. You are a winner.
Winner Well done
Non-HLA antibodies This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
If the crossmatch done in this clinical scenario is CDC crossmatch, then this assays uses donor lymphocytes and recipient serum.
After mixing donor’s and recipient’s components, followed by incubation period and addition of complement we observe the reaction. If DSA is present in the recipient serum then donor cell lysis is observed, the crossmatch will be positive. For donor’s lymphocytes, T cells express class I HLA molecules, and a positive T cell crossmatch indicates the presence of a class I DSA; B cells express both class I and class II HLA molecules, and a positive B cell crossmatch indicates class I and class II DSAs.
The CDC crossmatch depends on the amount of DSA present in recipient’s serum, isotype of immunoglobulin, and cell surface density of the target HLA antigen.
Results of crossmatch could be influenced by several factors:
-Amount of antibody or titer is important because low titer antibody may not be able to activate complement cascade and induce cell lysis and crossmatch would be negative.
-Isotype of immunoglobulin is important because the presence of IgM antibody, which could be either anti-HLA or non-HLA, could cause false positive crossmatch.
-Non-HLA IgG binding to cell surface antigens found on lymphocytes
-Positive CDC crossmatch could result from interference of some drugs with the assay. Drugs like Rituximab, ATG, alemtuzumab and IVIG could bind to donor’s lymphocytes and the test is considered positive.
-Antibodies to specific loci, not routinely reported by HLA laboratory, could also cause positive result.
DSA detection by solid phase assays, recognizes only IgG and do not detect anti-HLA IgM antibodies, even though IgM antibodies cause a positive crossmatch, similar to our case here. Solid phase assays could show false-positive results because of reactivity against the latex beads, denatured HLA antigen, or non-HLA protein used to coat the beads.
So in the setting of positive cross match (both T and B cells are positive), and negative DSA, my first impression that the recipient could have non-IgG antibodies against HLA class I which is present in both B and T lymphocytes. In this case it could be IgM anti-HLA antibodies. But non-HLA antibodies to components found on both T and B lymphocytes could be the case here.
References:
1. Cai J, Terasaki PI, Anderson N, et al. Intact HLA not beta2m-free heavy chain-specific HLA class I antibodies are predictive of graft failure. Transplantation 2009; 88:226.
2. Pereira S, Perkins S, Lee JH, et al. Donor-specific antibody against denatured HLA-A1: clinically nonsignificant? Hum Immunol 2011; 72:492.
3. Gombos P, Opelz G, Scherer S, et al. Influence of test technique on sensitization status of patients on the kidney transplant waiting list. Am J Transplant 2013; 13:2075.
Non-HLA antibodies This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Positive cross match ( both T and B cells),but no DSA…not a classic match…or association ,with some justification required. Judicious interpretation must be there aiming not losing suitable kidney offer .Revising validity that positivity of cross match and negativity of DSA is a reasonable thinking . False positive CDCXM may be due to autoantibodies(suggested by negative DSA)or lab errors. Addition of DTT can prevent the effect of IgM as an autoantibody .Other measures include heating recipient serum to 55 C ,washing, auto crossmatch or prolongation of incubation period. Also, FCXM should be done being more sensitive than CDCXM. False positive FCXM can occur in with non specific IgGs or monoclonal AB(eg rituximab)therapy ;which can be reduced by pronase.On the other hand, SAB is not detecting IgM autoantibodies and non-HLA autoantibodies. False negative results happens when C1 and IgM autoantibodies bind to beads ,preventing HLA antigen-antibody interaction(prozone phenomenon).other situations include use of IV IG infusion, inaccessible HLA Ag on the beads ,shred epitope by many beads resulting in decrease of MFI and if certain HLA Ag is not included on the SAB kit. Positive CDCXM and/or FCXM with negative L-SAB indicates standard risk immunological level.
A conclusive approach is to correlate clinical background in association with confirmation of crossmatch assay validity, abolishing lab errors and application of phenotype beads flow c PRA screen to avoid loss of good kidney offer.
References:
Chughahi,SA et al, Interpretation of crossmatch reports in a patient with lupus nephritis ,Arch Organ Transplant,2017,023:029
Chari,M et al: Cross matching in renal transplantation by non-immunologist for non-immunologist ,2017,Urol & Nephrol, July,15,257-261
Geier,SS et al 😛 271 False positive HLA antibody tests can lose organ offers, Human Immunology, Vol :78,Suppl Sept.,2017,P:252
Impressed with your explantation, well done. It represents deep thought. Do not forget that the lab will exclude autoantibodies and repeat the test if ant technical errors are suspected.
Winner Well done
Non-HLA antibodies This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Positive both B and T lymphocyte cross match but No DSA
1- A false positive flow cytometry may result from non specific binding of Ig G to Fc receptors by certain substances in recipient serum in case of :
A- consider recent treatment with rituximab ( anti CD20) and in this case , results can be confirmed by treating donor lymphocyte with pronase
B- These may be auto Ab, confirm the results by autocross match
C- Non-HLA antibody.
2- False negative SAB for DSA may result from
a- the positive MFI cut off value differs between different transplantation labs so DSA may exist but are less than this value so reported as negative
b- Lab error if the donor antigen is not represented in the particular SAB kit used
Schinstock, C. A., Gandhi, M. J., & Stegall, M. D. (2016). Interpreting Anti-HLA Antibody Testing Data: A Practical Guide for Physicians. Transplantation, 100(8), 1619–1628. https://doi.org/10.1097/TP.0000000000001203
Non-HLA antibodies This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Positive cross match (both T and B cells are positive), but no DSA.
Patient may have false positive crossmatche due to presence of IgM AB as in autoimmune diseases so CDC- DDT should be done to confirm absence of autoantibodies.
Another cause is that the patient might be desensitized .
Low number of antibodies that can’t activate complement to cause cell lysis
-Positive CDC crossmatch occurs with some drugs which interfers with the test . Drugs like Anti-CD20 monoclonal antibody (rituximab) has most commonly been implicated. Pre-treatment of donor lymphocytes with pronase can be helpful in removing the effect of CD 20 binding (23). , ATG, alemtuzumab and IVIG could bind to donor’s lymphocytes and the test is considered positive.
The SAB assay is only semi-quantitative, and the variation in MFI has been reported as high as 62%, especially when the MFI is relatively low (1000-3000)
So negative DSA might be false negative.
SABs may not include donor HLA antigen SO the test is false negative.
Carrie A. Schinstock, MD, Manish J. Gandhi, MD, and Mark D. Stegall, MD.Interpreting Anti-HLA Antibody Testing Data: A Practical Guide for Physicians.Transplantation. 2016 Aug; 100(8): 1619–1628.
Non-HLA antibodies This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
-Non- HLA antibodies .
-Technically error .
-Low level DSA antibodies detected by FCXM.
-false positive due to IgM antibodies.
Akram Abdullah
3 years ago
Cross-matching is the crucial step in kidney transplantation.There are Techniques used for cross-matching include:
Cell-based assay:
a- CDC: complement is added to the mixed recipient serum and donor lymphocytes and cell lysis of the lymphocytes is observed. Negative test when donor-specific anti-HLA antibodies are absent and complement activation does not occur. Positive test when donor-specific antibodies bind to lymphocytes activate complement, and cause cell lysis Advantages of CDC: The mainstay of pretransplant screening. Key role in preventing hyperacute rejection Detects only complement binding antibodies Drawbacks: Detects IgM and IgG antibodies and non HLA antibodies .b- Flow cytometry: it measures DSA by using an impedance flow cytometer. The measurement of DSA is done either by fluorescence intensity or by serial dilution of recipient serum that reacts with donor lymphocytes. If CDC negative & flow cytometry is positive it may be due to i. non-complement-fixing Abs ii. Non-HLA Abs iii. low level of Abs. So the test was used for sensitized patients. 2. Solid-phase Ab detection assay: a- ELISA: the serum of the recipient is mixed with specific Abs that bind to available HLA epitopes, then IgG is added to combined anti-HLA ABs. It is used in sensitized patients. b- bead technology (Luminex), by using beads labeled with fluorescein. The result was measured either by flow cytometry or Luminex ( the degree of fluorescein measured by MFI). 3 Virtual Cross-match: Mainly it is used for deceased donor transplantation to decrease ischemic time. By using bead technology the recipient anti-HLA Abs compared with donor HLA Ags.
Ahmed Fouad Omar
3 years ago
rossmatch techniques can be divided into cell based assays and solid phase assays. 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 acivating 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): 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 or a titred value of dilution required to get a negative result (for quantification). B) Solid Phase antibody detection assays: 1) 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) 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. 2. Please reflect on your practice if possible.
In our practice, which is mainly living donor transplant setting, CDC is the first test to be done. If there is no history of sensitization (prior transplant, pregnancy, blood transfusion), then we proceed with transplant without any further tests,
In patient with sensitization history, even if CDC crossmatch is negative, we get a single antigen bead (Luminex) assay for quantification of DSAs and proceed accordingly,
Mohammed Sobair
3 years ago
Introduction:
crossmatching was an attempt to identify those transplant recipients who had a higher
likelihood of acute vascular rejections after receiving the gratf from a given donor.
Tis hyperacute rejection is the result of the presence preformed antibodies in the donor
serum to one or many HLA (human leucocyte antigens).
Cell based technique:
1-CDC technique:
Complement dependent cytotoxicity crossmatching was first developed in the 1960.
Percentage of lymphocytes in the cell panel which has undergone lysis as a result of
complement activation or panel-reactive antibody (% PRA)
Twenty percent is usually taken as the minimum cut-of for a positive result thus
functioning as a qualitative and a semi-quantitative estimate of the strength of the
reaction.
. (2) Flow cytometry:
Flow cytometry for crossmatching was a technique first described in the context of pre
transplant in the early eighties. Here, the donor lymphocytes are mixed with the
recipient’s serum. Tese bind to the donor specific’s antibodies and are quantified by
detector
flowcytometer :
Interpretation of flow crossmatch results In the light of a negative CDC result, a positive
flow cytometry could be interpreted as indicating the presence of any of the following
Non-complement fixing antibodies
Non-HLA antibodies
Solid phase antibody detection assays :
Enzyme-linked immunosorbent assay (ELISA):
initial application of ELISA was to detect HLA in both bound and free forms but its
current utility is for the detection of HLA antibodies in serum. Te modifed assay uses HLA
glycoprotein immobilized into mictrotiter wells. Te recipient serum is added and specifc
antibodies bind the epitopes available.
Bead technology (Luminex):
HLA antibody testing was revolutionised by the introduction of beads labeled with
fourescein in the nineties.
Virtual Crossmatching:
technique is based on the comparison of the anti-HLA antibodies of the recipient to the
donor HLA antigens using bead technology. Tis method predicts the eventual crossmatch
and can assist in rapid identification of a suitable donor
Mohamed Essmat
3 years ago
CDC cross match – It involves incubation of donor’s lymphocytes in recipient serum, washing to remove unbound antibodies, then adding complement and after incubation period – If antibodies to donor HLA present complement activation occur and cell lysis . – It is highly specific, a positive cross match using CDC indicating the presence of clinically significant complement fixing antibodies thus if CDC positive donor should be excluded. – This test has low sensitivity, human anti- goblin can be added before complement that bind to Donor HLA antibodies, to increase sites for binding to complement and increase its sensitivity -False positive results can occur due to the presence of clinically non significant autoimmune IgM antibodies which can be eliminated either by heating or adding DTT . -False negative result can occur due to non-complement fixing, non HLA or due to low level of HLA antibodies -If antibodies are directed to only class II or if DSAs directed to HLA class II are low results may show positive B and negative T cell cross match, while if DSAs are directed to both class I, II it will give positive B and T cross match. – Positive T cell cross match has the worst prognosis and donor should be excluded. – Positive B cell cross match carry poor prognosis when compared to negative CDC cross match but better that positive T cell cross match. – CDC is used either in determining compatibility between donor and recipient or in determining sensitization in case of PRA – CDC should be repeated just before transplant since antibodies may be formed after antigen exposure such as blood transfusion or pregnancy Flowcytometry – It involves incubating donors lymphocytes with recipient serum, then addition fluorescein labeled anti- globulin that will bind to DSA present in the recipient serum that are attached to donor lymphocytes -FCM is used either in determining compatibility between donor and recipient or in determining -More sensitive than CDC as it can detect non complement fixing AB, non HLA antibodies and low level DSA, so CDC – FCM + cross match may indicate the presence of one of the previous mentioned ABS. – Positive T cell FLC (in highly sensitized patients only) is associated with poor graft survival, so it has a very big rule in sensitized recipients Solid phase assays – Bead assay (luminex) replace ELISA – Very sensitive very specific, can detect complement and non-complement fixing antibodies, low level DSA, non HLA antibodies. – Luminex give quantitative assessment of the strength of DSAs. Virtual cross match – By comparing anti HLA antibodies of recipient detected by luminex 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, – DSA may change over time due to sensitization from blood transfusion or pregnancy so luminex SAB should be repeated. In practice regarding living donors we do Virtual cross match and CDC to all patients and FCM or DTT cross match accordingly ⦁ If CDC positive we repeat using DTT or FCM if +ve excluded. ⦁ If FCM positive and CDC negative patient is assessed for desensitization using data from Solid phase assays.
AMAL Anan
3 years ago
Comparison between techniques:
**CDC
Advantage:
Low cost.
Play role in preventing hyper acute rejection.
Disadvantage:
Detect only complement fixing antibodies.
Less sensitive.
**Flowcytometry:
Advantage:
More sensitive
Detect low level of HLA IgG antibodies
Disadvantage:
Suitable for living donor than cadaveric.
**Solid phase assay:
More sensitive to detect donor specific HLA antibodies.
Identify all HLA alleles for recipients antibodies.
Disadvantage:
Positive assay with negative CDC/FCM ambiguous.
SAB detect both complement fixing and non fixing antibodies so recipients may be denied a graft without clinical significance.
AMAL Anan
3 years ago
*CDC technique (1960) : determine presence of donor specific HLA antibodies in recipients serum which help In prognosis of graft survival after transplantation.
*Involved donor lymphocytes ( B and T) with recipients serum and add complement to the mixture and cell lysis of lymphocytes is observed.
*Detect percentage of lymphocytes in cell panel .
Addition of AHG increase sensitivity of CDC .
*CDC may lead to false positive or false negative , so add DTT which help in preventing IgM, autoantibodies which mediate complement activation or allow only IgG DSA.
* False negative:
– low DSA level to cause complement activation.
– Type of antibodies not cause complement activation.
* New antibodies may develop if recipients become sensitised by new antigen exposure e.g pregnancy or blood transfusions.
* HLA encoded by gene on chromosome 6 , class I ( HLA A B C ) expressed on all nucleated cells .
HLA class II ( DR DP DQ) : expressed only on antigen presenting cells as macrophages and dendritic cells .
Vascular endothelial cells of transplant graf expressed both these antigens and antibodies respond to them predicting acute antibodies mediated Rejection.
* positive T and B cells :
Indicated presence of antibodies to HLA type I and HLA type II antigen .
* positive B cell indicate :
-DSA to type II antigen alone .
– low level of DSA to type I antigen .
* positive T cell alone :
– Technically error.
– Non- HLA antibodies.
– If you decide to transplant, may need aggressive therapy.
* Positive T cells crossmatch:
– After rule out of autoantibodies, transplantation can be performed but carry risk of poor outcomes
and high incidence of acute graft rejection.
* President positive T cell crossmatch post desensitisation is absolute contraindications of transplantation.
* False positive B cell crossmatch by luminex for DSA increase its specificity , true positive B cell crossmatch is less significant in acute and hyper acute graft rejection.
Flowcytometry: donor lymphocytes mixed with recipients serum these bind to DSA quantified by detectors in Flowcytometry.
FCXM measured by channel shift or serial dilution of recipients serum .
Solid phase antibodies detection assay :
By ELIZA or luminex ( SAB)
Virtual crossmatch:
Detect suitable donor rapidly, reduce cold ischemia time without increasing rejection or affect graft survival.
Abdullah Raoof
3 years ago
there is many crossmatch technique used to detect ant HLA ab in sensitized patients .
sensitization occur by
1- blood transfusion .
2- pregnancy .
3- previous kidney transplant .
detecting of these ab before transplantation is important to prevent hyper acute rejection .
it is of two type
1- cell based type .( CDC , flowcytometry )
2- solid phase .( uses beads instead of donor lymphocyte )
features of each one
1- CDC – complement dependent cytotoxicity .
developed since 1969 by terazaki .
most commonly used technique
advantages
available
simplicity
less costy
disadvantages
false positive ( IgM Ab, dtetects only complement dependent ab. non HLA ab )
false negative result ( low titre ab . non complement ab , non HLA ab )
2- FLOW CYTOMETRY .
uses patient serum mixed with lymphocyte , then TO add flourecent labled IG.
the positive result is given as a channel shift , or serial titration method .
it is vey sensitive with high false positive result
it is role is in assessing highly sensitized patients .
solid phase
1- ELISA TECHNIQUE
It is replaced by luminex – not used nowadays .
2- luminex technique .
uses beads with HLA ag instead of lymphocyte
it is vey sensitive and specific used for detection of DSA..
single ag beads
multiple ag beads ( for sreening )
the result is given as MFI ( MEAN FLOURECENT INTENSITY ).
Ahmed mehlis
3 years ago
●DSAs are commonly generated by:
(i) Blood transfusion
(ii) Previous transplantation
(iii) Pregnancy
●The role of crossmatching is to identify those transplant recipients who are at risk of acute vascular rejections after allograft transplantation.
-Hyperacute rejection is the result of preformed donor-specific antibodies(DSAs.
●There are three mthods of cross matching:
A) Cell based essay:
A mixture of donor lymphocytes and recipient serum is tests, searching for preformed DSA that if present may lead to acute graft rejection. The reaction is complement mediated and detected by lysis of cells. Greater than 20% is considered positive reaction.
A similar modality is flow cytometry cross matching, using either florescence intensity or serial dilution as a measure of the reaction.
b) Solid phase antibody detection:
– ELISA method, detecting HLA ab in the serum
– Luminex: using flourescein labeled beads and interpreting the tests using either flow cytometry or luminex methods.
Jamila Elamouri
3 years ago
An Update on Crossmatch Techniques in Transplantation
the crossmatch test for the presence of preformed DSAs in the recipient serum to HLA antigen. DSA puts the recipient at risk of acute rejection.
DSAs performed due to blood transfusion, previous crossmatch, or pregnancy. CDC Technique: complement-dependent cytotoxicity
Test: serum from the recipient is added to donor lymphocytes (T or B) separately in the presence of complement. Negative test: when donor-specific anti-HLA antibodies are absent, so, complement activation does occur, no cell lysis. Positive test: when DSAs are present, bind to lymphocytes, activate the complement, and cause cell lysis. Result: is the percentage of lymphocytes in the cell panel which has undergone lysis, or panel-reactive antibody (%PRA).
PRA of 20% is used as a minimum cut-off for a positive result. The strength of the test can be increased by:
1- Doubling dilutions of the recipient’s serum. The higher the dilution required to give negative results, the greater the strength of the immune reaction, and helps to determine the need for desensitization.
2- Addition of anti-human immunoglobulin (AHG) which increases complement activation, so the sensitivity of the test. Limitations: 1- False positive: due to IgM auto-antibodies. Addition of Dithiothreitol (DTT) removes IgM auto-Abs, allows only IgG (DSAs). 2- False negative: DSAs are either of low level or antibodies type does not cause complement activation.
· – vascular endothelial cells of the graft have both classes I, II.
· B-cells have both classes.
Ø If cross match to both T and B- cell was positive, this would indicate DSAs to HLA type I and type II Ags are present.
Ø Positive B cell cross match could indicate either (1) DSAs to type II Ag alone or (2) low levels of DSAs to type I Ag.
Ø Positive T cell crossmatch alone is usually due to Technical error.
· Positive T-cell crossmatch is associated with high incidence of acute graft rejection. Persistent positive T cell crossmatch result after desensitization is an absolute contraindication to renal transplant.
Ø Positive B-cell test is not as strongly associated with rejection as positive T-cell test, anti-class II HLA warrant desensitization before transplantation. And negative test is associated with better outcome.
Ø It is not so significant as it has high false positive results. Flow cytometry:
Donor’s lymphocytes are mixed; with recipient’s serum. If DSAs there, donor lymphocytes react with them. And quantified by:
1- Measurement of the fluorescence intensity as a ratio of the control (channel shifts).
2- Serial dilutions of the recipient’s serum made to react with donor lymphocytes and; the minimum dilution which yields a negative result gives a measurable estimate. Interpretation of flow crossmatch results:
– to fix the possibility of transplantation or the need for desensitization before transplantation. Negative CDC result with a positive flow cytometry indicate one of the following:
1- Non-complement fixing antibodies
2- Non-HLA antibodies
3- Low level antibodies Solid phase antibody detection assays: 1- Enzyme-linked immunosorbent assay (ELISA):
Uses microtiter wells fixed with HLA-glycoprotein. Recipient serum added. If; donor-specific antibodies present. They will bind the epitopes. after wash and IgG carrying alkaline phosphatase added. Wash again to remove the unfixed DSAs, and a substrate added, which, after phosphorylation by the passenger, undergo a color change. 2- Bead technology (Luminex):
These technic based on the use of beads that carry HLA molecules as antigen and soaked with fluorochromes. DSAs present in the recipient serum react with these HLA-Ag, resulting in a specific unique signal. Wash done and incubation with anti-human IgG labeled with phycoerythrin. Quantitation of the result:
1- flow cytometry: measure the channel shift results from binding of antibody.
2- Luminex method: The degree of fluorescence is calculated as mean fluorescence intensity (MFI) using laser. Interpretation of the flow crossmatch studies:
the cut-off values are not uniform cross laboratories. Low value would increase the sensitivity while affecting specificity and vice-versa. The virtual crossmatch:
rapid identification of a suitable donor, and predicts the eventual crossmatch. Technic
beads carry HLA-Ags are mixed with recipient serum. Each bead has specific dye. anti-HLA antibodies bind to the specific bead. Detector antibody then bind and sequester a reporter dye checked for using a laser beam. The test makes up a profile for the recipient antibodies. Which compared with the HLA structure of a potential donor so; predicting the result of the crossmatch. Technics comparison: Complement dependent cytotoxicity:
Advantage: availability, simplicity, low cost.
Drawback:
1- Detect only complement binding antibodies
2- Less sensitive than newer test
3- Detects both IgM and IgG Abs, autoantibodies, and non-HLA antibodies against Ags that are not relevant to transplantation. Flow cytometry:
Advantages:
1- more sensitive than CDC
2- can detect low levels of IgG HLA antibodies.
3- Less operator variability
Drawback:
Suitable for living donors rather than cadaveric donors. Solid-phase assays (ELISA and bead technology):
Advantages: 1- The most sensitive methods for DSA detection. 2- Allow the identification of all HLA alleles for which the recipient have antibodies. 3- No false positive from non-HLA antigens. 4- Specifically identify the class of HLA. Drawbacks: 1- positive assay with negative CDC/Flow crossmatch has an unclear interpretation. 2- the link of low-level antibodies to lower significant antigen is controversial. 3- Detect both complement-fixing and non-complement fixing HLA antibodies, may prevent transplantation without significant clinical reason.
Asmaa Khudhur
3 years ago
Review of crossmatch techniques:
Cell based assays:-
CDC crossmatch / the significance of CDC lies in its ability to determine the presence of DSA in the serum of the recipient.
The B and T cells are separately tested against serum from the recipients.
Outcome of positive T cell crossmatch result is poor with high incidence of acute graft rejection.
Outcome of positive B-cell crossmatch result the significance of anti-class ll HLA AB is of less significant in acute /hyperacute rejection and still need desensitization of the recipient prior to transplantation
Flow cytometry /if negative CDC result ,positive flow cytometry could be interpreted as indicating the presence of any of the following
1 non -complement fixing AB
2 non -HLA AB
3 low level antibodies
So the value of it is in using for sensitization.
Solid phase AB detection assays
– Eliza assay
– -Bead technology (luminax)
The virtual crossmatch
This method predict the eventual crossmatch and can assist in rapid identification of suitable donor.
Ramy Elshahat
3 years ago
Crossmatch in Transplantation is like rehearsal in acting.
You have akidney from person and you are going to put it in another body risking hyperacute rejection so you do cross match outside recipient body to make sure that recipient plasma doesn’t contain antibodies against donor’s tissue
There’s three main types of crossmatch
CDC, flow cytometry crossmatch and solid phase crossmatch.
They are complete each other like taking 3different angles by 3different cameras to get full 3d picture of the same person
So sometimes you need to do the 3 types together
Cdc crossmatch is a technique detect complement fixing antibodies
1st developed by Terasaki in which he mixed recipient serum(antibodies) with donor lymphocytes both bcells and tcells(HLA class 1 and 2) then added complement.then some modifications were done to improve its specificity like adding dithiothreitol(DTT) to prevent IgM from giving false positive results
Positive tcell crossmatch after excluding of autoantibodies us absolute contraindication for kidney transplantation and associated with hyperacute rejection
Positive b cell only and negative to tcell is not consistent associated with humoral rejection and its significant is uncertain but it considered as arisk and needs explanation using the other types of crossmatch and maybe low titre antibodies as b cells express more antigen on its surface
Flow cytometry crossmatch: detect larger spectrum of antibodies either complement or non complement fixing
Its very good negative test in which donor’s lymphocytes (HLA class 1 and 2) mixed with recipient plasma (antibodies) plus anti immunoglobulins antibodies
Positive reaction is detected by detector in the empendence flow cytometry and quantification of antibodies titre is done by 2methods …1) fluorescent channel shift in relation to control 2) serial dilution until become negative
Interpretation -ve is excluding any kind of antibodies but positive flow cytometry crossmatch in negative CDC DTT cross match may be related to no complement fixing,non HLA antibodies or low titre as its very sensitive test then you will need to proceed to solid phase crossmatch to decide what is the next step.
Large retrospective study showed that positive flow cytometry crossmatch against tcell associated with poorer 5y graft survival so some centers policy is reject donor if positive flow cytometry crossmatch against tcell.
Solid phase crossmatch
Includes ELIZA and luminex
ELIZA solid phase crossmatch in which HLA glycoprotein immobilized into micro titre well then recipient serum is added… antibodies bund the epitopes available…wash…anti IgG with reporterd molecules (alkaline phosphatase)…wash…. substrate which give colour by the reporter if still attached not removed by washing is detected.
Bead technology
Beads labelled with two floroscen dye in different ratio resulting in signal unique for each bead.each bead may have single HLA molecule(single antigen bead) or multiple (mixed antigen bead)
Recipient serum incubated with beads … antibodies react with beads and colour detected by either flow cytometry(mean channel shift) or luminex(MFI)
negative solid phase crossmatch is good
Positive solid phase crossmatch maybe due to non HLA antibodies or non complement fixing or very low level OF antibodies….the cutoff of antibodies significant is ambiguous and not uniform just centered base.
Mahmoud Hamada
3 years ago
There are three mthods of cross matching:
A) Cell based essay:
A mixture of donor lymphocytes and recipient serum is tests, searching for preformed DSA that if present may lead to acute graft rejection. The reaction is complement mediated and detected by lysis of cells. Greater than 20% is considered positive reaction.
A similar modality is flow cytometry cross matching, using either florescence intensity or serial dilution as a measure of the reaction.
b) Solid phase antibody detection:
– ELISA method, detecting HLA ab in the serum
– Luminex: using fourescein labeled beads and interpreting the tests using either flow cytometry or luminex methods.
Tahani Hadi
3 years ago
Cross match is used to prevent and detect the risk of posttransplant rejection alot of techniques used to for this purpose.
CELL BASED ASSAYS
Including CDC technique and flow cytometry
CDC is done by mixing recipient serum with donor lymphocytes T and B cells are tested alone after that adding complement .
Cell lysis means positive crossmatch through activation of classical pathway.
It’s basic test easy to be done but less sensitive .
FLOW CYTOMETRY:this is the same as CDC but differs by adding detectors .
SOLID PHASE ANTIBODIES DETECTION ASSAYS
(ELISA ,BEAD TECHNOLOGY LUMINEX )
VIRTUAL CROSS Match
Fatima AlTaher
3 years ago
Pretransplant assessment of the sensitization status of kidney transplant recipient is essential
For predicting graft outcome. several crossmatch techniques are available as
I- Cell based crossmatch : by mixing recipient serum with donor B and T lymphocytes and if the recipient has complement fixing Ab againt donor cells this will result in cell heamolysis
The test become more accurate by adding anti human immunoglobulin .
Drawbacks of this technique are
a- False positive results in case of autoantibodies , this can be avoided by adding DDT
b- False negative results in case of non complement fixing Ab or very low level of Abs
Flow cytometry: by mixing recipient serum with donor B and T lymphocytes to detect donor specific antibodes by Measuring the fluorescence intensity. It is more sensitive than CDC crossmatch
Advantage of flow cytometry it can detect non complement fixing Ab , non HLA Abs and low level Ab
II- Solid phase antibody detection assays
1- ELISA technique to detect free and bound form of Anti HLA Ab via mixing recipient serum with specific antibodies
2- Bead technology (Luminex) : can differentiate which HLA class are detected III- The virtual crossmatch :
This depends on comparing recipient Anti HLA Abs To donor HLa ags using bead technology
In our protocol (Zagazig university hospital ) , first we perform crossmatch CDC with DDT , then virtual crossmatch by luminex and an other final CDC crossmatch just prior to transplantation .
Hinda Hassan
3 years ago
Please reflect on your practice if possible.
In Sudan we do first HLA typing for both recipient and doner for A, B, C, DR,DP ,DQ.
And we do PRA and initial CDC. Recently we do only final CDC
We only proceed for FCX for patients with suspected DSA,historical positive PRA, highly sensitized patients , patients with very high PRA but negativeCDC
Cell based assay
1-Cdc techniques: doner lymphocytes and recipient serum were mixed and a complement is added. If the recipient serum contains antibodies the cells lysis will occur. The percentage of cells which underwent lysis is calculated in a panel ; hence the name PRA. 20% is considered positive result. The test is qualitative and semiquantitative. Some improvement in it increased its benefits :
I.titered crossmatch: through doubling dilution of the recipient serum. The more dilution required for making the test negative the higher the concentration of antibodies
II.AHG enhanced: these antibodies bind to recipient antibodies ,enhancing the complement binding . this is useful for cases with low titre of DSA
The test can have false positive results if there is IgM antibodies and this can be overcome by DTT. It can also have false negative results with low titre of antibodies or non complement fixing antibodies.
HLA class 1antigens are present in all nucleated cells and class 2 are present in APCs. The greft endothelium contains both types. So for anticipation of rejection t cells, which include only class 2, or b cells ,which include both classes , are used in cross match. Positive both t and b indicates antibodies to class1 and2. Positive T alone is usually an error while positive B alone may be due to anti class2 only or low level of anti class1 not detectd by t cells.
Positive T cell will end with acute graft rejection and is considered as an absolute contraindication for transplant. For positive B cell test, first rule out the false positive results. ,then with luminex look for DSA.
2- flowcytometry: recipient serum is mixed with doner lymphocyts and fluorescent labelled anti IgG . if a rection occur ,either the intensity of fluorescence is recorded or serial dilution are used. Because of the high sensitivity of this test,it is useful in sensitized patient to decide the reisk of rejection pretransplant . if CDC is negayive and flow cytometry is positive this can be due to low level of antibodies, non complemt or non HLA antibodies. T cell positive flowcytometry have less 5 year graft survival. Solid phase antibody detection assays
1- ELISA: used to detect sensetiztion through detection of bound and free antibodies. Recently its use is for the serum with use of anti IgG.
2- Luminex:using beads labeled with antigens mixed with the recipient serum and labeled anti IgG. It can detect whole class antibodies or specifically.
Virtual crossmatch
Use the patient serum against a set of antigen to predict the possible acceptable doners. Some researchers suggest using it instead of flowcytomty . it reduce cold ischemia time which has its effect on graft survival
Reem Younis
3 years ago
-The role of crossmatching is to identify those transplant recipients who are at risk of acute vascular rejections after allograft transplantation.
-Hyperacute rejection is the result of preformed donor-specific antibodies(DSAs).
-DSAs are generated by: blood transfusion, previous transplantation, pregnancy.
-DSAs can be identified with cell-based assays, solid phase, and more recently virtual crossmatch. Cell-based assays : 1. Complement dependent cytotoxicity: CDC technique:
-It is abasic and cornerstone cross matches in transplantation due to availability, cost, and simplicity but it is less sensitive than other newer assays.
-It can determine the presence of DSAs in the serum of the recipient.
– CDC technique is done by adding serum from the recipient to donor lymphocytes(T or B ) in the presence of complement.
-CDC detects only complement binding antibodies.
–The result is presented in terms of the percentage of lymphocytes in the cell panel which has undergone lysis as a result of complement activation or panel-reactive antibodies (PRA%).
–Titred crossmatch method in which a crossmatch of the donor lymphocytes is performed using serial doubling dilutions of the recipient, s serum. Higher dilution required to give a negative result means greater strength of the immune reaction that also helps to determine the need for desensitization.
-Addition of Anti-human immunoglobulin help increase the sensitivity of the test.
–False-positive CDC due to autoantibodies in recipient serum.
–False-negative CDC due to low level of DSA that cannot activate complement cascade or if antibodies are of the type that doesn’t cause complement activation.
-Antibody level of the recipient may vary with time so, matching must be done from a recently drawn recipient serum sample with donor lymphocytes especially in cadaveric allograft transplantation.
-T-cells express HLA –class I while B cells express both so, positive T cells and B cells crossmatch would indicate presence of antibodies to HLA-I and II while a positive B cell crossmatch could indicate either:
1.DSAs to type II antigen alone or
2..low level of DSAs to type I antigens.
-A positive T cell crossmatch alone is usually due to a technical error.
– When the renal transplant is performed in a recipient who has a positive T cell crossmatch will result in poor outcome and a high incidence of acute graft rejection as shown in several studies.
–Persistent positive T –cells crossmatch after desensitization is an absolute contraindication to renal transplantation.
-Negative B cell crossmatch is associated with better outcomes but its high false-positive rate makes its significance uncertain but this is improved by Luminex testing which increases its specificity.
– In the case of positive B cell cross matches, the significance of anti-class II HLA antibodies is less significant in acute/hyperacute rejection but still needs desensitization of the recipient before transplant. 2. Flow cytometry :
-Here, the donor lymphocytes are mixed with the recipient, s serum in the presence of anti-IgG fluorescein-labeled antibodies, and quantified either by :
1. Measurement of the fluorescence intensity as a ratio of the control.
2. Serial dilutions of the recipient, s serum are made to react with donor lymphocytes and the minimum dilution which yields a negative result gives a measurable estimate. Interpretation of flow crossmatch results: -Negative CDC result and positive flow cytometry means :
1. Non –complement-fixing antibodies.
2. Non –HLA antibodies
3. Low-level antibodies.
-The value of this test in using it for cross-matching of sensitized patients to determine transplant feasibility or need for desensitization before transplant.
-It is suitable for living donors rather than cadaveric donors. Solid-phase antibody detection assays Enzyme-linked immunosorbent assay (ELISA):
-Here, the patient serum is added and specific antibodies bind the epitopes available then a wash is performed and anti-IgG is added with substrate that undergoes a color change.
-It was replaced by bead technology. Bead technology (Luminex) :
-Each bead may have one or more HLA molecules types incorporated. The basic steps involve:
1 -incubation of patient serum with beads.HLA antibodies of the serum will react with HLA antigens on the beads.
2 -Then, beads are washed and incubated with anti-human IgG labeled with phycoerythrin. Quantification of the result -It is achieved by 2 methods:
1. Flow cytometry
2. luminex method
-The degree of fluorescence is calculated as mean fluorescence intensity (MFI)
Interpreting flow crossmatch studies:
-Positive test and corresponding CDC crossmatch negative may be due to -non –complement-fixing antibodies, non –HLA antibodies, Low-level antibodies.
-Low cut–off values increase sensitivity while affecting specificity and vice–versa.
The virtual crossmatch
-It is based on the comparison of the anti-HLA antibodies of the recipient to the donor HLA antigen using bead technology or microsphere that coated with multiple HLA antigens.
–Final transplant decision could be taken based on virtual crossmatch irrespective of the flow cytometry without significant difference in clinical outcome in those who were flow cytometry positive but virtual crossmatch negative.
Ben Lomatayo
3 years ago
Differential diagnosis of positive crossmatch and negative DSA are ;
Autoantibodies e.g. IgM
Non-HLA antibodies
Sensitization
Rarely technical error
Abdulrahman Ishag
3 years ago
Cross match techniques are;
-cell based assay.
CDC technique.
flow cytometry .
-solid phase antibody detection assay.
enzyme-linked immune sorbent assay (ELISA) .
virtual crossmatch.
CDC cross match;
It is a functional test that involve cells(donor lymphocytes) and serum antibodies ( recipient serum )in the presence of complement.
The percentage of lymphocytes in the cell panel which has undergone lysis as a result of complement activation is calculated and the result is represented as panel-reactive antibody (%PRA).
It simple , available and cost effective test .
It is the main stay of pre transplant screening for HLA antibodies .
It has a key role in prevention of hyper acute rejection.
It is less sensitive than other newer assays.
It detects only complement binding antibodies.
It detects IgM and IgG antibodies simultaneously, autoantibodies and non-HLA antibodies against antigens that are irrelevant as far as the transplant is concerned.
Human globulin (ATG) is added to increase the sensitivity of the test as multiple AHGs bind a single DSA, amplifying its complement activation at smaller titres.
False positive result due to auto antibodies can be reduced by addition of dithiothreitol (DTT) which helps to prevent IgM mediated complement activation and allow IgG (DSA).
Flow cytometry:
The donor lymphocytes are mixed with the recipient’s serum.
The reactive lymphocytes (that bind to the donor specific antibodies) are quantified by detectors in the impendence flow cytometery.
The result can be quantified by measurement of the fluorescence intensity as a ratio of the control (channel shifts) or serial dilutions of the recipient’s serum are made to react with donor lymphocytes and the minimum dilution which yields a negative result gives a measurable estimate.
It is more sensitive than CDC cross match .
It can detect lesser levels of IgG HLA antibodies and has less variability.
In the light of a negative CDC result, a positive flow cytometry could be interpreted as indicating the presence of any of the following non-complement fixing antibodies, non-HLA antibodies and low-level antibodies .
The value of this test’s sensitivity lies in using it for cross matching of sensitized patients determine transplant feasibility or need for desensitization protocols prior to transplant.
Solid phase assays (ELISA and bead technology);
They are the most sensitive tests in detecting DSA.
They allow identification of lower titre ,possibly clinically significant anti HLA Abs
They allow identification of all HLA alleles for which recipient harbours antibodies .
They eliminate confusion regarding the class of HLA that are detected (false positive of antibody binding to non –HLA antigens ) by CDC.
Single antigen bead assays detect both complement- fixing and none complement –fixing HLA antibodies.
Limitations of the tests including;
-the relevance of low level Abs to low detectable antigen level.
– the interpretation of a positive assay in the presence of negative CDC and flow cross match is ambiguous.
-interference by IgM ,incomplete of antigens in the bead sets and variability of HLA density on the density.
Enzyme-linked immunosorbent assay (ELISA):
The recipient serum is added and specific antibodies
Bead technology (Luminex):
Beads are;
– impregnated with different ratios of two fluorochromes resulting in a signal that is unique to the specific bead.
-each bead may have one or more HLA molecule types incorporated.
The basic steps involve first, incubation of recipient serum with the beads.
HLA antibodies of the serum will react with HLA antigens on the bead. The beads are washed and incubated with a second antibody, usually anti-human IgG labeled with phycoerythrin.
Quantification of the test is achieved by flow cytometory or luminex.
Virtual cross match;
This technique is based on the comparison of the anti-HLA antibodies of the recipient to the donor HLA antigen using bead technology .This method assist rapid identification of a suitable donor .Multiple synthetic microspheres are each given a single HLA antigen coating and incubated with recipient serum. Alternatively a microsphere or bead may be coated with multiple antigen to allow more efficient screening.
This method allows for sensitivity of flow cross match and identifying specific antibody.
The combination of these various assay allow us to exclude insignificant antibodies from risk assessment and allowing better preparation when more significant antibodies are identified that can potentially complicate tough not preclude transplant .
More studies are needed to understand the significance of low level DSA , non HLA Abs and non-complement fixing antibodies.
Last edited 3 years ago by Professor Ahmed Halawa
Ban Mezher
3 years ago
Using of cross-matched in pre transplantation assessment is very important to decrease the incidence of rejection due to antibodies against HLA. techniques used for cross-matching include:
Cell based assay:
a- CDC: it is important in determination of the presence of donor specific Abs in recipients to assess prognosis of graft survival. This test depend on complement activation.T & B cells of donor mixed with recipient serum with addition of complement, then observe the percentage of cell lysis ( 20% is the minimum cut off of positive result). To increase the sensitivity of the test adding of AHG. This test had several limitation as false positive ( presence pf autoAbs in recipient serum which can be abolished by adding DTT to prevent IgM complement activation), false positive ( due to presence of Low level of DSA or due to Abs not activated by complement). persistence positive DCD after sensitization is an absolute contraindication for transplantation. But B cells crossmatch positivity no necessarily associated with humeral rejections T cell, but negative B cell cross-match is associated with better prognosis.
b- Flow cytometry: it measure DSA by using impedance flow cytometer. The measurement of DSA is done either by fluorescence intensity or by serial dilution of recipient serum that react with donor lymphocytes. If CDC negative & flow cytometry is positive it may be due to
i. non complement fixing Abs
ii. non HLA Abs
iii. low level of Abs.
So the test used for sensitized patients.
2. Solid phase Ab detection assay:
a- ELISA: the serum of recipient is mixed with specific Abs that bind to available HLA epitopes, then IgG added to combined anti-HLA ABs. It used in sensitized patients.
b- bead technology (Luminex), by using beads labelled with fluorescein. The result measured either by flow cytometry or Luminex ( the degree of fluorescein measured by MFI).It can predict pre transplant risk & used for post-transplantation DSA monitoring.
3 Virtual Cross-match:
Mainly it used for deceased donor transplantation to decrease ischemic time.By using bead technology the recipient anti-HLA Abs compared with donor HLA Ags. Johnson et al found that transplantation can be done depending on virtual cross-match. If the flow cytometry result is positive & virtual cross-match is negative there is no change in graft outcome.
Winner Well done Non-HLA antibodies This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Assafi Mohammed
3 years ago
A Review of Crossmatch Techniques
The genesis of crossmatching was an attempt to identify those transplant recipients who had a higher likelihood of acute vascular rejections after receiving the graft from a given donor( Hyperacute Rejection).
DSAs are commonly generated by:
(i) Blood transfusion
(ii) Previous transplantation
(iii) Pregnancy. One important implication of this is that wives often develop HLA antibodies against their husband’s HLA in the course of conception. This may preclude the spouse as a donor in the event of renal failure. DSAs are conventionally identified with cell based assays, solid phase and more recently the virtual crossmatch.
Crossmatch Techniques: (a) Cell based assays 1.CDC technique; Complement dependent cytotoxicity crossmatching technique involved:
(i) Isolating donor lymphocytes (cadaveric/ living).
(ii) The B and T cells are separately tested against serum from the recipient.
(iii) Humeral immunological response is mediated though activation of the complement system by the classical pathway.
(iv)To demonstrate the effects of this cascade, complement is added to the mixed recipient serum and donor lymphocytes and cell lysis of the lymphocytes is observed.
(v) Negative test when donor-specific anti-HLA antibodies are absent and complement activation does not occur. Positive test when donor-specific antibodies bind to lymphocytes, activate complement and cause cell lysis.
How to quantify the strength of reaction in CDC technique:
% PRA : the result is represented in terms of the percentage of lymphocytes in the cell panel which has undergone lysis as a result of complement activation or panel-reactive antibody (% PRA). 20% is usually taken as the minimum cut-off fora positive result thus functioning as a qualitative and a semi-quantitative estimate of the strength of the reaction.
“Titred crossmatch” method: in which a crossmatch of the donor lymphocytes is performed using serial doubling dilutions of the recipient’s serum. Thus, higher the dilution required to give a negative result, greater the strength of the immune reaction. This also helps determine the need for a desensitization protocol for the recipient prior to transplant.
How to increase sensitivity of CDC technique:
Addition of anti-human immunoglobulin (AHG) helps increase the sensitivity of CDC as multiple AHGs bind to a single donor specific antibody, amplifying its complement activation response at smaller titres.
Limitations of CDC : 1.false positive reaction: often a result of autoantibodies in the recipient serum. This can be overcome by addition of Dithiothreitol (DTT) which helps prevent predominantly IgM autoantibody mediated complement activation and allows only IgG (donor specific antibodies).
2.false negative reaction: may occur when DSA levels are too low to result in activation of the complement cascade or if the antibodies are of the type that does not cause complement activation.
3.Antibody levels of the recipient may vary with time due to new antigen exposures and immunological sensitization that may occur after antigen exposure in the form of blood transfusions, pregnancy.This emphasized the importance of matching the most recently drawn recipient serum sample with the donor lymphocytes, especially in the context of patients on a donor transplant list for cadaveric transplant.
(2) Flow cytometry: in the Flow cytometry for crossmatching, the donor lymphocytes are mixed with the recipient’s serum. These bind to the donor specific antibodes and are quantified by detectors in the impedence flow cytometer. Two methods of quantification can be used:
(i) Measurement of the fluorescence intensity as a ratio of the control (channel shifts).
(ii) Serial dilutions of the recipient’s serum are made to react with donor lymphocytes and the minimum dilution which yields
a negative result gives a measurable estimate.
Interpretation of flow crossmatch results:
In the light of a negative CDC result, a positive flow cytometry could be interpreted as indicating the presence of any of the following :
The value of this test’s sensitivity lies in using it for cross matching of sensitized patients who have an inherently higher risk of acute graft rejection to determine transplant feasibility or need for desensitization protocols prior to transplant.
A large retrospective study based on data from the organ procurement and transplant network registry showed that CDC crossmatch negative patients who had positive T-cell flow cytometry results had significantly poorer absolute 5 year graft survival rates that those who were both CDC and flow cytometry crossmatch negative.{Graff RJ, Buchanan PM, Dzebisashvili N, Schnitzler MA, Tuttle-Newhall J, et al. (2010) The clinical importance of flow cytometry crossmatch in the context of CDC crossmatch results. Transplant Proc 42: 3471-3474.}
(b) Solid phase antibody detection assays: 1.Enzyme-linked immunosorbent assay (ELISA):
Enzyme-linked immunosorbent assay (ELISA): The initial application of ELISA was to detect HLA in both bound and free forms but its current utility is for the detection of HLA antibodies in serum.
(i) The modified assay uses HLA glycoprotein immobilized into mictrotiter wells.
(ii) The recipient serum is added and specific antibodies bind the epitopes available.
(iii) A wash is performed and anti-IgG with a passenger reported molecule (alkaline phosphatase) is added that combines
with the anti-HLA antibody.
(iv) After further washing to remove unbound antibody, a substrate is added which after dephosphorylation by the reporter
molecule undergoes a color change.
This method while effective in detecting sensitization in transplant candidates has been replaced by bead technology.
2.Bead technology (Luminex):
HLA antibody testing was revolutionised by the introduction of beads labeled with flourescein in the nineties.
Generally beads are impregnated with different ratios of two fluorochromes resulting in a signal that is unique to the specific bead. Each bead may have one or more HLA molecule types incorporated.
The basic steps for Luminex involve :
(i) incubation of recipient serum with the beads.
(ii) HLA antibodies of the serum will react with HLA antigens on the bead.
(iii) The beads are washed and incubated with a second antibody, usually anti-human IgG labeled with phycoerythrin.
Levels of testing:
(i) Screening: Beads are incorporated with a many molecules, both class I and II derived from cell lines.
(ii)Testing against a set of alleles from an individual genome: Beads have HLA molecules from a single cell line with two
molecules for each HLA locus. These results can be expressed as PRA.
(iii) Single antigen beads: These beads have a single type of HLA antigen molecules bound to them. The result is that a
conglomerate of heterogenous antibodies can be tested and specific HLA antigens can be identified.
Interpreting flow crossmatch studies:
The main significance of a positive test if the corresponding CDC crossmatch was negative. In this scenario, the reason for the positive match could be due to:
a low level antibody.
a non-HLA antibody.
a non-complement binding antibody.
Another issue is that the cut-off values are not uniform cross laboratories. Low cut offs would increase sensitivity while affecting specificity and vice-versa .
Single antigen beads are employed in allowing semiquantitative evaluation of HLA
antibodies in order to predict pretransplant risk as well as for donor specific antibody monitoring after transplant.
The virtual crossmatch
This technique is based on the comparison of the anti-HLA antibodies of the recipient to the donor HLA antigens using bead technology.
This method predicts the eventual crossmatch and can assist in rapid identification of a suitable donor. Multiple synthetic microspheres are each given a single HLA antigen coating and incubated with recipient serum.
Steps of Virtual crossmatch techniques:
(i) Serum from the recipient is added to synthetic beads with either a set of antigens or a single antigen, each bead can be
identified by an independent dye signature.
(ii) Anti-HLA antibodies if present will bind to the specific bead.
(iii) A detector antibody will then bind and sequester a reporter dye.
(iv) Beads can be checked for the reporter dye using a laser beam, this builds a profile of the antibodies present in the
recipient that can be compared with the HLA construct of a potential donor thus predicting the result of crossmatch.
This method allows for the sensitivity of flow crossmatch combined with identifying specific antibody. Johnson et al. found that the final transplant decision could be taken on the basis of the virtual crossmatch irrespective of the flow cytometry without significant difference in the clinical outcome in those who were flow cytometry positive but virtual crossmatch negative.
Comparison of Techniques
1.Complement dependent cytotoxicity Advantages:
It represents a functional test that involves cells and serum containing antibodies.
A basic and durable test still used as the foundation of crossmatch due to availability, cost and simplicity.
It has played a key role in preventing hyperacute rejection as the science of transplant immunology progressed.
It detects only complement binding antibodies.
Drawbacks:
It is less sensitive than other newer assays.
It detects IgM and IgG antibodies simultaneously, autoantibodies and non-HLA antibodies against antigens that are irrelevant as far as the transplant is concerned.
2.Flow cytometry Advantages:
More sensitive than CDC crossmatch.
It can detect lesser levels of IgG HLA antibodies and has less inter-observer variability.
Drawbacks:
Slow turnaround times meant this technique was suitable for living donors scenarios rather than for cadaveric donors.
3.Solid phase assays (ELISA and bead technology) Advantages:
They are the most sensitive of all the techniques for detecting donor specific HLA antibodies.
Enhanced sensitivity has allowed improved rates of success in retransplants where detection of pre-sensitization from previous grafts allow the avoidance of those HLA markers on subsequent grafts.
They allow the identification of all HLA alleles for which the recipient harbours antibodies.
They avoid false positives of antibody binding to non-HLA antigens and eliminates confusion regarding the class of HLA that are detected (CDC matching has an overlap between T and B cell matching, T with Class I and B with both class I and II).
Drawbacks:
The interpretation of a positive assay in the presence of negative CDC/flow crossmatch is ambiguous.
The relevance of low level antibodies to low significance antigens is debatable.
Single antigen bead assays detect both complement-fixing and non- complement-fixing HLA antibodies. On the basis of these results, prospective recipient may be denied a graft without established clinical significance.
Other limitations include (i) interference by IgM, (ii) incomplete library of antigens in the bead sets, (iii) variability of HLA density on the beads.
Wessam Moustafa
3 years ago
The idea of cross matching came to detect patients at higher risks for developing acute rejections .
These tests tries for detect any DSA present in the serum of the recepient.
**CDC techniques **
It was implemented in early 60s , donor s lymphocytes are mixed with serum of recepients , then complement is added .
Presence of DSAs will lead to complement activation through the classic pathway .
Limitations of CDC techniques include false positive results due to presence of autoantibodies ( which may be improved by addition of DTT ) , and false negative results due to very low Titres of Abs ( addition AHG may amplify immune response from those low Titre Abs )
T cells expresses only class I antigens while B cells expresses both MHC Class I and II antigens.
So positive B cross matching means either that there is DSAs against class II antigens , or low Titres of DSAs against class I antigens,
Also this is not always associated with acute rejections
While patients with Positive T cell cross match , usually have poor outcomes , so a persistent positive T cell cross match is absolute contraindication for transplantation
** flow cytometry techniques **
Donors lymphocytes are patients serum are mixed together , and the immune reaction is quantified using flow cytometer .
This technique is used in the high risk patients
Positive flow cytometer with negative CDC means that :
DSAs are not complement fixing
DSAs are non HLA antibodies
Very low Titres DSAs
** solid phase assays **
*) ELISA
*) luminex
Patients serum are mixed with flourecin beads , that are labelled with MHC class I and II antigens , incubated , then beads are washed and another incubation with anti human IG is done .
**) Virtual Cross match.
Using the bead technology, detected HLA antibodies are compared to donors HLA antigens .
Studies showed that a transplantation decision could be taken on basis of Virtual Cross match , even if flow cytometry results were positive
Winner Well done Non-HLA antibodies This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Esmat MD
3 years ago
Crossmatch is a cornerstone of transplantation sciences. Initial methods relied on complement dependent cytotoxicity. However, recently cell based and solid phase assays have been utilized. Flow cytometry is useful in previously sensitized patients while bead technology is an advanced technique for HLA antibody testing. Virtual cross match uses bead technology to detect specific recipient antibodies without requiring donor serum. Crossmatch techniques include cell-based assays, solid phase antibody detecting assays, and the virtual crossmatch.
Cell-base assays
CDC technique determines the presence of DSA. Serum from the recipient is added to separated T and B cells of donors in the presence of complement. A positive test is characterized by cell lysis. The results are represented in terms of the percentage of lymphocytes that have undergone lysis in the cell panel as a result of panel reactive antibody. 20% is a minimum cut off for positive results. Addition of AHG provides help to increase the sensitivity of CDC test. Titrated cross match method is another method for quantification of strength of reactions. The limitation of this technique is false positive and false negative results. False positive results due to autoantibodies can be overcome by addition of DTT and elimination of Ig M antibody. False negative results may occur with a very low level of DSA or if antibodies don’t cause complement activation. Because of the variation in antibody level of recipients, it is very important to match the most recently drawn recipient serum sample with the donor lymphocytes. Vascular endothelial cells express both class I and class II HLA, hence antibody responses to them can predict antibody mediated rejection.
A positive T cell and B cell cross match would indicate antibodies to both HLA type I and HLA type II antigens. A positive B cell cross match alone would indicate presence of DSA to type II antigens or low levels of DSA to type I antigens, while a positive T cell cross match alone is usually due to technical errors.
A positive T cell cross match results in a unacceptably high incidence of acute antibody mediated rejection and the recipient needs to undergo desensitization treatment. Persistence of positive T cell cross match after desensitization is an absolute contraindication of kidney transplantation.
On the other hand, a positive B cell cross match is not consistently associated with humoral rejection. High false positive results can be overcome by Luminex testing. In positive B cell cross match, the significance of HLA type II antibodies is less in acute or hyper acute rejection.
Flowcytometry crossmatch
Serum from the recipient is added to donor lymphocytes in the presence of anti IgG fluorescein-labelled antibodies. Quantification can be done by measurement of the channel shift or by serial dilution of the recipient’s serum.
A negative CDC result with a positive flow cytometry can indicate: Non-complement fixing antibodies or Non-HLA antibodies or Low-level antibodies, and correlates with poorer graft survival compared to both negative CDC and flow cytometry tests.
Solid phase antibody detection assays
ELISA: its current utility is for the detection of HLA antibodies. In this method, the recipient serum is added to immobilized HLA glycoproteins and specific antibodies bind to available epitopes. This method has been replaced by bead technology.
Bead technology (Luminex): This technique uses beads labeled with fluorescein and may have one or more HLA molecule types. Incubation of recipient serum with beads, leads to reaction with HLA antigens on the bead.
This test is used for screening, testing a set of alleles from an individual genome, and single antigen bead. Quantification is achieved by calculation of MFI in the Luminex method. Cut off value is different between laboratories and low cutoffs increases sensitivity while affecting specificity. Single bead assay provides semi quantitative evaluation of HLA antibodies.
The virtual cross match
The recipient serum is added to synthetic beads with a set of antigens or a single bead antigen and anti HLA antibodies if present will bind to the specific bead, then the dye will be checked by laser beam.
This method builds a profile of the antibodies of a given recipient that can be compared with the HLA construct of a potential donor. In negative virtual cross match and positive flow cytometry circumstances, there is no significant influence on clinical outcome. Using virtual cross match will reduce cold ischemia time and is valuable for sharing organ program.
Complement dependent cytotoxicity:
Advantages:
Mainstay of pretransplant screening.
Key role in preventing hyper acute rejection
Detects only complement binding antibodies
Drawbacks:
Detects IgM and IgG antibodies and non HLA antibodies
Flow cytometry:
advantages:
More sensitive than CDC
Less inter-observer variability
Drawbacks:
Slow turnaround times, thus suitable for living donors scenarios
Solid phase assays (ELISA and bead technology):
Advantages:
Most sensitive
Improved rates of success in retransplants
Identification of all HLA alleles for which the recipient harbours antibodies
Avoid false positives of antibody binding to non-HLA antigens
Eliminates confusion regarding the class of HLA that are detected
Drawbacks:
Interpretation of a positive assay in the presence of negative CDC/flow crossmatch is ambiguous
Relevance of low level antibodies to low significance antigens is debatable
Detects both complement-fixing and non complement-fixing HLA antibodies (may be denied a graft without established clinical significance.
Interference by IgM
Incomplete library of antigens in the bead sets
Variability of HLA density on the beads
Nasrin Esfandiar
3 years ago
Positive CDC-XM may be as a result of IgM autoimmune disease and can be omitted by adding DTT, auto crossmatch, washing and heating recipient’s serum or increased incubation time.
Positive FCXM but negative CDC-XM may be as a result of low titer or non-complement fixing Abs. Positive FCXM without DSA detected by Luminex may be as a result of prozone phenomenon, inaccessible HLA-Ag on the Luminex beads sharing same epitope by many beads and low MFI or absence of a specific HLA-Ag on the Luminx kit.
Thanks Nasrin What is the picture non-HLA antibodies when you do crossmatch (FCXM) in the absence of DSA?
Mahmud Islam
3 years ago
Cross-match testing is key in transplantation. the better the estimation of immunologic risk the best the prediction of outcome. There may be no stand-alone technique. All are integral to each other.
Crossmatching techniques development aimed to identify recipients who are likely to develop vascular rejection of grafts.
Various exposures may cause sensitization. Pregnancy, blood transfusions, and even previous infections may result in antibody formation.
Over more than 70 years many techniques came to the surface. Classical and still the mainstay is the complement-dependent cytotoxicity crossmatch (CDC). Followed by FLowcytometry this has the advantage of detecting lower antibody levels more specifically and sometimes more efficiently are called cell-based techniques. Aside from virtual crossmatch in which no real recipient blood samples are utilized, rather patients HLA antibody specificities with donors HLA type are matched we have the newer solid-phase techniques. ELIZA and Luminex which is a special type of flowcytometry-specific beads are used.
CDC crossmatch
First developed in the late 60s by Terazaki and Patels, recipients serum is used. Next, lymphocytes are incubated. The third complement is added. In case of antibodşes presence, the lymphocytes will lyse and this will be tested under the microscope. Here %20 cell lysis is the cutoff for positive results.
As T cells have class-I antigens while B cells have both, a positive T and B cross-match will point to class-I antibodies. Both t and B crossmatch negativity is favored. Only T cell positivity is a risk but B crossmatch only will refer to technical error as the share presence of class-I presentation with T cells.
Flow cytometry assays:
Flow cytometry is a very sensitive assay introduced in the 80s. Here the recipient’s serum is incubated with the donor’s lymphocytes. Fluorescence dye is applied and cells are markered (anti-cd3 pan marker, anti-cd-19 or -20 or B cells). the cells bound to
A positive crossmatch in case of negative CDC crossmatch may be due to very low levels of antibodies or other non-complement fixing or nonHLA antibodies. The role of flowcytometry then is essential in highly sensitized or those who have a high risk of rejection.
Solid-phase assays:
Here purified or engineered HLA antigens are used. ELIZA has been replaced with bead technology (LUMINEX). Either single antigen bead, phenotype bead, or mixed beads are used. The mean fluorescence intensity (MFI) is detected. As Luminex is a type of flowcytometry its positivity in case of negative CDC CX is also either due to on-HLA antibodies or very low levels.
Virtual crossmatch:
Here the need for a final physical crossmatch is replaced virtually. One handicap of this is that it is dependent on information supplied to the computer. Still have a big role in some patients especially to decided the suitable recipients in case of cadaveric transplants.
Heba Wagdy
3 years ago
Cell based assays: CDC technique:
Serum from recipient is added to donor B and T lymphocytes in presence of complement.
The result is assessed by percentage of lymphocytes destructed due to complement activation or panel reactive antibody
It provides qualitative and semi quantitative estimate of the strength of the reaction
have a main role in preventing hyperacute rejection
Titred crossmatch:
provides more quantification of the strength of the reaction by using doubling dilutions of recipient serum which help to decide the desensitization protocol.
False positive results due to autoantibodies in recipient serum , avoided by adding DTT which prevent IgM antibody mediated complement activation and allow only IgG DSA.
less sensitive as may detect non-HLA antibodies against antigens irrelevant to transplant.
False negative: due to very low DSA level which can’t activate complement cascade or due to presence of antibodies which don’t activate complement.
Studies showed that positive T cell crossmatch is associated with poor outcome and increased incidence of acute graft rejection
persistent positive T cell crossmatch post desensitization is absolute contraindication to transplantation
B cell crossmatch significance is still uncertain due to high rate of false positive result but
true positive result still require desensitization before transplantation.
Flow cytometry:
Donor lymphocytes are mixed with recipient serum in presence of anti IgG fluorescein labelled antibodies
Quantification of binding of DSA to lymphocytes done by:
fluorescence intensity as a ratio of the control (channel shifts)
serial dilution which give a measurable estimate
Negative CDC and positive flowcytometry indicate non complement fixing antibodies, non-HLA antibodies or low level of antibodies.
flow cytometry is more sensitive than CDC, used for crossmatch in sensitized patients at high risk of acute rejection to determine the need for desensitization before transplantation
It requires long time so more suitable for living donors
Solid phase antibody detection assay:
The most sensitive method for detecting DSA ELISA:
used for detection of HLA antibodies in serum, replaced by bead technology Bead technology (Luminex)
Each bead may have one or more HLA molecule, HLA antibodies of serum will react with antigen on beads
level of testing:
Screening: beads are mixed with many molecules of both class I and II
Testing against a set of alleles from an individual genome which can be expressed as PRA
Single antigen beads: single type of HLA antigen molecule is bound to the beads
So a combination of heterogenous antibodies can be tested and specific HLA antigen can be identified
Quantification of results done by flow cytometry measuring channel shift associated with binding of antibodies or Luminex method using mean fluorescence intensity.
Single antigen bead assay:
allow semiquantitative evaluation of HLA antibodies pre transplant and used for DSA monitoring post transplant.
disadvantage: detect complement and non complement fixing HLA antibodies so prospective recipient may be denied a graft without established clinical significance
The virtual crossmatch
Anti-HLA antibodies of the recipient are compared to donor HLA antigens using bead technology, it predict eventual crossmatch
recipient serum is incubated with beads each has either a unique HLA antigen or set of antigens on its surface
anti-HLA antibodies if present will bind to specific bead so provide a profile of antibodies present in a potential donor so predict the result of crossmatch
can allow more efficient screening when a bead is coated with multiple HLA antigens.
Shereen Yousef
3 years ago
Crossmatch techniques started a half century ago remain the base of transplantation science.
Its mainly done to identify recipients who are at higher risk for hyperacute and acute rejection
Hyperacute rejection occurs due to presence of preformed antibodies in the recipient’s serum to one or many HLA (human leucocyte antigens). referred to as DSAs (donor specific antibodies)
DSAs formation known as sensitization occurs due (i) Blood transfusion (ii) Previous transplantation (iii) Pregnancy.
One important implication of this is that wives develop HLA antibodies against their husband’s HLA during pregnancy.
Crossmatch Techniques
First Cell based assays
1-Complement dependent cytotoxicity crossmatching.
2 -Flow cytometry.
▪︎Complement dependent cytotoxicity crossmatching ( CDC) ;
Terasaki and Patel in 1969 observed the presence of DSAs in transplant recipiects were associated with a higher incidence of acute and hyperacute graft rejection
Technique of CDC:
it is done by adding donor lymphocytes to recipient serum with addition of complement .
B and T cells are separately tested.
if DSAs are present in recipient’s serum it will bind to lymphocytes activating the complements lead to lysis of donor’s lymphocytes
Reading results :
The results presented in the form of percentage of lymphocytes that undergoes lysis which is the percentage of panel-reactive antibody (% PRA).
20% is considered as the minimum cut off for positive results .
titred crossmatch is used to increase the strength of the test done by testing donor lymphocytes with serial doubling dilutions of the recipient’s serum. The higher serum dilution required to give a negative result, the greater the strength of the immune reaction against donors cells which indicates need for a desensitization of recipient.
To increase the sensitivity of CDC-XM anti-human immunoglobulin (AHG) can be add so multiple AHGs bind to a single donor specific antibody, amplifying its complement activation response at smaller titres.
Limitations of CDC-XM
1- false positive due to detection of IgM autoantibodies in the recipient serum. It can be avoided by addition of Dithiothreitol (DTT) which helps prevent IgM autoantibody complement activation and allows only IgG
2- false negative occurs with very low DSAs titre or non complement activating AB
3- CDC-XM is changing due to exposure to new different antigens.
Interpretation of results:
Vascular endothelial cells of the transplant graft express both classes of HLA antigens.
T cells express only class I antigens while B cells express both types .
persistent positive T cell crossmatch after desensitization is according to current guidelines, an absolute contraindication to renal transplant as it is associated with humoral rejection and lead to hyperacute rejection.
positive B-cell crossmatch results:
•high rate of false positive makes its significance uncertain.
•negative B cell crossmatch has better results
•False positive results can be avoided concurrent Luminex testing for DSAs which increases its specificity.
•Not consistently associated with humoral rejection as positive T cell crossmatches.
In case of true positive B cell crossmatches, anti-class II HLA antibodies are less signifcantl in acute/ hyperacute rejection but the patient
will need desensitization befor transplantation .
▪︎Flow cytometry crossmatch :
Also done by mixing serum from the recipient with donor lymphocytes (T or B) with addition of of anti-IgG fluorescein-labelled antibodies.
Positive test when DSAs bind to lymphocytes that are detected by flow cytometry once the anti-IgG fluorescein-labelled antibodies tag the lymphocytes.
Positive Flow cytometry with negative CDC-XM indicates either
Non-complement fixing antibodies,Non-HLA antibodies or Low-level antibodies.
This test is used for cross match of highly sensitized patients who are at higher risk of acute rejection to determine transplant feasibility or need for desensitization .
CDC crossmatch negative patients who had positive T-cell flow cytometry results had significantly poorer absolute 5 year graft survival rates than patients with both CDC and flow cytometry crossmatch negative.
Solid phase antibody detection
They are the most sensitive of all the techniques for detecting donor specific HLA antibodies.
It help in identification of all HLA alleles to which the recipient has antibodies.
They avoid false positive antibody binding to non-HLA antigens and eliminates confusion regarding the class of HLA that are detected in contrast to CDC-XM. Done by ELISA OR beads ;
ELISA is used for the detection of HLA antibodies in serum.
recipient serum is added and specific antibodies bind the epitopes available. A wash is performed and anti-IgG with alkaline phosphatase is added that combines with the anti-HLA antibody. further washing to remove unbound antibody, a substrate is added which after dephosphorylation by the reporter molecule undergoes a color change.
Although is effective to detect sensitization but it has been replaced by bead technology .
Bead technology (Luminex):
Generally beads are impregnated with different ratios of two fluorochromes resulting in a signal that is unique to the specific bead.
Each bead may have one or more HLA antigens types incorporated. basic steps involve first, incubation of recipient serum with the beads. HLA antibodies of the serum will react with HLA antigens on the bead.
beads are washed and incubated with a second antibody, usually anti-human IgG labeled with phycoerythrin
It is important in :
(i) Screening: Beads are incorporated with a many molecules, both class I and II derived from cell lines.
(ii) Testing against a set of alleles from an individual genome results can be expressed as PRA.
(iii) Single antigen beads: beads have a single type of HLA antigen molecules bound to them. specific HLA antibody can be identified .
Quantitation of the results is done by
Flow cytometry
Luminex method Two lasers are used to excite the fluorochrome of the bead and the phycoerythrin bound to the antibody and degree of fluorescence is calculated as a mean fluorescence intensity (MFI) each centre may determine according to its experience the MFI number that is accepted to transplant the patient .
Interpretation of results
CDC crossmatch negative with positive luminex indicate the presence of low level antibody, non-HLA antibody or a non-complement binding antibody.
Single antigen bead assays detect both complement-fixing and non-complement-fixing HLA antibodies so a doner can be refused without established clinical significance
The Virtual crossmatch ;
The test compares HLA antibodies in the recipient’s serum to the donor’s antigens using single antigen bead assays. It helps detection of suitable donor.
this builds a profile of the antibodies present in the recipient that can be compared with the HLA antigens of a potential donor predicting the result of crossmatch.
Transplantation can be done while positive flow cytometry and negative virtual crossmatch.
Nasrin Esfandiar
3 years ago
Nowadays crossmatch tests before transplantation is necessary to prevent hyperacute rejection conducted by preformed DSA .Different XM techniques are cell based and solid phase assays. Cell based assays include:
1–complement dependent cytotoxicity (CDC-XM): Recipient’s serum is added to donor’s lymphocytes. If donor lymphocytes are lysed after adding complement, the result of test will be positive and shows presence of DSA. More than 20% shows positive rest. Titered XM is done by serial dilution of patient serum that is needed to have negative result. AHG -CDC-XM: Adding anti-human immunoglobulin improve CDC sensitivity. False positive results are seen when autoantibody are present that could be omitted by Dithiothreitol (DTT) adding. False negative results is associated with low titer DSA or non- complement fixing Abs. The result of this test is expressed as T cell and B cell XM. Since T cells show class I antigenes and B cell both class I and II positivity of either of them has different meanings:
Both T cell and B cell XM positive: antibodies to HLA type I and II.
Positive B cell XM:
1-DSA to class II Ags
2- low level class I Ags.
3 – Necessitated Luminax for specificity
Positive T cell XM : persistence of positive T cell XM after desensitization is absolute contradiction to renal transplant .
4- Persistence of Anti-HLA Ab class II needs desensitization before TX.
2- Flow cytometry: Donor lymphocytes are added to patient’s serum. Then by measuring florescence intensity as a ratio of the control (channel shifts) or serial dilution its intensity is determined. Negative CDC-XM and positive flowcytometry may be related to:
1- Non-complement fixing Abs
2- 2-Non-HLA Abs
3- 3-low level Abs. It’s positivity may show need for desensitization.
3- Virtual Xm using solid phase assays:
1 – ElISA
2- Luminex: uses beads that contain HLA molecules labeled with fluorescein and are incubated with patient’s serum .Then beads are washed and an anti-IgG Ab labeled with phycoerythrin is added. This test may be used as screening by beads that contain many molecules or specific single Ag beads to detect specific HLA Ag and it’s intensity is express as MFI (means flow intensity) for virtual Xm anti-HLA Abs are determined by Lumina method and in recipient’s serum and are compared with donor’s HLA to define DSA and predict the result of XM. If the result of XM is negative, then irrespective of flowcytometry, TX can be done that decreases cold ischemia time and results in a better graft outcome especially in decreased donors and pre-sensitized recipients.
Thanks Nasrin You mentioned the answer of the question here which is non-HLA antibodies. I will consider it the answer of the question and you deserve the reward.
Sherif Yusuf
3 years ago
Positive cross match (both T and B cells are positive), but no DSA can occur in the following situations :
1- In autoimmune diseases ; due to either the presence of clinically non significant IgM antibodies as in rheumatoid arthritis and SLE or sometimes the antibodies are complement-fixing IgG autoantibodies as in SLE
2- In HIV positive patients: non-specific positive crossmatches can occur in HIV positive patients
3- Those with prior intake of rituximab : use of rituximab is associated with false positive cross match result
4- Highly sensitized recipients due to previous transplantation: as they may have positive cross match abd low or undetected DSA
These patients can be transplanted with good outcome (risk of ABMR, CAMR is low) especially if auto cross match is positive (indicating autoimmune disease), but monitoring of DSA post transplantation is important since the incidence of development of denovo DSA post transplant is increased.
But in highly sensitized recipients (cPRA> 80%) the risk of ABMR, CAMR is greater even if there is no DSA
Thanks Dr Sherif
autoantibodies will get excluded automatically by the atocrossmatch and also DTT, augmented CDC, etc
You are right. Rituximab can cause positive crossmatch. Low level DSA can cause negative CDC but not FCXM
Your answer is partially correct, but you derive the present as you mentioned rituximab.
Winner
Non-HLA antibodies This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Ben Lomatayo
3 years ago
In my practice currently we still depend on CDCXM and we we are doing well with it. Recently our hospital bought new Luminex machine but is not yet functioning and hopefully next year it will start working and adding complexity to our understanding.
Ben Lomatayo
3 years ago
Abstract ; Crossmatch test is the cornerstone for practicing safe transplantation. The test evolved from the era of the standard CDCXM to more sensitive tests such as flow cytometry, solid phase assays and virtual crossmatch. Introduction ; The idea of the crossmatch is to identify patients at high risk of hyper-acute rejections. This occurs because of preformed antibodies in the recipient’s serum as a results of sensitisation. Sensitization evens includes ; pregnancy, blood transfusion and previous transplantation. Cell based assay ; In 1969, Terasaki and Patel demonstrated that the presence of preformed antibodies were associated with hyper-acute rejection(1). The principle of CDCXM is mixing donor’s lymphocytes with recipient’s serum, complement is added and if antibodies are presence ,it binds donor’s lymphocytes resulting in cell lysis. The test is interpreted by % of lymphocytes which undergone lysis. 20% is considered to be positive(2). The strength of antibodies can also be detected by serial dilutions of the recipient’s serum. The sensitivity of the CDCXM can be improved by addition of anti-human globulin antibodies. Limitations ; are false positive test due the autoantibodies(IgM). This is overcome by adding Dithiothreitol(DTT) which prevent IgM from binding complements(3). False negatives are also seen due to either low levels antibodies or non-complement binding antibodies(4). Outcomes in positive T cell crossmatch results ; positive cell crossmatch is the only contraindication to transplantation unless the patient is sensitised. Outcomes in positive B cell crossmatch results ; positive B cell crossmatch is not clearly associated with hyper-acute rejection. Despite that, desensitisation is required before transplantation in the setting of truly positive B cell crossmatch results. Flow cytometry ; Recipient’s serum is mixed with donor’s lymphocytes, and fluorescence -labelled antibodies are added. If anti-donor’s antibodies are present, it will bind donor’s lymphocytes and the strength of the reaction is quantified by detectors in the impedence flow cytometer. Quantitation is done by 1. measurement of the fluorescence intensity as ratio of control = Channel shift. 2. Serial dilution of the recipient’s serum reacting with donor’s lymphocytes . The minimum dilution that gives negative results is measured. Interpretation of the crossmatch results ; Negative CDCXM and Positive Flow cytometry; 3 possibilities 1. non-complement binding Ab 2. non-HLA Ab 3. low level Ab
The flow cytometry crossmatch is useful in sensitised patients to make decisions regarding desensitisation protocols(9) Solid phase assays ;
ELISA ; tests HLA antibodies in the serum, currently replaced by Bead Technology(11)
Bead technology( Luminex); This technology uses purified HLA antigens beads which will react with HLA antibodies. There is a wash phase followed by addition of anti-human globulin IgG labelled with phycoerythrin. The results are expressed as Channel shift(12) or the degree of the fluorescence intensity known as Mean Fluorescence Intensity(MFI) (13)
The Virtual crossmatch ; The test compares HLA antibodies in the recipient’s serum to the donor’s antigens using single antigen bead assays. It aids in the detection of suitable donor(16). Transplantation can still proceeds with positive flow cytometry and negative virtual crossmatch Comparison of techniques ;
CDCXM ; Advantages ; 1. Simple 2. Available 3. Cheap.
Disadvantages ; 1. Low sensitivity 2. Detects autoantibodies(IgM)
2.Flow cytometry ; Advantages ; More sensitive than CDC
Disadvantages ; Slow turn around time
3.Solid phase assays ; Advantages ; Highest sensitivity
Disadvantages ; 1. False positive 2. Un-necessary exclusions of patients from transplantation 3. Difficult to interpret positive results if both CDCXM and Flow cytometry XM were negative Conclusion ; CDCXM is still the main crossmatch test for screening pre-transplant HLA antibodies. The recent tests are more sensitive than CDCXM. Combination of tests are essential to make decisions about transplantation.
INCUBATION PATIENT SERUM WITH PANEL OF LYMPHOCYTES (T OR B)THAT REPRESENT HLAs OF THE LOCAL DONOR POPULATION.BINDING OCCURS BETWEEN HLA ABs IN SERUM AND TARGETED LYMPHOCYTES AND THE UNBOUNDANTIBODIES ARE WASHED.THIS ISFOLLOWED BY ADDITION OF COMPLEMENT AND VITAL DYE,RESULTING IN INITIATION OF COMPLEMENT CASCADE ENDING BY CELL LYSIS.RESULTS ARE QUALITATIVE/SEMIQUANTITATIVE .POSITIVE RESULTS ARE PRESENT WHEN CELLS TAKE THE DYE DENOTING STRONG COMPLEMENT -BINDING HLA DSA.
PANEL REACTIVE PERCENTAGE: IS THE PERCENTAGE RATIO OF POSITIVE WELLS TO THE TOTAL WELLS.PERCENTAGE OF NEGATIVE WELLS WILL REPRESENT THE CHANCE OF HAVING NEGATIVE CYTOTOXIC CROSS MATCH.THE ASSAY HAS INABILITY TO DETERMINE AB SPECIFITIES IF PATIENTS ARE BROADLY SENSITIZED.
IT INCREASES THE SENSITIVITY WITH DETECTION OF LOWER TITRE ABs.
-FLOW CYTOMETRY:
DSAs ARE DETERMINED BY IMPEDEDANCE FLOW CYTOMETER.QUANTIFICATION IS DONE BY MEASUREMENT OF FLOURESCENCE INTENSITY ANDSERIAL DILUTIONS OF THE RECIPIENT SERUM.ITS VALUE OF SENSITIVITY VALIDATES ITS USE IN SENSITIZED PATIENTS TO DETERMINE TRANSPLANT FEASIBILITY OR OMPLEMENTATION OF DESENSITIZATION.
– SOLID PHASE PRA: AND MONITORING FOR DE NOVO HLA ABs FOLLOWING Tx.
ELISA ASSAYS ARE MORE SENSITIVE THAN CDC-BASED ASSAYS BUT SUPERSEDED BY LUMINEX TECHNOLOGY USING HL-COATED BEADS FOR IDENTIFICATION OF PREFORMED HLA ABs BEFORE Tx. 2PLATFORMS ARE USED :-SCREENING BEADS : TO SHOW HLA CLASS I OR II ANTIGENS.A MICROSPHERE IS AFFIXED TO NATIVE COMPOSITON HLA ANTIGENS.
-SINGLE -ANTIGEN BEADS: EACH MICROSPHERE IS AFFIXED TO ONE RECOMBINENT HLA MOLECULE. PATIENT SERUM IS MIXED WITH ANTIGEN -COATED BEADS THEN HLA ABs BIND TO BEADS WHICH ARE WASHED FOLLOWED BY ADDITION OF ANTI-IgG DETECTION REAGENT.LUMINEXI ANALYZER IS USED FOR IDENTIFICATION OF HLA-ABs BOUND TO THE BEADS.SEMIQUANTIFICATION RESULTS REVEAL MEAN FLUORESENCE INTENSITY(MFI)RELATED TO STRENGTH ABs AND LIMITED BY THE DYNAMIC RANGE OF THE TEST.OTHER LIMITATIONS INCLUDE THAT IgG IS IDENTIFIED TOTALLY WITHOUT SUBCLSSES SPECIFICATIONS AND ORIGINALLY BY THE ONLY Ag INCLUDED IN THE PANEL.%PRA represents the percentage of reactive microspheres.
MODIFICATION OF THE SINGLE AG- BASED ASSAY:
– SERUM DILUTION PROZONE PHENOMENON:
TESTING OF DILUTED SERA ADDS TO THE ACCURACY OF ASSESSMENT OF STRONG ABs SATURATING SINGLE ANTIGEN BEADSUSUALLY SEEN IN HIGHLY SENSITIZED RECIPIENTS ;PROZONE OR EFFECT.THAT EFFECT CAN BE OVERCOME BY SERUM DILUTION ,ADDITION OF DTT OR EDTA OR SERUM HEATING.
C1q ASSAY:USED FOR SELECTIVE IDENTIFICATION OF DSA BINDING C1q,WHICH IS MORE PREDICTIVE OF ALLOGRAFT INJURY.COST AND TECHNICAL ISSUES LIMITED THE ROUTINE USE.
-EPITOPE-BASED MATCHING(EBM):
TO AVOID FALSE POSITIVE MATCHING RESULTS INDUCED BY PUBLIC EPITOPES ,PRIVATE EPITOPES ARE TARGETED WITH THE OUTCOMEOF MORE PREDICTIVE RESULTS.EBM CAN BE DONE IN-VITRO OR IN -SILICO(COMPUTED -BASED SYSTEM).IN-VITRO EBM SYSTEM IS THE HIGHLY SPECIFIC SAB ONE.HLA MATCHMAKER ALGORITHM ;A COMPUTER AND WEB-BASED;IS THE MOST COMMONLY USED ESPECIALLY IN HIGHLY SENSITIZED RECIPIENTS.
– VIRTUAL CROSS MATCHING: IT IS THE PREDICTION OF ACTUAL CROSS MATCH DEPENDING ON SCREENING OF RECIPIENT DSA WITH COMPARISON TO HLA TYPE OF POTENTIAL DONOR ,USING LUMINEX-SAB WITHOUT DONOR AND RECIPIENT SEUM SAMPLING;USING IN SILICO COMUTER -BASED VXM.CDC XMATCH IS THE ESSENTIAL TEST IN PRETRANSPLANT SCREENING.IT DETECTS ONLY COMPLEMENT BINDING ABs.
FLOW CYTOMETRY IS MORE SENSITIVE THAN CDC X MATCHING AND MORE SUITABLE FOR LIVING RATHER CADAVERIC DONORS.
LUMINEX SAB IS THE MOSTSPECIFIC AND SENSTIVE OF ALL ASSAYS.HOWEVER,NO SINGLE XMATCHING IS CONSIDERED ADEQUATE FOR A CLINICALLY CHALLENGING RECIPIENT.
EPITOPE LOAD AND AFFINITY CONSTANTS:
USING SURFACE PLASMON RESONANCE ALLOWED MEASURING DIFFERENT AFFINITY CONSTANTS OF ABs TOWARDS HLA PROTEINS(UP TO 25 AMINOACID RESIDUES INVOLVED).AFFINITY CONSTANTS MY EXPLAIN DIFERRENCES IN MFI RESULTS OF MICROSPHERES WITH SIMILAR EPITOPE TARGETS.
REFERENCES:
KUMAR,A ET AL ; AN UPDATE ON CROSSMATCH TECHNIQUES IN TRANSPLANTATION,J KIDNEY, 2017VOL 3,ISSUE 4,
GUNAWANSA,N ET AL :CROSSMATCH STRATEGIES IN RENAL TRANSPLANTATION: A PRACTICAL GUIDE FOR THE PRACTICING CLINICIAN,J TRANSPLANT SURG,2017,1(1):8-15
ROSSER,C AND SAGE,D:APPROACHES FOR THE CHARACTERIZATION OF CLINICALLY RELEVENT PRE-TRANSPLANT HUMAN LEUCOCYTE ANTIGEN(HLA) ANTIBODIES IN SOLID ORGAN TRANSPLANT PATIENTS:INT J IMMUNOGENET,2021;48:385-402
Well done and thank you for the hard work.
Please not in capital letters. It is not user friendly and difficult to read
Last edited 3 years ago by Professor Ahmed Halawa
Dalia Ali
3 years ago
crossmatch was used to diagnose transplant recipients who at higher risk for acute rejections and hyperacute rejection
hyperacute rejection result from preformed antibodies in the donor serum which named as donor specific antibiotics (DSA)
These antibodies result from the following causes
1- Blood transfusion
2- Previous transplantation
3- Pregnancy
There are 3 methods used to measure the DSA titer
Cell based assays
solid phase
virtual crossmatch
*Cell based assays
Cell based assays done by
-CDC technique
-Flow cytometry
The benefit of CDC technique is in identifying the presence of DSA in serum of transplant recipients so it will help to determine the graft survival after transplantation
The procedure of CDC
The technique done by isolating donor lymphocytes. The B and T cells are tested in separate manner against serum from the recipient. Humeral immunological response is mediated though activation of the complement system by the classical pathway.then complement is added to the mixed recipient serum and donor lymphocytes and cell lysis of the lymphocytes is observed.
The result
The result can be obtained depending on percentage of lymphocytes that undergo lysis
20% is considered as the minimum cut off for positive result
To increase test sensitivity we can added antihuman immunoglobulin (AHG) because multiple AHGs can bind to a single donor specific antibody and this will amplify the complement activation response at smaller titres.
Limitation of CDC
CDC test is less sensitive and detect only complement binding antibodies.
false positive result from presence of autoantibodies in the recipient serum .this result can be prevented by addition of Dithiothreitol (DTT) which prevent IgM autoantibody mediated complement activation and allows only IgG (donor specific antibodies)
False negative reactions may occur when DSA levels are too low to result in activation of the complement cascade or if the antibodies are of the type that does not cause complement activation
Interpretation of CDC test
T cells express class I HLA antigens (A,B,C) . B cells express both class I and II HLA antigen(A,B,C,DR,DP,DQ)
positive T cell and B cells cross match indicate presence of antibodies to HLA type I and II antigens while a positive B cell cross match could indicate either DSAs to type II antigens alone or Low levels of DSAs to type 1 antigens
Outcome in positive T-cell crossmatch
Poor outcome and high risk of rejection occurs in positive T-cell crossmatch recipients so desensitization must be done before transplantation and repeat the test again after that.however if persistent positive T-cell test even after desensitization is considered as an absolute contraindication to renal transplantation
Outcome in positive B-cell crossmatch
positive B cell crossmatch is not as associated with humoral rejection like positive T cell crossmatches. a negative B cell crossmatch is associated with better outcomes but the there is high rate of false positive result so we can overcome this problem by using luminex test for DSA.
In case of true positive B cell crossmatches the significance of anti-class II HLA antibodies is of little importance in acute/ hyperacute rejection but still need desensitization of the recipient prior to transplant
Flow cytometry
More sensitive than CDC test and can detect lower titer of DSA so it
Used in transplant recipients with high risk of rejection
Procedure
Serum from the recipient is added to donor lymphocytes (T or B) in the presence of anti-IgG f fluorescein-labelled antibodies.
Negative test when donorspecific antibodies are absent and binding does not occur. Positive test when donor-specific antibodies bind to lymphocytes that are detected by flow cytometry once the anti-IgG fluorescein labelled antibodies bind to the lymphocytes
Interpretation of flow crossmatch results
negative CDC result with positive flow cytometry indicate the following conditions
CDC crossmatch negative with positive T-cell flow cytometry indicate poorer absolute 5 year graft survival rates than those who have both CDC and f low cytometry crossmatch negative
*Solid phase antibody detection assays
Most sensitive methods in detecting DSA and can identify all HLA alleles
Done by
-Enzyme-linked immunosorbent assay (ELISA)
-Bead technology (Luminex)
Enzyme-linked immunosorbent assay (ELISA)
The recipient serum is added and specific antibodies bind the epitopes available. A wash is performed and anti-IgG with alkaline phosphatase molecule is added to combine with the anti-HLA antibody. further washing needed to remove unbound antibody, a substrate is added and then after dephospho-rylation by the reporter molecule undergoes a color change
Although this method is effective but it replaced by the Bead technology (Luminex)
Bead technology (Luminex)
Done by incubating the recipient serum with the beads then the HLA antibodies of the serum will react with HLA antigens on the bead. The beads are washed and incubated with a second antibody, usually anti-human IgG labeled with phycoerythrin
Quantitation of the result
The test done by
Flow cytometry
Luminex method
Interpreting flow crossmatch studies
CDC crossmatch negative with positive luminex indicate the presence of low level antibody, non-HLA antibody or a non-complement binding antibody.
* The virtual crossmatch
T his technique is used to detecting the presence of anti-HLA antibodies of the recipient to the donor HLA antigens using bead technology.
Multiple synthetic microspheres are coated with a single HLA antigen and incubated with recipient serum.
The test give more efficient screening in renal transplantation. final transplant decision should be taken on the basis of the virtual crossmatch irrespective of the flow cytometry without significant difference in the clinical outcome in those who have flow cytometry positive but virtual crossmatch negative
Mujtaba Zuhair
3 years ago
CDC crossmatch : The test is simple , and non expensive. But it had low sensitivity . The sensitivitycan be increased with the addition of anti human globulin.
False positive results due the presence of auto antibodies , and false negative results could be due to :
low antibody titer.
non complement fixing antibodies.
If the CDC cross match is positive and transplantation is done there is high risk of hyperacute rejection.
Flow cytometry crossmatch : It has better sensitivity than CDC crossmatch . but it’s more expensive and more time consuming. IT can detect non complement fixing antibodies and can detect low antibodies level.
CDC XM -ve , Flow cytometry crossmatch +ve patients had poor graft outcome when compared to CDC -ve , Flow crossmatch -ve patients.
Virtual crossmatch : By using Luminex technology , we can detect anti HLA antibody in the recipient serum and comparing it with the HLA of the donor, we can have virtual crossmatch. With the use of virtual crossmatch , the time to transplantation of highly sensitized is reduced and cold ischemia time also reduced.
In our center we have living donor transplantation only . we do CDC , if positive , the donor is excluded.
If cdc is negative , we proceed with Flow cytometry, and Luminex solid bead assy, if FCM XM, and anti HLA antibodies is negative we can proceed with Transplantation.
if the FCM XM is positive , we do plasmapheresis , and repeat the FCM XM if negative , we proceed with transplantation.
Doaa Elwasly
3 years ago
CDC technique:
It determines the presence of donor specifc antibodies in the serum of the recipient which is indicative of graft survival after transplantation.
It is done by isolating donorlymphocytes (cadaveric/ living). B and T cells are separately tested against serum from the recipient. The immunological response is mediated though classical pathway of the complement system. The complement is added to the mixed recipient serum and donor lymphocytes and cell lysis is observed.The percentage of lysed lymphocytes in the cell panel by complement activation or panel-reactive antibody represent the result.If more than 20% is considered positive.
Another way is titred cross match which is the more dilution needed to the recipient’s serum the more the immune response is.
Its wek points is the occurnce of false positive due to autoantibodies overcomed by DTT, where as false negative can occur due to low titers of
DSA.
Its advantage is being simple cost effective and its draw back is being less sensitive .
T cells express only class I antigens while
B cells express both.
-positive T cell and B cells cross match means presence of antibodies to HLA type I and II antigens
-positive B cell cross match means either DSAs to type II antigens alone or Low levels of DSAs to type 1 antigens.
– positive T cell crossmatch alone is usually due to technical error.
Flow cytometry:The donor lymphocytes are mixed with the recipient’s serum and are assessed by detectors in the impedence fow cytometer
If we have negative CDC result, a positive flow cytometry means the presence of either -Non-complement fxing antibodies -Non-HLA antibodies -Low-level antibodies
So it is usualy used in cross matching of sensitized patients to determine transplant possibility or the need of desensitization before transplantation to lower rejection possibility . Its advantage is being more sensitive and its disdvanatge is being suitable for living donors not cadaveric ones .
Solid phase antibody detection assays Enzyme-linked immunosorbent assay (ELISA): used to detect HLA in both bound and free forms . This method while being effective in detecting sensitization in transplant patients has been replaced by bead technology Bead technology (Luminex): improved the HLA antibody testing by the using of beads labeled with fluorescein
Quantitation of the result is quantified by Flow cytometry and Luminex method .
Its advantage is being most sensitive and avoids false positive results.
Its draw back is that single antigen bead assays detect both complement-fxing and noncomplement-fxing HLA antibodies so a graft can be denied without having clincaly insignificant variation .
The virtual crossmatch
Is done by comparison of the anti-HLA antibodies of the recipient to the donor HLA antigens using bead technology. It predicts the eventual crossmatch and can identify rapidly the suitable donor.
Mahmoud Rabie
3 years ago
CROSSMATCH TECHNIQUES INCLUDE:
1- Complement dependant cytotoxicity ( CDC ):
It is the most cost effective, available , and simple technique and detect only the complement fixing Abs.
It is done by mixing the recipient serum with T&B cells of the donor with adding of complement to detect the effect of complement activation after binding of DSAs with lymphocytes.
The result is estimated as percentage of lymphocytes destructed as a result of complement activation. Another method used to determine the result of the test is by serial double dilution of the serum of the recipient till being negative. The higher the dilution needed, the higher the positivity of the reaction.
False positive CDC may result due to the presence of IgM in the patient serum and this can be avoided by adding DTT.
False negative CDC may occur due to low level of complement or the antibodies are unable to activate the complement system.
** Interpretation of CDC:
1- positive CDC against both T&B means the antibodies are against both class I&II.
2- positive against B cell alone means either antibodies against class II alone or with low levels of antibodies against class I antigens.
3- positive against T cells alone means probable technical error.
High risk of acute rejection is associated with positive T cells crossmatch and the need for multiple PP treatment is needed to decrease this risk. But in case of persistent positivity post PP treatment , it is an absolute contraindication of TX.
However, positive B cells crossmatch is not highly associated with acute and hyperacute rejection as positive T cells crossmatch, but desensitization prior to transplantation should be done.
Negative B cells crossmatch is associated with better rates of graft survival.
2- Flowcytometry:
It is done by mixing the recipient serum with donor lymphocytes in presence of anti IgG fluorescein labelled Abs. Positive tests when the labelled Abs attach the lymphocyte and detected by flowcytometry.
Flowcytometry is more sensitive than CDC and can be used in sensitized patients. Negative CDC with positive flowcytometry indicate the presence of non complement fixing Abs, low titre of anti HLA Abs , or non HLA Abs.
3- Virtual crossmatch:
It is done using bead technology by detection of single or set of antigens on the surface of beads that the recipient formed Abs against and comparing the result with the donor specific antigens to determine the significant anti HLA antibodies.
It is the most sensitive technique.
Theepa Mariamutu
3 years ago
Crossmatch techniques in transplantations
Cell based assays
CDC
• lies in ability to determine the presence of DSA anti-HLA antibodies
In the recipient’s serum
• helping in determination of prognosis of graft survival after transplantation
Techniques
• isolating lymphocytes from donor
• B and T cells are separated tested against serum from recipient
• serum from recipient is added to donor lymphocytes (T or B cells) in the presence of complement
• complement is added to mixed recipient serum and donor lymphocyte and cell lysis of the lymphocytes.This humeral Immunological response is mediated thru activation of the complement system by classical pathway
• results is in percentage and >20% is considered as positive
• other methods is titred crossmatch – preformed by doubling dilutions of the recipient’s serum
• additional of AHG – increases the sensitivity of CDC as multiple AHGs bind to Single DSA
limitations
• false positive (due to autoantibodies)-overcome by adding Dithiothreitol (DTT) which help prevent IgM autoantibody mediated complement activation and allows only IgG
• negative results -happens when DSA too low or if antibodies are not the type that can activate complement
Advantages
• Functional test that involves cells and serum containing antibodies
• Still a foundation test – cheap, availability and simple
• Key role in preventing hyper acute rejection
• Detects only complement binding antibodies
Drawbacks
• Detect IgG and IgM antibodies, autoantibodies and non-HLA antibodies – irrelevant to transplant
Outcomes in positive T cell crossmatch results
• Positive T cell cross match – poor outcome and unacceptably high incidence of acute graft rejection
• Positive – multiple PP lead to reproducible desensitisation ands low acute and hyper acute graft rejection
• Persistent positive post desensitisation – absolute contraindication
Outcomes in positive B cell Crossmatch
• Positive B cell – does not equal to hum oral rejection
• Negative – better outcome
• False positive can be overcome by Luminex
Flow cytometry
Technique
• Donor lymphocytes are mixed with recipient’s serum and these bind to DSA.
• Quantified by detectors in the impedance flow cytometer
• Methods
o Measurement of fluorescence intensity as ratio of the control
o Serial dilutions of recipient’s serum are added to react with donor lymphocytes and the minimum dilution which yields a negative result give a measurable estimate
Advantages
• More sensitive than CDC crossmatch
• Able to detect low levels of IgG HLA
• Less inter-observer variability
Drawbacks
• Slow turnaround times – suitable for Living related transplant than deceased donor transplant
Interpretation of flow crossmatch results
• Negative CDC and positive Flow
o Non complement fixing antibodies
o Non-HLA antibodies
o Low level antibodies
CDC negative and positive T cell flow cytometry -poorer 5 year survival rates
Solid phase antibody detection
Enzyme-linked immunosorbent assay (ELISA)
• For detection of HLA antibodies in serum
• Modified assay uses HLA glycoprotein immobilised into microtiter wall
Techniques
• Recipient serum added and specific antibodies bind to epitopes that available
• Wash is performed and anti-IgG with a passenger reported alkaline phosphatase is added which combines with anti-HLA antibody
• After additional washing to remove unbound antibody , materials is added after dephosphorylased by reporter molecule undergoes a colour change
• Best in detecting sensitisation in transplant candidates but has been replaced by bead technology
Bead Technology (Luminex)
• Beads are impregnated with different ratios of 2 fluorochromes that results in a signal that unique to the specific bead
• Every bead may have one or more HLA molecule types that incorporated
Techniques
• Incubate the recipient serum with beads
• HLA antibodies with react with HLA antigens on the beads
• Bead are washed and incubated with a second antibody which usually is anti human IgG labelled with phycoerythrin
Advantages
• most sensitive for detecting DSA for HLA antibodies
• enhanced sensitivity leads to improved rates of success in subsequent transplantations which allowed avoidance of HLA markers on subsequent grafts
• allow identification of all HLA alleles which antibodies harboured in recipient
• avoid false positive binding to non HLA antigens and avoid confusion pertaining class of HLA that are detected
Drawbacks
• when CDC and Flow crossmatch is negative, positive make it suspicious
• low level antibodies remains a debatable topic
• detect both complement -fixing and non – complement fixing HLA antibodies
• interference by IgM, incomplete library of antigens in bead sets, variability of HLA density on the beads
In our centre, we do both CDC and FCXM as a screening for every kidney transplantation recipient. If CDC/ FCXM found to be positive, the immunology lab will automatically proceed with Luminex to identify mean fluorescence intensity (MFI). The cost is an issue but the responsibility is on government as it’s allocated by ministry of health. Patient does not required to pay for the crossmatch. The immunology lab will take the responsibility to decide to proceed to Luminex or not based on CDC/FCXM.
Ibrahim Omar
3 years ago
1- Summary :
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.
A- 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.
B- 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.
C- ELISA :
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.
D- Bead technology : it has replaced ELISA
2- Regarding reflection on my practise, I will adhere to the local polcies then negotiate with senior colleagues about trying new techniques in at least studying other than considering the clinical significance of these non-specific and low titres of DSA.
Weam Elnazer
3 years ago
Advantages of complement-dependent cytotoxicity:
It is a functional test that uses cells and antibody serum. The availability, affordability, and simplicity of this test make it the cornerstone of crossmatch.
It has helped avoid hyperacute rejection as transplant immunology has evolved.
It exclusively finds complement binding antibodies.
It is less sensitive than recent tests.
It detects IgM and IgG antibodies, autoantibodies, and non-HLA antibodies against non-transplantable antigens.
Flow cytometry has advantages over CDC crossmatch. It has lower inter-observer variability and detects fewer IgG HLA antibodies.
Cons: Slow turnaround times made this approach more appropriate for living donors than cadavers.
ADVANTAGES OF SOLID PHASE ASSAYS All approaches for identifying donor-specific HLA antibodies are sensitive.
Detection of pre-sensitization from earlier grafts allows avoidance of those HLA markers on subsequent grafts.
They avoid antibody binding to non-HLA antigens and misunderstanding about the HLA class discovered (CDC matching overlaps T and B cell matching, T with Class I and B with Class I and II).
Cons: A positive assay with a negative CDC/flow crossmatch is unclear. Is low-level antibodies against minor antigens relevant.
Single antigen bead tests identify HLA antibodies that are complement-fixing or not. These findings may exclude a prospective recipient from receiving a graft. 3. Other limitations include IgM interference, a lack of antigens in the bead sets, and variable HLA density.
Mohamed Fouad
3 years ago
The main target for cross match techniques is to identify the recipients who have anti HLA antibodies against a specific donor (Donor specific antibodies) and they have a higher chance for hyperacute rejection as result of the presence preformed antibodies. DSAs are commonly generated by previous transplantation, frequent blood transfusion and pregnancy.
Different cross match techniques
I-Cell based assays
1-CDC technique: Complement dependent lymphocytotoxicity crossmatching: Mixing recipient serum with donor lymphocytes (living and cadaveric) in presence of complement. The donor B and T cells are separately tested against serum from the recipient.
Negative cross match that means there is no anti HLA antibodies (no cell lysis). Positive cross match that means there is anti HLA antibodies (more than 20 % cell lysis).
Addition of anti-human immunoglobulin (AHG) helps increase the sensitivity of CDC
-The drawback of CDC is include : False positive results which is due to IgM autoantibodies in the recipient serum. False negative results due to either there is very low levels of DSAs to result in activation of the complement cascade or the type of DSAs not causing complement activation.
A positive T cell cross match results in poor outcome and unacceptably high incidence of acute graftt rejection. a persistent positive T cell crossmatch result post desensitization is according to current guidelines, an absolute contraindication to renal transplant.
A positive B cell crossmatch is not as consistently associated with ABMR as positive T cell crossmatches. Although a negative B cell crossmatch is associated with better outcomes.
2- Flow cytometry: the donor lymphocytes are mixed with the recipient’s serum. in the presence of anti-IgG fluorescein-labelled antibodies. These bind to the donor specific antibodies and are quantified by detectors in the impedance flow cytometer. A negative CDC result and a positive flow cytometry indicating that there is non complement fixing antibodies, Non-HLA antibodies or Low level antibodies.
The drawback of the Flow cytometry: Time consuming that mean this technique is suitable for living donors scenarios rather than for cadaveric donors.
II) Solid phase antibody detection assays
1-Enzyme-linked immunosorbent assay (ELISA): Used for the detection of HLA antibodies in serum. Technique: The recipient serum is added and specific antibodies bind the epitopes available. A wash is performed and anti-IgG with a passenger reported molecule (alkaline phosphatase) is added that combines with the anti-HLA antibody. After further washing to remove unbound antibody, a substrate is added which after dephosphorylation by the reporter molecule undergoes a colour change. This method has been replaced by bead technology.
2- Bead technology (Luminex): Technique: Incubation of recipient serum with the beads. HLA antibodies of the serum will react with HLA antigens on the bead. the beads are washed and incubated with a second antibody, usually anti-human IgG labelled with phycoerythrin.
Quantitation of the result This is achieved by two methods: -Flow cytometry – Luminex method
The low positivity cut-off of 300 MFI (mean-fluorescence intensity) is predictive of graft survival and some used the higher cut-off of 1,000 MFI.
3-The virtual crossmatch: The Wait Is Over
The Physical cross matching is costly and time consuming.
This technique is based on the comparison between donor and recipients without requiring viable donor cells, depending on complete HLA typing of the donor and anti-HLA antibodies of the recipient. The use of the virtual crossmatch as a final decision to proceed with transplant across transplant centres can lead to decreased cold ischemia time and lower DGF rates without increased risk of rejection. These cross match results must be updated every 3 months especially in patients on cadaveric transplant list.
Mohamad Habli
3 years ago
Summary of the various crossmatch techniques
Cross match between donor lymphocytes and recipient serum is an essential step in identifying the risk of rejection and possible outcomes. The response of host immunity to the allograft occurs through two major mechanisms: T cell-mediated (cellular) responses and antibody-mediated (humoral) responses. This classification into 2 entities oversimplify the response as we already understand that the function of T and B lymphocytes are interconnected and activation of one will activate the other.
Complement dependent cytotoxic cross match:
In this technique, lymphocytes from the donor are added to recipipient’s serum which may contain antibodies against the host cells. but yo identify against which MHC (HLA) the antibodies are direct, there is initially separation of donor’s lymphocytes into CD 19+ <–>B and CD 3= <–>T cells. after mixing the recipients serum with the separated lymphocytes, complement is added with viability dye. so if there are DSA against MHC I , B and T lymphocytes will undergo lysis, If only against MHC II, only B cells will undergo lysis, if absent -no DSAs are present.
Flow cytometry crossmatch : The flow cytometry crossmatch assay is capable of detecting IgG DSA without the need iof complement activation.in this technoque recipient serum is added to the donor lymphocytes, followed by the addition of a secondary fluorochrome-conjugated antibody that detects IgG. In this assay, B and T cells are not separated. Rather, additional fluorochrome-conjugated antibodies are added to distinguish between them. Samples are analyzed using flow cytometer. The results are presented as median fluorescence intensity (MFI).
Solid phase antibody detection assays include Enzyme-linked immunosorbent assay (ELISA) and Bead based techniques. ELISA is efective in detecting sensitization in transplant candidates but has been replaced by bead technology.
In Single Antigen Bead-based screening assays, color-coded microspheres coated with HLA are used to identify both HLA class I and II antibodies in recipient serum. The Luminex anti-HLA antibody detection assay is reportedly more sensitive and specific than either the CDC or flow cytometric crossmatches. Using single-antigen technology, the Luminex technology can predict a patient’s sensitization to particular HLA types before transplantation without performing a physical CDC or flow cytometric crossmatch which is termed as virtual crossmatch.
So The term “virtual” crossmatch refers to a process in which the results of anti-HLA screening results and the HLA type of the donor and recipient are compared to look for mismatched antigens and against what the recipient has preformed antibodies.
Reflect on my practice
Actually, I had no experience in kidney transplantation since fellowship. In the hospital where I used to work, transplant programs not available. But what I remember from the few living donor kidney transplantation cases during my fellowship, that we did complement dependent cytotoxic crossmatch as first step. If the CDC is negative and DSA ( I couldn’t recall which method was used for DSA identification) are negative, no further evaluation was done. If the patient has history of sensitization, then flow cytometry cross match was performed for better identification of target HLA antibodies.
Ala Ali
Admin
3 years ago
Dear all, the most critical issue here is reflections on your current practice, how it differs from other practice areas, and why? For example, is it the logistics, finance, expertise, or other factors that affect your practice decision?
MICHAEL Farag
3 years ago
Aim of cross-match techniques:
To identify the antibodies in the donor serum to one or many HLA (human leukocyte antigens). These are referred to as DSAs (donor-specific antibodies) to prevent hyperacute rejection.
DSAs are commonly generated by: (i) Blood transfusion (ii) Previous transplantation
(iii) Pregnancy.
Crossmatch techniques: I) Cell-based assays 1- Complement dependent cytotoxicity (CDC) technique:
CDC crossmatch (A) Serum from the recipient is added to donor lymphocytes (T or B) in the presence of complement.
(B) Negative test when donor-specific anti-HLA antibodies are absent and complement activation does not occur.
(C) Positive test when donor-specific antibodies bind to lymphocytes, activate complement and cause cell lysis.
Twenty percent is usually taken as the minimum cut-off for a positive result thus functioning as a qualitative and a semi-quantitative estimate of the strength of the reaction.
Limitations of CDC: false positive and negative results
Outcomes in positive T-cell crossmatch results: renal transplant performed when the recipient has a positive T cell cross match results in poor outcome and unacceptably high incidence of acute graft rejection. a persistent positive T cell crossmatch result post desensitization is according to current guidelines, an absolute contraindication to renal transplant.
Outcomes in positive B-cell crossmatch results: A positive B cell crossmatch is not as consistently associated with humoral rejection as positive T cell crossmatches. Although a negative B cell crossmatch is associated with better outcomes, the test’s high false positive rate
makes its significance uncertain. This can be overcome by concurrent Luminex testing for DSAs which increases its specificity.
Advantages: A basic and durable test still used as the foundation of crossmatch due to availability, cost and simplicity.
It detects only complement binding antibodies.
Drawbacks: It is less sensitive than other newer assays. It detects IgM and IgG antibodies simultaneously, autoantibodies and non-HLA antibodies against antigens that are irrelevant as far as the transplant is concerned
2- Flow cytometry:
(A) Serum from the recipient is added to donor lymphocytes (T or B) in the presence of anti-IgG
fluorescein-labelled antibodies. (B) Negative test when donor specific antibodies are absent and binding does not occur. (C) Positive test when donor-specific antibodies bind to lymphocytes
that are detected by flow cytometry once the anti-IgG fluorescein labelled antibodies tag the lymphocytes.
Hence, the value of this test’s sensitivity lies in using it for cross matching of sensitized patients who have an inherently higher risk of acute graft rejection to determine transplant feasibility or need for desensitization protocols prior to transplant
Advantages: More sensitive than CDC crossmatch. It can detect lesser levels of IgG HLA antibodies and has less inter-observer variability.
Drawbacks: Slow turnaround times meant this technique was suitable for living donors scenarios rather than for cadaveric donors.
II) Solid phase antibody detection assays 1)Enzyme-linked immunosorbent assay (ELISA):
The modified assay uses HLA glycoprotein immobilized into mictrotiter wells. The recipient serum is added and specific antibodies bind the epitopes available. A wash is performed and anti-IgG with a passenger reported molecule (alkaline phosphatase) is added that combines with the anti-HLA antibody. After further washing to remove unbound antibody, a substrate is added which after dephosphorylation by the reporter molecule undergoes a color change.
This method while effective in detecting sensitization in transplant candidates has been replaced by bead technology.
2) Bead technology (Luminex):
The basic steps involve first, incubation of recipient serum with the beads. HLA antibodies of the serum will react with HLA antigens on the bead. The beads are washed and incubated with a second antibody; usually anti-human IgG labeled with phycoerythrin.
Advantages: They are the most sensitive of all the techniques for detecting donor specific HLA antibodies.
Enhanced sensitivity has allowed improved rates of success in retransplants where detection of pre-sensitization from previous grafts allow the avoidance of those HLA markers on subsequent grafts.
They allow the identification of all HLA alleles for which the recipient harbors antibodies.
They avoid false positives of antibody binding to non-HLA antigens and eliminates confusion regarding the class of HLA that are detected (CDC matching has an overlap between T and B cell matching, T with Class I and B with both class I and II).
Drawbacks: The interpretation of a positive assay in the presence of negative CDC/flow crossmatch is ambiguous.
The relevance of low-level antibodies to low significance antigens is debatable.
Single antigen bead assays detect both complement-fixing and non-complement-fixing HLA antibodies. On the basis of these results, prospective recipient may be denied a graft without established clinical significance. Other limitations include interference by IgM,
incomplete library of antigens in the bead sets, variability of HLA density on the beads
III) The virtual crossmatch (A) Serum from the recipient is added to synthetic beads with either a set of antigens or a single antigen, each bead can be identified by an independent dye signature.
(B) Anti-HLA antibodies if present will bind to the specific bead.
(C) A detector antibody will then bind and sequester a reporter dye.
(D) Beads can be checked for the reporter dye using a laser beam, this builds a profile of the antibodies present in the recipient that can be compared with the HLA construct of a potential donor thus predicting the result of crossmatch.
Alternatively, a microsphere or bead may be coated with multiple HLA antigens – this iteration of the test allows for more efficient screening. HLA antibodies bind specifically with the bead and are detected by isotype specific antibodies using flow cytometry. This method allows for the sensitivity of flow crossmatch combined with identifying specific antibody.
My opinion Complement dependent cytotoxicity remains the mainstay of pretransplant screening for HLA specific antibodies. Newer methods have helped significantly increase the sensitivity and specificity of detecting antibodies of significance in acute humoral graft rejection in the
recipient when interpreted in the light of the patient’s clinical picture and CDC crossmatch testing whilst accounting for inter-lab variations and technical errors
So in some patients we need to use combination of those techniques to take the right decision
Huda Al-Taee
3 years ago
Cell based techniques:
CDC technique:
first developed in 1960
determine the presence of DSA
the technique based on mixing donor lymphocytes( B and T cells) with the recipient serum, complement is added to the mix and cell lysis is observed. Lysis of more than 20% of the cells is considered as a positive result.
addition of anti-human immunoglobulin increases the sensitivity of CDC.
titred crossmatch is a method at which crossmatching of donor lymphocytes is performed using serial dilutions of the recipient”s serum, the higher the dilution required to get a negative result, the greater the strength of immune reaction.
Limitation of this technique:
false positive result: due to autoantibodies in the recipient serum( overcomed by the addition of DDT).
false negative result: when DSA level is low
non complement fixing ab
T cell express only class I antigen while B cells express both class I & II
positive T cell crossmatch: results in ( after ruling out the presence of autoantibodies) high incidence of acute allograft rejection, a persistent positive crossmatch after desensitization is considered as contraindication to transplantation.
positive B cell crossmatch: high false positive rate and this can be overcomed by luminex testing for DSA, the significance of anti-HLA class II ab is of less importance for acute/ hyperacute rejection.
Flow cytometry:
first detected in the early eighties
the donor lymphocytes mixed with the recipient serum, binding to donor specific antibodies and quantification by detectors in the impedence flow cytometer will happen.
two methods of quantification are used:
I. measurement of florescence intensity( channel shift)
II. serial dilution of recipient serum
In CDC negative result, a positive flow cytometry result indicates either:
non complement fixing ab
low level ab
non-HLA ab
so the importance of flow cytometry is to test sensitized patients.
solid phase antibody detection assays:
ELISA: the recipient serum and specific ab.s bind the epitopes available. A wash is performed and anti IgG with a passenger reported molecule is added that combine with anti HLA antibody.
this method has been replaced by bead technology.
Bead technology( Luminex):
patient serum incubated with the beads, HLA ab will react with HLA ag on the bead, the beads are washed and incubated in another ab. usually antihuman IgG labeled with phycoerythrin.
this method can be used for screening for antibodies or testing against set of alleles or as single antigen beads.
the result is quantified either through flow cytometry to measure channel shift or through Luminex method to calculate the MFI.
The main significance of the test is when CDC is negative, and this is due to either non HLA ab.s or non complement fixing ab.s or low level ab.s also low cut off values.
Virtual crossmatch:
based on the comparison of the anti HLA ab of the recipient to the donor HLA ag using bead technology.
allow rapid identification of a suitable donor
recipient serum is added to a synthetic beads with either a set of antigen or single antigen, Anti HLA antibodies if present will bind to the specific bead and this reaction can be detected using a laser beam.
Sherif Yusuf
3 years ago
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
– It is highly specific, a positive cross match using CDC indicating the presence of clinically significant complement fixing antibodies thus if CDC positive donor should be excluded.
– This test has low sensitivity, human anti- goblin can be added before complement that bind to Donor HLA antibodies, to increase sites for binding to complement and increase its sensitivity
– False positive results can occur due to the presence of clinically non significant autoimmune IgM antibodies which can be eliminated either by heating or adding reducing agent.
– Rituximab can cause false positive CDC.
False negative result can occur due to non complement fixing, non HLA or due to low level of HLA antibodies
– T cell express only HLA class I while B cell (as it act as APC) express HLA class I and II, so if antibodies are directed to only class II or if DSA directed to HLA class II are week it will give positive B and negative T cell cross match, on the other hand if DSA are directed to both class I, II it will give positive B and T cross match, positive T and negative B cross match indicates technical error.
– Positive T cell cross match has the worst prognosis and donor should be excluded
– Positive B cell cross match carry poor prognosis when compared to negative CDC cross match but better that positive T cell cross match , positive B cell cross match commonly occur due to false positive result (not due to DSA), but true positive can occur due to DSA against class II HLA, luminex is done at this situation to confirm the presence of DSA against class II if present desensitization can be done.
– CDC is used either in determining compatibility between donor and recipient or in determining sensitization in case of PRA
– CDC give semiquantitative assumption of the strength of DSA through the following methods :
⦁ Score is given according to percentage of ruptured lymphocytes from 2 (20% of cells ruptured) to 8 (indicating strongest reaction), below 2 the test is negative
⦁ Titred crossmatch method using serial dilution of recipients serum, so the higher the dilution needed to render the test negative the greater the strength of DSA.
– CDC should be repeated just before transplant since antibodies may be formed after antigen exposure such as blood transfusion or pregnancy
II- Flowcytometry
– It involves incubating donors lymphocytes with recipient serum , then add fluorescein labeled anti- globulin that will bind to DSA present in the recipient serum that are attached to donor lymphocytes
-FCM is used either in determining compatibility between donor and recipient or in determining sensitization in case of cPRA
– FCM give quantitative assessment of the strength of DSA through the following methods :
⦁ Measurement of fluorescence intensity and compare it to control (channel shift)
⦁ Titred crossmatch method using serial dilution of recipients serum, so determine the minimum dilution needed to render the test negative this give us the strength of DSA
– More sensitive than CDC as it can detect non complement fixing AB, non HLA antibodies and low level DSA, so CDC – FCM + cross match indicates the presence of one of the previous mentioned ABS.
– Positive T cell FLC (in highly sensitized patients only) is associated with poor graft survival, so it has a very big rule in sensitized recipients
– False positive results can occur due to binding of non specific IgG antibody to B cell, also rituximab was found to cause false positive results
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 fourescein, each bead either have single (SAB) or multiple HLA molecule then anti human globulin labelled with phycoirythrin is added.
– Very sensitive very specific, can detect complement and non complement fixing antibodies, low level DSA, non HLA antibodies including those against MiHC antigens
– Luminex give quantitative assessment of the strength of DSA through the following methods
⦁ Flow cytometry method, that measure fuorescence intensity and compare it to control (median channel shift- MCS)
⦁ Luminex method, that calculate the degree of fluorescence (median fluorescence intensity -MFI) through the use of 2 lasers to excite the fluorochrome of the beed and the phycoerythrin bound to the antibody.
– The major obstacle is usually that it is expensive
– False positive result can occur due to denatured antigens attached to beeds
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
– DSA may change over time due to sensitization from blood transfusion or pregnancy so luminex SAB should be repeated every 3 m
In practice my experience is only in living related donors
we do both CDC and FCM cross match in all transplant recipients
⦁ If CDC positive donor is excluded
⦁ If FCM positive and CDC negative patient is assesd for desensitization using data from luminex teqnique
⦁ If CDC negative, FCM negative we proceed for transplantation
Please summarise the various crossmatch techniques.
Please reflect on your practice if possible.
1-CELL BASED ASSAY
CDC technique ( complement dependent cytotoxicity ) this type of assay depend on mixing the recipient sera with the donor lymophoctes ( T, B Cells separatly ) in the precence of complement .
postive CDC indicate presnce of donor sepcific antiHLA ab that induce cell lysis due to complement activation, this serollogy method have been used since 1960 , developed by terasaki and patel , the result interpretaion by the % of PRA with minimum cutoff value of 20% for postive results
titred cross-match by using serial double dilution , the higher the dilution will help to quantify the strenght of the reaction and give negative results
limitations of the CDC crossmatch :
1- false postive results with autoAB like IgM mediated complement activation
2-False negative results for non-HLA- AB or when the DSA presnet in very low titer
also should keep in mind that the recpient AB may vary within time due to possible new antigen exposure espcialy with recent blood transfusion , , pregnancy so we should repeat the CDC crososmatch and depend the most recent one
postive T cell CDC crossmatch is absolute containdications to perform the transplantation
2- Flowcytometry crossmatch
this technique started in the early eighties by using molecular genetic testing also depend on mixing the recipent sera with panle of donor lymphocytes ( T cell , B cell),in the presence of anti IgG fouricence labelled antibodies and are quantified by detectors in the impendece flow cytometer
two methods used for Quantifications of DSAs
A Meauremnet of the flourcence intesity as a ratio of control ( MFI)
b-seria dilutions and by esitimated the minimum dilution which give negtaive results
its more sensitive and specific assay for detection of noncomplement fixing AB , non-HLA AB low level DSA so its prefered test for sensitized patients and help to decide about the need of desensitization protocal prior to transplant .
3- Solid phase antibody detection assay
a- enzyme linked immunosorbent assay(ELIZA), the use of modified ELIZA test for dectection of sepcific anti IgG anti HLA ab in recipient serum at the level of epitopes
b single bead antigen ( luminex )
the basic of bead technolgy which started since ninties , this test involve incubation of recipient serum with the beads , then the HLA -ab of the recipient will react with the HLA antigens on the beads , the beads are washed and incubated wtih a second anti human IgG labeled with phycoerthrin, whic bound to the AB ,the result interpretaion as PRA and calculated by mean flourcence intensity MFI , help in identication of very sepcific ant iHLA antigens ab in sensitized patient even with low MFI value , the cut off value of MFI not standadrazied among laberotieris
the single antigen bead test( SAB ) also help in monitoring DSA level pre and post transplantation for sensitazed recipients , its use limited due to the cost and avialablity
in SQUH its university tertiary centre we have our genetic lab, that doing all the HLA TYPING for LD program , we dont have DD program and we still using CDC Assay , we dont have flowcytometry and we just introduce the SABlumenix assay recently for DSA monitoring for sensitazed kidney transplantaionwith MFI cut off value for postive test of1000 and above to decide for desensatization protocal
we have more commercial kidney transplant recipients with out information about their donors HLA status whic is very challenging to us , we adopt SAB – lumenix assay as best method to monitor for DSA after transplantaion .
Good and transparent reflections on practice, thank you
Riham Marzouk
3 years ago
Various techniques of cross match: A- Cell based assays
1- CDC /ADCC ( complement dependent cytotoxicity) : T and B cells of the donor (living or deceased) are isolated and separated and tested against serum of the recipient, complement is added to detect if cascade will be activated or not ; more than 20% cell lysis will be considered positive CM.
Titred cross match : done for more detection of antibodies through serial doubling dilution of the recipient serum. High dilution required for negative result. More intense immune response is present with high dilution.
False positive can happened because of presence of autoantibodies IgM in the recipient serum, can be overcome by addition of DTT (dithiothretol) which eliminate IgM complement cascade activation leaving only IgG which are DSA.
Also can be false negative ; in presence of non complement activation antibodies or low titre of DSA.
There are T and B cell cross match , T cell express HLA I, B cell express HLA I and II, doing CM against both of them separately can specify the type of antibody to which antigen.
Of course positive T cell cross match will negatively affect graft function and survival , desensitization protocol should be applied before transplantation, persistent positive T cell CM is an absolute contraindication to transplantation.
Positive T and B cell CM indicates presence of antibodies against class II and I.
Positive B cell CM alone is insignificant because of B cell viability, so should be combined with Luminex detection of DSA.
CDC technique is cheap and available, used as preventive measure against hyperacute rejection, but it is less sensitive.
2- Flow cytometry CM donor lymphocytes with recipient serum, can be quantified by:
Fluorescence intensity or serial dilution of recipient serum
Positive FCXM (flow cytometry) with negative CDC indicates :
non HLA antibodies
Non complement activation antibodies
Low titre of DSA
It is more sensitive than CDC , used more in living donor. B- Solid phase antibody detection assays: it is the most sensitive technique because it detects antibodies against all HLA alleles, this will improve graft survival and will help in deciding pretransplant desensitization protocol.
a- ELISA indicated by color changes
b- Bead technology (Luminex) beads labelled with fluoresceine each bead has one or more type of HLA molecule incorporated .
It may used as screening, PRA (panel reactive antibody) , SAB (single antigen bead).
c- Virtual CM basic concept is comparison between recipient anti HLA antibodies with donor HLA antigens, it uses bead technology.
SAB technique can detect complement and non complement fixing antibody
Amit Sharma
3 years ago
Crossmatches are used to identify the risk of graft rejection in a prospective transplant recipient.
Please summarise the various crossmatch techniques.
Crossmatch techniques can be divided into cell based assays and solid phase assays.
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 acivating 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): 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 or a titred value of dilution required to get a negative result (for quantification).
B) Solid Phase antibody detection assays:
1) 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) 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.
2. Please reflect on your practice if possible.
In our practice, which is mainly living donor transplant setting, CDC is the first test to be done. If there is no history of sensitization (prior transplant, pregnancy, blood transfusion), then we proceed with transplant without any further tests, except in case of spousal (husband to wife) donation where we advise a single antigen bead (Luminex) assay. But due to the cost involved, it is common that the patients refuse to undergo this test.
In patient with sensitization history, even if CDC crossmatch is negative, we get a single antigen bead (Luminex) assay for quantification of DSAs and proceed accordingly,
Excellent reflection on your practice Amit This what we need from colleagues. We have to make evidence based medicine viable by looking at our resources, rather suggesting treatment plan which is not practical.
Positive cross match (both T and B cells are positive), but no DSA
When HLA titres are high and prozone phenomenon occurs-
high titre antibodies lead to complement activation and deposition of complement proteins on the bead which then prevents HLA antibody from binding to the HLA antigen on the bead.
same occurs with IgM antibodies or other serum factors binding
epitope sharing
different HLA antigens on different beads
share common antibody binding epitopes leading to
binding of an anti‑HLA antibody to more than one bead
with consequent reduction in the MFI on a single bead.usually MFI>1000 is considered significant.
during the process of attachment of HLA molecule,the bead, immunologically relevant epitope, can become hidden and un-accessible to antibody binding.
in our centre it was not a high volume centre for transplant with about 25 transplants per year.also patients affordability was not great so we never went beyond CDC crossmatch with transplant if negative and rejection if positive.
However personally I would do CDC and flow cross match in all and if flow crossmatch positive then DSA,as that would dictate decisions about induction and desensitization strategies if needed,unless patient is not willing to take the additional cost.CDC positive then avoid transplant .
Cell based assays
CDC technique:
Determine the presence of donor specifc anti- HLA antibodies in the serum of the recipient.
Result is given in terms of percentage of lymphocytes in the cell panel which has undergone lysis as a result of complement activation or panel-reactive antibody (% PRA) . Twenty percent is usually taken as the minimum cut-of for a positive result.
AHG helps increase the sensitivity of CDC.
False positive- due to autoantibody
False negative-
DSA levels too low
If the antibody is not complement fixing.
Class I antigens (HLA-A, B,C) are expressed on all nucleated cells
class II antigens HLA-DR,DP, DQ) are expressed on antigen presenting cells like macrophages
and dendritic cells.
Vascular endothelial cells of the transplant graft express both these antigens.
T cells express only class I antigens
B cells express both.
a positive T cell and B cells cross match would indicate presence of antibodies to HLA type I and II antigens
a positive B cell cross match could indicate either (i) DSAs to type II antigens alone or (ii)
Low levels of DSAs to type 1 antigens.
A positive T cell crossmatch alone is usually due to technical error.
positive T cell crossmatch result is an absolute contraindication to renal transplant.
negative B cell crossmatch is associated with better outcomes, the test’s high false positive rate
makes its signifcance uncertain. Concurrent Luminex testing for DSAs can increase its specificity.
Drawbacks:
less sensitive than other newer assays.
It detects IgM and IgG antibodies
autoantibodies
and non-HLA antibodies
(2) Flow cytometry
Is more sensitive than CDC test
In the light of a negative CDC result, a positive fow cytometry indicates –
Non-complement fxing antibodies
Non-HLA antibodies
Low-level antibodies
CDC crossmatch negative patients who had positive T-cell fow cytometry results had signifcantly poorer absolute 5 year graft survival rates that those who were both CDC and flow cytometry crossmatch negative.
Solid phase antibody detection assays:
1)Enzyme-linked immunosorbent assay (ELISA)
2) Bead technology (Luminex)
Beads impregnated with HLA antigens are used
A) Screening beads – contains both I and II antigens
B) Set – contains either I or II antigens
C) Single antigen beads- contains single antigens.
Quantified by flow cytometry or Luminex method
CDC negative and a positive DSA could be due to due to a low level antibody, a non-HLA antibody or a non-complement binding antibody.
Drawbacks:
The interpretation of a positive assay in the presence of negative CDC/fow crossmatch is ambiguous. The relevance of low level antibodies to low signifcance antigens is debatable.
Single antigen bead assays detect both complement-fxing and non complement-fxing HLA antibodies.
interference by IgM
incomplete library of antigens in the bead sets
variability of HLA density on the beads.
Positive cross match with negative DSA :
Due to presence of other non HLA such as MICA, angiotensin II type 1 receptor, H-Y antigen, antibodies against graft endothelium, antibodies against nsSNPs, and antibodies against LIMS1 proteins. Also presence of IgM antibody in the context of autoimmune disease or using rituximab in the CDC test.absence of rare antigen among the HLA antigen profile of bead assays and so DSA will not be detected.
Crossmatch techniques can be divided into cell based assays and solid phase assays.
A. Cell-based assays: These are of 2 types.
1) CDC technique:
a mixture of recipient serum and donor lymphocytes (T and B cells separately) and complement is added , If DSA is present, it will bind with the lymphocytes, this will activate complement and cell lysis will occur , giving a positive result.
To increase its sensitivity, anti human immunoglobulin (AHG) can be added .
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).
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.
False positive :
Presence of autoantibodies
Low level of DSA
presence of non-HLA acivating antibodies.
We can add DTT (ditheothreitol) inhibits IgM mediated complement activation.
2) Flowcytometry Crossmatch (FCXM):
By adding fluorescein labelled anti IgG antibody 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 can detect low level of DSA, non-HLA antibodies and non-complement binding antibodies.
B) Solid Phase antibody detection assays:
1) ELISA:
It is a 3 step procedure :
* recipient serum is added to HLA glycoprotein labelled microtiter wells
*after giving a wash, anti IgG with a passenger reporter molecule (alkaline phosphatase) is added followed by a wash.
* addition of a substrate which undergoes a color change if antibodies are present.
2) 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.
Dear All
How would you explain the following result
Positive cross match (both T and B cells are positive), but no DSA.
There is a present for the correct answers. I will announce it On Saturday morning
It is Recent Advances and Clinical Outcomes of Kidney Transplantation
Volume 1 and 2 2020 EDITION, Original copy.
presence of non-HLA antibodies like angiotensin II type 1 receptor antibody (IgG) in the recipient.
can be positive crossmatch in case of donor treated by rituximab recently.
history of prior desensitization of the patient.
very low level of DSA not detected by SAB single antigen bead because of complement interference.
B cell positive alone is considered false in more than 50 % of cases, and can not be relied on, so if no DSA ….consider it false.
WILLIAM R MULLEY, JOHN KANELLIS. Understanding crossmatch testing in organ transplantation: A case-based guide for the general nephrologist. 19 October 2010
Judith Desoutter,1 Marie-Joëlle Apithy,1 Ségolène Bartczak,1 and Nicolas Guillaume. False Positive B-Cells Crossmatch after Prior Rituximab Exposure of the Kidney Donor. Hindawi. Case reports in transplantation.2016.
Winner
Well done
Non-HLA antibodies
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
How would you explain the following result
Positive cross match (both T and B cells are positive), but no DSA.
we should be very carful in interpreting this results as still could be false postive results if done by the CDC assay either due to techincal erro so better to repeat by using DTT assayby fruther dilutions to cleave out the auto-AB by reduces the disulfide bonds in IgM ,also we can do auto-crossmatch test in which the recipient serum is crossmatched against self- lymphocytes not the donor thereby preventing IgM antibodies from generating a positive result. IgM antibodies are generally regarded as having no pathological significance in transplantation.
if this crossmatch results done by flowcytomtery assay
so still could be false postive due to the presence of following :
1- non- HLA AB
2- non-complement fixing AB
3- low level of the DSA which calculated by MFI with the variable cutoff value for postive results between different laberotories usualy postive flow cytometry in senesitzed recipient more senesitive and specific and some centres even with low values will go for desensitazation or allocated for pair donation program .
Winner
Well done
Non-HLA antibodies
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
A positive cross match in the absence of DSA needs to be evaluated properly.
History:
Any history of immunological disease like SLE
Any history of injection rituximab (but only B cell cross match should be positive)
Any history of desensitization.
Method of crossmatch:
False positive CDC cross match can be seen in presence of autoantibodies. Hence DTT should be used to remove autoantibody related false positives.
The test kit used for DSA detection, if is not representative of the HLA prevalent in the community, it is possible that the single antigen bead test might miss the antibodies present in the recipient serum due to lack of corresponding HLA antigen on the beads, giving a false negative DSA report.
Positive cross match (both T and B cells are positive), but no DSA.
This result can be seen in lupus patients, usually crossmatching by CDC method will be positive( false positive) due to the presence of IgM antibodies, but when we do flow crossmatch the result will be negative and DSA testing by solid phase assay will be negative.
Another explanation is the presence of non-HLA antibodies.
Reference:
Chughtai Sh. A., Sharma A.K., Halawa A. Interpretation of Crossmatch reports in a patient with Lupus Nephritis. Arch Organ Transplant 2(1): 023-029.
Winner
Well done
Non-HLA antibodies
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
The result should be interpreted with caution.
This can be due to autoantibodies or due to non HLA antibodies .
Or due to incomplete typing of the HLA of the donor HLA C ,
or HLA antibody to rare HLA antigen that not expressed in the Luminex SAB panel
Reference :
Carrie A. Schinstock, MD, Manish J. Gandhi, MD, and Mark D. Stegall, MD
Interpreting Anti-HLA Antibody Testing Data: A Practical Guide for Physicians
Transplantation 2016;100: 1619–1628DOI: 10.1097/TP.0000000000001203
Winner
Well done
Non-HLA antibodies
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Dear Dr Ahmed,
Regarding the question
“How would you explain the following result
Positive crossmatch (both T and B cells are positive), but no DSA.”
Possible causes include autoantibodies, non- HLA antibodies, IgM antibodies or history of ATG or Alemtuzumab use (1).
I found a very comprehensive and organized table that collected causes of the positive crossmatch and their clinical interpretation, and I attached a screenshot of it in the attached photo (1). The table will guide us to differentiate each of the above mentioned possible causes of the proposed scenario.
References:
1) A. Chandraker et al. (eds.), Core Concepts in Renal Transplantation, DOI 10.1007/978-1-4614-0008-0_2, © Springer Science+Business Media, LLC 2012
Winner
Well done
Non-HLA antibodies
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
As crossmatch may have false-positive results due to autoantibodşes and those of IgM type.
DDT will resolve the problem of IgM. Despite all measures some patients still have false-positive CDC crossmatch and or Flowcytometry. This is mainly due to autoantibodies. Some therapies like rituximab will induce complement-dependent cytotoxicity thereby interfering with CDC. Also, it may be recognized by anti-human antibodies used in the flow cytometry technique.
Non-HLA antibodies
You are right. Non-HLA antibodies such as drugs (Rituximab) or MICA MICB, anti-endothelial antibodies, Angiotensin II receptors antibodies.
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Winner
Please think of common things first.
There is a reported case of positive flow cytometry crossmatch (FCXM) in a renal transplant candidate following a recent COVID-19 vaccination. Interestingly, the donor-specific antibody (DSA) was not detectable with the sensitive solid-phase single-antigen beads (SAB) assay. Infection and vaccination have been reported to be associated with allo-sensitization either in healthy population or in solid organ transplant recipients.
Positive flow cytometry crossmatch with discrepant antibody testing results following COVID-19 vaccinationQingyong Xu,Puneet Sood,Dennis Helmick,Jon S. Lomago,Amit D. Tevar,Adriana Zeevi
First published: 09 July 2021 https://doi.org/10.1111/ajt.16753
Non-HLA antibodies
You looked for unusual cases. What you described is non-HLA antibodies such as drugs (Rituximab) for MICA MICB and anti-endothelial antibodies, Angiotensin II receptors antibodies.
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
I will considered you a winner as you thought outside the box, but remember we are looking for common events. Do not think of the rarities first.
This could be explained by presence of non-IgG Ab like IgM (rarely encountered)
the IgM-antibodies were responsible for the positive AHG-CDC-XM result. The patient with positive CDC XM has a good outcome in the absence of DSA. Further work is needed to determine under what circumstances CDCXM positive transplants can be performed with FCXM and DSA negative.
{Case Reports RenFail.2013 Aug;35(7):102730.doi:10.3109/0886022X.2013.810539. Epub 2013 Jul 5.
Kidney transplantation with positive complement-dependent lymphocytotoxicity crossmatch with negative flow crossmatching and Luminexx donor-specific antibodiesVivek B Kute 1, Aruna V Vanikar, Manoj R Gumber, Varsha B Trivedi, Pankaj R Shah, Himanshu V Patel, Manish R Balwani, Pranjal R Modi, Hargovind L Trivedi
PMID: 23829775 DOI: 10.3109/0886022X.2013.810539}
a positive T cell and B cells cross match would indicate presence of antibodies to HLA type I and II antigens while a positive B cell cross match could indicate either:
(i) DSAs to type II antigens alone or
(ii) Low levels of DSAs to type 1 antigens.
A positive T cell crossmatch alone is usually due to technical error.
persistent positive T cell crossmatch result post desensitization is according to current guidelines, an absolute contraindication to renal transplant.
a positive T cell and B cells cross match would indicate presence of antibodies to HLA type I and II antigens while a positive B cell cross match could indicate either:
(i) DSAs to type II antigens alone or
(ii) Low levels of DSAs to type 1 antigens.
A positive T cell crossmatch alone is usually due to technical error.
persistent positive T cell crossmatch result post desensitization is according to current guidelines, an absolute contraindication to renal transplant.
Winner
Well done
Non-HLA antibodies
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Donor specific antibodies are against HLA antibodies and the absence of DSA doesn’t mean that other non-HLA antibodies that can lead to positive cross match are absent, such as MICA, angiotensin II type 1 receptor, H-Y antigen, antibodies against graft endothelium, antibodies against nsSNPs, and antibodies against LIMS1 proteins.
In addition, false positive results may be an explanation for positive crossmatch and negative DSA results such as presence of IgM antibody in the context of autoimmune disease or using rituximab in the CDC test.
Another reason may be the absence of rare antigen among the HLA antigen profile of bead assays and so DSA will not be detected.
You made it very complicated, but well done. Yes, this is a typical presentation of non-HLA antibodies. Unlikely due to IgM. It is a standard practice now that the HLA lap will run the autocross match to exclude autoantibodies.
Very impressed with your knowledge. You are a winner.
Winner
Well done
Non-HLA antibodies
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
If the crossmatch done in this clinical scenario is CDC crossmatch, then this assays uses donor lymphocytes and recipient serum.
After mixing donor’s and recipient’s components, followed by incubation period and addition of complement we observe the reaction. If DSA is present in the recipient serum then donor cell lysis is observed, the crossmatch will be positive. For donor’s lymphocytes, T cells express class I HLA molecules, and a positive T cell crossmatch indicates the presence of a class I DSA; B cells express both class I and class II HLA molecules, and a positive B cell crossmatch indicates class I and class II DSAs.
The CDC crossmatch depends on the amount of DSA present in recipient’s serum, isotype of immunoglobulin, and cell surface density of the target HLA antigen.
Results of crossmatch could be influenced by several factors:
-Amount of antibody or titer is important because low titer antibody may not be able to activate complement cascade and induce cell lysis and crossmatch would be negative.
-Isotype of immunoglobulin is important because the presence of IgM antibody, which could be either anti-HLA or non-HLA, could cause false positive crossmatch.
-Non-HLA IgG binding to cell surface antigens found on lymphocytes
-Positive CDC crossmatch could result from interference of some drugs with the assay. Drugs like Rituximab, ATG, alemtuzumab and IVIG could bind to donor’s lymphocytes and the test is considered positive.
-Antibodies to specific loci, not routinely reported by HLA laboratory, could also cause positive result.
DSA detection by solid phase assays, recognizes only IgG and do not detect anti-HLA IgM antibodies, even though IgM antibodies cause a positive crossmatch, similar to our case here. Solid phase assays could show false-positive results because of reactivity against the latex beads, denatured HLA antigen, or non-HLA protein used to coat the beads.
So in the setting of positive cross match (both T and B cells are positive), and negative DSA, my first impression that the recipient could have non-IgG antibodies against HLA class I which is present in both B and T lymphocytes. In this case it could be IgM anti-HLA antibodies. But non-HLA antibodies to components found on both T and B lymphocytes could be the case here.
References:
1. Cai J, Terasaki PI, Anderson N, et al. Intact HLA not beta2m-free heavy chain-specific HLA class I antibodies are predictive of graft failure. Transplantation 2009; 88:226.
2. Pereira S, Perkins S, Lee JH, et al. Donor-specific antibody against denatured HLA-A1: clinically nonsignificant? Hum Immunol 2011; 72:492.
3. Gombos P, Opelz G, Scherer S, et al. Influence of test technique on sensitization status of patients on the kidney transplant waiting list. Am J Transplant 2013; 13:2075.
Winner
Well done
Non-HLA antibodies
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Positive cross match ( both T and B cells),but no DSA…not a classic match…or association ,with some justification required. Judicious interpretation must be there aiming not losing suitable kidney offer .Revising validity that positivity of cross match and negativity of DSA is a reasonable thinking . False positive CDCXM may be due to autoantibodies(suggested by negative DSA)or lab errors. Addition of DTT can prevent the effect of IgM as an autoantibody .Other measures include heating recipient serum to 55 C ,washing, auto crossmatch or prolongation of incubation period. Also, FCXM should be done being more sensitive than CDCXM. False positive FCXM can occur in with non specific IgGs or monoclonal AB(eg rituximab)therapy ;which can be reduced by pronase.On the other hand, SAB is not detecting IgM autoantibodies and non-HLA autoantibodies. False negative results happens when C1 and IgM autoantibodies bind to beads ,preventing HLA antigen-antibody interaction(prozone phenomenon).other situations include use of IV IG infusion, inaccessible HLA Ag on the beads ,shred epitope by many beads resulting in decrease of MFI and if certain HLA Ag is not included on the SAB kit. Positive CDCXM and/or FCXM with negative L-SAB indicates standard risk immunological level.
A conclusive approach is to correlate clinical background in association with confirmation of crossmatch assay validity, abolishing lab errors and application of phenotype beads flow c PRA screen to avoid loss of good kidney offer.
References:
Chughahi,SA et al, Interpretation of crossmatch reports in a patient with lupus nephritis ,Arch Organ Transplant,2017,023:029
Chari,M et al: Cross matching in renal transplantation by non-immunologist for non-immunologist ,2017,Urol & Nephrol, July,15,257-261
Geier,SS et al 😛 271 False positive HLA antibody tests can lose organ offers, Human Immunology, Vol :78,Suppl Sept.,2017,P:252
sorry for the writing mistake ….correction is 2017 without that not intended yellow symbol….really a mistake so so so sorry
Moyo,O, Sharma,AK and Halawa,A,: Case -based review of cross -match technique in kidney transplantation,2018,J Egyptian Soc Nephrol transplant,107-114
Impressed with your explantation, well done. It represents deep thought. Do not forget that the lab will exclude autoantibodies and repeat the test if ant technical errors are suspected.
Winner
Well done
Non-HLA antibodies
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Positive both B and T lymphocyte cross match but No DSA
1- A false positive flow cytometry may result from non specific binding of Ig G to Fc receptors by certain substances in recipient serum in case of :
A- consider recent treatment with rituximab ( anti CD20) and in this case , results can be confirmed by treating donor lymphocyte with pronase
B- These may be auto Ab, confirm the results by autocross match
C- Non-HLA antibody.
2- False negative SAB for DSA may result from
a- the positive MFI cut off value differs between different transplantation labs so DSA may exist but are less than this value so reported as negative
b- Lab error if the donor antigen is not represented in the particular SAB kit used
Schinstock, C. A., Gandhi, M. J., & Stegall, M. D. (2016). Interpreting Anti-HLA Antibody Testing Data: A Practical Guide for Physicians. Transplantation, 100(8), 1619–1628. https://doi.org/10.1097/TP.0000000000001203
Winner
Well done
Non-HLA antibodies
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Positive cross match (both T and B cells are positive), but no DSA.
Patient may have false positive crossmatche due to presence of IgM AB as in autoimmune diseases so CDC- DDT should be done to confirm absence of autoantibodies.
Another cause is that the patient might be desensitized .
Low number of antibodies that can’t activate complement to cause cell lysis
-Positive CDC crossmatch occurs with some drugs which interfers with the test . Drugs like Anti-CD20 monoclonal antibody (rituximab) has most commonly been implicated. Pre-treatment of donor lymphocytes with pronase can be helpful in removing the effect of CD 20 binding (23). , ATG, alemtuzumab and IVIG could bind to donor’s lymphocytes and the test is considered positive.
The SAB assay is only semi-quantitative, and the variation in MFI has been reported as high as 62%, especially when the MFI is relatively low (1000-3000)
So negative DSA might be false negative.
SABs may not include donor HLA antigen SO the test is false negative.
Carrie A. Schinstock, MD, Manish J. Gandhi, MD, and Mark D. Stegall, MD.Interpreting Anti-HLA Antibody Testing Data: A Practical Guide for Physicians.Transplantation. 2016 Aug; 100(8): 1619–1628.
Winner
Well done
Non-HLA antibodies
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
This due to either autoantibodies , or non-HLA AB or low level AB or due to previous desensitization
Presence of Non HLA Antibodies
-Non- HLA antibodies .
-Technically error .
-Low level DSA antibodies detected by FCXM.
-false positive due to IgM antibodies.
Cross-matching is the crucial step in kidney transplantation.There are Techniques used for cross-matching include:
a- CDC: complement is added to the mixed recipient serum and donor lymphocytes and cell lysis of the lymphocytes is observed.
Negative test when donor-specific anti-HLA antibodies are absent and complement activation does not occur.
Positive test when donor-specific antibodies bind to lymphocytes activate complement, and cause cell lysis
Advantages of CDC:
The mainstay of pretransplant screening.
Key role in preventing hyperacute rejection
Detects only complement binding antibodies
Drawbacks:
Detects IgM and IgG antibodies and non HLA antibodies
.b- Flow cytometry: it measures DSA by using an impedance flow cytometer. The measurement of DSA is done either by fluorescence intensity or by serial dilution of recipient serum that reacts with donor lymphocytes. If CDC negative & flow cytometry is positive it may be due to
i. non-complement-fixing Abs
ii. Non-HLA Abs
iii. low level of Abs.
So the test was used for sensitized patients.
2. Solid-phase Ab detection assay:
a- ELISA: the serum of the recipient is mixed with specific Abs that bind to available HLA epitopes, then IgG is added to combined anti-HLA ABs. It is used in sensitized patients.
b- bead technology (Luminex), by using beads labeled with fluorescein. The result was measured either by flow cytometry or Luminex ( the degree of fluorescein measured by MFI).
3 Virtual Cross-match:
Mainly it is used for deceased donor transplantation to decrease ischemic time. By using bead technology the recipient anti-HLA Abs compared with donor HLA Ags.
rossmatch techniques can be divided into cell based assays and solid phase assays.
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 acivating 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): 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 or a titred value of dilution required to get a negative result (for quantification).
B) Solid Phase antibody detection assays:
1) 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) 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.
2. Please reflect on your practice if possible.
In our practice, which is mainly living donor transplant setting, CDC is the first test to be done. If there is no history of sensitization (prior transplant, pregnancy, blood transfusion), then we proceed with transplant without any further tests,
In patient with sensitization history, even if CDC crossmatch is negative, we get a single antigen bead (Luminex) assay for quantification of DSAs and proceed accordingly,
Introduction:
crossmatching was an attempt to identify those transplant recipients who had a higher
likelihood of acute vascular rejections after receiving the gratf from a given donor.
Tis hyperacute rejection is the result of the presence preformed antibodies in the donor
serum to one or many HLA (human leucocyte antigens).
Cell based technique:
1-CDC technique:
Complement dependent cytotoxicity crossmatching was first developed in the 1960.
Percentage of lymphocytes in the cell panel which has undergone lysis as a result of
complement activation or panel-reactive antibody (% PRA)
Twenty percent is usually taken as the minimum cut-of for a positive result thus
functioning as a qualitative and a semi-quantitative estimate of the strength of the
reaction.
. (2) Flow cytometry:
Flow cytometry for crossmatching was a technique first described in the context of pre
transplant in the early eighties. Here, the donor lymphocytes are mixed with the
recipient’s serum. Tese bind to the donor specific’s antibodies and are quantified by
detector
flowcytometer :
Interpretation of flow crossmatch results In the light of a negative CDC result, a positive
flow cytometry could be interpreted as indicating the presence of any of the following
Non-complement fixing antibodies
Non-HLA antibodies
Solid phase antibody detection assays :
Enzyme-linked immunosorbent assay (ELISA):
initial application of ELISA was to detect HLA in both bound and free forms but its
current utility is for the detection of HLA antibodies in serum. Te modifed assay uses HLA
glycoprotein immobilized into mictrotiter wells. Te recipient serum is added and specifc
antibodies bind the epitopes available.
Bead technology (Luminex):
HLA antibody testing was revolutionised by the introduction of beads labeled with
fourescein in the nineties.
Virtual Crossmatching:
technique is based on the comparison of the anti-HLA antibodies of the recipient to the
donor HLA antigens using bead technology. Tis method predicts the eventual crossmatch
and can assist in rapid identification of a suitable donor
CDC cross match
– It involves incubation of donor’s lymphocytes in recipient serum, washing to remove unbound antibodies, then adding complement and after incubation period
– If antibodies to donor HLA present complement activation occur and cell lysis .
– It is highly specific, a positive cross match using CDC indicating the presence of clinically significant complement fixing antibodies thus if CDC positive donor should be excluded.
– This test has low sensitivity, human anti- goblin can be added before complement that bind to Donor HLA antibodies, to increase sites for binding to complement and increase its sensitivity
-False positive results can occur due to the presence of clinically non significant autoimmune IgM antibodies which can be eliminated either by heating or adding DTT .
-False negative result can occur due to non-complement fixing, non HLA or due to low level of HLA antibodies
-If antibodies are directed to only class II or if DSAs directed to HLA class II are low results may show positive B and negative T cell cross match, while if DSAs are directed to both class I, II it will give positive B and T cross match.
– Positive T cell cross match has the worst prognosis and donor should be excluded.
– Positive B cell cross match carry poor prognosis when compared to negative CDC cross match but better that positive T cell cross match.
– CDC is used either in determining compatibility between donor and recipient or in determining sensitization in case of PRA
– CDC should be repeated just before transplant since antibodies may be formed after antigen exposure such as blood transfusion or pregnancy
Flowcytometry
– It involves incubating donors lymphocytes with recipient serum, then addition fluorescein labeled anti- globulin that will bind to DSA present in the recipient serum that are attached to donor lymphocytes
-FCM is used either in determining compatibility between donor and recipient or in determining
-More sensitive than CDC as it can detect non complement fixing AB, non HLA antibodies and low level DSA, so CDC – FCM + cross match may indicate the presence of one of the previous mentioned ABS.
– Positive T cell FLC (in highly sensitized patients only) is associated with poor graft survival, so it has a very big rule in sensitized recipients
Solid phase assays
– Bead assay (luminex) replace ELISA
– Very sensitive very specific, can detect complement and non-complement fixing antibodies, low level DSA, non HLA antibodies.
– Luminex give quantitative assessment of the strength of DSAs.
Virtual cross match
– By comparing anti HLA antibodies of recipient detected by luminex 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,
– DSA may change over time due to sensitization from blood transfusion or pregnancy so luminex SAB should be repeated.
In practice regarding living donors
we do Virtual cross match and CDC to all patients and FCM or DTT cross match accordingly
⦁ If CDC positive we repeat using DTT or FCM if +ve excluded.
⦁ If FCM positive and CDC negative patient is assessed for desensitization using data from Solid phase assays.
Comparison between techniques:
**CDC
Advantage:
Low cost.
Play role in preventing hyper acute rejection.
Disadvantage:
Detect only complement fixing antibodies.
Less sensitive.
**Flowcytometry:
Advantage:
More sensitive
Detect low level of HLA IgG antibodies
Disadvantage:
Suitable for living donor than cadaveric.
**Solid phase assay:
More sensitive to detect donor specific HLA antibodies.
Identify all HLA alleles for recipients antibodies.
Disadvantage:
Positive assay with negative CDC/FCM ambiguous.
SAB detect both complement fixing and non fixing antibodies so recipients may be denied a graft without clinical significance.
*CDC technique (1960) : determine presence of donor specific HLA antibodies in recipients serum which help In prognosis of graft survival after transplantation.
*Involved donor lymphocytes ( B and T) with recipients serum and add complement to the mixture and cell lysis of lymphocytes is observed.
*Detect percentage of lymphocytes in cell panel .
Addition of AHG increase sensitivity of CDC .
*CDC may lead to false positive or false negative , so add DTT which help in preventing IgM, autoantibodies which mediate complement activation or allow only IgG DSA.
* False negative:
– low DSA level to cause complement activation.
– Type of antibodies not cause complement activation.
* New antibodies may develop if recipients become sensitised by new antigen exposure e.g pregnancy or blood transfusions.
* HLA encoded by gene on chromosome 6 , class I ( HLA A B C ) expressed on all nucleated cells .
HLA class II ( DR DP DQ) : expressed only on antigen presenting cells as macrophages and dendritic cells .
Vascular endothelial cells of transplant graf expressed both these antigens and antibodies respond to them predicting acute antibodies mediated Rejection.
* positive T and B cells :
Indicated presence of antibodies to HLA type I and HLA type II antigen .
* positive B cell indicate :
-DSA to type II antigen alone .
– low level of DSA to type I antigen .
* positive T cell alone :
– Technically error.
– Non- HLA antibodies.
– If you decide to transplant, may need aggressive therapy.
* Positive T cells crossmatch:
– After rule out of autoantibodies, transplantation can be performed but carry risk of poor outcomes
and high incidence of acute graft rejection.
* President positive T cell crossmatch post desensitisation is absolute contraindications of transplantation.
* False positive B cell crossmatch by luminex for DSA increase its specificity , true positive B cell crossmatch is less significant in acute and hyper acute graft rejection.
Flowcytometry: donor lymphocytes mixed with recipients serum these bind to DSA quantified by detectors in Flowcytometry.
FCXM measured by channel shift or serial dilution of recipients serum .
Solid phase antibodies detection assay :
By ELIZA or luminex ( SAB)
Virtual crossmatch:
Detect suitable donor rapidly, reduce cold ischemia time without increasing rejection or affect graft survival.
there is many crossmatch technique used to detect ant HLA ab in sensitized patients .
sensitization occur by
1- blood transfusion .
2- pregnancy .
3- previous kidney transplant .
detecting of these ab before transplantation is important to prevent hyper acute rejection .
it is of two type
1- cell based type .( CDC , flowcytometry )
2- solid phase .( uses beads instead of donor lymphocyte )
features of each one
1- CDC – complement dependent cytotoxicity .
developed since 1969 by terazaki .
most commonly used technique
advantages
disadvantages
2- FLOW CYTOMETRY .
uses patient serum mixed with lymphocyte , then TO add flourecent labled IG.
the positive result is given as a channel shift , or serial titration method .
it is vey sensitive with high false positive result
it is role is in assessing highly sensitized patients .
solid phase
1- ELISA TECHNIQUE
It is replaced by luminex – not used nowadays .
2- luminex technique .
uses beads with HLA ag instead of lymphocyte
it is vey sensitive and specific used for detection of DSA..
the result is given as MFI ( MEAN FLOURECENT INTENSITY ).
●DSAs are commonly generated by:
(i) Blood transfusion
(ii) Previous transplantation
(iii) Pregnancy
●The role of crossmatching is to identify those transplant recipients who are at risk of acute vascular rejections after allograft transplantation.
-Hyperacute rejection is the result of preformed donor-specific antibodies(DSAs.
●There are three mthods of cross matching:
A) Cell based essay:
A mixture of donor lymphocytes and recipient serum is tests, searching for preformed DSA that if present may lead to acute graft rejection. The reaction is complement mediated and detected by lysis of cells. Greater than 20% is considered positive reaction.
A similar modality is flow cytometry cross matching, using either florescence intensity or serial dilution as a measure of the reaction.
b) Solid phase antibody detection:
– ELISA method, detecting HLA ab in the serum
– Luminex: using flourescein labeled beads and interpreting the tests using either flow cytometry or luminex methods.
An Update on Crossmatch Techniques in Transplantation
the crossmatch test for the presence of preformed DSAs in the recipient serum to HLA antigen. DSA puts the recipient at risk of acute rejection.
DSAs performed due to blood transfusion, previous crossmatch, or pregnancy.
CDC Technique: complement-dependent cytotoxicity
Test: serum from the recipient is added to donor lymphocytes (T or B) separately in the presence of complement.
Negative test: when donor-specific anti-HLA antibodies are absent, so, complement activation does occur, no cell lysis.
Positive test: when DSAs are present, bind to lymphocytes, activate the complement, and cause cell lysis.
Result: is the percentage of lymphocytes in the cell panel which has undergone lysis, or panel-reactive antibody (%PRA).
PRA of 20% is used as a minimum cut-off for a positive result.
The strength of the test can be increased by:
1- Doubling dilutions of the recipient’s serum. The higher the dilution required to give negative results, the greater the strength of the immune reaction, and helps to determine the need for desensitization.
2- Addition of anti-human immunoglobulin (AHG) which increases complement activation, so the sensitivity of the test.
Limitations:
1- False positive: due to IgM auto-antibodies. Addition of Dithiothreitol (DTT) removes IgM auto-Abs, allows only IgG (DSAs).
2- False negative: DSAs are either of low level or antibodies type does not cause complement activation.
· – vascular endothelial cells of the graft have both classes I, II.
· B-cells have both classes.
Ø If cross match to both T and B- cell was positive, this would indicate DSAs to HLA type I and type II Ags are present.
Ø Positive B cell cross match could indicate either (1) DSAs to type II Ag alone or (2) low levels of DSAs to type I Ag.
Ø Positive T cell crossmatch alone is usually due to Technical error.
· Positive T-cell crossmatch is associated with high incidence of acute graft rejection. Persistent positive T cell crossmatch result after desensitization is an absolute contraindication to renal transplant.
Ø Positive B-cell test is not as strongly associated with rejection as positive T-cell test, anti-class II HLA warrant desensitization before transplantation. And negative test is associated with better outcome.
Ø It is not so significant as it has high false positive results.
Flow cytometry:
Donor’s lymphocytes are mixed; with recipient’s serum. If DSAs there, donor lymphocytes react with them. And quantified by:
1- Measurement of the fluorescence intensity as a ratio of the control (channel shifts).
2- Serial dilutions of the recipient’s serum made to react with donor lymphocytes and; the minimum dilution which yields a negative result gives a measurable estimate.
Interpretation of flow crossmatch results:
– to fix the possibility of transplantation or the need for desensitization before transplantation.
Negative CDC result with a positive flow cytometry indicate one of the following:
1- Non-complement fixing antibodies
2- Non-HLA antibodies
3- Low level antibodies
Solid phase antibody detection assays:
1- Enzyme-linked immunosorbent assay (ELISA):
Uses microtiter wells fixed with HLA-glycoprotein. Recipient serum added. If; donor-specific antibodies present. They will bind the epitopes. after wash and IgG carrying alkaline phosphatase added. Wash again to remove the unfixed DSAs, and a substrate added, which, after phosphorylation by the passenger, undergo a color change.
2- Bead technology (Luminex):
These technic based on the use of beads that carry HLA molecules as antigen and soaked with fluorochromes. DSAs present in the recipient serum react with these HLA-Ag, resulting in a specific unique signal. Wash done and incubation with anti-human IgG labeled with phycoerythrin.
Quantitation of the result:
1- flow cytometry: measure the channel shift results from binding of antibody.
2- Luminex method: The degree of fluorescence is calculated as mean fluorescence intensity (MFI) using laser.
Interpretation of the flow crossmatch studies:
the cut-off values are not uniform cross laboratories. Low value would increase the sensitivity while affecting specificity and vice-versa.
The virtual crossmatch:
rapid identification of a suitable donor, and predicts the eventual crossmatch.
Technic
beads carry HLA-Ags are mixed with recipient serum. Each bead has specific dye. anti-HLA antibodies bind to the specific bead. Detector antibody then bind and sequester a reporter dye checked for using a laser beam. The test makes up a profile for the recipient antibodies. Which compared with the HLA structure of a potential donor so; predicting the result of the crossmatch.
Technics comparison:
Complement dependent cytotoxicity:
Advantage: availability, simplicity, low cost.
Drawback:
1- Detect only complement binding antibodies
2- Less sensitive than newer test
3- Detects both IgM and IgG Abs, autoantibodies, and non-HLA antibodies against Ags that are not relevant to transplantation.
Flow cytometry:
Advantages:
1- more sensitive than CDC
2- can detect low levels of IgG HLA antibodies.
3- Less operator variability
Drawback:
Suitable for living donors rather than cadaveric donors.
Solid-phase assays (ELISA and bead technology):
Advantages:
1- The most sensitive methods for DSA detection.
2- Allow the identification of all HLA alleles for which the recipient have antibodies.
3- No false positive from non-HLA antigens.
4- Specifically identify the class of HLA.
Drawbacks:
1- positive assay with negative CDC/Flow crossmatch has an unclear interpretation.
2- the link of low-level antibodies to lower significant antigen is controversial.
3- Detect both complement-fixing and non-complement fixing HLA antibodies, may prevent transplantation without significant clinical reason.
Review of crossmatch techniques:
Cell based assays:-
CDC crossmatch / the significance of CDC lies in its ability to determine the presence of DSA in the serum of the recipient.
The B and T cells are separately tested against serum from the recipients.
Outcome of positive T cell crossmatch result is poor with high incidence of acute graft rejection.
Outcome of positive B-cell crossmatch result the significance of anti-class ll HLA AB is of less significant in acute /hyperacute rejection and still need desensitization of the recipient prior to transplantation
Flow cytometry /if negative CDC result ,positive flow cytometry could be interpreted as indicating the presence of any of the following
1 non -complement fixing AB
2 non -HLA AB
3 low level antibodies
So the value of it is in using for sensitization.
Solid phase AB detection assays
– Eliza assay
– -Bead technology (luminax)
The virtual crossmatch
This method predict the eventual crossmatch and can assist in rapid identification of suitable donor.
Crossmatch in Transplantation is like rehearsal in acting.
You have akidney from person and you are going to put it in another body risking hyperacute rejection so you do cross match outside recipient body to make sure that recipient plasma doesn’t contain antibodies against donor’s tissue
There’s three main types of crossmatch
CDC, flow cytometry crossmatch and solid phase crossmatch.
They are complete each other like taking 3different angles by 3different cameras to get full 3d picture of the same person
So sometimes you need to do the 3 types together
Cdc crossmatch is a technique detect complement fixing antibodies
1st developed by Terasaki in which he mixed recipient serum(antibodies) with donor lymphocytes both bcells and tcells(HLA class 1 and 2) then added complement.then some modifications were done to improve its specificity like adding dithiothreitol(DTT) to prevent IgM from giving false positive results
Positive tcell crossmatch after excluding of autoantibodies us absolute contraindication for kidney transplantation and associated with hyperacute rejection
Positive b cell only and negative to tcell is not consistent associated with humoral rejection and its significant is uncertain but it considered as arisk and needs explanation using the other types of crossmatch and maybe low titre antibodies as b cells express more antigen on its surface
Flow cytometry crossmatch: detect larger spectrum of antibodies either complement or non complement fixing
Its very good negative test in which donor’s lymphocytes (HLA class 1 and 2) mixed with recipient plasma (antibodies) plus anti immunoglobulins antibodies
Positive reaction is detected by detector in the empendence flow cytometry and quantification of antibodies titre is done by 2methods …1) fluorescent channel shift in relation to control 2) serial dilution until become negative
Interpretation -ve is excluding any kind of antibodies but positive flow cytometry crossmatch in negative CDC DTT cross match may be related to no complement fixing,non HLA antibodies or low titre as its very sensitive test then you will need to proceed to solid phase crossmatch to decide what is the next step.
Large retrospective study showed that positive flow cytometry crossmatch against tcell associated with poorer 5y graft survival so some centers policy is reject donor if positive flow cytometry crossmatch against tcell.
Solid phase crossmatch
Includes ELIZA and luminex
ELIZA solid phase crossmatch in which HLA glycoprotein immobilized into micro titre well then recipient serum is added… antibodies bund the epitopes available…wash…anti IgG with reporterd molecules (alkaline phosphatase)…wash…. substrate which give colour by the reporter if still attached not removed by washing is detected.
Bead technology
Beads labelled with two floroscen dye in different ratio resulting in signal unique for each bead.each bead may have single HLA molecule(single antigen bead) or multiple (mixed antigen bead)
Recipient serum incubated with beads … antibodies react with beads and colour detected by either flow cytometry(mean channel shift) or luminex(MFI)
negative solid phase crossmatch is good
Positive solid phase crossmatch maybe due to non HLA antibodies or non complement fixing or very low level OF antibodies….the cutoff of antibodies significant is ambiguous and not uniform just centered base.
There are three mthods of cross matching:
A) Cell based essay:
A mixture of donor lymphocytes and recipient serum is tests, searching for preformed DSA that if present may lead to acute graft rejection. The reaction is complement mediated and detected by lysis of cells. Greater than 20% is considered positive reaction.
A similar modality is flow cytometry cross matching, using either florescence intensity or serial dilution as a measure of the reaction.
b) Solid phase antibody detection:
– ELISA method, detecting HLA ab in the serum
– Luminex: using fourescein labeled beads and interpreting the tests using either flow cytometry or luminex methods.
Cross match is used to prevent and detect the risk of posttransplant rejection alot of techniques used to for this purpose.
CELL BASED ASSAYS
Including CDC technique and flow cytometry
CDC is done by mixing recipient serum with donor lymphocytes T and B cells are tested alone after that adding complement .
Cell lysis means positive crossmatch through activation of classical pathway.
It’s basic test easy to be done but less sensitive .
FLOW CYTOMETRY:this is the same as CDC but differs by adding detectors .
SOLID PHASE ANTIBODIES DETECTION ASSAYS
(ELISA ,BEAD TECHNOLOGY LUMINEX )
VIRTUAL CROSS Match
Pretransplant assessment of the sensitization status of kidney transplant recipient is essential
For predicting graft outcome. several crossmatch techniques are available as
I- Cell based crossmatch : by mixing recipient serum with donor B and T lymphocytes and if the recipient has complement fixing Ab againt donor cells this will result in cell heamolysis
The test become more accurate by adding anti human immunoglobulin .
Drawbacks of this technique are
a- False positive results in case of autoantibodies , this can be avoided by adding DDT
b- False negative results in case of non complement fixing Ab or very low level of Abs
Flow cytometry: by mixing recipient serum with donor B and T lymphocytes to detect donor specific antibodes by Measuring the fluorescence intensity. It is more sensitive than CDC crossmatch
Advantage of flow cytometry it can detect non complement fixing Ab , non HLA Abs and low level Ab
II- Solid phase antibody detection assays
1- ELISA technique to detect free and bound form of Anti HLA Ab via mixing recipient serum with specific antibodies
2- Bead technology (Luminex) : can differentiate which HLA class are detected
III- The virtual crossmatch :
This depends on comparing recipient Anti HLA Abs To donor HLa ags using bead technology
In our protocol (Zagazig university hospital ) , first we perform crossmatch CDC with DDT , then virtual crossmatch by luminex and an other final CDC crossmatch just prior to transplantation .
In Sudan we do first HLA typing for both recipient and doner for A, B, C, DR,DP ,DQ.
And we do PRA and initial CDC. Recently we do only final CDC
We only proceed for FCX for patients with suspected DSA,historical positive PRA, highly sensitized patients , patients with very high PRA but negativeCDC
Thanks Hinda for your reflection
Cell based assay
1-Cdc techniques: doner lymphocytes and recipient serum were mixed and a complement is added. If the recipient serum contains antibodies the cells lysis will occur. The percentage of cells which underwent lysis is calculated in a panel ; hence the name PRA. 20% is considered positive result. The test is qualitative and semiquantitative. Some improvement in it increased its benefits :
I.titered crossmatch: through doubling dilution of the recipient serum. The more dilution required for making the test negative the higher the concentration of antibodies
II.AHG enhanced: these antibodies bind to recipient antibodies ,enhancing the complement binding . this is useful for cases with low titre of DSA
The test can have false positive results if there is IgM antibodies and this can be overcome by DTT. It can also have false negative results with low titre of antibodies or non complement fixing antibodies.
HLA class 1antigens are present in all nucleated cells and class 2 are present in APCs. The greft endothelium contains both types. So for anticipation of rejection t cells, which include only class 2, or b cells ,which include both classes , are used in cross match. Positive both t and b indicates antibodies to class1 and2. Positive T alone is usually an error while positive B alone may be due to anti class2 only or low level of anti class1 not detectd by t cells.
Positive T cell will end with acute graft rejection and is considered as an absolute contraindication for transplant. For positive B cell test, first rule out the false positive results. ,then with luminex look for DSA.
2- flowcytometry: recipient serum is mixed with doner lymphocyts and fluorescent labelled anti IgG . if a rection occur ,either the intensity of fluorescence is recorded or serial dilution are used. Because of the high sensitivity of this test,it is useful in sensitized patient to decide the reisk of rejection pretransplant . if CDC is negayive and flow cytometry is positive this can be due to low level of antibodies, non complemt or non HLA antibodies. T cell positive flowcytometry have less 5 year graft survival.
Solid phase antibody detection assays
1- ELISA: used to detect sensetiztion through detection of bound and free antibodies. Recently its use is for the serum with use of anti IgG.
2- Luminex:using beads labeled with antigens mixed with the recipient serum and labeled anti IgG. It can detect whole class antibodies or specifically.
Virtual crossmatch
Use the patient serum against a set of antigen to predict the possible acceptable doners. Some researchers suggest using it instead of flowcytomty . it reduce cold ischemia time which has its effect on graft survival
-The role of crossmatching is to identify those transplant recipients who are at risk of acute vascular rejections after allograft transplantation.
-Hyperacute rejection is the result of preformed donor-specific antibodies(DSAs).
-DSAs are generated by: blood transfusion, previous transplantation, pregnancy.
-DSAs can be identified with cell-based assays, solid phase, and more recently virtual crossmatch.
Cell-based assays :
1. Complement dependent cytotoxicity: CDC technique:
-It is abasic and cornerstone cross matches in transplantation due to availability, cost, and simplicity but it is less sensitive than other newer assays.
-It can determine the presence of DSAs in the serum of the recipient.
– CDC technique is done by adding serum from the recipient to donor lymphocytes(T or B ) in the presence of complement.
-CDC detects only complement binding antibodies.
–The result is presented in terms of the percentage of lymphocytes in the cell panel which has undergone lysis as a result of complement activation or panel-reactive antibodies (PRA%).
–Titred crossmatch method in which a crossmatch of the donor lymphocytes is performed using serial doubling dilutions of the recipient, s serum. Higher dilution required to give a negative result means greater strength of the immune reaction that also helps to determine the need for desensitization.
-Addition of Anti-human immunoglobulin help increase the sensitivity of the test.
–False-positive CDC due to autoantibodies in recipient serum.
–False-negative CDC due to low level of DSA that cannot activate complement cascade or if antibodies are of the type that doesn’t cause complement activation.
-Antibody level of the recipient may vary with time so, matching must be done from a recently drawn recipient serum sample with donor lymphocytes especially in cadaveric allograft transplantation.
-T-cells express HLA –class I while B cells express both so, positive T cells and B cells crossmatch would indicate presence of antibodies to HLA-I and II while a positive B cell crossmatch could indicate either:
1.DSAs to type II antigen alone or
2..low level of DSAs to type I antigens.
-A positive T cell crossmatch alone is usually due to a technical error.
– When the renal transplant is performed in a recipient who has a positive T cell crossmatch will result in poor outcome and a high incidence of acute graft rejection as shown in several studies.
–Persistent positive T –cells crossmatch after desensitization is an absolute contraindication to renal transplantation.
-Negative B cell crossmatch is associated with better outcomes but its high false-positive rate makes its significance uncertain but this is improved by Luminex testing which increases its specificity.
– In the case of positive B cell cross matches, the significance of anti-class II HLA antibodies is less significant in acute/hyperacute rejection but still needs desensitization of the recipient before transplant.
2. Flow cytometry :
-Here, the donor lymphocytes are mixed with the recipient, s serum in the presence of anti-IgG fluorescein-labeled antibodies, and quantified either by :
1. Measurement of the fluorescence intensity as a ratio of the control.
2. Serial dilutions of the recipient, s serum are made to react with donor lymphocytes and the minimum dilution which yields a negative result gives a measurable estimate.
Interpretation of flow crossmatch results:
-Negative CDC result and positive flow cytometry means :
1. Non –complement-fixing antibodies.
2. Non –HLA antibodies
3. Low-level antibodies.
-The value of this test in using it for cross-matching of sensitized patients to determine transplant feasibility or need for desensitization before transplant.
-It is suitable for living donors rather than cadaveric donors.
Solid-phase antibody detection assays
Enzyme-linked immunosorbent assay (ELISA):
-Here, the patient serum is added and specific antibodies bind the epitopes available then a wash is performed and anti-IgG is added with substrate that undergoes a color change.
-It was replaced by bead technology.
Bead technology (Luminex) :
-Each bead may have one or more HLA molecules types incorporated. The basic steps involve:
1 -incubation of patient serum with beads.HLA antibodies of the serum will react with HLA antigens on the beads.
2 -Then, beads are washed and incubated with anti-human IgG labeled with phycoerythrin.
Quantification of the result
-It is achieved by 2 methods:
1. Flow cytometry
2. luminex method
-The degree of fluorescence is calculated as mean fluorescence intensity (MFI)
Interpreting flow crossmatch studies:
-Positive test and corresponding CDC crossmatch negative may be due to -non –complement-fixing antibodies, non –HLA antibodies, Low-level antibodies.
-Low cut–off values increase sensitivity while affecting specificity and vice–versa.
The virtual crossmatch
-It is based on the comparison of the anti-HLA antibodies of the recipient to the donor HLA antigen using bead technology or microsphere that coated with multiple HLA antigens.
–Final transplant decision could be taken based on virtual crossmatch irrespective of the flow cytometry without significant difference in clinical outcome in those who were flow cytometry positive but virtual crossmatch negative.
Differential diagnosis of positive crossmatch and negative DSA are ;
Cross match techniques are;
-cell based assay.
CDC technique.
flow cytometry .
-solid phase antibody detection assay.
enzyme-linked immune sorbent assay (ELISA) .
virtual crossmatch.
CDC cross match;
It is a functional test that involve cells(donor lymphocytes) and serum antibodies ( recipient serum )in the presence of complement.
The percentage of lymphocytes in the cell panel which has undergone lysis as a result of complement activation is calculated and the result is represented as panel-reactive antibody (%PRA).
It simple , available and cost effective test .
It is the main stay of pre transplant screening for HLA antibodies .
It has a key role in prevention of hyper acute rejection.
It is less sensitive than other newer assays.
It detects only complement binding antibodies.
It detects IgM and IgG antibodies simultaneously, autoantibodies and non-HLA antibodies against antigens that are irrelevant as far as the transplant is concerned.
Human globulin (ATG) is added to increase the sensitivity of the test as multiple AHGs bind a single DSA, amplifying its complement activation at smaller titres.
False positive result due to auto antibodies can be reduced by addition of dithiothreitol (DTT) which helps to prevent IgM mediated complement activation and allow IgG (DSA).
Flow cytometry:
The donor lymphocytes are mixed with the recipient’s serum.
The reactive lymphocytes (that bind to the donor specific antibodies) are quantified by detectors in the impendence flow cytometery.
The result can be quantified by measurement of the fluorescence intensity as a ratio of the control (channel shifts) or serial dilutions of the recipient’s serum are made to react with donor lymphocytes and the minimum dilution which yields a negative result gives a measurable estimate.
It is more sensitive than CDC cross match .
It can detect lesser levels of IgG HLA antibodies and has less variability.
In the light of a negative CDC result, a positive flow cytometry could be interpreted as indicating the presence of any of the following non-complement fixing antibodies, non-HLA antibodies and low-level antibodies .
The value of this test’s sensitivity lies in using it for cross matching of sensitized patients determine transplant feasibility or need for desensitization protocols prior to transplant.
Solid phase assays (ELISA and bead technology);
They are the most sensitive tests in detecting DSA.
They allow identification of lower titre ,possibly clinically significant anti HLA Abs
They allow identification of all HLA alleles for which recipient harbours antibodies .
They eliminate confusion regarding the class of HLA that are detected (false positive of antibody binding to non –HLA antigens ) by CDC.
Single antigen bead assays detect both complement- fixing and none complement –fixing HLA antibodies.
Limitations of the tests including;
-the relevance of low level Abs to low detectable antigen level.
– the interpretation of a positive assay in the presence of negative CDC and flow cross match is ambiguous.
-interference by IgM ,incomplete of antigens in the bead sets and variability of HLA density on the density.
Enzyme-linked immunosorbent assay (ELISA):
The recipient serum is added and specific antibodies
Bead technology (Luminex):
Beads are;
– impregnated with different ratios of two fluorochromes resulting in a signal that is unique to the specific bead.
-each bead may have one or more HLA molecule types incorporated.
The basic steps involve first, incubation of recipient serum with the beads.
HLA antibodies of the serum will react with HLA antigens on the bead. The beads are washed and incubated with a second antibody, usually anti-human IgG labeled with phycoerythrin.
Quantification of the test is achieved by flow cytometory or luminex.
Virtual cross match;
This technique is based on the comparison of the anti-HLA antibodies of the recipient to the donor HLA antigen using bead technology .This method assist rapid identification of a suitable donor .Multiple synthetic microspheres are each given a single HLA antigen coating and incubated with recipient serum. Alternatively a microsphere or bead may be coated with multiple antigen to allow more efficient screening.
This method allows for sensitivity of flow cross match and identifying specific antibody.
The combination of these various assay allow us to exclude insignificant antibodies from risk assessment and allowing better preparation when more significant antibodies are identified that can potentially complicate tough not preclude transplant .
More studies are needed to understand the significance of low level DSA , non HLA Abs and non-complement fixing antibodies.
Using of cross-matched in pre transplantation assessment is very important to decrease the incidence of rejection due to antibodies against HLA. techniques used for cross-matching include:
a- CDC: it is important in determination of the presence of donor specific Abs in recipients to assess prognosis of graft survival. This test depend on complement activation.T & B cells of donor mixed with recipient serum with addition of complement, then observe the percentage of cell lysis ( 20% is the minimum cut off of positive result). To increase the sensitivity of the test adding of AHG. This test had several limitation as false positive ( presence pf autoAbs in recipient serum which can be abolished by adding DTT to prevent IgM complement activation), false positive ( due to presence of Low level of DSA or due to Abs not activated by complement). persistence positive DCD after sensitization is an absolute contraindication for transplantation. But B cells crossmatch positivity no necessarily associated with humeral rejections T cell, but negative B cell cross-match is associated with better prognosis.
b- Flow cytometry: it measure DSA by using impedance flow cytometer. The measurement of DSA is done either by fluorescence intensity or by serial dilution of recipient serum that react with donor lymphocytes. If CDC negative & flow cytometry is positive it may be due to
i. non complement fixing Abs
ii. non HLA Abs
iii. low level of Abs.
So the test used for sensitized patients.
2. Solid phase Ab detection assay:
a- ELISA: the serum of recipient is mixed with specific Abs that bind to available HLA epitopes, then IgG added to combined anti-HLA ABs. It used in sensitized patients.
b- bead technology (Luminex), by using beads labelled with fluorescein. The result measured either by flow cytometry or Luminex ( the degree of fluorescein measured by MFI).It can predict pre transplant risk & used for post-transplantation DSA monitoring.
3 Virtual Cross-match:
Mainly it used for deceased donor transplantation to decrease ischemic time.By using bead technology the recipient anti-HLA Abs compared with donor HLA Ags. Johnson et al found that transplantation can be done depending on virtual cross-match. If the flow cytometry result is positive & virtual cross-match is negative there is no change in graft outcome.
Winner
Well done
Non-HLA antibodies
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
A Review of Crossmatch Techniques
The genesis of crossmatching was an attempt to identify those transplant recipients who had a higher likelihood of acute vascular rejections after receiving the graft from a given donor( Hyperacute Rejection).
DSAs are commonly generated by:
(i) Blood transfusion
(ii) Previous transplantation
(iii) Pregnancy. One important implication of this is that wives often develop HLA antibodies against their husband’s HLA in the course of conception. This may preclude the spouse as a donor in the event of renal failure. DSAs are conventionally identified with cell based assays, solid phase and more recently the virtual crossmatch.
Crossmatch Techniques:
(a) Cell based assays
1.CDC technique; Complement dependent cytotoxicity crossmatching technique involved:
(i) Isolating donor lymphocytes (cadaveric/ living).
(ii) The B and T cells are separately tested against serum from the recipient.
(iii) Humeral immunological response is mediated though activation of the complement system by the classical pathway.
(iv)To demonstrate the effects of this cascade, complement is added to the mixed recipient serum and donor lymphocytes and cell lysis of the lymphocytes is observed.
(v) Negative test when donor-specific anti-HLA antibodies are absent and complement activation does not occur.
Positive test when donor-specific antibodies bind to lymphocytes, activate complement and cause cell lysis.
How to quantify the strength of reaction in CDC technique:
How to increase sensitivity of CDC technique:
Addition of anti-human immunoglobulin (AHG) helps increase the sensitivity of CDC as multiple AHGs bind to a single donor specific antibody, amplifying its complement activation response at smaller titres.
Limitations of CDC :
1.false positive reaction: often a result of autoantibodies in the recipient serum. This can be overcome by addition of Dithiothreitol (DTT) which helps prevent predominantly IgM autoantibody mediated complement activation and allows only IgG (donor specific antibodies).
2.false negative reaction: may occur when DSA levels are too low to result in activation of the complement cascade or if the antibodies are of the type that does not cause complement activation.
3.Antibody levels of the recipient may vary with time due to new antigen exposures and immunological sensitization that may occur after antigen exposure in the form of blood transfusions, pregnancy.This emphasized the importance of matching the most recently drawn recipient serum sample with the donor lymphocytes, especially in the context of patients on a donor transplant list for cadaveric transplant.
(2) Flow cytometry: in the Flow cytometry for crossmatching, the donor lymphocytes are mixed with the recipient’s serum. These bind to the donor specific antibodes and are quantified by detectors in the impedence flow cytometer.
Two methods of quantification can be used:
(i) Measurement of the fluorescence intensity as a ratio of the control (channel shifts).
(ii) Serial dilutions of the recipient’s serum are made to react with donor lymphocytes and the minimum dilution which yields
a negative result gives a measurable estimate.
Interpretation of flow crossmatch results:
In the light of a negative CDC result, a positive flow cytometry could be interpreted as indicating the presence of any of the following :
The value of this test’s sensitivity lies in using it for cross matching of sensitized patients who have an inherently higher risk of acute graft rejection to determine transplant feasibility or need for desensitization protocols prior to transplant.
(b) Solid phase antibody detection assays:
1.Enzyme-linked immunosorbent assay (ELISA):
Enzyme-linked immunosorbent assay (ELISA): The initial application of ELISA was to detect HLA in both bound and free forms but its current utility is for the detection of HLA antibodies in serum.
(i) The modified assay uses HLA glycoprotein immobilized into mictrotiter wells.
(ii) The recipient serum is added and specific antibodies bind the epitopes available.
(iii) A wash is performed and anti-IgG with a passenger reported molecule (alkaline phosphatase) is added that combines
with the anti-HLA antibody.
(iv) After further washing to remove unbound antibody, a substrate is added which after dephosphorylation by the reporter
molecule undergoes a color change.
This method while effective in detecting sensitization in transplant candidates has been replaced by bead technology.
2.Bead technology (Luminex):
HLA antibody testing was revolutionised by the introduction of beads labeled with flourescein in the nineties.
Generally beads are impregnated with different ratios of two fluorochromes resulting in a signal that is unique to the specific bead. Each bead may have one or more HLA molecule types incorporated.
The basic steps for Luminex involve :
(i) incubation of recipient serum with the beads.
(ii) HLA antibodies of the serum will react with HLA antigens on the bead.
(iii) The beads are washed and incubated with a second antibody, usually anti-human IgG labeled with phycoerythrin.
Levels of testing:
(i) Screening: Beads are incorporated with a many molecules, both class I and II derived from cell lines.
(ii)Testing against a set of alleles from an individual genome: Beads have HLA molecules from a single cell line with two
molecules for each HLA locus. These results can be expressed as PRA.
(iii) Single antigen beads: These beads have a single type of HLA antigen molecules bound to them. The result is that a
conglomerate of heterogenous antibodies can be tested and specific HLA antigens can be identified.
Interpreting flow crossmatch studies:
The main significance of a positive test if the corresponding CDC crossmatch was negative. In this scenario, the reason for the positive match could be due to:
Single antigen beads are employed in allowing semiquantitative evaluation of HLA
antibodies in order to predict pretransplant risk as well as for donor specific antibody monitoring after transplant.
The virtual crossmatch
Steps of Virtual crossmatch techniques:
(i) Serum from the recipient is added to synthetic beads with either a set of antigens or a single antigen, each bead can be
identified by an independent dye signature.
(ii) Anti-HLA antibodies if present will bind to the specific bead.
(iii) A detector antibody will then bind and sequester a reporter dye.
(iv) Beads can be checked for the reporter dye using a laser beam, this builds a profile of the antibodies present in the
recipient that can be compared with the HLA construct of a potential donor thus predicting the result of crossmatch.
This method allows for the sensitivity of flow crossmatch combined with identifying specific antibody. Johnson et al. found that the final transplant decision could be taken on the basis of the virtual crossmatch irrespective of the flow cytometry without significant difference in the clinical outcome in those who were flow cytometry positive but virtual crossmatch negative.
Comparison of Techniques
1.Complement dependent cytotoxicity
Advantages:
Drawbacks:
2.Flow cytometry
Advantages:
Drawbacks:
3.Solid phase assays (ELISA and bead technology)
Advantages:
Drawbacks:
The idea of cross matching came to detect patients at higher risks for developing acute rejections .
These tests tries for detect any DSA present in the serum of the recepient.
**CDC techniques **
It was implemented in early 60s , donor s lymphocytes are mixed with serum of recepients , then complement is added .
Presence of DSAs will lead to complement activation through the classic pathway .
Limitations of CDC techniques include false positive results due to presence of autoantibodies ( which may be improved by addition of DTT ) , and false negative results due to very low Titres of Abs ( addition AHG may amplify immune response from those low Titre Abs )
T cells expresses only class I antigens while B cells expresses both MHC Class I and II antigens.
So positive B cross matching means either that there is DSAs against class II antigens , or low Titres of DSAs against class I antigens,
Also this is not always associated with acute rejections
While patients with Positive T cell cross match , usually have poor outcomes , so a persistent positive T cell cross match is absolute contraindication for transplantation
** flow cytometry techniques **
Donors lymphocytes are patients serum are mixed together , and the immune reaction is quantified using flow cytometer .
This technique is used in the high risk patients
Positive flow cytometer with negative CDC means that :
DSAs are not complement fixing
DSAs are non HLA antibodies
Very low Titres DSAs
** solid phase assays **
*) ELISA
*) luminex
Patients serum are mixed with flourecin beads , that are labelled with MHC class I and II antigens , incubated , then beads are washed and another incubation with anti human IG is done .
**) Virtual Cross match.
Using the bead technology, detected HLA antibodies are compared to donors HLA antigens .
Studies showed that a transplantation decision could be taken on basis of Virtual Cross match , even if flow cytometry results were positive
Winner
Well done
Non-HLA antibodies
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
Crossmatch is a cornerstone of transplantation sciences. Initial methods relied on complement dependent cytotoxicity. However, recently cell based and solid phase assays have been utilized. Flow cytometry is useful in previously sensitized patients while bead technology is an advanced technique for HLA antibody testing. Virtual cross match uses bead technology to detect specific recipient antibodies without requiring donor serum. Crossmatch techniques include cell-based assays, solid phase antibody detecting assays, and the virtual crossmatch.
Cell-base assays
CDC technique determines the presence of DSA. Serum from the recipient is added to separated T and B cells of donors in the presence of complement. A positive test is characterized by cell lysis. The results are represented in terms of the percentage of lymphocytes that have undergone lysis in the cell panel as a result of panel reactive antibody. 20% is a minimum cut off for positive results. Addition of AHG provides help to increase the sensitivity of CDC test. Titrated cross match method is another method for quantification of strength of reactions. The limitation of this technique is false positive and false negative results. False positive results due to autoantibodies can be overcome by addition of DTT and elimination of Ig M antibody. False negative results may occur with a very low level of DSA or if antibodies don’t cause complement activation. Because of the variation in antibody level of recipients, it is very important to match the most recently drawn recipient serum sample with the donor lymphocytes. Vascular endothelial cells express both class I and class II HLA, hence antibody responses to them can predict antibody mediated rejection.
A positive T cell and B cell cross match would indicate antibodies to both HLA type I and HLA type II antigens. A positive B cell cross match alone would indicate presence of DSA to type II antigens or low levels of DSA to type I antigens, while a positive T cell cross match alone is usually due to technical errors.
A positive T cell cross match results in a unacceptably high incidence of acute antibody mediated rejection and the recipient needs to undergo desensitization treatment. Persistence of positive T cell cross match after desensitization is an absolute contraindication of kidney transplantation.
On the other hand, a positive B cell cross match is not consistently associated with humoral rejection. High false positive results can be overcome by Luminex testing. In positive B cell cross match, the significance of HLA type II antibodies is less in acute or hyper acute rejection.
Flowcytometry crossmatch
Serum from the recipient is added to donor lymphocytes in the presence of anti IgG fluorescein-labelled antibodies. Quantification can be done by measurement of the channel shift or by serial dilution of the recipient’s serum.
A negative CDC result with a positive flow cytometry can indicate: Non-complement fixing antibodies or Non-HLA antibodies or Low-level antibodies, and correlates with poorer graft survival compared to both negative CDC and flow cytometry tests.
Solid phase antibody detection assays
ELISA: its current utility is for the detection of HLA antibodies. In this method, the recipient serum is added to immobilized HLA glycoproteins and specific antibodies bind to available epitopes. This method has been replaced by bead technology.
Bead technology (Luminex): This technique uses beads labeled with fluorescein and may have one or more HLA molecule types. Incubation of recipient serum with beads, leads to reaction with HLA antigens on the bead.
This test is used for screening, testing a set of alleles from an individual genome, and single antigen bead. Quantification is achieved by calculation of MFI in the Luminex method. Cut off value is different between laboratories and low cutoffs increases sensitivity while affecting specificity. Single bead assay provides semi quantitative evaluation of HLA antibodies.
The virtual cross match
The recipient serum is added to synthetic beads with a set of antigens or a single bead antigen and anti HLA antibodies if present will bind to the specific bead, then the dye will be checked by laser beam.
This method builds a profile of the antibodies of a given recipient that can be compared with the HLA construct of a potential donor. In negative virtual cross match and positive flow cytometry circumstances, there is no significant influence on clinical outcome. Using virtual cross match will reduce cold ischemia time and is valuable for sharing organ program.
Complement dependent cytotoxicity:
Advantages:
Mainstay of pretransplant screening.
Key role in preventing hyper acute rejection
Detects only complement binding antibodies
Drawbacks:
Detects IgM and IgG antibodies and non HLA antibodies
Flow cytometry:
advantages:
More sensitive than CDC
Less inter-observer variability
Drawbacks:
Slow turnaround times, thus suitable for living donors scenarios
Solid phase assays (ELISA and bead technology):
Advantages:
Most sensitive
Improved rates of success in retransplants
Identification of all HLA alleles for which the recipient harbours antibodies
Avoid false positives of antibody binding to non-HLA antigens
Eliminates confusion regarding the class of HLA that are detected
Drawbacks:
Interpretation of a positive assay in the presence of negative CDC/flow crossmatch is ambiguous
Relevance of low level antibodies to low significance antigens is debatable
Detects both complement-fixing and non complement-fixing HLA antibodies (may be denied a graft without established clinical significance.
Interference by IgM
Incomplete library of antigens in the bead sets
Variability of HLA density on the beads
Positive CDC-XM may be as a result of IgM autoimmune disease and can be omitted by adding DTT, auto crossmatch, washing and heating recipient’s serum or increased incubation time.
Positive FCXM but negative CDC-XM may be as a result of low titer or non-complement fixing Abs. Positive FCXM without DSA detected by Luminex may be as a result of prozone phenomenon, inaccessible HLA-Ag on the Luminex beads sharing same epitope by many beads and low MFI or absence of a specific HLA-Ag on the Luminx kit.
Thanks Nasrin
What is the picture non-HLA antibodies when you do crossmatch (FCXM) in the absence of DSA?
Cross-match testing is key in transplantation. the better the estimation of immunologic risk the best the prediction of outcome. There may be no stand-alone technique. All are integral to each other.
Crossmatching techniques development aimed to identify recipients who are likely to develop vascular rejection of grafts.
Various exposures may cause sensitization. Pregnancy, blood transfusions, and even previous infections may result in antibody formation.
Over more than 70 years many techniques came to the surface. Classical and still the mainstay is the complement-dependent cytotoxicity crossmatch (CDC). Followed by FLowcytometry this has the advantage of detecting lower antibody levels more specifically and sometimes more efficiently are called cell-based techniques. Aside from virtual crossmatch in which no real recipient blood samples are utilized, rather patients HLA antibody specificities with donors HLA type are matched we have the newer solid-phase techniques. ELIZA and Luminex which is a special type of flowcytometry-specific beads are used.
CDC crossmatch
First developed in the late 60s by Terazaki and Patels, recipients serum is used. Next, lymphocytes are incubated. The third complement is added. In case of antibodşes presence, the lymphocytes will lyse and this will be tested under the microscope. Here %20 cell lysis is the cutoff for positive results.
As T cells have class-I antigens while B cells have both, a positive T and B cross-match will point to class-I antibodies. Both t and B crossmatch negativity is favored. Only T cell positivity is a risk but B crossmatch only will refer to technical error as the share presence of class-I presentation with T cells.
Flow cytometry assays:
Flow cytometry is a very sensitive assay introduced in the 80s. Here the recipient’s serum is incubated with the donor’s lymphocytes. Fluorescence dye is applied and cells are markered (anti-cd3 pan marker, anti-cd-19 or -20 or B cells). the cells bound to
A positive crossmatch in case of negative CDC crossmatch may be due to very low levels of antibodies or other non-complement fixing or nonHLA antibodies. The role of flowcytometry then is essential in highly sensitized or those who have a high risk of rejection.
Solid-phase assays:
Here purified or engineered HLA antigens are used. ELIZA has been replaced with bead technology (LUMINEX). Either single antigen bead, phenotype bead, or mixed beads are used. The mean fluorescence intensity (MFI) is detected. As Luminex is a type of flowcytometry its positivity in case of negative CDC CX is also either due to on-HLA antibodies or very low levels.
Virtual crossmatch:
Here the need for a final physical crossmatch is replaced virtually. One handicap of this is that it is dependent on information supplied to the computer. Still have a big role in some patients especially to decided the suitable recipients in case of cadaveric transplants.
Cell based assays:
CDC technique:
Serum from recipient is added to donor B and T lymphocytes in presence of complement.
The result is assessed by percentage of lymphocytes destructed due to complement activation or panel reactive antibody
It provides qualitative and semi quantitative estimate of the strength of the reaction
have a main role in preventing hyperacute rejection
Titred crossmatch:
provides more quantification of the strength of the reaction by using doubling dilutions of recipient serum which help to decide the desensitization protocol.
False positive results due to autoantibodies in recipient serum , avoided by adding DTT which prevent IgM antibody mediated complement activation and allow only IgG DSA.
less sensitive as may detect non-HLA antibodies against antigens irrelevant to transplant.
False negative: due to very low DSA level which can’t activate complement cascade or due to presence of antibodies which don’t activate complement.
Studies showed that positive T cell crossmatch is associated with poor outcome and increased incidence of acute graft rejection
persistent positive T cell crossmatch post desensitization is absolute contraindication to transplantation
B cell crossmatch significance is still uncertain due to high rate of false positive result but
true positive result still require desensitization before transplantation.
Flow cytometry:
Donor lymphocytes are mixed with recipient serum in presence of anti IgG fluorescein labelled antibodies
Quantification of binding of DSA to lymphocytes done by:
fluorescence intensity as a ratio of the control (channel shifts)
serial dilution which give a measurable estimate
Negative CDC and positive flowcytometry indicate non complement fixing antibodies, non-HLA antibodies or low level of antibodies.
flow cytometry is more sensitive than CDC, used for crossmatch in sensitized patients at high risk of acute rejection to determine the need for desensitization before transplantation
It requires long time so more suitable for living donors
Solid phase antibody detection assay:
The most sensitive method for detecting DSA
ELISA:
used for detection of HLA antibodies in serum, replaced by bead technology
Bead technology (Luminex)
Each bead may have one or more HLA molecule, HLA antibodies of serum will react with antigen on beads
level of testing:
So a combination of heterogenous antibodies can be tested and specific HLA antigen can be identified
Quantification of results done by flow cytometry measuring channel shift associated with binding of antibodies or Luminex method using mean fluorescence intensity.
Single antigen bead assay:
The virtual crossmatch
Anti-HLA antibodies of the recipient are compared to donor HLA antigens using bead technology, it predict eventual crossmatch
recipient serum is incubated with beads each has either a unique HLA antigen or set of antigens on its surface
anti-HLA antibodies if present will bind to specific bead so provide a profile of antibodies present in a potential donor so predict the result of crossmatch
can allow more efficient screening when a bead is coated with multiple HLA antigens.
Crossmatch techniques started a half century ago remain the base of transplantation science.
Its mainly done to identify recipients who are at higher risk for hyperacute and acute rejection
Hyperacute rejection occurs due to presence of preformed antibodies in the recipient’s serum to one or many HLA (human leucocyte antigens). referred to as DSAs (donor specific antibodies)
DSAs formation known as sensitization occurs due (i) Blood transfusion (ii) Previous transplantation (iii) Pregnancy.
One important implication of this is that wives develop HLA antibodies against their husband’s HLA during pregnancy.
Crossmatch Techniques
First Cell based assays
1-Complement dependent cytotoxicity crossmatching.
2 -Flow cytometry.
▪︎Complement dependent cytotoxicity crossmatching ( CDC) ;
Terasaki and Patel in 1969 observed the presence of DSAs in transplant recipiects were associated with a higher incidence of acute and hyperacute graft rejection
Technique of CDC:
it is done by adding donor lymphocytes to recipient serum with addition of complement .
B and T cells are separately tested.
if DSAs are present in recipient’s serum it will bind to lymphocytes activating the complements lead to lysis of donor’s lymphocytes
Reading results :
The results presented in the form of percentage of lymphocytes that undergoes lysis which is the percentage of panel-reactive antibody (% PRA).
20% is considered as the minimum cut off for positive results .
titred crossmatch is used to increase the strength of the test done by testing donor lymphocytes with serial doubling dilutions of the recipient’s serum. The higher serum dilution required to give a negative result, the greater the strength of the immune reaction against donors cells which indicates need for a desensitization of recipient.
To increase the sensitivity of CDC-XM anti-human immunoglobulin (AHG) can be add so multiple AHGs bind to a single donor specific antibody, amplifying its complement activation response at smaller titres.
Limitations of CDC-XM
1- false positive due to detection of IgM autoantibodies in the recipient serum. It can be avoided by addition of Dithiothreitol (DTT) which helps prevent IgM autoantibody complement activation and allows only IgG
2- false negative occurs with very low DSAs titre or non complement activating AB
3- CDC-XM is changing due to exposure to new different antigens.
Interpretation of results:
Vascular endothelial cells of the transplant graft express both classes of HLA antigens.
T cells express only class I antigens while B cells express both types .
persistent positive T cell crossmatch after desensitization is according to current guidelines, an absolute contraindication to renal transplant as it is associated with humoral rejection and lead to hyperacute rejection.
positive B-cell crossmatch results:
•high rate of false positive makes its significance uncertain.
•negative B cell crossmatch has better results
•False positive results can be avoided concurrent Luminex testing for DSAs which increases its specificity.
•Not consistently associated with humoral rejection as positive T cell crossmatches.
In case of true positive B cell crossmatches, anti-class II HLA antibodies are less signifcantl in acute/ hyperacute rejection but the patient
will need desensitization befor transplantation .
▪︎Flow cytometry crossmatch :
Also done by mixing serum from the recipient with donor lymphocytes (T or B) with addition of of anti-IgG fluorescein-labelled antibodies.
Positive test when DSAs bind to lymphocytes that are detected by flow cytometry once the anti-IgG fluorescein-labelled antibodies tag the lymphocytes.
Positive Flow cytometry with negative CDC-XM indicates either
Non-complement fixing antibodies,Non-HLA antibodies or Low-level antibodies.
This test is used for cross match of highly sensitized patients who are at higher risk of acute rejection to determine transplant feasibility or need for desensitization .
CDC crossmatch negative patients who had positive T-cell flow cytometry results had significantly poorer absolute 5 year graft survival rates than patients with both CDC and flow cytometry crossmatch negative.
Solid phase antibody detection
They are the most sensitive of all the techniques for detecting donor specific HLA antibodies.
It help in identification of all HLA alleles to which the recipient has antibodies.
They avoid false positive antibody binding to non-HLA antigens and eliminates confusion regarding the class of HLA that are detected in contrast to CDC-XM. Done by ELISA OR beads ;
ELISA is used for the detection of HLA antibodies in serum.
recipient serum is added and specific antibodies bind the epitopes available. A wash is performed and anti-IgG with alkaline phosphatase is added that combines with the anti-HLA antibody. further washing to remove unbound antibody, a substrate is added which after dephosphorylation by the reporter molecule undergoes a color change.
Although is effective to detect sensitization but it has been replaced by bead technology .
Bead technology (Luminex):
Generally beads are impregnated with different ratios of two fluorochromes resulting in a signal that is unique to the specific bead.
Each bead may have one or more HLA antigens types incorporated. basic steps involve first, incubation of recipient serum with the beads. HLA antibodies of the serum will react with HLA antigens on the bead.
beads are washed and incubated with a second antibody, usually anti-human IgG labeled with phycoerythrin
It is important in :
(i) Screening: Beads are incorporated with a many molecules, both class I and II derived from cell lines.
(ii) Testing against a set of alleles from an individual genome results can be expressed as PRA.
(iii) Single antigen beads: beads have a single type of HLA antigen molecules bound to them. specific HLA antibody can be identified .
Quantitation of the results is done by
Flow cytometry
Luminex method Two lasers are used to excite the fluorochrome of the bead and the phycoerythrin bound to the antibody and degree of fluorescence is calculated as a mean fluorescence intensity (MFI) each centre may determine according to its experience the MFI number that is accepted to transplant the patient .
Interpretation of results
CDC crossmatch negative with positive luminex indicate the presence of low level antibody, non-HLA antibody or a non-complement binding antibody.
Single antigen bead assays detect both complement-fixing and non-complement-fixing HLA antibodies so a doner can be refused without established clinical significance
The Virtual crossmatch ;
The test compares HLA antibodies in the recipient’s serum to the donor’s antigens using single antigen bead assays. It helps detection of suitable donor.
this builds a profile of the antibodies present in the recipient that can be compared with the HLA antigens of a potential donor predicting the result of crossmatch.
Transplantation can be done while positive flow cytometry and negative virtual crossmatch.
Nowadays crossmatch tests before transplantation is necessary to prevent hyperacute rejection conducted by preformed DSA .Different XM techniques are cell based and solid phase assays. Cell based assays include:
1–complement dependent cytotoxicity (CDC-XM): Recipient’s serum is added to donor’s lymphocytes. If donor lymphocytes are lysed after adding complement, the result of test will be positive and shows presence of DSA. More than 20% shows positive rest. Titered XM is done by serial dilution of patient serum that is needed to have negative result. AHG -CDC-XM: Adding anti-human immunoglobulin improve CDC sensitivity. False positive results are seen when autoantibody are present that could be omitted by Dithiothreitol (DTT) adding. False negative results is associated with low titer DSA or non- complement fixing Abs. The result of this test is expressed as T cell and B cell XM. Since T cells show class I antigenes and B cell both class I and II positivity of either of them has different meanings:
Both T cell and B cell XM positive: antibodies to HLA type I and II.
Positive B cell XM:
1-DSA to class II Ags
2- low level class I Ags.
3 – Necessitated Luminax for specificity
Positive T cell XM : persistence of positive T cell XM after desensitization is absolute contradiction to renal transplant .
4- Persistence of Anti-HLA Ab class II needs desensitization before TX.
2- Flow cytometry: Donor lymphocytes are added to patient’s serum. Then by measuring florescence intensity as a ratio of the control (channel shifts) or serial dilution its intensity is determined. Negative CDC-XM and positive flowcytometry may be related to:
1- Non-complement fixing Abs
2- 2-Non-HLA Abs
3- 3-low level Abs. It’s positivity may show need for desensitization.
3- Virtual Xm using solid phase assays:
1 – ElISA
2- Luminex: uses beads that contain HLA molecules labeled with fluorescein and are incubated with patient’s serum .Then beads are washed and an anti-IgG Ab labeled with phycoerythrin is added. This test may be used as screening by beads that contain many molecules or specific single Ag beads to detect specific HLA Ag and it’s intensity is express as MFI (means flow intensity) for virtual Xm anti-HLA Abs are determined by Lumina method and in recipient’s serum and are compared with donor’s HLA to define DSA and predict the result of XM. If the result of XM is negative, then irrespective of flowcytometry, TX can be done that decreases cold ischemia time and results in a better graft outcome especially in decreased donors and pre-sensitized recipients.
Thanks Nasrin
You mentioned the answer of the question here which is non-HLA antibodies. I will consider it the answer of the question and you deserve the reward.
Positive cross match (both T and B cells are positive), but no DSA can occur in the following situations :
1- In autoimmune diseases ; due to either the presence of clinically non significant IgM antibodies as in rheumatoid arthritis and SLE or sometimes the antibodies are complement-fixing IgG autoantibodies as in SLE
2- In HIV positive patients: non-specific positive crossmatches can occur in HIV positive patients
3- Those with prior intake of rituximab : use of rituximab is associated with false positive cross match result
4- Highly sensitized recipients due to previous transplantation: as they may have positive cross match abd low or undetected DSA
These patients can be transplanted with good outcome (risk of ABMR, CAMR is low) especially if auto cross match is positive (indicating autoimmune disease), but monitoring of DSA post transplantation is important since the incidence of development of denovo DSA post transplant is increased.
But in highly sensitized recipients (cPRA> 80%) the risk of ABMR, CAMR is greater even if there is no DSA
Thanks Dr Sherif
autoantibodies will get excluded automatically by the atocrossmatch and also DTT, augmented CDC, etc
You are right. Rituximab can cause positive crossmatch. Low level DSA can cause negative CDC but not FCXM
Your answer is partially correct, but you derive the present as you mentioned rituximab.
Winner
Non-HLA antibodies
This is the typical presentation. Of course technical errors could be a factor, but the HLA lap will repeat the test twice before coming to a conclusion.
In my practice currently we still depend on CDCXM and we we are doing well with it. Recently our hospital bought new Luminex machine but is not yet functioning and hopefully next year it will start working and adding complexity to our understanding.
Abstract ; Crossmatch test is the cornerstone for practicing safe transplantation. The test evolved from the era of the standard CDCXM to more sensitive tests such as flow cytometry, solid phase assays and virtual crossmatch.
Introduction ; The idea of the crossmatch is to identify patients at high risk of hyper-acute rejections. This occurs because of preformed antibodies in the recipient’s serum as a results of sensitisation. Sensitization evens includes ; pregnancy, blood transfusion and previous transplantation.
Cell based assay ; In 1969, Terasaki and Patel demonstrated that the presence of preformed antibodies were associated with hyper-acute rejection(1). The principle of CDCXM is mixing donor’s lymphocytes with recipient’s serum, complement is added and if antibodies are presence ,it binds donor’s lymphocytes resulting in cell lysis. The test is interpreted by % of lymphocytes which undergone lysis. 20% is considered to be positive(2). The strength of antibodies can also be detected by serial dilutions of the recipient’s serum. The sensitivity of the CDCXM can be improved by addition of anti-human globulin antibodies. Limitations ; are false positive test due the autoantibodies(IgM). This is overcome by adding Dithiothreitol(DTT) which prevent IgM from binding complements(3). False negatives are also seen due to either low levels antibodies or non-complement binding antibodies(4).
Outcomes in positive T cell crossmatch results ; positive cell crossmatch is the only contraindication to transplantation unless the patient is sensitised.
Outcomes in positive B cell crossmatch results ; positive B cell crossmatch is not clearly associated with hyper-acute rejection. Despite that, desensitisation is required before transplantation in the setting of truly positive B cell crossmatch results.
Flow cytometry ; Recipient’s serum is mixed with donor’s lymphocytes, and fluorescence -labelled antibodies are added. If anti-donor’s antibodies are present, it will bind donor’s lymphocytes and the strength of the reaction is quantified by detectors in the impedence flow cytometer. Quantitation is done by 1. measurement of the fluorescence intensity as ratio of control = Channel shift. 2. Serial dilution of the recipient’s serum reacting with donor’s lymphocytes . The minimum dilution that gives negative results is measured.
Interpretation of the crossmatch results ; Negative CDCXM and Positive Flow cytometry ; 3 possibilities 1. non-complement binding Ab 2. non-HLA Ab 3. low level Ab
The flow cytometry crossmatch is useful in sensitised patients to make decisions regarding desensitisation protocols(9)
Solid phase assays ;
The Virtual crossmatch ; The test compares HLA antibodies in the recipient’s serum to the donor’s antigens using single antigen bead assays. It aids in the detection of suitable donor(16). Transplantation can still proceeds with positive flow cytometry and negative virtual crossmatch
Comparison of techniques ;
Disadvantages ; 1. Low sensitivity 2. Detects autoantibodies(IgM)
2.Flow cytometry ; Advantages ; More sensitive than CDC
Disadvantages ; Slow turn around time
3.Solid phase assays ; Advantages ; Highest sensitivity
Disadvantages ; 1. False positive 2. Un-necessary exclusions of patients from transplantation 3. Difficult to interpret positive results if both CDCXM and Flow cytometry XM were negative
Conclusion ; CDCXM is still the main crossmatch test for screening pre-transplant HLA antibodies. The recent tests are more sensitive than CDCXM. Combination of tests are essential to make decisions about transplantation.
VARIOUS CROSS MATCH TECHNIQUES:
Well done and thank you for the hard work.
Please not in capital letters. It is not user friendly and difficult to read
crossmatch was used to diagnose transplant recipients who at higher risk for acute rejections and hyperacute rejection
hyperacute rejection result from preformed antibodies in the donor serum which named as donor specific antibiotics (DSA)
These antibodies result from the following causes
1- Blood transfusion
2- Previous transplantation
3- Pregnancy
There are 3 methods used to measure the DSA titer
Cell based assays
solid phase
virtual crossmatch
*Cell based assays
Cell based assays done by
-CDC technique
-Flow cytometry
CDC technique (Complement dependent cytotoxicity crossmatching)
The benefit of CDC technique is in identifying the presence of DSA in serum of transplant recipients so it will help to determine the graft survival after transplantation
The procedure of CDC
The technique done by isolating donor lymphocytes. The B and T cells are tested in separate manner against serum from the recipient. Humeral immunological response is mediated though activation of the complement system by the classical pathway.then complement is added to the mixed recipient serum and donor lymphocytes and cell lysis of the lymphocytes is observed.
The result
The result can be obtained depending on percentage of lymphocytes that undergo lysis
20% is considered as the minimum cut off for positive result
To increase test sensitivity we can added antihuman immunoglobulin (AHG) because multiple AHGs can bind to a single donor specific antibody and this will amplify the complement activation response at smaller titres.
Limitation of CDC
CDC test is less sensitive and detect only complement binding antibodies.
false positive result from presence of autoantibodies in the recipient serum .this result can be prevented by addition of Dithiothreitol (DTT) which prevent IgM autoantibody mediated complement activation and allows only IgG (donor specific antibodies)
False negative reactions may occur when DSA levels are too low to result in activation of the complement cascade or if the antibodies are of the type that does not cause complement activation
Interpretation of CDC test
T cells express class I HLA antigens (A,B,C) . B cells express both class I and II HLA antigen(A,B,C,DR,DP,DQ)
positive T cell and B cells cross match indicate presence of antibodies to HLA type I and II antigens while a positive B cell cross match could indicate either DSAs to type II antigens alone or Low levels of DSAs to type 1 antigens
Outcome in positive T-cell crossmatch
Poor outcome and high risk of rejection occurs in positive T-cell crossmatch recipients so desensitization must be done before transplantation and repeat the test again after that.however if persistent positive T-cell test even after desensitization is considered as an absolute contraindication to renal transplantation
Outcome in positive B-cell crossmatch
positive B cell crossmatch is not as associated with humoral rejection like positive T cell crossmatches. a negative B cell crossmatch is associated with better outcomes but the there is high rate of false positive result so we can overcome this problem by using luminex test for DSA.
In case of true positive B cell crossmatches the significance of anti-class II HLA antibodies is of little importance in acute/ hyperacute rejection but still need desensitization of the recipient prior to transplant
Flow cytometry
More sensitive than CDC test and can detect lower titer of DSA so it
Used in transplant recipients with high risk of rejection
Procedure
Serum from the recipient is added to donor lymphocytes (T or B) in the presence of anti-IgG f fluorescein-labelled antibodies.
Negative test when donorspecific antibodies are absent and binding does not occur. Positive test when donor-specific antibodies bind to lymphocytes that are detected by flow cytometry once the anti-IgG fluorescein labelled antibodies bind to the lymphocytes
Interpretation of flow crossmatch results
negative CDC result with positive flow cytometry indicate the following conditions
Non-complement fixing antibodies
Non-HLA antibodies
Low-level antibodies
CDC crossmatch negative with positive T-cell flow cytometry indicate poorer absolute 5 year graft survival rates than those who have both CDC and f low cytometry crossmatch negative
*Solid phase antibody detection assays
Most sensitive methods in detecting DSA and can identify all HLA alleles
Done by
-Enzyme-linked immunosorbent assay (ELISA)
-Bead technology (Luminex)
Enzyme-linked immunosorbent assay (ELISA)
The recipient serum is added and specific antibodies bind the epitopes available. A wash is performed and anti-IgG with alkaline phosphatase molecule is added to combine with the anti-HLA antibody. further washing needed to remove unbound antibody, a substrate is added and then after dephospho-rylation by the reporter molecule undergoes a color change
Although this method is effective but it replaced by the Bead technology (Luminex)
Bead technology (Luminex)
Done by incubating the recipient serum with the beads then the HLA antibodies of the serum will react with HLA antigens on the bead. The beads are washed and incubated with a second antibody, usually anti-human IgG labeled with phycoerythrin
Quantitation of the result
The test done by
Flow cytometry
Luminex method
Interpreting flow crossmatch studies
CDC crossmatch negative with positive luminex indicate the presence of low level antibody, non-HLA antibody or a non-complement binding antibody.
* The virtual crossmatch
T his technique is used to detecting the presence of anti-HLA antibodies of the recipient to the donor HLA antigens using bead technology.
Multiple synthetic microspheres are coated with a single HLA antigen and incubated with recipient serum.
The test give more efficient screening in renal transplantation. final transplant decision should be taken on the basis of the virtual crossmatch irrespective of the flow cytometry without significant difference in the clinical outcome in those who have flow cytometry positive but virtual crossmatch negative
CDC crossmatch : The test is simple , and non expensive. But it had low sensitivity . The sensitivity can be increased with the addition of anti human globulin.
False positive results due the presence of auto antibodies , and false negative results could be due to :
If the CDC cross match is positive and transplantation is done there is high risk of hyperacute rejection.
Flow cytometry crossmatch : It has better sensitivity than CDC crossmatch . but it’s more expensive and more time consuming. IT can detect non complement fixing antibodies and can detect low antibodies level.
CDC XM -ve , Flow cytometry crossmatch +ve patients had poor graft outcome when compared to CDC -ve , Flow crossmatch -ve patients.
Virtual crossmatch : By using Luminex technology , we can detect anti HLA antibody in the recipient serum and comparing it with the HLA of the donor, we can have virtual crossmatch. With the use of virtual crossmatch , the time to transplantation of highly sensitized is reduced and cold ischemia time also reduced.
In our center we have living donor transplantation only . we do CDC , if positive , the donor is excluded.
If cdc is negative , we proceed with Flow cytometry, and Luminex solid bead assy, if FCM XM, and anti HLA antibodies is negative we can proceed with Transplantation.
if the FCM XM is positive , we do plasmapheresis , and repeat the FCM XM if negative , we proceed with transplantation.
CDC technique:
It determines the presence of donor specifc antibodies in the serum of the recipient which is indicative of graft survival after transplantation.
It is done by isolating donor lymphocytes (cadaveric/ living). B and T cells are separately tested against serum from the recipient. The immunological response is mediated though classical pathway of the complement system. The complement is added to the mixed recipient serum and donor lymphocytes and cell lysis is observed. The percentage of lysed lymphocytes in the cell panel by complement activation or panel-reactive antibody represent the result.If more than 20% is considered positive.
Another way is titred cross match which is the more dilution needed to the recipient’s serum the more the immune response is.
Its wek points is the occurnce of false positive due to autoantibodies overcomed by DTT, where as false negative can occur due to low titers of
DSA.
Its advantage is being simple cost effective and its draw back is being less sensitive .
T cells express only class I antigens while
B cells express both.
-positive T cell and B cells cross match means presence of antibodies to HLA type I and II antigens
-positive B cell cross match means either DSAs to type II antigens alone or Low levels of DSAs to type 1 antigens.
– positive T cell crossmatch alone is usually due to technical error.
Flow cytometry: The donor lymphocytes are mixed with the recipient’s serum and are assessed by detectors in the impedence fow cytometer
If we have negative CDC result, a positive flow cytometry means the presence of either
-Non-complement fxing antibodies
-Non-HLA antibodies
-Low-level antibodies
So it is usualy used in cross matching of sensitized patients to determine transplant possibility or the need of desensitization before transplantation to lower rejection possibility .
Its advantage is being more sensitive and its disdvanatge is being suitable for living donors not cadaveric ones .
Solid phase antibody detection assays
Enzyme-linked immunosorbent assay (ELISA): used to detect HLA in both bound and free forms .
This method while being effective in detecting sensitization in transplant patients has been replaced by bead technology
Bead technology (Luminex): improved the HLA antibody testing by the using of beads labeled with fluorescein
Quantitation of the result is quantified by Flow cytometry and Luminex method .
Its advantage is being most sensitive and avoids false positive results.
Its draw back is that single antigen bead assays detect both complement-fxing and noncomplement-fxing HLA antibodies so a graft can be denied without having clincaly insignificant variation .
The virtual crossmatch
Is done by comparison of the anti-HLA antibodies of the recipient to the donor HLA antigens using bead technology. It predicts the eventual crossmatch and can identify rapidly the suitable donor.
CROSSMATCH TECHNIQUES INCLUDE:
1- Complement dependant cytotoxicity ( CDC ):
It is the most cost effective, available , and simple technique and detect only the complement fixing Abs.
It is done by mixing the recipient serum with T&B cells of the donor with adding of complement to detect the effect of complement activation after binding of DSAs with lymphocytes.
The result is estimated as percentage of lymphocytes destructed as a result of complement activation. Another method used to determine the result of the test is by serial double dilution of the serum of the recipient till being negative. The higher the dilution needed, the higher the positivity of the reaction.
False positive CDC may result due to the presence of IgM in the patient serum and this can be avoided by adding DTT.
False negative CDC may occur due to low level of complement or the antibodies are unable to activate the complement system.
** Interpretation of CDC:
1- positive CDC against both T&B means the antibodies are against both class I&II.
2- positive against B cell alone means either antibodies against class II alone or with low levels of antibodies against class I antigens.
3- positive against T cells alone means probable technical error.
High risk of acute rejection is associated with positive T cells crossmatch and the need for multiple PP treatment is needed to decrease this risk. But in case of persistent positivity post PP treatment , it is an absolute contraindication of TX.
However, positive B cells crossmatch is not highly associated with acute and hyperacute rejection as positive T cells crossmatch, but desensitization prior to transplantation should be done.
Negative B cells crossmatch is associated with better rates of graft survival.
2- Flowcytometry:
It is done by mixing the recipient serum with donor lymphocytes in presence of anti IgG fluorescein labelled Abs. Positive tests when the labelled Abs attach the lymphocyte and detected by flowcytometry.
Flowcytometry is more sensitive than CDC and can be used in sensitized patients. Negative CDC with positive flowcytometry indicate the presence of non complement fixing Abs, low titre of anti HLA Abs , or non HLA Abs.
3- Virtual crossmatch:
It is done using bead technology by detection of single or set of antigens on the surface of beads that the recipient formed Abs against and comparing the result with the donor specific antigens to determine the significant anti HLA antibodies.
It is the most sensitive technique.
Crossmatch techniques in transplantations
Cell based assays
CDC
• lies in ability to determine the presence of DSA anti-HLA antibodies
In the recipient’s serum
• helping in determination of prognosis of graft survival after transplantation
Techniques
• isolating lymphocytes from donor
• B and T cells are separated tested against serum from recipient
• serum from recipient is added to donor lymphocytes (T or B cells) in the presence of complement
• complement is added to mixed recipient serum and donor lymphocyte and cell lysis of the lymphocytes.This humeral Immunological response is mediated thru activation of the complement system by classical pathway
• results is in percentage and >20% is considered as positive
• other methods is titred crossmatch – preformed by doubling dilutions of the recipient’s serum
• additional of AHG – increases the sensitivity of CDC as multiple AHGs bind to Single DSA
limitations
• false positive (due to autoantibodies)-overcome by adding Dithiothreitol (DTT) which help prevent IgM autoantibody mediated complement activation and allows only IgG
• negative results -happens when DSA too low or if antibodies are not the type that can activate complement
Advantages
• Functional test that involves cells and serum containing antibodies
• Still a foundation test – cheap, availability and simple
• Key role in preventing hyper acute rejection
• Detects only complement binding antibodies
Drawbacks
• Detect IgG and IgM antibodies, autoantibodies and non-HLA antibodies – irrelevant to transplant
Outcomes in positive T cell crossmatch results
• Positive T cell cross match – poor outcome and unacceptably high incidence of acute graft rejection
• Positive – multiple PP lead to reproducible desensitisation ands low acute and hyper acute graft rejection
• Persistent positive post desensitisation – absolute contraindication
Outcomes in positive B cell Crossmatch
• Positive B cell – does not equal to hum oral rejection
• Negative – better outcome
• False positive can be overcome by Luminex
Flow cytometry
Technique
• Donor lymphocytes are mixed with recipient’s serum and these bind to DSA.
• Quantified by detectors in the impedance flow cytometer
• Methods
o Measurement of fluorescence intensity as ratio of the control
o Serial dilutions of recipient’s serum are added to react with donor lymphocytes and the minimum dilution which yields a negative result give a measurable estimate
Advantages
• More sensitive than CDC crossmatch
• Able to detect low levels of IgG HLA
• Less inter-observer variability
Drawbacks
• Slow turnaround times – suitable for Living related transplant than deceased donor transplant
Interpretation of flow crossmatch results
• Negative CDC and positive Flow
o Non complement fixing antibodies
o Non-HLA antibodies
o Low level antibodies
CDC negative and positive T cell flow cytometry -poorer 5 year survival rates
Solid phase antibody detection
Enzyme-linked immunosorbent assay (ELISA)
• For detection of HLA antibodies in serum
• Modified assay uses HLA glycoprotein immobilised into microtiter wall
Techniques
• Recipient serum added and specific antibodies bind to epitopes that available
• Wash is performed and anti-IgG with a passenger reported alkaline phosphatase is added which combines with anti-HLA antibody
• After additional washing to remove unbound antibody , materials is added after dephosphorylased by reporter molecule undergoes a colour change
• Best in detecting sensitisation in transplant candidates but has been replaced by bead technology
Bead Technology (Luminex)
• Beads are impregnated with different ratios of 2 fluorochromes that results in a signal that unique to the specific bead
• Every bead may have one or more HLA molecule types that incorporated
Techniques
• Incubate the recipient serum with beads
• HLA antibodies with react with HLA antigens on the beads
• Bead are washed and incubated with a second antibody which usually is anti human IgG labelled with phycoerythrin
Advantages
• most sensitive for detecting DSA for HLA antibodies
• enhanced sensitivity leads to improved rates of success in subsequent transplantations which allowed avoidance of HLA markers on subsequent grafts
• allow identification of all HLA alleles which antibodies harboured in recipient
• avoid false positive binding to non HLA antigens and avoid confusion pertaining class of HLA that are detected
Drawbacks
• when CDC and Flow crossmatch is negative, positive make it suspicious
• low level antibodies remains a debatable topic
• detect both complement -fixing and non – complement fixing HLA antibodies
• interference by IgM, incomplete library of antigens in bead sets, variability of HLA density on the beads
In our centre, we do both CDC and FCXM as a screening for every kidney transplantation recipient. If CDC/ FCXM found to be positive, the immunology lab will automatically proceed with Luminex to identify mean fluorescence intensity (MFI). The cost is an issue but the responsibility is on government as it’s allocated by ministry of health. Patient does not required to pay for the crossmatch. The immunology lab will take the responsibility to decide to proceed to Luminex or not based on CDC/FCXM.
1- Summary :
A- Complement dependent cytotoxicity (CDC) :
B- Flow cytometry: using impedence flow cytometer
C- ELISA :
D- Bead technology : it has replaced ELISA
2- Regarding reflection on my practise, I will adhere to the local polcies then negotiate with senior colleagues about trying new techniques in at least studying other than considering the clinical significance of these non-specific and low titres of DSA.
Advantages of complement-dependent cytotoxicity:
It is a functional test that uses cells and antibody serum. The availability, affordability, and simplicity of this test make it the cornerstone of crossmatch.
It has helped avoid hyperacute rejection as transplant immunology has evolved.
It exclusively finds complement binding antibodies.
It is less sensitive than recent tests.
It detects IgM and IgG antibodies, autoantibodies, and non-HLA antibodies against non-transplantable antigens.
Flow cytometry has advantages over CDC crossmatch. It has lower inter-observer variability and detects fewer IgG HLA antibodies.
Cons: Slow turnaround times made this approach more appropriate for living donors than cadavers.
ADVANTAGES OF SOLID PHASE ASSAYS All approaches for identifying donor-specific HLA antibodies are sensitive.
Detection of pre-sensitization from earlier grafts allows avoidance of those HLA markers on subsequent grafts.
They avoid antibody binding to non-HLA antigens and misunderstanding about the HLA class discovered (CDC matching overlaps T and B cell matching, T with Class I and B with Class I and II).
Cons: A positive assay with a negative CDC/flow crossmatch is unclear. Is low-level antibodies against minor antigens relevant.
Single antigen bead tests identify HLA antibodies that are complement-fixing or not. These findings may exclude a prospective recipient from receiving a graft. 3. Other limitations include IgM interference, a lack of antigens in the bead sets, and variable HLA density.
The main target for cross match techniques is to identify the recipients who have anti HLA antibodies against a specific donor (Donor specific antibodies) and they have a higher chance for hyperacute rejection as result of the presence preformed antibodies. DSAs are commonly generated by previous transplantation, frequent blood transfusion and pregnancy.
Different cross match techniques
I-Cell based assays
1-CDC technique: Complement dependent lymphocytotoxicity crossmatching: Mixing recipient serum with donor lymphocytes (living and cadaveric) in presence of complement. The donor B and T cells are separately tested against serum from the recipient.
Negative cross match that means there is no anti HLA antibodies (no cell lysis).
Positive cross match that means there is anti HLA antibodies (more than 20 % cell lysis).
Addition of anti-human immunoglobulin (AHG) helps increase the sensitivity of CDC
-The drawback of CDC is include :
False positive results which is due to IgM autoantibodies in the recipient serum.
False negative results due to either there is very low levels of DSAs to result in activation of the complement cascade or the type of DSAs not causing complement activation.
A positive T cell cross match results in poor outcome and unacceptably high incidence of acute graftt rejection. a persistent positive T cell crossmatch result post desensitization is according to current guidelines, an absolute contraindication to renal transplant.
A positive B cell crossmatch is not as consistently associated with ABMR as positive T cell crossmatches. Although a negative B cell crossmatch is associated with better outcomes.
2- Flow cytometry: the donor lymphocytes are mixed with the recipient’s serum. in the presence of anti-IgG fluorescein-labelled antibodies. These bind to the donor specific antibodies and are quantified by detectors in the impedance flow cytometer.
A negative CDC result and a positive flow cytometry indicating that there is non complement fixing antibodies, Non-HLA antibodies or Low level antibodies.
The drawback of the Flow cytometry: Time consuming that mean this technique is suitable for living donors scenarios rather than for cadaveric donors.
II) Solid phase antibody detection assays
1-Enzyme-linked immunosorbent assay (ELISA): Used for the detection of HLA antibodies in serum.
Technique: The recipient serum is added and specific antibodies bind the epitopes available. A wash is performed and anti-IgG with a passenger reported molecule (alkaline phosphatase) is added that combines with the anti-HLA antibody. After further washing to remove unbound antibody, a substrate is added which after dephosphorylation by the reporter molecule undergoes a colour change. This method has been replaced by bead technology.
2- Bead technology (Luminex):
Technique: Incubation of recipient serum with the beads. HLA antibodies of the serum will react with HLA antigens on the bead. the beads are washed and incubated with a second antibody, usually anti-human IgG labelled with phycoerythrin.
Quantitation of the result This is achieved by two methods: -Flow cytometry – Luminex method
The low positivity cut-off of 300 MFI (mean-fluorescence intensity) is predictive of graft survival and some used the higher cut-off of 1,000 MFI.
3-The virtual crossmatch: The Wait Is Over
The Physical cross matching is costly and time consuming.
This technique is based on the comparison between donor and recipients without requiring viable donor cells, depending on complete HLA typing of the donor and anti-HLA antibodies of the recipient. The use of the virtual crossmatch as a final decision to proceed with transplant across transplant centres can lead to decreased cold ischemia time and lower DGF rates without increased risk of rejection. These cross match results must be updated every 3 months especially in patients on cadaveric transplant list.
Summary of the various crossmatch techniques
Cross match between donor lymphocytes and recipient serum is an essential step in identifying the risk of rejection and possible outcomes. The response of host immunity to the allograft occurs through two major mechanisms: T cell-mediated (cellular) responses and antibody-mediated (humoral) responses. This classification into 2 entities oversimplify the response as we already understand that the function of T and B lymphocytes are interconnected and activation of one will activate the other.
Complement dependent cytotoxic cross match:
In this technique, lymphocytes from the donor are added to recipipient’s serum which may contain antibodies against the host cells. but yo identify against which MHC (HLA) the antibodies are direct, there is initially separation of donor’s lymphocytes into CD 19+ <–>B and CD 3= <–>T cells. after mixing the recipients serum with the separated lymphocytes, complement is added with viability dye. so if there are DSA against MHC I , B and T lymphocytes will undergo lysis, If only against MHC II, only B cells will undergo lysis, if absent -no DSAs are present.
Flow cytometry crossmatch : The flow cytometry crossmatch assay is capable of detecting IgG DSA without the need iof complement activation.in this technoque recipient serum is added to the donor lymphocytes, followed by the addition of a secondary fluorochrome-conjugated antibody that detects IgG. In this assay, B and T cells are not separated. Rather, additional fluorochrome-conjugated antibodies are added to distinguish between them. Samples are analyzed using flow cytometer. The results are presented as median fluorescence intensity (MFI).
Solid phase antibody detection assays include Enzyme-linked immunosorbent assay (ELISA) and Bead based techniques. ELISA is efective in detecting sensitization in transplant candidates but has been replaced by bead technology.
In Single Antigen Bead-based screening assays, color-coded microspheres coated with HLA are used to identify both HLA class I and II antibodies in recipient serum. The Luminex anti-HLA antibody detection assay is reportedly more sensitive and specific than either the CDC or flow cytometric crossmatches. Using single-antigen technology, the Luminex technology can predict a patient’s sensitization to particular HLA types before transplantation without performing a physical CDC or flow cytometric crossmatch which is termed as virtual crossmatch.
So The term “virtual” crossmatch refers to a process in which the results of anti-HLA screening results and the HLA type of the donor and recipient are compared to look for mismatched antigens and against what the recipient has preformed antibodies.
Reflect on my practice
Actually, I had no experience in kidney transplantation since fellowship. In the hospital where I used to work, transplant programs not available. But what I remember from the few living donor kidney transplantation cases during my fellowship, that we did complement dependent cytotoxic crossmatch as first step. If the CDC is negative and DSA ( I couldn’t recall which method was used for DSA identification) are negative, no further evaluation was done. If the patient has history of sensitization, then flow cytometry cross match was performed for better identification of target HLA antibodies.
Dear all, the most critical issue here is reflections on your current practice, how it differs from other practice areas, and why? For example, is it the logistics, finance, expertise, or other factors that affect your practice decision?
Aim of cross-match techniques:
To identify the antibodies in the donor serum to one or many HLA (human leukocyte antigens). These are referred to as DSAs (donor-specific antibodies) to prevent hyperacute rejection.
DSAs are commonly generated by: (i) Blood transfusion (ii) Previous transplantation
(iii) Pregnancy.
Crossmatch techniques:
I) Cell-based assays
1- Complement dependent cytotoxicity (CDC) technique:
CDC crossmatch (A) Serum from the recipient is added to donor lymphocytes (T or B) in the presence of complement.
(B) Negative test when donor-specific anti-HLA antibodies are absent and complement activation does not occur.
(C) Positive test when donor-specific antibodies bind to lymphocytes, activate complement and cause cell lysis.
Twenty percent is usually taken as the minimum cut-off for a positive result thus functioning as a qualitative and a semi-quantitative estimate of the strength of the reaction.
Limitations of CDC: false positive and negative results
Outcomes in positive T-cell crossmatch results: renal transplant performed when the recipient has a positive T cell cross match results in poor outcome and unacceptably high incidence of acute graft rejection. a persistent positive T cell crossmatch result post desensitization is according to current guidelines, an absolute contraindication to renal transplant.
Outcomes in positive B-cell crossmatch results: A positive B cell crossmatch is not as consistently associated with humoral rejection as positive T cell crossmatches. Although a negative B cell crossmatch is associated with better outcomes, the test’s high false positive rate
makes its significance uncertain. This can be overcome by concurrent Luminex testing for DSAs which increases its specificity.
Advantages: A basic and durable test still used as the foundation of crossmatch due to availability, cost and simplicity.
It detects only complement binding antibodies.
Drawbacks: It is less sensitive than other newer assays. It detects IgM and IgG antibodies simultaneously, autoantibodies and non-HLA antibodies against antigens that are irrelevant as far as the transplant is concerned
2- Flow cytometry:
(A) Serum from the recipient is added to donor lymphocytes (T or B) in the presence of anti-IgG
fluorescein-labelled antibodies. (B) Negative test when donor specific antibodies are absent and binding does not occur. (C) Positive test when donor-specific antibodies bind to lymphocytes
that are detected by flow cytometry once the anti-IgG fluorescein labelled antibodies tag the lymphocytes.
Interpretation of flow crossmatch results
Non-complement fixing antibodies
Non-HLA antibodies
Low-level antibodies
Hence, the value of this test’s sensitivity lies in using it for cross matching of sensitized patients who have an inherently higher risk of acute graft rejection to determine transplant feasibility or need for desensitization protocols prior to transplant
Advantages: More sensitive than CDC crossmatch. It can detect lesser levels of IgG HLA antibodies and has less inter-observer variability.
Drawbacks: Slow turnaround times meant this technique was suitable for living donors scenarios rather than for cadaveric donors.
II) Solid phase antibody detection assays
1) Enzyme-linked immunosorbent assay (ELISA):
The modified assay uses HLA glycoprotein immobilized into mictrotiter wells. The recipient serum is added and specific antibodies bind the epitopes available. A wash is performed and anti-IgG with a passenger reported molecule (alkaline phosphatase) is added that combines with the anti-HLA antibody. After further washing to remove unbound antibody, a substrate is added which after dephosphorylation by the reporter molecule undergoes a color change.
This method while effective in detecting sensitization in transplant candidates has been replaced by bead technology.
2) Bead technology (Luminex):
The basic steps involve first, incubation of recipient serum with the beads. HLA antibodies of the serum will react with HLA antigens on the bead. The beads are washed and incubated with a second antibody; usually anti-human IgG labeled with phycoerythrin.
Advantages: They are the most sensitive of all the techniques for detecting donor specific HLA antibodies.
Enhanced sensitivity has allowed improved rates of success in retransplants where detection of pre-sensitization from previous grafts allow the avoidance of those HLA markers on subsequent grafts.
They allow the identification of all HLA alleles for which the recipient harbors antibodies.
They avoid false positives of antibody binding to non-HLA antigens and eliminates confusion regarding the class of HLA that are detected
(CDC matching has an overlap between T and B cell matching, T with Class I and B with both class I and II).
Drawbacks: The interpretation of a positive assay in the presence of negative CDC/flow crossmatch is ambiguous.
The relevance of low-level antibodies to low significance antigens is debatable.
Single antigen bead assays detect both complement-fixing and non-complement-fixing HLA antibodies. On the basis of these results, prospective recipient may be denied a graft without established clinical significance. Other limitations include interference by IgM,
incomplete library of antigens in the bead sets, variability of HLA density on the beads
III) The virtual crossmatch
(A) Serum from the recipient is added to synthetic beads with either a set of antigens or a single antigen, each bead can be identified by an independent dye signature.
(B) Anti-HLA antibodies if present will bind to the specific bead.
(C) A detector antibody will then bind and sequester a reporter dye.
(D) Beads can be checked for the reporter dye using a laser beam, this builds a profile of the antibodies present in the recipient that can be compared with the HLA construct of a potential donor thus predicting the result of crossmatch.
Alternatively, a microsphere or bead may be coated with multiple HLA antigens – this iteration of the test allows for more efficient screening. HLA antibodies bind specifically with the bead and are detected by isotype specific antibodies using flow cytometry. This
method allows for the sensitivity of flow crossmatch combined with identifying specific antibody.
My opinion Complement dependent cytotoxicity remains the mainstay of pretransplant screening for HLA specific antibodies. Newer methods have helped significantly increase the sensitivity and specificity of detecting antibodies of significance in acute humoral graft rejection in the
recipient when interpreted in the light of the patient’s clinical picture and CDC crossmatch testing whilst accounting for inter-lab variations and technical errors
So in some patients we need to use combination of those techniques to take the right decision
Cell based techniques:
CDC technique:
first developed in 1960
determine the presence of DSA
the technique based on mixing donor lymphocytes( B and T cells) with the recipient serum, complement is added to the mix and cell lysis is observed. Lysis of more than 20% of the cells is considered as a positive result.
addition of anti-human immunoglobulin increases the sensitivity of CDC.
titred crossmatch is a method at which crossmatching of donor lymphocytes is performed using serial dilutions of the recipient”s serum, the higher the dilution required to get a negative result, the greater the strength of immune reaction.
Limitation of this technique:
false positive result: due to autoantibodies in the recipient serum( overcomed by the addition of DDT).
false negative result: when DSA level is low
non complement fixing ab
T cell express only class I antigen while B cells express both class I & II
positive T cell crossmatch: results in ( after ruling out the presence of autoantibodies) high incidence of acute allograft rejection, a persistent positive crossmatch after desensitization is considered as contraindication to transplantation.
positive B cell crossmatch: high false positive rate and this can be overcomed by luminex testing for DSA, the significance of anti-HLA class II ab is of less importance for acute/ hyperacute rejection.
Flow cytometry:
first detected in the early eighties
the donor lymphocytes mixed with the recipient serum, binding to donor specific antibodies and quantification by detectors in the impedence flow cytometer will happen.
two methods of quantification are used:
I. measurement of florescence intensity( channel shift)
II. serial dilution of recipient serum
In CDC negative result, a positive flow cytometry result indicates either:
non complement fixing ab
low level ab
non-HLA ab
so the importance of flow cytometry is to test sensitized patients.
solid phase antibody detection assays:
ELISA: the recipient serum and specific ab.s bind the epitopes available. A wash is performed and anti IgG with a passenger reported molecule is added that combine with anti HLA antibody.
this method has been replaced by bead technology.
Bead technology( Luminex):
patient serum incubated with the beads, HLA ab will react with HLA ag on the bead, the beads are washed and incubated in another ab. usually antihuman IgG labeled with phycoerythrin.
this method can be used for screening for antibodies or testing against set of alleles or as single antigen beads.
the result is quantified either through flow cytometry to measure channel shift or through Luminex method to calculate the MFI.
The main significance of the test is when CDC is negative, and this is due to either non HLA ab.s or non complement fixing ab.s or low level ab.s also low cut off values.
Virtual crossmatch:
based on the comparison of the anti HLA ab of the recipient to the donor HLA ag using bead technology.
allow rapid identification of a suitable donor
recipient serum is added to a synthetic beads with either a set of antigen or single antigen, Anti HLA antibodies if present will bind to the specific bead and this reaction can be detected using a laser beam.
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
– It is highly specific, a positive cross match using CDC indicating the presence of clinically significant complement fixing antibodies thus if CDC positive donor should be excluded.
– This test has low sensitivity, human anti- goblin can be added before complement that bind to Donor HLA antibodies, to increase sites for binding to complement and increase its sensitivity
– False positive results can occur due to the presence of clinically non significant autoimmune IgM antibodies which can be eliminated either by heating or adding reducing agent.
– Rituximab can cause false positive CDC.
False negative result can occur due to non complement fixing, non HLA or due to low level of HLA antibodies
– T cell express only HLA class I while B cell (as it act as APC) express HLA class I and II, so if antibodies are directed to only class II or if DSA directed to HLA class II are week it will give positive B and negative T cell cross match, on the other hand if DSA are directed to both class I, II it will give positive B and T cross match, positive T and negative B cross match indicates technical error.
– Positive T cell cross match has the worst prognosis and donor should be excluded
– Positive B cell cross match carry poor prognosis when compared to negative CDC cross match but better that positive T cell cross match , positive B cell cross match commonly occur due to false positive result (not due to DSA), but true positive can occur due to DSA against class II HLA, luminex is done at this situation to confirm the presence of DSA against class II if present desensitization can be done.
– CDC is used either in determining compatibility between donor and recipient or in determining sensitization in case of PRA
– CDC give semiquantitative assumption of the strength of DSA through the following methods :
⦁ Score is given according to percentage of ruptured lymphocytes from 2 (20% of cells ruptured) to 8 (indicating strongest reaction), below 2 the test is negative
⦁ Titred crossmatch method using serial dilution of recipients serum, so the higher the dilution needed to render the test negative the greater the strength of DSA.
– CDC should be repeated just before transplant since antibodies may be formed after antigen exposure such as blood transfusion or pregnancy
II- Flowcytometry
– It involves incubating donors lymphocytes with recipient serum , then add fluorescein labeled anti- globulin that will bind to DSA present in the recipient serum that are attached to donor lymphocytes
-FCM is used either in determining compatibility between donor and recipient or in determining sensitization in case of cPRA
– FCM give quantitative assessment of the strength of DSA through the following methods :
⦁ Measurement of fluorescence intensity and compare it to control (channel shift)
⦁ Titred crossmatch method using serial dilution of recipients serum, so determine the minimum dilution needed to render the test negative this give us the strength of DSA
– More sensitive than CDC as it can detect non complement fixing AB, non HLA antibodies and low level DSA, so CDC – FCM + cross match indicates the presence of one of the previous mentioned ABS.
– Positive T cell FLC (in highly sensitized patients only) is associated with poor graft survival, so it has a very big rule in sensitized recipients
– False positive results can occur due to binding of non specific IgG antibody to B cell, also rituximab was found to cause false positive results
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 fourescein, each bead either have single (SAB) or multiple HLA molecule then anti human globulin labelled with phycoirythrin is added.
– Very sensitive very specific, can detect complement and non complement fixing antibodies, low level DSA, non HLA antibodies including those against MiHC antigens
– Luminex give quantitative assessment of the strength of DSA through the following methods
⦁ Flow cytometry method, that measure fuorescence intensity and compare it to control (median channel shift- MCS)
⦁ Luminex method, that calculate the degree of fluorescence (median fluorescence intensity -MFI) through the use of 2 lasers to excite the fluorochrome of the beed and the phycoerythrin bound to the antibody.
– The major obstacle is usually that it is expensive
– False positive result can occur due to denatured antigens attached to beeds
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
– DSA may change over time due to sensitization from blood transfusion or pregnancy so luminex SAB should be repeated every 3 m
In practice my experience is only in living related donors
we do both CDC and FCM cross match in all transplant recipients
⦁ If CDC positive donor is excluded
⦁ If FCM positive and CDC negative patient is assesd for desensitization using data from luminex teqnique
⦁ If CDC negative, FCM negative we proceed for transplantation
⦁ We do not use virtual cross match
Good response, thank you
1-CELL BASED ASSAY
CDC technique ( complement dependent cytotoxicity ) this type of assay depend on mixing the recipient sera with the donor lymophoctes ( T, B Cells separatly ) in the precence of complement .
postive CDC indicate presnce of donor sepcific antiHLA ab that induce cell lysis due to complement activation, this serollogy method have been used since 1960 , developed by terasaki and patel , the result interpretaion by the % of PRA with minimum cutoff value of 20% for postive results
titred cross-match by using serial double dilution , the higher the dilution will help to quantify the strenght of the reaction and give negative results
limitations of the CDC crossmatch :
1- false postive results with autoAB like IgM mediated complement activation
2-False negative results for non-HLA- AB or when the DSA presnet in very low titer
also should keep in mind that the recpient AB may vary within time due to possible new antigen exposure espcialy with recent blood transfusion , , pregnancy so we should repeat the CDC crososmatch and depend the most recent one
postive T cell CDC crossmatch is absolute containdications to perform the transplantation
2- Flowcytometry crossmatch
this technique started in the early eighties by using molecular genetic testing also depend on mixing the recipent sera with panle of donor lymphocytes ( T cell , B cell),in the presence of anti IgG fouricence labelled antibodies and are quantified by detectors in the impendece flow cytometer
two methods used for Quantifications of DSAs
A Meauremnet of the flourcence intesity as a ratio of control ( MFI)
b-seria dilutions and by esitimated the minimum dilution which give negtaive results
its more sensitive and specific assay for detection of noncomplement fixing AB , non-HLA AB low level DSA so its prefered test for sensitized patients and help to decide about the need of desensitization protocal prior to transplant .
3- Solid phase antibody detection assay
a- enzyme linked immunosorbent assay(ELIZA), the use of modified ELIZA test for dectection of sepcific anti IgG anti HLA ab in recipient serum at the level of epitopes
b single bead antigen ( luminex )
the basic of bead technolgy which started since ninties , this test involve incubation of recipient serum with the beads , then the HLA -ab of the recipient will react with the HLA antigens on the beads , the beads are washed and incubated wtih a second anti human IgG labeled with phycoerthrin, whic bound to the AB ,the result interpretaion as PRA and calculated by mean flourcence intensity MFI , help in identication of very sepcific ant iHLA antigens ab in sensitized patient even with low MFI value , the cut off value of MFI not standadrazied among laberotieris
the single antigen bead test( SAB ) also help in monitoring DSA level pre and post transplantation for sensitazed recipients , its use limited due to the cost and avialablity
in SQUH its university tertiary centre we have our genetic lab, that doing all the HLA TYPING for LD program , we dont have DD program and we still using CDC Assay , we dont have flowcytometry and we just introduce the SABlumenix assay recently for DSA monitoring for sensitazed kidney transplantaionwith MFI cut off value for postive test of1000 and above to decide for desensatization protocal
we have more commercial kidney transplant recipients with out information about their donors HLA status whic is very challenging to us , we adopt SAB – lumenix assay as best method to monitor for DSA after transplantaion .
Good and transparent reflections on practice, thank you
Various techniques of cross match:
A- Cell based assays
1- CDC /ADCC ( complement dependent cytotoxicity) : T and B cells of the donor (living or deceased) are isolated and separated and tested against serum of the recipient, complement is added to detect if cascade will be activated or not ; more than 20% cell lysis will be considered positive CM.
Titred cross match : done for more detection of antibodies through serial doubling dilution of the recipient serum. High dilution required for negative result. More intense immune response is present with high dilution.
False positive can happened because of presence of autoantibodies IgM in the recipient serum, can be overcome by addition of DTT (dithiothretol) which eliminate IgM complement cascade activation leaving only IgG which are DSA.
Also can be false negative ; in presence of non complement activation antibodies or low titre of DSA.
There are T and B cell cross match , T cell express HLA I, B cell express HLA I and II, doing CM against both of them separately can specify the type of antibody to which antigen.
Of course positive T cell cross match will negatively affect graft function and survival , desensitization protocol should be applied before transplantation, persistent positive T cell CM is an absolute contraindication to transplantation.
Positive T and B cell CM indicates presence of antibodies against class II and I.
Positive B cell CM alone is insignificant because of B cell viability, so should be combined with Luminex detection of DSA.
CDC technique is cheap and available, used as preventive measure against hyperacute rejection, but it is less sensitive.
2- Flow cytometry CM donor lymphocytes with recipient serum, can be quantified by:
Fluorescence intensity or serial dilution of recipient serum
Positive FCXM (flow cytometry) with negative CDC indicates :
non HLA antibodies
Non complement activation antibodies
Low titre of DSA
It is more sensitive than CDC , used more in living donor.
B- Solid phase antibody detection assays: it is the most sensitive technique because it detects antibodies against all HLA alleles, this will improve graft survival and will help in deciding pretransplant desensitization protocol.
a- ELISA indicated by color changes
b- Bead technology (Luminex) beads labelled with fluoresceine each bead has one or more type of HLA molecule incorporated .
It may used as screening, PRA (panel reactive antibody) , SAB (single antigen bead).
c- Virtual CM basic concept is comparison between recipient anti HLA antibodies with donor HLA antigens, it uses bead technology.
SAB technique can detect complement and non complement fixing antibody
Crossmatches are used to identify the risk of graft rejection in a prospective transplant recipient.
Crossmatch techniques can be divided into cell based assays and solid phase assays.
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 acivating 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): 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 or a titred value of dilution required to get a negative result (for quantification).
B) Solid Phase antibody detection assays:
1) 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) 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.
2. Please reflect on your practice if possible.
In our practice, which is mainly living donor transplant setting, CDC is the first test to be done. If there is no history of sensitization (prior transplant, pregnancy, blood transfusion), then we proceed with transplant without any further tests, except in case of spousal (husband to wife) donation where we advise a single antigen bead (Luminex) assay. But due to the cost involved, it is common that the patients refuse to undergo this test.
In patient with sensitization history, even if CDC crossmatch is negative, we get a single antigen bead (Luminex) assay for quantification of DSAs and proceed accordingly,
Excellent reflection on your practice Amit
This what we need from colleagues. We have to make evidence based medicine viable by looking at our resources, rather suggesting treatment plan which is not practical.
Positive cross match (both T and B cells are positive), but no DSA
When HLA titres are high and prozone phenomenon occurs-
high titre antibodies lead to complement activation and deposition of complement proteins on the bead which then prevents HLA antibody from binding to the HLA antigen on the bead.
same occurs with IgM antibodies or other serum factors binding
epitope sharing
different HLA antigens on different beads
share common antibody binding epitopes leading to
binding of an anti‑HLA antibody to more than one bead
with consequent reduction in the MFI on a single bead.usually MFI>1000 is considered significant.
during the process of attachment of HLA molecule,the bead, immunologically relevant epitope, can become hidden and un-accessible to antibody binding.
in our centre it was not a high volume centre for transplant with about 25 transplants per year.also patients affordability was not great so we never went beyond CDC crossmatch with transplant if negative and rejection if positive.
However personally I would do CDC and flow cross match in all and if flow crossmatch positive then DSA,as that would dictate decisions about induction and desensitization strategies if needed,unless patient is not willing to take the additional cost.CDC positive then avoid transplant .
The above for low risk individuals,in high risk patients advise CDC,Flow cytometry,DSA .
Cell based assays
CDC technique:
Determine the presence of donor specifc anti- HLA antibodies in the serum of the recipient.
Result is given in terms of percentage of lymphocytes in the cell panel which has undergone lysis as a result of complement activation or panel-reactive antibody (% PRA) . Twenty percent is usually taken as the minimum cut-of for a positive result.
AHG helps increase the sensitivity of CDC.
False positive- due to autoantibody
False negative-
DSA levels too low
If the antibody is not complement fixing.
Class I antigens (HLA-A, B,C) are expressed on all nucleated cells
class II antigens HLA-DR,DP, DQ) are expressed on antigen presenting cells like macrophages
and dendritic cells.
Vascular endothelial cells of the transplant graft express both these antigens.
T cells express only class I antigens
B cells express both.
a positive T cell and B cells cross match would indicate presence of antibodies to HLA type I and II antigens
a positive B cell cross match could indicate either (i) DSAs to type II antigens alone or (ii)
Low levels of DSAs to type 1 antigens.
A positive T cell crossmatch alone is usually due to technical error.
positive T cell crossmatch result is an absolute contraindication to renal transplant.
negative B cell crossmatch is associated with better outcomes, the test’s high false positive rate
makes its signifcance uncertain. Concurrent Luminex testing for DSAs can increase its specificity.
Drawbacks:
less sensitive than other newer assays.
It detects IgM and IgG antibodies
autoantibodies
and non-HLA antibodies
(2) Flow cytometry
Is more sensitive than CDC test
In the light of a negative CDC result, a positive fow cytometry indicates –
Non-complement fxing antibodies
Non-HLA antibodies
Low-level antibodies
CDC crossmatch negative patients who had positive T-cell fow cytometry results had signifcantly poorer absolute 5 year graft survival rates that those who were both CDC and flow cytometry crossmatch negative.
Solid phase antibody detection assays:
1)Enzyme-linked immunosorbent assay (ELISA)
2) Bead technology (Luminex)
Beads impregnated with HLA antigens are used
A) Screening beads – contains both I and II antigens
B) Set – contains either I or II antigens
C) Single antigen beads- contains single antigens.
Quantified by flow cytometry or Luminex method
CDC negative and a positive DSA could be due to due to a low level antibody, a non-HLA antibody or a non-complement binding antibody.
Drawbacks:
The interpretation of a positive assay in the presence of negative CDC/fow crossmatch is ambiguous. The relevance of low level antibodies to low signifcance antigens is debatable.
Single antigen bead assays detect both complement-fxing and non complement-fxing HLA antibodies.
interference by IgM
incomplete library of antigens in the bead sets
variability of HLA density on the beads.
Positive cross match with negative DSA :
Due to presence of other non HLA such as MICA, angiotensin II type 1 receptor, H-Y antigen, antibodies against graft endothelium, antibodies against nsSNPs, and antibodies against LIMS1 proteins. Also presence of IgM antibody in the context of autoimmune disease or using rituximab in the CDC test.absence of rare antigen among the HLA antigen profile of bead assays and so DSA will not be detected.
Crossmatch techniques can be divided into cell based assays and solid phase assays.
A. Cell-based assays: These are of 2 types.
1) CDC technique:
a mixture of recipient serum and donor lymphocytes (T and B cells separately) and complement is added , If DSA is present, it will bind with the lymphocytes, this will activate complement and cell lysis will occur , giving a positive result.
To increase its sensitivity, anti human immunoglobulin (AHG) can be added .
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).
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.
False positive :
Presence of autoantibodies
Low level of DSA
presence of non-HLA acivating antibodies.
We can add DTT (ditheothreitol) inhibits IgM mediated complement activation.
2) Flowcytometry Crossmatch (FCXM):
By adding fluorescein labelled anti IgG antibody 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 can detect low level of DSA, non-HLA antibodies and non-complement binding antibodies.
B) Solid Phase antibody detection assays:
1) ELISA:
It is a 3 step procedure :
* recipient serum is added to HLA glycoprotein labelled microtiter wells
*after giving a wash, anti IgG with a passenger reporter molecule (alkaline phosphatase) is added followed by a wash.
* addition of a substrate which undergoes a color change if antibodies are present.
2) 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.