it is a molecular assay which includes amplification of DNA sequence. called Sequence-Specific Oligonucleotide probe (SSOP)
this method can give more details as it test to split antigen and the epitope level
so this method as compared to the serological one is more accurate and safer in detecting the best donor
dina omar
2 years ago
Its Molecular assay via Sequence Specific Oligonucleotide Probe (SSOP).It is more specific and accurate than serological HLA method.
Nazik Mahmoud
2 years ago
There’s two technique to did HLA typing
Serology and molecular (PCR)
The PCR is a most accurate and can detect split antigens and their epitope , it done by DNA amplification and sequencing called sequence specific oligonucletide probes(SSOP)
Wee Leng Gan
2 years ago
HLA typing assess whether donors are suitably and safely matched to a transplant recipient.
The HLA typing method in this question is : Molecular assay via Sequence Specific Oligonucleotide Probe
Other possible HLA methods.
1)Serology assay : Microlymphocytotoxicity test.
2)Cellular assay : Mixed Lymphocyte culture.
Mahmoud Hamada
2 years ago
this method of hla typing is called Sequence-specific oligonucleotide probes (SSOP) typing. In this method, DNA amplification occur .
it is more suited for large number of samples as it can test 180 sample in 2 days.
it is more accurate and specific than serological methods.
Ahmed Fouad Omar
2 years ago
This is a molecular HLA typing method called SSOP ( Sequence specific oligonucleotide probe).This molecular technique has low or intermediate resolution.
There are two ways of HLA typing:
1) Serological typing: By mixing donor lymphocytes with the recipient serum in the presence of complement and dye. Binding of the AB with the antigen on the lymphocyte surface results in complement activation and cell lysis. Dead cells are identified under phase contrast microscopy.
o Advantages: quickly available results, more important role in deceased donors(short cold ischemia time), can identify Null alleles.
o Disadvantages: Cannot detect differences in HLA protein small amino acids. It is poor at HLA Cw, DQ and DP .Additionally, there is lack of sera with antibody specificities which can identify the increasing number of HLA alleles.
2) Molecular typing: There are 3 methods using PCR and provide more reliable results
i. Sequence-specific oligonucleotide probes (SSOPs)
ii. Sequence-specific primers (SSPs).
iii. Sequencing-based typing (SBT).
Both SSP and SSOP can provide low or intermediate resolution of typing required in solid organ transplantation.
o Advantages:
Can identify specific alleles without cross reactivity
Can identify differences in HLA antigens between the donor and recipient, thus avoiding the risks with antigen mismatch
Can provide details to the amino acid level
o Disadvantages:
May fail to identify novel alleles not on the HLA data sequence
Reference:
Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and cross match: A comprehensive review. World J Transplant. 2017 Dec 24;7(6):339-348.
Nandita Sugumar
2 years ago
HLA TYPING
The method of HLA typing in the given picture is molecular typing – sequence specific oligonucleotides probes used.
Sequence specific primer polymerase chain reaction – Extracted DNA is amplified in wells. Primers in the wells are complementary to specific HLA alleles. Electrophoresis is done leading to appearance of a band. HLA typing is allocated based on matching primers to amplification product of DNA sequence.
Sequence specific oligonucleotides probes – Amplified DNA is mixed with oligonucleotides complementary to DNA segments of different alleles
DIrect DNA sequencing – Precise order of nucleotides is determined in a given gene and then HLA type is assigned by comparison with already published HLA allele sequences.
Molecular typing clearly identifies differences in HLA antigen between donor and recipient. This can help in identifying risks associated with antigen mismatch. PCR based HLA typing is extremely accurate for identifying specific alleles with no cross reactivity. However, new alleles may not be identified.
The other technique is serological typing. A tray containing sera with antibodies to known HLA alleles is used. Complement pathways are stimulated to act and when cell death occurs, dye enters the cell and under phase contrast microscopy this is clearly visible.
Serological typing is quick and can be arranged in a short period of time for emergent surgeries. The short time frame also links with lesser cold ischemia time in kidney transplant. HLA alleles that have identifiable DNA sequences with molecular typing but not cell surface antigen expression can be differentiated clearly. HLA Cw, DQ and DP cannot be identified with this method because of the lack of sera with antibody specificities that are capable of identifying the growing number of HLA alleles.
In addition, serological typing cannot readily distinguish HLA protein small amino acids. It has not used in many centers in recent times.
Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant. 2017 Dec 24;7(6):339-348. doi: 10.5500/wjt.v7.i6.339. PMID: 29312863; PMCID: PMC5743871.
Akram Abdullah
2 years ago
HLA typing is the most important step for successful
of kidney transplantation .The method mentioned above is the molecular technique of HLA typing ,PCR-reverse SSOP (Lumiex) ,
The molecular technique is more sensitive and accurate , depends on amplification of DNA , & can identify the HLA antigens
The other method is serological technique which depends on the interaction between the sera of the recipient & the donor`s cells, which is less accurate than the molecular one.
Manal Malik
2 years ago
HLA typing is molecular typing for class1 and 2 and PCR -Reverse SSOP(Luminex) in this case.
HLA typing is a crucial step in renal transplantation, as recognition of foreign HLA by recipient T lymphocytes would trigger an immune response .
two types of HLA typing:
1- molecular typing
identify different in HLA Ag between donor and recipient with details of amino acids
typing also resolve the public epitopes and distinguish the common null alleles from expressed alleles
DNA based HLA typing include:
Reverse sequence -specific oligonucleotide (rSSO) probe-hybridization, sequence -specific primer(SSP)-directed polymerase chain reaction (PCR)amplification are the typical method used for HLA typing of deceased donor.
The SSP method is a commonly used technique in which multiple pairs of allele -specific primers are used to determine the HLA alleles present in a DNA sample.
SSP method is fast and can be completed within 2-3 h
RT PCR method has a simple work flow without need for gel electrophoresis or hybridization and provide result within 90 min
HLA typing RT-PCR method is not accurate .
2- Serological typing
recipient lymphocytes are introduced to try wells contain sera of known HLA alleles with complement and dye so after Ag-complement reaction take place the dye enter the cell this cell death identify by phase contrast
this method take short time so can be used in deceased donor. REFERENCES1. Mittal KK, Mickey MR, Singal DP, Terasaki PI. Serotyping for homotransplantation. 18. Refinement of microdroplet lymphocyte cytotoxicity test. Transplantation 1968; 6(8): 913.
2. Bidwell JL, Bidwell EA, Savage DA, Middleton D, Klouda PT, Bradley BA. A DNA-RFLP typing system that positively identifies serologically well-defined and ill-defined HLA-DR and DQ alleles, including DRw10. Transplantation 1988; 45(3): 640.
This HLA typing is a form of molecular typing:
PCR-reverse SSOP; Molecular HLA typing done for class I,II HLA
sequence-specific oligonucleotide probes (SSOPs)
HLA typing techniques include
Serologic methods
Lymphocytes from the donor or recipient are added to several wells of plates containing different sera, incubated then complement is added to wells, the occurrence of reaction leading to dead cells is a positive test. Buy it has some limitations which is inability to recognize HLA large sepicifities. DNA based molecular methods Sequence-specific primers (SSP) typing
It recognize a particular HLA sequence (allele) or group of similar alleles.
DNA is extracted and amplified by PCR.
The pattern of amplicons allows for the assignment of the HLA genotype.
The SSP method can be used for either low-resolution typing, by identifying allele groups of a particular antigen, or for high-resolution typing, for a specific allele. Suitable in the typing of deceased donors because it is rapid . However, it is not well suited for typing large numbers of samples Sequence-specific oligonucleotide probes (SSOP) typing
With SSOP typing, DNA is amplified using a set of primers that recognize a particular HLA locus. As with SSP typing, it amplify the most polymorphic regions of the HLA gene (exons 2 and 3 for class I genes and exon 2 for class II genes).
Primers used in SSOP for DNA amplification are less specific than those used in SSP and cannot distinguish between groups of similar alleles. To discriminate between alleles within a locus, a labeled SSOPs that recognize specific sequences are added . Oligonucleotide probes can also be linked to beads to allow for multiplex analysis of HLA typing using the Luminex platform.
SSOP typing is well suited for typing large numbers of samples.
For less samples, reverse-SSO (rSSO) can be used where oligonucleotide probes are bound to the membranes, with each membrane having all the SSOs bound that are required for typing of a particular HLA locus rSSO typing still has the same drawbacks but is faster Real-time PCR (RT-PCR)-based typing
It uses allele-specific PCR similar to SSP methods ,amplicons are detected in real time with the use of fluorescent dyes or probes. Fluorescent readings of each well are obtained at different temperatures.and the pattern of reactive wells identifies the HLA typing.
Alternatively, labeled SSOPs can be used instead of the less specific cyanine dye. Probe-based approach allows for different fluorescent reporter molecules to be used in the same reaction well, allowing for a greater degree of multiplexing.
RT PCR is a rapid method Sequence-based typing (SBT)
Depends on direct amplification and sequencing of the relevant exons using fluorescently labeled dideoxynucleotides , allowing for a high resolution typing.
Next-generation sequencing (NGS)
It permitted high-resolution typing with significantly reduced ambiguity as it allows for base calls to be assigned to the same or different alleles.
AMAL Anan
3 years ago
Different techniques for HLA typing:
-Serological or Molecular.
-Molecular is sensitive and accurate than serological.
-Soluble or recombinant HLA molecules based assay which not target lymphocytes as lymphocytes express HLA and non-HLA antigens.
*** Molecular DNA-based HLA typing techniques It includes different types:
1- sequence specific oligonucleotide probes( PCR-SSOP) which is based on first amplifying genomic DNA using locus- or group specific primers and then detecting the hybridization of specific oligonucleotide probes tagged with enzymatic or fluorescent markers to the amplified product, specific segments of the DNA of different alleles. Unique HLA alleles are then identified using fluorescent tags in commercial kit.
2-sequence-specific primer amplification(SSPs)
determined by DNA augmentation using group- or allele-specific primers and Sensing an amplified product of the correct size by gel electrophoresis.
3- Direct DNA sequencing( (SBT) typing determines the precise order of nucleotides in the gene of interest, allowing HLA type comparison, through the amino acid level, to provide insight to the risk monitoring.
-SSP and SSOP can easily provide low or intermediate levels of typing of different HLA antigens at allel level ,however Irrespective of the method, molecular typing can detect differences in HLA- Ag between donor and recipient specifics to the amino acid level with no cross-reactivity.
-Limitations:
Novel alleles not currently on the HLA sequence database will be not classified.
moving to next-generation sequencing with high resolation.
AMAL Anan
3 years ago
The technique used her is reverse SSOP with PCR
IT is one of molecular tissue typing techniques .
Balaji Kirushnan
3 years ago
The technique used in the above HLA typing is sequence specific oligonucleotide probe with use of Luminex technology
HLA typing has improved from the primitive serological methods to the current NGS (next generation sequencing), which has lead to the discovery of further alleles
In the serological typing method the recipient lymphocytes are introduced into the wells containing sera, complement and dye. If the antibodies can bind to the antigens on the surface of the lymphocytes the complement is activated. The cell lysis is identified and through comparison and elimination of positive wells the HLA typing is determined. The advantages are that it is a cheaper method, results are available in shorter time. The disadvantages are that the sera used dose not include antibodies for an ever growing number of the newer HLA alleles. The sera do not detect HLA Cw,DQ and DP antigens. they cannot readily detect differences in small protein amino acids.
Molecular methods for HLA typing use polymerase chain technology to identify HLA alleles. They are more precise in that they identify even small HLA alleles upto the amino acid level. They can identify the HLA alleles with no cross reactivity. They are 2 major methods of molecular methods of typing. One is sequence specific primer polymerase reaction and sequence specific oligonucleotide probes.
ahmed saleeh
3 years ago
The technique used her is reverse Sequence specific oligonucleotide probes with PCR.
Sequence specific oligonucleotide probes:
Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles. Unique HLA alleles are then identified using fluorescent tags. For a particular gene of interest, the precise order of nucleotides is determined through sequencing. HLA type is then assigned using available HLA allele sequences.
Molecular typing
1-Sequence-specific primer polymerase chain reaction
2-Sequence specific oligonucleotide probes
3-Direct DNA sequencing
CARLOS TADEU LEONIDIO
3 years ago
This is a molecular Technique :
PCR – reverse SSOP (Luminex) was used – which means that a sequence-specific oligonucleotide probes were identified characterizing locus or group specific of primers through microparticles read in a Luminex machine. Thus, it can easily provide low or intermediate levels of typing, identifying the recognized HLA antigens and major allele groups.
– recognizes possible antibodies from structural epitopes
– No require viable cells
The other one, is a serologic Tchnique: it compares cells deaths identified in microscopy will provide the result to be evaluated.
– No specific for HLA antibodies
– Require a suficiente supply of viable lymphocytes
Murad Hemadneh
3 years ago
The HLA typing showed on this case scenario is single antigen beads SAB method which is molecular method of HLA typing and is considered the best because it’s accurate, rapid and reproducible.
HLA typing can be done by different methods, Those are classified to either serological or molecular methods.
Serologic method where the recipient lymphocytes are introduced to a tray wells containing sera with antibodies to a multitude of known HLA alleles then complement and dye will be added. If antibodies bind to the antigen a complement activation will occur and finally the dye will enter the cell. Phase contrast microscopy is then used to identify wells with significant cell death.
Advantages of serologic methods are they are fast with results in short time which is important in deceased donor transplantation and the ability to identify non HLA alleles which have identifiable DNA sequences with molecular typing but no cell surface antigen expression.
Disadvantaged of serologic methods are the inability to detect the increasing number of HLA alleles because of the lake of sera which contains specific antibodies. In addition that serologic method is not able to detect differences in HLA protein small amino acids that may be antigenic enough to induce potent immune response.
There are three molecular HLA typing methods. First, sequence-specific primer PCR where extracted DNA is amplified in several wells where DNA probes are complementary to the specific sequence of HLA molecule, then is instilled into an agarose gel and undergo electrophoresis and appear as a band. Second, Sequence-specific oligonucleotide probes where amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles and unique HLA alleles identified with fluorescent tags. Finally, Direct DNA sequencing where HLA type is assigned by comparison to published HLA allele sequences.
There are many pros of molecular methods of HLA typing, they can clearly identify the differences in HLA antigen between the donor and the recipient regardless of the method up to the amino acids level which make them highly specific methods. PCR typing can identify specific alleles with no cross reactivity – identification of allele which is essentially similar to the allele of interest – although a gene may occur in two or more forms (alleles). While this is highly specific it has the disadvantage that new alleles not on HLA databank will not be identified.
Reference:
Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant. 2017 Dec 24;7(6):339-348. doi: 10.5500/wjt.v7.i6.339. PMID: 29312863; PMCID: PMC5743871.
amiri elaf
3 years ago
#Serological method:
Benefits:
Preliminary or supportive method for molecular assays, fast and cheap.
Drawbacks:
Low resolution, requires viable cells, poor reagent supply and not the current standad
# Cellular method:
Benefits:
Used for HLA class 11
Drawbacks:
Low resolution, requires viable cells, informative and rarely used currently
# SSP Sequence specific priming:
Benefits :
Nowadays used to distinguish cis/trans ambiguities
Drawbacks:
Low or intermediate resolution, limited to previous known polymorphisms and restricted to selected exons
# SBT Sequencing based typing:
Benefits:
High resolution
Drawback:
Dose not distinguish cis/trans ambiguities and restricted to selected exons
# NGS next generstion sequencing
Brnefits:
High resolution, high throughput typing and increases rate of resolved ambiguities.
Drawback :
Complicated workflow and data analysis, noval technique,could become reasonably priced when used in centralized facilities
# SSOP Sequence specific oligonucleotide probes
A new high throughput, high resolution genotyping method developed for the detection of alleles at the human leukocyte antigen (HLA)A, B, C, and DRB1 loci by combining polymerase chain reaction (PCR) and sequence-specific oligonucleotide probes (SSOPs) protocols with the Luminex 100 xMAP flow cytometry dual laser system to quantitate fluorescently labeled oligonucleotides attached to color coded microbeads.
The PCR-SSOP-Luminex method provides a simple, accurate, and rapid approach toward multiplex genotyping of HLA alleles to the four-digit or higher level of resolution It takes only approximately 5 h from DNA extraction to the definition of HLA four-digit alleles at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci for 96 samples when handled by a single typist
– Itoh Y, Mizuki N, Shimada T, Azuma F, Itakura M, Kashiwase K, Kikkawa E, Kulski JK, Satake M, Inoko H. High-throughput DNA typing of HLA-A, -B, -C, and -DRB1 loci by a PCR-SSOP-Luminex method in the Japanese population. Immunogenetics. 2005 Nov;57(10):717-29. doi: 10.1007/s00251-005-0048-3. Epub 2005 Nov 8. PMID: 16215732.
– Project: Analysis of immune cell infiltrates and biomarkers during clinical acute gastro intesinal graft vs host disease
Authors:
Mateja Kralj Juric
Medical University of Vienna
Sakhila GhimireUniversity Hospital Regensburg Justyna Ogonek Medigene
Eva M Mischak-Weissinger Hannover Medical School
Molecular HLA typing using SSOP (Luminex)
HLA typing done by I-Serological
Depends on Mixing the lymphocytes of the tested person with selected antisera then add the complement and a vital dye is added and the percentage of cell lysis is examined under microscopy. II DNA -Molecular typing – sequence-specific oligonucleatide probes. – Sequence -specific primer – Direct DNA sequencing
Sahar elkharraz
3 years ago
HLA typing are important for successful of graft survival.
This technique is DNA molecular typing PCR with reverse SSOP (Luminex)
Other techniques:
HLA typing are
– [ ] Serology
– [ ] Molecular (PCR) help to identify unique HLA alleles
– [ ] DNA molecular typing:
SSP ( sequence specific primer)
SSO ( sequence specific oligonucleotide)
Sequence based typing
mai shawky
3 years ago
·this method of HLA typing is SSOP ( Sequence specific oligonucleotide probe), one of molecular techniques with low or intermediate resolution.
as HLA typing is done either by:
o Serological method: adding recipient lymphocytes to sera containing multiple wells with addition of complement, then detection of complement dependent cell lysis. It is rapid and useful in deceased donor, but does not detect HLA type Cw, DP or DQ which may affect transplant outcome.
o Molecular technique: more accurate and use DNA probes and PCR reactions. It includes:
§ Sequence specific primer PCR.
§ Sequence specific oligonucleotide probe.
§ Direct DNA sequencing.
o Mainly we stress on HLA A, B and DR.
Asmaa Khudhur
3 years ago
Molecular HLA typing using SSOP (Luminex)
HLA typing is a crucial step in renal transplantation ,HLA laboratories currently perform serological and molecular typing methods.
1-)Serological typing :her a tray containing sera with Abs to a many known types of HLA alleles to which recipient lymphocytes are introduced in edition to complement and dye .
Cons:
1- the results are available in short period especially in deceased donor renal transplantation which mean less cold ischemia times.
2-the other benefit is that it offer the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigens expression which is called NULL HLA alleles with less immunological significance.
Prons:
1-) is the lake of sera with Ab specificities that are capable of identifying the ever-growing number of HLA alleles.
2-)do not readily detect differences in HLA proteins small amino acids which may be antigenic enough to trigger potent immunological responses.
Serological typing has fallen into disuse with more advanced methods of typing currently available.
2-) molecular typing
– sequence-specific oligonucleatide probes.
– Sequence -specific primer
– Direct DNA sequencing
Advantages:
1-) regardless of the method, molecular typing can identify differences in HLA Ag between donor and recipient with details to the amino acid level.
2-) HLA typing by PCR is highly specific with no cross-reactivity.
Disadvantages:
New alleles not currently on the HLA sequence databank will fail to be identified.
DNA typing is more fast and reliable than serology , in addition some alleles not expressed on cell surface
dalia
3 years ago
This typing is done by (SSOP )using Luminex machine Other techniques for tissue typing are I-Serological
This are first techniques used for HLA typing , not commonly used nowadays. Depends on Mixing the lymphocytes from the tested individual with selected antisera then add the complement and a vital dye is added and the percentage of cell lysis is examined under microscopy
Disdvantages ;
1- the HLA-typing antisera are rare so we can miss several HLA Ages .
2- Cross reaction between Abs lead to non specific results II DNA -Molecular typing
1- sequence-specific oligonucleotide probe (SSOP) : can easily provide low or intermediate resolution HLA typing .
2- sequence-specific primer (SSP) : Identify high resolution typing (allel level resolution
3- SBT (sequence-based typing) : high-resolution HLA typing
4– NGS (next-generation sequencing )
Abdulrahman Ishag
3 years ago
The technique used her is reverse SSOP with PCR.
Sequence specific oligonucleotide probes:
Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles. Unique HLA alleles are then identified using fluorescent tags. For a particular gene of interest, the precise order of nucleotides is determined through sequencing. HLA type is then assigned using available HLA allele sequences.
Molecular typing 1-Sequence-specific primer polymerase chain reaction 2-Sequence specific oligonucleotide probes 3-Direct DNA sequencing
1-Molecular typing regardless of the method can clearly identify differences in HLA antigen between donor and recipient.
2-HLA typing based on polymerase chain reaction (PCR) is highly specific where specific alleles are identified with no cross-reactivity.
3-The disadvantage it poses is that new alleles not currently on the HLA sequence databank will fail to be identified.
Serological typing;
In this approach, a tray containing sera with antibodies to a multitude of known HLA alleles is used.
1-The benefit of serologic typing is that results are available in a short period. This is particularly important in deceased donor renal transplantation. Quick results mean less cold ischemia times.
2-This method also offers the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression.
Reference ;
Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant.
The technique used for HLA assessment is DNA typing methods, specifically a polymerase chain reaction conjunction identifying sequence-specific oligonucleotide probes (SSOP)
Other methods are sequence-specific primers (SSPs), and sequencing-based typing (SBT).
SSOP – Amplifying genomic DNA using primers and then detecting the hybridization on specific oligonucleotide probes tagged with enzymatic or fluorescent markers to the amplified product.
SSP – Depends on DNA amplification using group or allele-specific primers and detecting an amplified product by gel electrophoresis.
SBT – gene-specific primers to sequence polymorphic regions of the gene, and alleles can be assigned based on the nucleotides identified at key positions in the sequence.
SBT is apparently a more complete method, but the first two tend to be sufficient in most cases of kidney transplantation and are more easily reproduced.
The technique used her is reverse SSOP with PCR
IT is one of molecular tissue typing techniques .
* Different tissue typing techniques :
●Serological methods
In this approach, a tray containing sera with antibodies to a known HLA alleles is used. These are commercially available. For typing, recipient lymphocytes are introduced into the tray wells contacting sera, complement and dye. In tray wells where antibodies can bind to the antigens on the surface of lymphocytes; complement is activated. This results in complement pathways triggered resulting in cell death, ultimately allowing the dye to enter the cell. Tray wells with significant cell death are then identified under phase contrast microscopy.
benefit of serologic typing :
Fast results so mainly important in deceased donor to decreasecold ischemia times
But its not specific and HLA-Cw, DQ, and DP antigen may have clinically significant effects on the outcomes of allografts. However, serologic assays are scarce for these loci.
●Molecular typing
Molecular typing can identify differences in HLA antigen between donor and recipient with detail to the amino acid level
The recent technical developments in the field of molecular methods and DNA based typing have led to continuous modification of HLA nomenclature and better understanding of rejection based on mismatching at very specific amino acids.
*Sequence-specific primer polymerase chain reaction: extracted DNA is amplified in several wells. Each well has primers that are complementary to specific HLA alleles. In wells where DNA probes are complementary to the specific sequence of the HLA molecule, an amplification product is formed. This is then instilled into an agarose gel and undergoes electrophoresis where they appear as a band. HLA typing is then allocated by matching the primers of the amplification product to DNA sequences of several candidate alleles.
*Sequence specific oligonucleotide probes: Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles. Unique HLA alleles are then identified using fluorescent tags. For a particular gene of interest, the precise order of nucleotides is determined through sequencing. HLA type is then assigned using available HLA allele sequences.
*Direct DNA sequencing: This method determines the precise order of nucleotides in the gene of interest. Using published HLA allele sequences, HLA type is subsequently assigned by comparison.
HLA typing based on polymerase chain reaction (PCR) is highly specific where specific alleles are identified ( but sometimes cross reactivity can occur, gene may occur in two or more forms called alleles. Cross-reactivity is the identification of an allele which is essentially similar to the allele of interest.
Human leukocyte antigen typing and crossmatch: A comprehensive review.Mohammed Mahdi Althaf, Mohsen El Kossi, and Ahmed Mostafa Halawa.
World J Transplant. 2017 Dec 24; 7(6): 339–348.
The technique used here is SSOP (sequence-specific oligonucleotide probe) one of the molecular typing techniques
the other molecular ones are : sequence-specific Primer PCR and direct DNA sequencing)
in the past the modality used was the serological method: but is not standard in the time being.. Still has the advantage of giving results in a short time; important in cadaveric transplantation because of shortened cold ischemia time etc.)
The molecular techniques give more information about alleles and give high-resolution results.
Here the technique used is HLA typing with PCR reverse SSOP( – By Luminex) .
HLA typing can be done by serological methods and molecular typing.
Serological Methods- These can be by antibody dependent cell mediated cytotoxicity or Compliment dependent cytotoxicity. The results are usually available quickly and so it is more important in deceased donors. It can identify Null alleles. It cannot detect differences in HLA protein small amino acids. It is poor at HLA Cw, DP and DQ
Molecular methods
These methods achieve standardization and variability between labs is deceased. These are DNA based HLA typing methods and include:
SSOP – Sequence specific oligonucleotide probe Hybridization
It can produce low or intermediate resolution HLA typing. It is based on amplifying genomic DNA and can used specific locus or group based primers to detect hybridization of specific nucleotide probe tagged with fluorescent and enzymatic markers to the amplified product. Micro particles read on luminex machine
SSP- sequence specific primer amplification.
It depends upon DNA amplification to detect amplified product by gel electrophoresis.
Real time PCR based typing
Sequence based typing
Next generation Sequencing
Comparison between techniques.
CDC was gold standard method in the past and currently it is rarely used due to growing number of new alleles which cannot be covered by sera and the antibodies are directed against more than one HLA molecules and the results may not be accurate. It has a role in deceased donors as results can obtained quickly. High resolution typing is not used in solid organ transplantation.
Reference :Mohammad Mehdi Althaf et al. Human leukocyte antigen typing and cross match: A comprehensive review. World Journal of Transplant. 2017 Dec 24; 7(6): 339–348.
The technique used was HLA Typing with polymerase chain reaction with sequence-specific oligo probe with use of the Luminex™ Technology
Serological typing
Recipient lymphocytes are introduced into the tray wells contacting sera, complement and dye. In tray wells where antibodies can bind to the antigens on the surface of lymphocytes; complement is activated. This results in complement pathways triggered resulting in cell death, ultimately allowing the dye to enter the cell.
Tray wells with significant cell death are then identified under phase contrast microscopy. Through a process of comparison and elimination of positive wells the HLA type is assigned
Advantages:
results are available in a short period
important in deceased donor renal transplantation-less cold ischemia times
offers the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression- “null” HLA alleles
Disadvantages:
the lack of sera with antibody specificities that can identify the ever-growing number of HLA alleles
scarce for HLA-Cw, DQ, and DP antigen
serologic methods do not
cannot readily detect differences in HLA protein small amino acids
Molecular typing
Sequence-specific primer polymerase chain reaction:
extracted DNA from the subject is amplified in several wells.
Each well has primers that are complementary to specific HLA alleles.
In wells where DNA probes are complementary to the specific sequence of the HLA molecule, an amplification product is formed.
This is then instilled into an agarose gel and undergoes electrophoresis where they appear as a band
HLA typing is then allocated by matching the primers of the amplification product to DNA sequences of several candidate alleles
Sequence specific oligonucleotide probes:
Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles.
Unique HLA alleles are then identified using fluorescent tags. For a particular gene of interest, the precise order of nucleotides is determined through sequencing.
HLA type is then assigned using available HLA allele sequences
Advantages
Clearly identify differences in HLA antigen between donor and recipient
detail to the amino acid level that can provide insight to the risk accompanying mismatched donor-recipient antigens, epitopes and amino acid
highly specific where specific alleles are identified with no cross-reactivity
Disadvantages
gene may occur in two or more forms called alleles. Cross-reactivity is the identification of an allele which is essentially similar to the allele of interest
Reference Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant. 2017 Dec 24;7(6):339-348. doi: 10.5500/wjt.v7.i6.339. PMID: 29312863; PMCID: PMC5743871.
This is a Molecular method which is dependent on DNA amplification by PCR.
Sequence Specific oligonecleotide probes are used .
Full HLA class I and II typing begins with drawing a patient’s blood and extracting DNA from the white blood cells.
SSOP. In this technique, the DNA undergoes PCR amplification, using a set of carefully selected primers designed to amplify the desired portion of DNA.
This procedure permits amplification of specific DNA fragments that are then sufficiently abundant for analysis. PCR technology has provided the basis for development of convenient and rapid methods of HLA typing, based upon the exact nucleotide sequences of individual alleles.
DNA-based typing with direct Sanger sequencing — DNA-based typing with direct Sanger sequencing, also called “sequence-based typing” (SBT), is a technique that consists of the amplification and direct sequencing of relevant exons.
Next-generation DNA-based typing — NGS refers to sequencing technologies that perform sequencing of millions of short DNA fragments in parallel.
SSOP typing is usually considered as “intermediate resolution typing”
High-resolution typing” is considered to distinguish all the alleles at a specific locus (at the four-digit level [eg, HLA-DRB1*04:01] or protein level).
DNA based typing can be done quickly and is more reliable
Serology based typing is cost effective though provide low resolution typing
☆ The technique:
___________________
▪︎This is a type of molecular methods for HLA class I& II typing: PCR – SSOP (Luminex).
▪︎This technique relies on[1]:
1. Target DNA is PCR-amplified using 5 ′ -biotin-labeled primers that are highly specific to certain sequences of HLA genes. 2. The amplified DNA is allowed to hybridize to complementary DNA probes coupled to microbeads which is labeled with SA – PE.
4. Measurement : Luminex apparatus identifies the fluorescent intensity of PE on each coded oligobead.
4. A software assists in determining the HLA genotype (alleles) of the sample DNA.
HLA typing approaches [2]:
__________________________
A. Serological :
▪︎Were used for defining the tissue types, or HLA, in humans (Thorsby, 2009) but, since the development of the PCR DNA-based PCR methods have predominated.
B. Molecular techniques:
▪︎ Include three basic methods used in conjunction with PCR:
1. Sequence-specific oligonucleotide probes
(SSOPs)
2. Sequence-specific primers (SSPs).
3. Sequencing-based typing (SBT).
▪︎SSOP was first applied to HLA class II typing because of the limitations of DR serology and of the better knowledge of allelic polymorphism at DR/DQ loci.
▪︎SSOP procedure is discussed above.
▪︎SSP depends on DNA amplification using group- or allele-specific primers and detecting an amplified product of the correct size by gel electrophoresis. The size is determined by running an agarose gel
that separates the PCR products according to their size.
▪︎SBT uses gene-specific primers to sequence polymorphic regions of the gene,
and alleles can be assigned based on the nucleotides identified at key positions in the sequence.
Drawbacks of molecular typing:
___________________________________
1. It is difficult to produce reagents that uniquely recognize each individual HLA Ag.
2. It is often necessary to identify patterns of primer and probe reactivity in order to determine the HLA type.
3. Computer programs assist in the analysis of primer and probe patterns, which are more difficult to analyze unaided because of
the added complexity of the HLA genes.
4. It is difficult to identify HLA alleles without performing SBT, because the differences between alleles may be determined by single nucleotide differences.
Advantages:
____________
▪︎ SSP and SSOP can easily provide low or intermediate levels of typing and this level of typing is sufficient for renal transplan in most cases.
▪︎Concerning SSOP approach:
_________________________________
▪︎It gives the advantages of: better reagent availability, lower cost, more rapid turn-around time, and greater accuracy, all of which would warrant its use as an HLA typing method of choice. But;
▪︎Limitations:
1. Not as fast as PCR‐SSP and batch testing
3. Results need careful analysis/review
4. Probes may miss new alleles
________________
Ref:
[1]http://medweb4.unige.ch/immunologie/home/HSC/donor/HLA_typing/SSO.php1].
[2] Paul P. J. et al. “Human leucocyte antigen typing: techniques and technology, a critical appraisal”. https://doi.org/10.1111/j.1744-13X.2011.01040.x
This HLA typing is a kind of molecular typing in the following way:
PCR-reverse SSOP is an abbreviation for PCR-reverse SSOP.
When using commercial kits, the process of SSOP is reversed, in that the specific oligonucleotide probes are attached to microparticles that can hybridize with labelled PCR products, yielding distinct fluorescent beads on hybridization, giving out specific patterns, which can be read by software to determine the HLA type.
Techniques for HLA typing include serological assays, cellular assays, and DNA-based molecular approaches, among others.
There are two major ways of determining HLA typing.
Method No. 1: Serologic
Using either antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity as a method of killing cancer cells (CDC).
Advantages include quick findings and a lower level of immunological significance.
With the rising relevance of HLA subtypes, there is a limitation in that it is difficult to get good quality blood.
DNA-based HLA typing approaches (also known as molecular typing) include the following:
DNA-based HLA typing may be classified as either low resolution or high resolution, and it comprises the following:
A- Sequence-specific oligonucleotide probe hybridization using the SSOP protocol (used in our patient)
B- Sequence-specific primer amplification using SSP primers
C- Real-time polymerase chain reaction (PCR) based typing (high-resolution typing)
D- Sequence-based typing is a kind of typing that is based on a sequence of letters (high-resolution typing)
E- Sequencing using next-generation technology (high-resolution typing)
In solid organ transplantation, high-resolution typing is not necessary, and merely medium resolution typing is generally acceptable in the majority of cases.
For dead donors, RT PCR is the technique of choice because of the very quick results that may be obtained in as little as 2-3 hours after the donor’s death. Typing for the recipient and live donor is done using the SSOP technique, which is the most generally used approach in this case, and the results are obtained in 2 days or less.
-Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant
HLA antigens are cell membrane glycoproteins with key roles in the initiation of the immune response. Current methods for HLA typing define HLA alleles and allele groups using DNA-based methods. Different DNA-based molecular techniques are used depending on the clinical application. Solid organ transplantation requires a low- to intermediate-level typing resolution to determine an individual’s HLA antigens.
Technique used for this assay :
Molecular HLA typing done for class I,II HLA using the SSOP (luminex)
sequence-specific oligonucleotide probes (SSOPs).
types Serological typing and cellular typing
Serological typing (Principle of microlymphocyto toxicity test technology).HLA cytotoxic antibodies are IgG and IgM isotypes. In the presence of complements, these antibodies are capable of binding with their corresponding antigens on the surface of lymphocytes and inducing holes on the membrane. There is no such effect if the lymphocytes do not carry the corresponding antigens. The principle for this reaction is shown in. Dead lymphocytes with damaged membrane can be observed in a number of ways, the simplest of which staining with eosin or trypan blue. Dead cells are stained and appear expanded due to incorporation of the dye; live cells are not stained. Generally, the extent of the antigen-antibody reaction is determined on the basis of the percentage of dead cells.
. DNA based molecular methods Sequence-specific primers (SSP) typing Sequence-specific oligonucleotide probes (SSOP) typing Sequence-based typing (SBT) Next-generation sequencing (NGS)
*PCR detecting HLA genes.
*PCR-RFLP
*Variable number tandem repeat typing.
*DNA sequence based typing.
*karyosome analysis.
now days serological typing is insufficient due to low expression of HLA antigen, lack of serological reagent ,misclassification of antigen within groups.
DNA typing is more fast and reliable than serology , in addition some alleles not expressed on cell surface .
{Article in Slovak]
E Kulcsarova et al. Bratisl Lek Listy. 2000.
Molecular HLA typing done for class I,II HLA using the SSOP (luminex)
sequence-specific oligonucleotide probes (SSOPs)
HLA-TYPING TECHNIQUES
The Microcytotoxicity Test
This serologic test is performed in small plastic trays with a grid of small flat-bottomed wells, each of which contains a selected antiserum to which lymphocytes from the individual to be typed are added, and incubated. Complement is added, and after another incubation, a vital dye is added to indicate the proportion of dead cells in each well when examined under the microscope. Using the products of an immune response (antibodies) to measure the targets of an immune response (HLA antigens) has a certain inherent logic.
If an antigen had an antibody response, its immunologic importance was demonstrated. However, the HLA-typing antisera are monospecific because they do not recognize a single private specificity so it is necessary to examine the patterns of reactivity with several antibodies to determine the HLA type.
DNA Typing Methods
Three basic methods used in conjunction with polymerase chain reaction (PCR) employ
Even with these molecular approaches to HLA typing, it is difficult to produce reagents that uniquely recognize each individual HLA antigen. As with serology, it is often necessary to identify patterns of primer and probe reactivity in order to determine the HLA type.
Computer programs assist in the analysis of primer and probe patterns, which are more difficult to analyze unaided because of the added complexity of the HLA genes
It is difficult to identify HLA alleles without performing SBT, because the differences between alleles may be determined by single nucleotide differences. However, SSP and SSOP can easily provide low or intermediate levels of typing, identifying the recognized HLA antigens and major allele groups, respectively. This level of typing is sufficient for renal transplantation in most cases.
Reference
Gabriel M. Danovitch, MD. Handbook of Kidney Transplantation. SIXTH EDITION
saja Mohammed
3 years ago
Please comment on the technique of HLA typing below comparing it with the other techniques.
PCR -SSOP molecular genetic DNA based HLA typing for class1, and class 2.
Molecular based HLA typing are more sensitive and accurate than serological typing. soluble or recombinant HLA molecules based assay not targeting lymphocytes as lymphocytes can express HLA and non-HLA antigens.
Molecular DNA-based HLA typing techniques It includes different types:
1- sequence specific oligonucleotide probes( PCR-SSOP) which is based on first amplifying genomic DNA using locus- or group specific primers and then detecting the hybridization of specific oligonucleotide probes tagged with enzymatic or fluorescent markers to the amplified product, specific segments of the DNA of different alleles. Unique HLA alleles are then identified using fluorescent tags in commercial kit
2-sequence-specific primer amplification(SSPs)
determined by DNA augmentation using group- or allele-specific primers and
Sensing an amplified product of the correct size by gel electrophoresis
3- Direct DNA sequencing( (SBT) typing determines the precise order of nucleotides in the gene of interest, allowing HLA type comparison, through the amino acid level, to provide insight to the risk monitoring.
SSP and SSOP can easily provide low or intermediate levels of typing of different HLA antigens at allel level ,however Irrespective of the method, molecular typing can detect differences in HLA- Ag between donor and recipient specifics to the amino acid level with no cross-reactivity.
Limitations:
Novel alleles not currently on the HLA sequence database will be not classified.
moving to next-generation sequencing with high resolation
References:
1- Kidney transplant hand book :Gabriel M. Danovitch, MD
2-Human leukocyte antigen typing and crossmatch: A comprehensive review:World J Transplant 2017 December 24; 7(6): 339-348
This a molecular method of HLA typing.
The specific molecular method used here is the Sequence-specific oligonucleotide probe (SSOP). This method used single-nucleotide polymorphisms (SNPS) rather than primers.
It allows for testing of large numbers of donors.
The SNPs lie within the probe instead of the 3’termnus position, the probe binds to the denatured amplicon.
Other methods used.
Serological methods; This is being phased out in many transplant centres with emphasis being put on molecular testing methods.
Lymphocytes are tested against a panel of well known HLA- specific alloantibodies. This is incubated and complement added. Cells with the specific HLA will be lysed which allows them to be permeable to fluorochrome and are then identified by microscopy.
This method has very low resolution as it can only identify the HLA gene. Eg HLA-A2.
HLA class I & II typed by molecular methods specifically ; Sequence Specific Oligo-nucleotide Probe, low resolution technique, HLA-mismatch 000. In this technique,amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of DNA of different alleles. Other molecular typing methods are ;
Sequence-specific primer polymerase chain reaction
Extracted DNA from the subject is amplified in several wells.
Each well has primer that are complementary to specific HLA alleles = amplification product is perform
2.Direct DNA sequencing ;
This method determine the precise order of nucleotide in the gene of interest.
Using published HLA allele sequences, HLA is type subsequently assigned by comparison.
In addition to molecular method there is also serology methods for typing HLA antigens. The principle is similar to CDC & is very limited due to lack of sera with antibody specificites that are capable of identifying the ever growing numbers of HLA alleles.
Amit Sharma
3 years ago
This HLA typing is a form of molecular typing:
PCR-reverse SSOP
Commercial kits have the process of SSOP reversed, whereby the specific oligoneucleotide probes are attached to microparticles which can be hybridized with labeled PCR products, giving rise to distinct fluorescent beads on hybridization, giving out specific paterns, read by softwares, determining the HLA type.
HLA typing techniques include serological assay, cellular assay and DNA based molecular methods.
a) Serological assay: This method utilizes antibody-dependent cell mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Recipient lymphocyte is added to tray with wells containing serum with antibodies, complement and a dye leading to cell lysis which is observed under a phase contrast microscope. Results are rapidly available leading to a decreased cold ischemia time. These methods are not used nowadays due to limitations like lack of commercially available serum containing specific antibodies against different HLA alleles.
b) Cellular assay: It is a mixed lymphocyte culture (MLC) which is usually utilized for HLA class II typing. It is more sensitive than the serological assay in catching the HLA differences.
c) Molecular methods: These are DNA based methods which are more sensitive, more accurate and have higher resolution, ultimately helping in better HLA typing. These methods include:
i. SSP (Sequence Specific Primer): Extracted DNA from patient is mixed with primers complementary to specific alleles in wells forming an amplification product (using PCR) which, on an agarose gel electrophoresis, forms a band helping in HLA typing. It is a very rapid and sensitive method. ii. SSOP (Sequence Specific Oligonucleotide Probe hybridization): Amplified DNA is mixed with oligonucleotide probe complementary to specific DNA segment of different alleles which are identified using fluorescent markers leading to HLA typing. It cannot distinguish between alleles, but is useful for typing large number of samples. iii. SBT (Sequence based typing): Exon specific primers (exon 2 for class II and exon 2 & 3 for class I) are used. It has highest reliability, detecting the nucleotide sequences of HLA alleles directly. iv. RSCA (Reference Strand based Conformation Analysis): In this, amplified sample is mixed with amplified reference allele and compared on agarose gel electrophoresis. v. NGS (Next Generation Sequencing): It involves clonal amplification and ability to sequence both introns and exons giving better resolution HLA typing. vi. STR (Short Tandem Repeat) genotyping: It is a rapid test used for screening sibling donors.
References:
1) Deshpande A. The human leukocyte antigen system … simplified. Glob J Transfus Med 2017;2:77-88.
2) Dunn PP. Human leucocyte antigen typing: techniques and technology, a critical appraisal. Int J Immunogenet. 2011 Dec;38(6):463-73. doi: 10.1111/j.1744-313X.2011.01040.x. PMID: 22059555.
3) Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant. 2017 Dec 24;7(6):339-348. doi: 10.5500/wjt.v7.i6.339. PMID: 29312863; PMCID: PMC5743871.
4) Mahdi BM. A glow of HLA typing in organ transplantation. Clin Transl Med. 2013 Feb 23;2(1):6. doi: 10.1186/2001-1326-2-6. PMID: 23432791; PMCID: PMC3598844.
a) Serological assay:
Advantage: Results are rapidly available leading to a decreased cold ischemia time.
Disadvantage: Not used nowadays due to limitations like lack of commercially available serum containing specific antibodies against different HLA alleles.
b) Cellular assay:
Advantage: More sensitive than the serological assay in catching the HLA differences.
Disadvantage: Less specific.
c) Molecular methods:
Advantage: More sensitive, more accurate and have higher resolution.
Disadvantage: More challenging technically, increased cost. New alleles not currently in the HLA sequence databank will not be recognized.
i. SSP (Sequence Specific Primer):
Advantage: Very rapid and sensitive method.
ii. SSOP (Sequence Specific Oligonucleotide Probe hybridization):
Advantage: Useful for typing large number of samples. Disadvantage: It cannot distinguish between alleles.
iii. SBT (Sequence based typing):
Advantage: Highest reliability, detecting the nucleotide sequences of HLA alleles directly.
Disadvantage: Time-consuming.
iv. RSCA (Reference Strand based Conformation Analysis):
Advantage: More specific
Disadvantage: High cost, time-consuming
vi. STR (Short Tandem Repeat) genotyping:
Advantage: Rapid test for screening sibling donors.
Ala Ali
Admin
3 years ago
Dear alL, in your responses; respond to the question first and then add theoretical knowledge
Doaa Elwasly
3 years ago
HLA typing of deceased donors is done using real-time polymerase chain reaction (RT-PCR) or premade, reverse sequence-specific oligonucleotide probes (rSSO) trays.
High-resolution typing methods can be used in living-donor evaluations.
Serologic methods
Lymphocytes from the donor or recipient are added to several wells of plates containing different sera, incubated then complement is added to wells, the occurrence of reaction leading to dead cells is a positive test. Buy it has some limitations which is inability to recognize HLA large sepicifities.
DNA based molecular methods
Sequence-specific primers (SSP) typing
It recognize a particular HLA sequence (allele) or group of similar alleles.
DNA is extracted and amplified by PCR.
The pattern of amplicons allows for the assignment of the HLA genotype.
The SSP method can be used for either low-resolution typing, by identifying allele groups of a particular antigen, or for high-resolution typing, for a specific allele. Suitable in the typing of deceased donors because it is rapid . However, it is not well suited for typing large numbers of samples
With SSOP typing, DNA is amplified using a set of primers that recognize a particular HLA locus. As with SSP typing, it amplify the most polymorphic regions of the HLA gene (exons 2 and 3 for class I genes and exon 2 for class II genes).
Primers used in SSOP for DNA amplification are less specific than those used in SSP and cannot distinguish between groups of similar alleles. To discriminate between alleles within a locus, a labeled SSOPs that recognize specific sequences are added. Oligonucleotide probes can also be linked to beads to allow for multiplex analysis of HLA typing using the Luminex platform.
SSOP typing is well suited for typing large numbers of samples.
For less samples, reverse-SSO (rSSO) can be used where oligonucleotide probes are bound to the membranes, with each membrane having all the SSOs bound that are required for typing of a particular HLA locus rSSO typing still has the same drawbacks but is faster
Real-time PCR (RT-PCR)-based typing
It uses allele-specific PCR similar to SSP methods ,amplicons are detected in real time with the use of fluorescent dyes or probes. Fluorescent readings of each well are obtained at different temperatures.and the pattern of reactive wells identifies the HLA typing.
Alternatively, labeled SSOPs can be used instead of the less specific cyanine dye. Probe-based approach allows for different fluorescent reporter molecules to be used in the same reaction well, allowing for a greater degree of multiplexing.
RT PCR is a rapid method
Sequence-based typing (SBT)
Depends on direct amplification and sequencing of the relevant exons using fluorescently labeled dideoxynucleotides , allowing for a high resolution typing.
Next-generation sequencing (NGS)
It permitted high-resolution typing with significantly reduced ambiguity as it allows for base calls to be assigned to the same or different alleles.
Reference
Surgical splenectomy has been used as part of desensitization protocol(antibodies removal )and also in ABOI transplantation (evidence is of low quality)but now recently Rituximab(anti-CD20) have taken its place(medical splenectomy).
2- Surgical splenectomy has also been used in refractory AMBR as salvage therapy after failed first line therapy( PLEX,IVIG, rituximab in previously highly sensitized patients).
The major drawback related to surgical splenectomy was that it removes a major source of lymphocytes, including antibody-secreting B cells, B cell precursor cells, and plasma cells and also the effect of splenectomy on the immune system is permanent, which may increase the risk for the development of life-threatening sepsis, especially from encapsulated bacteria. REFERENCE:
1-Montgomery RA, Locke JE, King KE, et al. ABO incompatible renal transplantation: a paradigm ready for broad implementation. Transplantation 2009; 87:124
2-Morath C., Zieir M., Dohler B.,et al. ABO- Incompatible Kidney Transplantation. Front Immunol :2017 Mar 6;8:234
3-Locke JE, Zachary AA, Haas M, et al. The utility of splenectomy as rescue treatment for severe acute antibody mediated rejection. Am J Transplant 2007; 7:842
Yes, this belongs to another question, it is a mistake.
Mohamed Saad
3 years ago
HLA-TYPING TECHNIQUES. The Micro cytotoxicity Test:
Its serologic old technique which done by selected antiserum to which lymphocytes from the individual to be typed are added, and incubated then Complement is added, and after another incubation, a vital dye is added to indicate the proportion of dead cells in each well when examined under the microscope, its less specific and sometime can miss several HLA antigens. DNA Typing Methods:
Three basic methods used in conjunction with polymerase chain reaction (PCR)
1-sequence-specific oligonucleotide probes (SSOPs).
2-sequence-specific primers (SSPs).
3-sequencing-based typing (SBT).
Even with these molecular approaches to HLA typing, it is difficult to produce reagents that uniquely recognize each individual HLA antigen. It is difficult to identify HLA alleles without performing SBT, because the differences between alleles may be determined by single nucleotide differences.
However, SSP and SSOP can easily provide low or intermediate levels of typing, identifying the recognized HLA antigens and major allele groups, respectively.
We still waiting the development of next-generation sequencing will eventually bring higher resolution HLA typing at all loci at decreased costs. REFERENCE:
Handbook of Kidney Transplantation, 6th Ed, Lippincott Williams & Wilkins (LWW) 2017
Last edited 3 years ago by Mohamed Saad
Mohamed Saad
3 years ago
HLA-TYPING TECHNIQUES. The Micro cytotoxicity Test:
Its serologic old technique which done by selected antiserum to which lymphocytes from the individual to be typed are added, and incubated then Complement is added, and after another incubation, a vital dye is added to indicate the proportion of dead cells in each well when examined under the microscope, its less specific and sometime can miss several HLA antigens.
DNA Typing Methods:
Three basic methods used in conjunction with polymerase chain reaction (PCR)
1-sequence-specific oligonucleotide probes (SSOPs).
2-sequence-specific primers (SSPs).
3-sequencing-based typing (SBT).
Even with these molecular approaches to HLA typing, it is difficult to produce reagents that uniquely recognize each individual HLA antigen.
It is difficult to identify HLA alleles without performing SBT, because the differences between alleles may be determined by single nucleotide differences. However, SSP and SSOP can easily provide low or intermediate levels of typing, identifying the recognized HLA antigens and major allele groups, respectively.
We still waiting the development of next-generation sequencing will eventually bring higher resolution HLA typing at all loci at decreased costs. REFERENCE:
Handbook of Kidney Transplantation, 6th Ed, Lippincott Williams & Wilkins (LWW) 2017
Please describe it in a little more detail. Also, comment on the technique shown in the question asked with more detail and depth.
Last edited 3 years ago by Hemant Sharma
Batool Butt
3 years ago
HLA typing can be done by two methods 1- Serologic method
Serological method –older method, uses serum containing known antibodies to specific HLA antigens. In this method , recipient lymphocytes are added to several wells of plates that contain sera of antibodies to all known HLA antigens. After incubation period, complement is added to produce complement mediated cell lysis in wells where known antibodies are bound to HLA antigens. The presence of lysis indicated the HLA antigen type. But this method would not identify typing at allele specific level or determine the polymorphism of different antigens and also can’t differentiate between HLA protein amino acids which may be antigenic & induce strong immune response. 2- DNA-based HLA typing methods (molecular typing):
Two methods:
A- SSOP sequence specific oligonucleotide probe hybridization (used in our patient)
SSOP is based on first amplifying DNA using specific primers and then detecting the hybridization of specific oligonucleotide probes tagged with fluorescent markers to the amplified product.The microparticles are read on a flow cytometer or Luminex machine.
B- SSP sequence-specific primer amplification
SSP depends on DNA amplification using group- or allele-specific primers and detecting an amplified product of the correct size by gel electrophoresis.
Primers used in SSOP for DNA amplification is less specific than SSP as it can’t differentiate between allele within a locus, or group of similar alleles.
C- RFLP-PCR (restriction fragment length polymorphism polymerase chain reaction)
D- Sequence based typing (high resolution typing)
SBT-considered gold standard ,it may produce uncertain results
E- Next generation sequencing (high resolution typing)
High resolution typing is not required in solid organ transplantation and only low resolution typing is usually sufficient. REFERENCE:
Handbook of Kidney Transplantation, 6th Ed, Lippincott Williams & Wilkins (LWW) 2017
Alaa eddin salamah
3 years ago
this is a PCR adjuncted with reverse SSOP using Luminex machine for microparticles reading
HLA-TYPING TECHNIQUES
The Microcytotoxicity Test
This serologic test is performed in small plastic trays with a grid of small flat-bottomed wells, each of which contains a selected antiserum to which lymphocytes from the individual to be typed are added, and incubated. Complement is added, and after another incubation, a vital dye is added to indicate the proportion of dead cells in each well when examined under the microscope. Using the products of an immune response (antibodies) to measure the targets of an immune response (HLA antigens) has a certain inherent logic. If an antigen had provoked an antibody response, its immunologic importance was demonstrated. However, the HLA-typing antisera are seldom monospecific (i.e., they do not recognize a single private specificity), so in most cases it is necessary to examine the patterns of reactivity with several antibodies to determine the HLA type.
DNA Typing Methods
it is now more common to type individuals by DNA-based methods. Three basic methods used in conjunction with polymerase chain reaction (PCR) employ sequence-specific oligonucleotide probes (SSOPs), sequence-specific primers (SSPs), and sequencing-based typing (SBT). SSOP is based on first amplifying genomic DNA using locus- or group-specific primers and then detecting the hybridization of specific oligonucleotide probes tagged with enzymatic or fluorescent markers to the amplified product. The microparticles are read on a flow cytometer or Luminex machine SSP depends on DNA amplification using group- or allele-specific primers and detecting an amplified product of the correct size by gel electrophoresis.
SBT uses gene-specific primers to sequence polymorphic regions of the gene, and alleles can be based on the nucleotides identified at key positions in the sequence. It is difficult to identify HLA alleles without performing SBT, because the differences between alleles may be determined by single nucleotide differences.
SSP and SSOP can easily provide low or intermediate levels of typing, identifying the recognized HLA antigens and major allele groups, respectively.
Reference
Handbook of Kidney Transplantation, 6th Ed,Lippincott Williams & Wilkins (LWW) 2017
Fatima AlTaher
3 years ago
This typing is done by sequence based oligonucleotide probe (SSOP) Other techniques for tissue typing are I-Serological (microcytotoxicity) Are the earliest techniques used for HLA typing , not commonly used nowadays. Depends on Mixing the lymphocytes from the tested person with different known antisera then add the complement and a vital dye is added and the percentage of cell lysis is examined under microscopy Disdvantages ; 1- Can miss several HLA Ags due to lack of antisera 2- Non specific results due to cross reaction between Abs II-Molecular typing a- sequence-specific oligonucleotide probe (SSOP) : can produce low or intermediate resolution HLA typing according to number of oligonucleotides used . b- sequence-specific primer (SSP) : not routemly used. Mainly used for ambiguous results as it can differniate between allele even if they only different in one nucleatide c- SBT (sequence-based typing) : high-resolution HLA typing d- NGS (next-generation sequencing )
Uses gene-specific primer followed by amplification (DNA polymerase) and
identification by agarose gel electrophoresis.
2-sequence-specific oligonucleotides:SSO
Uses a gene-specific primer with unique fluorescent tags, which are subsequently
identified using a fow cytometer.
Mohamed Mohamed
3 years ago
HLA Typing techniques are:
A.HLA typing by serology (Based on CDC or microlympocytotoxicity):
Advantages:
– simple
– low cost
Disadvantages:
– low resolution typing of HLA-A & HLA-B (discrimination between groups of related alleles
only) B.HLA typing DNA-based methods: ( more fast & reliable compared to serology methods)
Methods include:
(i) SSP- (sequence-specific primer)
(ii) SSO- (sequence-specific oligonucleotide); the one used in the case presented here
(iii) RFLP-PCR (restriction fragment length polymorphism polymerase chain reaction)
(iv) SBT (sequence-based typing) (Tait et al., 2009; Bontadini, 2012; Erlich, 2012).
SBT, although considered the gold-standard method for high-resolution HLA genotyping, it may produce uncertain results due to insufficient sequencing and ambiguous haplotype phasing (Erlich, 2012).
-This HLA typing is done by SSOP method. HLA typing methods:
1. Serologic Typing:
-This serologic test is performed in small plastic trays which contain a selected
antiserum to which lymphocytes from the individual to be typed are added, and incubated. Complement and a vital dye are added.Dye indicates the proportion of dead cells in each well when examined under the microscope.
-The HLA-typing antisera are not monospecific, so it is necessary to examine with several antibodies to determine the HLA type.
2.DNA Typing Methods:
– it is more common to type individuals by DNA-based methods than serological method
-Three basic methods used in conjunction with polymerase chain reaction (PCR) employ sequence-specific oligonucleotide probes (SSOPs), sequence-specific primers (SSPs), and sequencing-based typing (SBT). – SSOP is based on first amplifying genomic DNA using locus- or group-specific primers and then detecting the hybridization of specific oligonucleotide probes tagged with enzymatic or fluorescent markers to the amplified product.
-The microparticles are read on a flow cytometer or Luminex machine and sophisticated software programs assist in the interpretation of the patterns to determine the HLA type.
–SSP depends on DNA amplification using group- or allele-specific primers and detecting an amplified product of the correct size by gel electrophoresis.
–SBT uses gene-specific primers to sequence polymorphic regions of the gene,
and alleles can be assigned based on the nucleotides identified at key positions in the sequence.
– Even with these molecular approaches to HLA typing, it is difficult to produce reagents that uniquely recognize each HLA antigen.
-SSP and SSOP can easily provide low or intermediate levels of typing, identifying
the recognized HLA antigens and major allele groups, respectively.
-The type of technique used differs between laboratories and depends on experience and cost.
– DNA Typing is more sensitive and precise than serological typing.
Professor Ahmed Halawa
Admin
3 years ago
Dear All Please read the handbook I sent to you. HLA typing is well written and well explained.
Wael Jebur
3 years ago
Reverse_Sequence _specific oligonucleotide probes typing rSSOP:DNA based molecular method, in which DNA is amplified using a set of primer that recognize a particular HLA locus. As with Sequence Specific Primers SSP, primers are designed to amplify thevmist polymorphic regions of the HLA gene (exons 2 and 3 for class I and exon 2 for classvIi gene). Primers used ln
SSOP for DNA amplification isvless specific than SSP. It cant distinguish between allels within a locus, or group of similar allels. SSOP typing is well suited for typing large numbers of samples. For fewer samples reverse_SSO rSSO can be used. rSSO has faster turnaround time than SSP. In comparison, Real_time PCR based typing, shortens turnaround time to approximatly one hour and tequires much less hands on involvement of technicians in addition to automated data interpretation also simplifies the analysis significantly. In Next _generation sequencing(NGS), has enabled high resolution typing with significantly reduced ambiguity however limitations in throughput, scalability, cost, and spees restrict its use in some solid organs transplantation.
it is a molecular assay which includes amplification of DNA sequence. called Sequence-Specific Oligonucleotide probe (SSOP)
this method can give more details as it test to split antigen and the epitope level
so this method as compared to the serological one is more accurate and safer in detecting the best donor
Its Molecular assay via Sequence Specific Oligonucleotide Probe (SSOP).It is more specific and accurate than serological HLA method.
There’s two technique to did HLA typing
Serology and molecular (PCR)
The PCR is a most accurate and can detect split antigens and their epitope , it done by DNA amplification and sequencing called sequence specific oligonucletide probes(SSOP)
HLA typing assess whether donors are suitably and safely matched to a transplant recipient.
The HLA typing method in this question is : Molecular assay via Sequence Specific Oligonucleotide Probe
Other possible HLA methods.
1)Serology assay : Microlymphocytotoxicity test.
2)Cellular assay : Mixed Lymphocyte culture.
this method of hla typing is called Sequence-specific oligonucleotide probes (SSOP) typing. In this method, DNA amplification occur .
it is more suited for large number of samples as it can test 180 sample in 2 days.
it is more accurate and specific than serological methods.
This is a molecular HLA typing method called SSOP ( Sequence specific oligonucleotide probe).This molecular technique has low or intermediate resolution.
There are two ways of HLA typing:
1) Serological typing: By mixing donor lymphocytes with the recipient serum in the presence of complement and dye. Binding of the AB with the antigen on the lymphocyte surface results in complement activation and cell lysis. Dead cells are identified under phase contrast microscopy.
o Advantages: quickly available results, more important role in deceased donors(short cold ischemia time), can identify Null alleles.
o Disadvantages: Cannot detect differences in HLA protein small amino acids. It is poor at HLA Cw, DQ and DP .Additionally, there is lack of sera with antibody specificities which can identify the increasing number of HLA alleles.
2) Molecular typing: There are 3 methods using PCR and provide more reliable results
i. Sequence-specific oligonucleotide probes (SSOPs)
ii. Sequence-specific primers (SSPs).
iii. Sequencing-based typing (SBT).
Both SSP and SSOP can provide low or intermediate resolution of typing required in solid organ transplantation.
o Advantages:
Can identify specific alleles without cross reactivity
Can identify differences in HLA antigens between the donor and recipient, thus avoiding the risks with antigen mismatch
Can provide details to the amino acid level
o Disadvantages:
May fail to identify novel alleles not on the HLA data sequence
Reference:
Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and cross match: A comprehensive review. World J Transplant. 2017 Dec 24;7(6):339-348.
HLA TYPING
The method of HLA typing in the given picture is molecular typing – sequence specific oligonucleotides probes used.
Molecular typing clearly identifies differences in HLA antigen between donor and recipient. This can help in identifying risks associated with antigen mismatch. PCR based HLA typing is extremely accurate for identifying specific alleles with no cross reactivity. However, new alleles may not be identified.
The other technique is serological typing. A tray containing sera with antibodies to known HLA alleles is used. Complement pathways are stimulated to act and when cell death occurs, dye enters the cell and under phase contrast microscopy this is clearly visible.
Serological typing is quick and can be arranged in a short period of time for emergent surgeries. The short time frame also links with lesser cold ischemia time in kidney transplant. HLA alleles that have identifiable DNA sequences with molecular typing but not cell surface antigen expression can be differentiated clearly. HLA Cw, DQ and DP cannot be identified with this method because of the lack of sera with antibody specificities that are capable of identifying the growing number of HLA alleles.
In addition, serological typing cannot readily distinguish HLA protein small amino acids. It has not used in many centers in recent times.
Reference :
Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant. 2017 Dec 24;7(6):339-348. doi: 10.5500/wjt.v7.i6.339. PMID: 29312863; PMCID: PMC5743871.
HLA typing is the most important step for successful
of kidney transplantation .The method mentioned above is the molecular technique of HLA typing ,PCR-reverse SSOP (Lumiex) ,
The molecular technique is more sensitive and accurate , depends on amplification of DNA , & can identify the HLA antigens
The other method is serological technique which depends on the interaction between the sera of the recipient & the donor`s cells, which is less accurate than the molecular one.
HLA typing is molecular typing for class1 and 2 and PCR -Reverse SSOP(Luminex) in this case.
HLA typing is a crucial step in renal transplantation, as recognition of foreign HLA by recipient T lymphocytes would trigger an immune response .
two types of HLA typing:
1- molecular typing
identify different in HLA Ag between donor and recipient with details of amino acids
typing also resolve the public epitopes and distinguish the common null alleles from expressed alleles
DNA based HLA typing include:
Reverse sequence -specific oligonucleotide (rSSO) probe-hybridization, sequence -specific primer(SSP)-directed polymerase chain reaction (PCR)amplification are the typical method used for HLA typing of deceased donor.
The SSP method is a commonly used technique in which multiple pairs of allele -specific primers are used to determine the HLA alleles present in a DNA sample.
SSP method is fast and can be completed within 2-3 h
RT PCR method has a simple work flow without need for gel electrophoresis or hybridization and provide result within 90 min
HLA typing RT-PCR method is not accurate .
2- Serological typing
recipient lymphocytes are introduced to try wells contain sera of known HLA alleles with complement and dye so after Ag-complement reaction take place the dye enter the cell this cell death identify by phase contrast
this method take short time so can be used in deceased donor.
REFERENCES1. Mittal KK, Mickey MR, Singal DP, Terasaki PI. Serotyping for homotransplantation. 18. Refinement of microdroplet lymphocyte cytotoxicity test. Transplantation 1968; 6(8): 913.
2. Bidwell JL, Bidwell EA, Savage DA, Middleton D, Klouda PT, Bradley BA. A DNA-RFLP typing system that positively identifies serologically well-defined and ill-defined HLA-DR and DQ alleles, including DRw10. Transplantation 1988; 45(3): 640.
This HLA typing is a form of molecular typing:
PCR-reverse SSOP; Molecular HLA typing done for class I,II HLA
sequence-specific oligonucleotide probes (SSOPs)
HLA typing techniques include
Serologic methods
Lymphocytes from the donor or recipient are added to several wells of plates containing different sera, incubated then complement is added to wells, the occurrence of reaction leading to dead cells is a positive test. Buy it has some limitations which is inability to recognize HLA large sepicifities.
DNA based molecular methods
Sequence-specific primers (SSP) typing
It recognize a particular HLA sequence (allele) or group of similar alleles.
DNA is extracted and amplified by PCR.
The pattern of amplicons allows for the assignment of the HLA genotype.
The SSP method can be used for either low-resolution typing, by identifying allele groups of a particular antigen, or for high-resolution typing, for a specific allele. Suitable in the typing of deceased donors because it is rapid . However, it is not well suited for typing large numbers of samples
Sequence-specific oligonucleotide probes (SSOP) typing
With SSOP typing, DNA is amplified using a set of primers that recognize a particular HLA locus. As with SSP typing, it amplify the most polymorphic regions of the HLA gene (exons 2 and 3 for class I genes and exon 2 for class II genes).
Primers used in SSOP for DNA amplification are less specific than those used in SSP and cannot distinguish between groups of similar alleles. To discriminate between alleles within a locus, a labeled SSOPs that recognize specific sequences are added . Oligonucleotide probes can also be linked to beads to allow for multiplex analysis of HLA typing using the Luminex platform.
SSOP typing is well suited for typing large numbers of samples.
For less samples, reverse-SSO (rSSO) can be used where oligonucleotide probes are bound to the membranes, with each membrane having all the SSOs bound that are required for typing of a particular HLA locus rSSO typing still has the same drawbacks but is faster
Real-time PCR (RT-PCR)-based typing
It uses allele-specific PCR similar to SSP methods ,amplicons are detected in real time with the use of fluorescent dyes or probes. Fluorescent readings of each well are obtained at different temperatures.and the pattern of reactive wells identifies the HLA typing.
Alternatively, labeled SSOPs can be used instead of the less specific cyanine dye. Probe-based approach allows for different fluorescent reporter molecules to be used in the same reaction well, allowing for a greater degree of multiplexing.
RT PCR is a rapid method
Sequence-based typing (SBT)
Depends on direct amplification and sequencing of the relevant exons using fluorescently labeled dideoxynucleotides , allowing for a high resolution typing.
Next-generation sequencing (NGS)
It permitted high-resolution typing with significantly reduced ambiguity as it allows for base calls to be assigned to the same or different alleles.
Different techniques for HLA typing:
-Serological or Molecular.
-Molecular is sensitive and accurate than serological.
-Soluble or recombinant HLA molecules based assay which not target lymphocytes as lymphocytes express HLA and non-HLA antigens.
*** Molecular DNA-based HLA typing techniques It includes different types:
1- sequence specific oligonucleotide probes( PCR-SSOP) which is based on first amplifying genomic DNA using locus- or group specific primers and then detecting the hybridization of specific oligonucleotide probes tagged with enzymatic or fluorescent markers to the amplified product, specific segments of the DNA of different alleles. Unique HLA alleles are then identified using fluorescent tags in commercial kit.
2-sequence-specific primer amplification(SSPs)
determined by DNA augmentation using group- or allele-specific primers and Sensing an amplified product of the correct size by gel electrophoresis.
3- Direct DNA sequencing( (SBT) typing determines the precise order of nucleotides in the gene of interest, allowing HLA type comparison, through the amino acid level, to provide insight to the risk monitoring.
-SSP and SSOP can easily provide low or intermediate levels of typing of different HLA antigens at allel level ,however Irrespective of the method, molecular typing can detect differences in HLA- Ag between donor and recipient specifics to the amino acid level with no cross-reactivity.
-Limitations:
Novel alleles not currently on the HLA sequence database will be not classified.
moving to next-generation sequencing with high resolation.
The technique used her is reverse SSOP with PCR
IT is one of molecular tissue typing techniques .
The technique used in the above HLA typing is sequence specific oligonucleotide probe with use of Luminex technology
HLA typing has improved from the primitive serological methods to the current NGS (next generation sequencing), which has lead to the discovery of further alleles
In the serological typing method the recipient lymphocytes are introduced into the wells containing sera, complement and dye. If the antibodies can bind to the antigens on the surface of the lymphocytes the complement is activated. The cell lysis is identified and through comparison and elimination of positive wells the HLA typing is determined. The advantages are that it is a cheaper method, results are available in shorter time. The disadvantages are that the sera used dose not include antibodies for an ever growing number of the newer HLA alleles. The sera do not detect HLA Cw,DQ and DP antigens. they cannot readily detect differences in small protein amino acids.
Molecular methods for HLA typing use polymerase chain technology to identify HLA alleles. They are more precise in that they identify even small HLA alleles upto the amino acid level. They can identify the HLA alleles with no cross reactivity. They are 2 major methods of molecular methods of typing. One is sequence specific primer polymerase reaction and sequence specific oligonucleotide probes.
The technique used her is reverse Sequence specific oligonucleotide probes with PCR.
Sequence specific oligonucleotide probes:
Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles. Unique HLA alleles are then identified using fluorescent tags. For a particular gene of interest, the precise order of nucleotides is determined through sequencing. HLA type is then assigned using available HLA allele sequences.
Molecular typing
1-Sequence-specific primer polymerase chain reaction
2-Sequence specific oligonucleotide probes
3-Direct DNA sequencing
This is a molecular Technique :
PCR – reverse SSOP (Luminex) was used – which means that a sequence-specific oligonucleotide probes were identified characterizing locus or group specific of primers through microparticles read in a Luminex machine. Thus, it can easily provide low or intermediate levels of typing, identifying the recognized HLA antigens and major allele groups.
– recognizes possible antibodies from structural epitopes
– No require viable cells
The other one, is a serologic Tchnique: it compares cells deaths identified in microscopy will provide the result to be evaluated.
– No specific for HLA antibodies
– Require a suficiente supply of viable lymphocytes
The HLA typing showed on this case scenario is single antigen beads SAB method which is molecular method of HLA typing and is considered the best because it’s accurate, rapid and reproducible.
HLA typing can be done by different methods, Those are classified to either serological or molecular methods.
Serologic method where the recipient lymphocytes are introduced to a tray wells containing sera with antibodies to a multitude of known HLA alleles then complement and dye will be added. If antibodies bind to the antigen a complement activation will occur and finally the dye will enter the cell. Phase contrast microscopy is then used to identify wells with significant cell death.
Advantages of serologic methods are they are fast with results in short time which is important in deceased donor transplantation and the ability to identify non HLA alleles which have identifiable DNA sequences with molecular typing but no cell surface antigen expression.
Disadvantaged of serologic methods are the inability to detect the increasing number of HLA alleles because of the lake of sera which contains specific antibodies. In addition that serologic method is not able to detect differences in HLA protein small amino acids that may be antigenic enough to induce potent immune response.
There are three molecular HLA typing methods. First, sequence-specific primer PCR where extracted DNA is amplified in several wells where DNA probes are complementary to the specific sequence of HLA molecule, then is instilled into an agarose gel and undergo electrophoresis and appear as a band. Second, Sequence-specific oligonucleotide probes where amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles and unique HLA alleles identified with fluorescent tags. Finally, Direct DNA sequencing where HLA type is assigned by comparison to published HLA allele sequences.
There are many pros of molecular methods of HLA typing, they can clearly identify the differences in HLA antigen between the donor and the recipient regardless of the method up to the amino acids level which make them highly specific methods. PCR typing can identify specific alleles with no cross reactivity – identification of allele which is essentially similar to the allele of interest – although a gene may occur in two or more forms (alleles). While this is highly specific it has the disadvantage that new alleles not on HLA databank will not be identified.
Reference:
#Serological method:
Benefits:
Preliminary or supportive method for molecular assays, fast and cheap.
Drawbacks:
Low resolution, requires viable cells, poor reagent supply and not the current standad
# Cellular method:
Benefits:
Used for HLA class 11
Drawbacks:
Low resolution, requires viable cells, informative and rarely used currently
# SSP Sequence specific priming:
Benefits :
Nowadays used to distinguish cis/trans ambiguities
Drawbacks:
Low or intermediate resolution, limited to previous known polymorphisms and restricted to selected exons
# SBT Sequencing based typing:
Benefits:
High resolution
Drawback:
Dose not distinguish cis/trans ambiguities and restricted to selected exons
# NGS next generstion sequencing
Brnefits:
High resolution, high throughput typing and increases rate of resolved ambiguities.
Drawback :
Complicated workflow and data analysis, noval technique,could become reasonably priced when used in centralized facilities
# SSOP Sequence specific oligonucleotide probes
A new high throughput, high resolution genotyping method developed for the detection of alleles at the human leukocyte antigen (HLA)A, B, C, and DRB1 loci by combining polymerase chain reaction (PCR) and sequence-specific oligonucleotide probes (SSOPs) protocols with the Luminex 100 xMAP flow cytometry dual laser system to quantitate fluorescently labeled oligonucleotides attached to color coded microbeads.
The PCR-SSOP-Luminex method provides a simple, accurate, and rapid approach toward multiplex genotyping of HLA alleles to the four-digit or higher level of resolution It takes only approximately 5 h from DNA extraction to the definition of HLA four-digit alleles at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci for 96 samples when handled by a single typist
– Itoh Y, Mizuki N, Shimada T, Azuma F, Itakura M, Kashiwase K, Kikkawa E, Kulski JK, Satake M, Inoko H. High-throughput DNA typing of HLA-A, -B, -C, and -DRB1 loci by a PCR-SSOP-Luminex method in the Japanese population. Immunogenetics. 2005 Nov;57(10):717-29. doi: 10.1007/s00251-005-0048-3. Epub 2005 Nov 8. PMID: 16215732.
– Project: Analysis of immune cell infiltrates and biomarkers during clinical acute gastro intesinal graft vs host disease
Authors:
Mateja Kralj Juric
Medical University of Vienna
Sakhila GhimireUniversity Hospital Regensburg Justyna Ogonek Medigene
Eva M Mischak-Weissinger Hannover Medical School
Molecular HLA typing using SSOP (Luminex)
HLA typing done by
I-Serological
Depends on Mixing the lymphocytes of the tested person with selected antisera then add the complement and a vital dye is added and the percentage of cell lysis is examined under microscopy.
II DNA -Molecular typing
– sequence-specific oligonucleatide probes.
– Sequence -specific primer
– Direct DNA sequencing
HLA typing are important for successful of graft survival.
This technique is DNA molecular typing PCR with reverse SSOP (Luminex)
Other techniques:
HLA typing are
– [ ] Serology
– [ ] Molecular (PCR) help to identify unique HLA alleles
– [ ] DNA molecular typing:
SSP ( sequence specific primer)
SSO ( sequence specific oligonucleotide)
Sequence based typing
·this method of HLA typing is SSOP ( Sequence specific oligonucleotide probe), one of molecular techniques with low or intermediate resolution.
as HLA typing is done either by:
o Serological method: adding recipient lymphocytes to sera containing multiple wells with addition of complement, then detection of complement dependent cell lysis. It is rapid and useful in deceased donor, but does not detect HLA type Cw, DP or DQ which may affect transplant outcome.
o Molecular technique: more accurate and use DNA probes and PCR reactions. It includes:
§ Sequence specific primer PCR.
§ Sequence specific oligonucleotide probe.
§ Direct DNA sequencing.
o Mainly we stress on HLA A, B and DR.
Molecular HLA typing using SSOP (Luminex)
HLA typing is a crucial step in renal transplantation ,HLA laboratories currently perform serological and molecular typing methods.
1-)Serological typing :her a tray containing sera with Abs to a many known types of HLA alleles to which recipient lymphocytes are introduced in edition to complement and dye .
Cons:
1- the results are available in short period especially in deceased donor renal transplantation which mean less cold ischemia times.
2-the other benefit is that it offer the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigens expression which is called NULL HLA alleles with less immunological significance.
Prons:
1-) is the lake of sera with Ab specificities that are capable of identifying the ever-growing number of HLA alleles.
2-)do not readily detect differences in HLA proteins small amino acids which may be antigenic enough to trigger potent immunological responses.
Serological typing has fallen into disuse with more advanced methods of typing currently available.
2-) molecular typing
– sequence-specific oligonucleatide probes.
– Sequence -specific primer
– Direct DNA sequencing
Advantages:
1-) regardless of the method, molecular typing can identify differences in HLA Ag between donor and recipient with details to the amino acid level.
2-) HLA typing by PCR is highly specific with no cross-reactivity.
Disadvantages:
New alleles not currently on the HLA sequence databank will fail to be identified.
DNA typing is more fast and reliable than serology , in addition some alleles not expressed on cell surface
This typing is done by (SSOP )using Luminex machine
Other techniques for tissue typing are
I-Serological
This are first techniques used for HLA typing , not commonly used nowadays. Depends on Mixing the lymphocytes from the tested individual with selected antisera then add the complement and a vital dye is added and the percentage of cell lysis is examined under microscopy
Disdvantages ;
1- the HLA-typing antisera are rare so we can miss several HLA Ages .
2- Cross reaction between Abs lead to non specific results
II DNA -Molecular typing
1- sequence-specific oligonucleotide probe (SSOP) : can easily provide low or intermediate resolution HLA typing .
2- sequence-specific primer (SSP) : Identify high resolution typing (allel level resolution
3- SBT (sequence-based typing) : high-resolution HLA typing
4– NGS (next-generation sequencing )
The technique used her is reverse SSOP with PCR.
Sequence specific oligonucleotide probes:
Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles. Unique HLA alleles are then identified using fluorescent tags. For a particular gene of interest, the precise order of nucleotides is determined through sequencing. HLA type is then assigned using available HLA allele sequences.
Molecular typing
1-Sequence-specific primer polymerase chain reaction
2-Sequence specific oligonucleotide probes
3-Direct DNA sequencing
1-Molecular typing regardless of the method can clearly identify differences in HLA antigen between donor and recipient.
2-HLA typing based on polymerase chain reaction (PCR) is highly specific where specific alleles are identified with no cross-reactivity.
3-The disadvantage it poses is that new alleles not currently on the HLA sequence databank will fail to be identified.
Serological typing;
In this approach, a tray containing sera with antibodies to a multitude of known HLA alleles is used.
1-The benefit of serologic typing is that results are available in a short period. This is particularly important in deceased donor renal transplantation. Quick results mean less cold ischemia times.
2-This method also offers the ability to differentiate HLA alleles that have identifiable DNA sequences with molecular typing but with no cell surface antigen expression.
Reference ;
Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant.
Thanks
The technique used for HLA assessment is DNA typing methods, specifically a polymerase chain reaction conjunction identifying sequence-specific oligonucleotide probes (SSOP)
Other methods are sequence-specific primers (SSPs), and sequencing-based typing (SBT).
SSOP – Amplifying genomic DNA using primers and then detecting the hybridization on specific oligonucleotide probes tagged with enzymatic or fluorescent markers to the amplified product.
SSP – Depends on DNA amplification using group or allele-specific primers and detecting an amplified product by gel electrophoresis.
SBT – gene-specific primers to sequence polymorphic regions of the gene, and alleles can be assigned based on the nucleotides identified at key positions in the sequence.
SBT is apparently a more complete method, but the first two tend to be sufficient in most cases of kidney transplantation and are more easily reproduced.
Thanks
The technique used her is reverse SSOP with PCR
IT is one of molecular tissue typing techniques .
* Different tissue typing techniques :
●Serological methods
In this approach, a tray containing sera with antibodies to a known HLA alleles is used. These are commercially available. For typing, recipient lymphocytes are introduced into the tray wells contacting sera, complement and dye. In tray wells where antibodies can bind to the antigens on the surface of lymphocytes; complement is activated. This results in complement pathways triggered resulting in cell death, ultimately allowing the dye to enter the cell. Tray wells with significant cell death are then identified under phase contrast microscopy.
benefit of serologic typing :
Fast results so mainly important in deceased donor to decreasecold ischemia times
But its not specific and HLA-Cw, DQ, and DP antigen may have clinically significant effects on the outcomes of allografts. However, serologic assays are scarce for these loci.
●Molecular typing
Molecular typing can identify differences in HLA antigen between donor and recipient with detail to the amino acid level
The recent technical developments in the field of molecular methods and DNA based typing have led to continuous modification of HLA nomenclature and better understanding of rejection based on mismatching at very specific amino acids.
*Sequence-specific primer polymerase chain reaction: extracted DNA is amplified in several wells. Each well has primers that are complementary to specific HLA alleles. In wells where DNA probes are complementary to the specific sequence of the HLA molecule, an amplification product is formed. This is then instilled into an agarose gel and undergoes electrophoresis where they appear as a band. HLA typing is then allocated by matching the primers of the amplification product to DNA sequences of several candidate alleles.
*Sequence specific oligonucleotide probes: Amplified DNA is mixed with oligonucleotide probes that are complementary to specific segments of the DNA of different alleles. Unique HLA alleles are then identified using fluorescent tags. For a particular gene of interest, the precise order of nucleotides is determined through sequencing. HLA type is then assigned using available HLA allele sequences.
*Direct DNA sequencing: This method determines the precise order of nucleotides in the gene of interest. Using published HLA allele sequences, HLA type is subsequently assigned by comparison.
HLA typing based on polymerase chain reaction (PCR) is highly specific where specific alleles are identified ( but sometimes cross reactivity can occur, gene may occur in two or more forms called alleles. Cross-reactivity is the identification of an allele which is essentially similar to the allele of interest.
Human leukocyte antigen typing and crossmatch: A comprehensive review.Mohammed Mahdi Althaf, Mohsen El Kossi, and Ahmed Mostafa Halawa.
World J Transplant. 2017 Dec 24; 7(6): 339–348.
Thanks
The technique used here is SSOP (sequence-specific oligonucleotide probe) one of the molecular typing techniques
the other molecular ones are : sequence-specific Primer PCR and direct DNA sequencing)
in the past the modality used was the serological method: but is not standard in the time being.. Still has the advantage of giving results in a short time; important in cadaveric transplantation because of shortened cold ischemia time etc.)
The molecular techniques give more information about alleles and give high-resolution results.
Thanks
Here the technique used is HLA typing with PCR reverse SSOP( – By Luminex) .
HLA typing can be done by serological methods and molecular typing.
Serological Methods- These can be by antibody dependent cell mediated cytotoxicity or Compliment dependent cytotoxicity. The results are usually available quickly and so it is more important in deceased donors. It can identify Null alleles. It cannot detect differences in HLA protein small amino acids. It is poor at HLA Cw, DP and DQ
Molecular methods
These methods achieve standardization and variability between labs is deceased. These are DNA based HLA typing methods and include:
SSOP – Sequence specific oligonucleotide probe Hybridization
It can produce low or intermediate resolution HLA typing. It is based on amplifying genomic DNA and can used specific locus or group based primers to detect hybridization of specific nucleotide probe tagged with fluorescent and enzymatic markers to the amplified product. Micro particles read on luminex machine
SSP- sequence specific primer amplification.
It depends upon DNA amplification to detect amplified product by gel electrophoresis.
Real time PCR based typing
Sequence based typing
Next generation Sequencing
Comparison between techniques.
CDC was gold standard method in the past and currently it is rarely used due to growing number of new alleles which cannot be covered by sera and the antibodies are directed against more than one HLA molecules and the results may not be accurate. It has a role in deceased donors as results can obtained quickly. High resolution typing is not used in solid organ transplantation.
Reference :Mohammad Mehdi Althaf et al. Human leukocyte antigen typing and cross match: A comprehensive review. World Journal of Transplant. 2017 Dec 24; 7(6): 339–348.
Thanks
The technique used was HLA Typing with polymerase chain reaction with sequence-specific oligo probe with use of the Luminex™ Technology
Serological typing
Recipient lymphocytes are introduced into the tray wells contacting sera, complement and dye. In tray wells where antibodies can bind to the antigens on the surface of lymphocytes; complement is activated. This results in complement pathways triggered resulting in cell death, ultimately allowing the dye to enter the cell.
Tray wells with significant cell death are then identified under phase contrast microscopy. Through a process of comparison and elimination of positive wells the HLA type is assigned
Advantages:
Disadvantages:
Molecular typing
Sequence-specific primer polymerase chain reaction:
Sequence specific oligonucleotide probes:
Advantages
Disadvantages
gene may occur in two or more forms called alleles. Cross-reactivity is the identification of an allele which is essentially similar to the allele of interest
Reference
Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant. 2017 Dec 24;7(6):339-348. doi: 10.5500/wjt.v7.i6.339. PMID: 29312863; PMCID: PMC5743871.
Excellent
This is a Molecular method which is dependent on DNA amplification by PCR.
Sequence Specific oligonecleotide probes are used .
Full HLA class I and II typing begins with drawing a patient’s blood and extracting DNA from the white blood cells.
SSOP. In this technique, the DNA undergoes PCR amplification, using a set of carefully selected primers designed to amplify the desired portion of DNA.
This procedure permits amplification of specific DNA fragments that are then sufficiently abundant for analysis. PCR technology has provided the basis for development of convenient and rapid methods of HLA typing, based upon the exact nucleotide sequences of individual alleles.
DNA-based typing with direct Sanger sequencing — DNA-based typing with direct Sanger sequencing, also called “sequence-based typing” (SBT), is a technique that consists of the amplification and direct sequencing of relevant exons.
Next-generation DNA-based typing — NGS refers to sequencing technologies that perform sequencing of millions of short DNA fragments in parallel.
SSOP typing is usually considered as “intermediate resolution typing”
High-resolution typing” is considered to distinguish all the alleles at a specific locus (at the four-digit level [eg, HLA-DRB1*04:01] or protein level).
DNA based typing can be done quickly and is more reliable
Serology based typing is cost effective though provide low resolution typing
Thanks
☆ The technique:
___________________
▪︎This is a type of molecular methods for HLA class I& II typing: PCR – SSOP (Luminex).
▪︎This technique relies on[1]:
1. Target DNA is PCR-amplified using 5 ′ -biotin-labeled primers that are highly specific to certain sequences of HLA genes. 2. The amplified DNA is allowed to hybridize to complementary DNA probes coupled to microbeads which is labeled with SA – PE.
4. Measurement : Luminex apparatus identifies the fluorescent intensity of PE on each coded oligobead.
4. A software assists in determining the HLA genotype (alleles) of the sample DNA.
HLA typing approaches [2]:
__________________________
A. Serological :
▪︎Were used for defining the tissue types, or HLA, in humans (Thorsby, 2009) but, since the development of the PCR DNA-based PCR methods have predominated.
B. Molecular techniques:
▪︎ Include three basic methods used in conjunction with PCR:
1. Sequence-specific oligonucleotide probes
(SSOPs)
2. Sequence-specific primers (SSPs).
3. Sequencing-based typing (SBT).
▪︎SSOP was first applied to HLA class II typing because of the limitations of DR serology and of the better knowledge of allelic polymorphism at DR/DQ loci.
▪︎SSOP procedure is discussed above.
▪︎SSP depends on DNA amplification using group- or allele-specific primers and detecting an amplified product of the correct size by gel electrophoresis. The size is determined by running an agarose gel
that separates the PCR products according to their size.
▪︎SBT uses gene-specific primers to sequence polymorphic regions of the gene,
and alleles can be assigned based on the nucleotides identified at key positions in the sequence.
Drawbacks of molecular typing:
___________________________________
1. It is difficult to produce reagents that uniquely recognize each individual HLA Ag.
2. It is often necessary to identify patterns of primer and probe reactivity in order to determine the HLA type.
3. Computer programs assist in the analysis of primer and probe patterns, which are more difficult to analyze unaided because of
the added complexity of the HLA genes.
4. It is difficult to identify HLA alleles without performing SBT, because the differences between alleles may be determined by single nucleotide differences.
Advantages:
____________
▪︎ SSP and SSOP can easily provide low or intermediate levels of typing and this level of typing is sufficient for renal transplan in most cases.
▪︎Concerning SSOP approach:
_________________________________
▪︎It gives the advantages of: better reagent availability, lower cost, more rapid turn-around time, and greater accuracy, all of which would warrant its use as an HLA typing method of choice. But;
▪︎Limitations:
1. Not as fast as PCR‐SSP and batch testing
3. Results need careful analysis/review
4. Probes may miss new alleles
________________
Ref:
[1]http://medweb4.unige.ch/immunologie/home/HSC/donor/HLA_typing/SSO.php1].
[2] Paul P. J. et al. “Human leucocyte antigen typing: techniques and technology, a critical appraisal”.
https://doi.org/10.1111/j.1744-13X.2011.01040.x
Excellent
Thanks
This HLA typing is a kind of molecular typing in the following way:
PCR-reverse SSOP is an abbreviation for PCR-reverse SSOP.
When using commercial kits, the process of SSOP is reversed, in that the specific oligonucleotide probes are attached to microparticles that can hybridize with labelled PCR products, yielding distinct fluorescent beads on hybridization, giving out specific patterns, which can be read by software to determine the HLA type.
Techniques for HLA typing include serological assays, cellular assays, and DNA-based molecular approaches, among others.
There are two major ways of determining HLA typing.
Method No. 1: Serologic
Using either antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity as a method of killing cancer cells (CDC).
Advantages include quick findings and a lower level of immunological significance.
With the rising relevance of HLA subtypes, there is a limitation in that it is difficult to get good quality blood.
DNA-based HLA typing approaches (also known as molecular typing) include the following:
DNA-based HLA typing may be classified as either low resolution or high resolution, and it comprises the following:
A- Sequence-specific oligonucleotide probe hybridization using the SSOP protocol (used in our patient)
B- Sequence-specific primer amplification using SSP primers
C- Real-time polymerase chain reaction (PCR) based typing (high-resolution typing)
D- Sequence-based typing is a kind of typing that is based on a sequence of letters (high-resolution typing)
E- Sequencing using next-generation technology (high-resolution typing)
In solid organ transplantation, high-resolution typing is not necessary, and merely medium resolution typing is generally acceptable in the majority of cases.
For dead donors, RT PCR is the technique of choice because of the very quick results that may be obtained in as little as 2-3 hours after the donor’s death. Typing for the recipient and live donor is done using the SSOP technique, which is the most generally used approach in this case, and the results are obtained in 2 days or less.
-Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant
Excellent
HLA antigens are cell membrane glycoproteins with key roles in the initiation of the immune response. Current methods for HLA typing define HLA alleles and allele groups using DNA-based methods. Different DNA-based molecular techniques are used depending on the clinical application. Solid organ transplantation requires a low- to intermediate-level typing resolution to determine an individual’s HLA antigens.
Technique used for this assay :
Molecular HLA typing done for class I,II HLA using the SSOP (luminex)
sequence-specific oligonucleotide probes (SSOPs).
types
Serological typing and cellular typing
Serological typing (Principle of microlymphocyto toxicity test technology).HLA cytotoxic antibodies are IgG and IgM isotypes. In the presence of complements, these antibodies are capable of binding with their corresponding antigens on the surface of lymphocytes and inducing holes on the membrane. There is no such effect if the lymphocytes do not carry the corresponding antigens. The principle for this reaction is shown in. Dead lymphocytes with damaged membrane can be observed in a number of ways, the simplest of which staining with eosin or trypan blue. Dead cells are stained and appear expanded due to incorporation of the dye; live cells are not stained. Generally, the extent of the antigen-antibody reaction is determined on the basis of the percentage of dead cells.
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DNA based molecular methods
Sequence-specific primers (SSP) typing
Sequence-specific oligonucleotide probes (SSOP) typing
Sequence-based typing (SBT)
Next-generation sequencing (NGS)
Thanks
Molecular genotyping Method PCR -reverse SSOP(Luminex ):
Method of HLA typing :
A-phenotypic Method
*Serology – micro cytotoxicity
*tissue typing -mixed lymphocyte reaction .
B- Genotypic Method :
*PCR detecting HLA genes.
*PCR-RFLP
*Variable number tandem repeat typing.
*DNA sequence based typing.
*karyosome analysis.
now days serological typing is insufficient due to low expression of HLA antigen, lack of serological reagent ,misclassification of antigen within groups.
DNA typing is more fast and reliable than serology , in addition some alleles not expressed on cell surface .
{Article in Slovak]
E Kulcsarova et al. Bratisl Lek Listy. 2000.
Great
Short and sweet
Molecular HLA typing done for class I,II HLA using the SSOP (luminex)
sequence-specific oligonucleotide probes (SSOPs)
HLA-TYPING TECHNIQUES
The Microcytotoxicity Test
This serologic test is performed in small plastic trays with a grid of small flat-bottomed wells, each of which contains a selected antiserum to which lymphocytes from the individual to be typed are added, and incubated. Complement is added, and after another incubation, a vital dye is added to indicate the proportion of dead cells in each well when examined under the microscope. Using the products of an immune response (antibodies) to measure the targets of an immune response (HLA antigens) has a certain inherent logic.
If an antigen had an antibody response, its immunologic importance was demonstrated. However, the HLA-typing antisera are monospecific because they do not recognize a single private specificity so it is necessary to examine the patterns of reactivity with several antibodies to determine the HLA type.
DNA Typing Methods
Three basic methods used in conjunction with polymerase chain reaction (PCR) employ
1- sequence-specific oligonucleotide probes (SSOPs)
2- sequence-specific primers (SSPs)
3- sequencing-based typing (SBT).
Even with these molecular approaches to HLA typing, it is difficult to produce reagents that uniquely recognize each individual HLA antigen. As with serology, it is often necessary to identify patterns of primer and probe reactivity in order to determine the HLA type.
Computer programs assist in the analysis of primer and probe patterns, which are more difficult to analyze unaided because of the added complexity of the HLA genes
It is difficult to identify HLA alleles without performing SBT, because the differences between alleles may be determined by single nucleotide differences. However, SSP and SSOP can easily provide low or intermediate levels of typing, identifying the recognized HLA antigens and major allele groups, respectively. This level of typing is sufficient for renal transplantation in most cases.
Reference
Gabriel M. Danovitch, MD. Handbook of Kidney Transplantation. SIXTH EDITION
Please comment on the technique of HLA typing below comparing it with the other techniques.
PCR -SSOP molecular genetic DNA based HLA typing for class1, and class 2.
Molecular based HLA typing are more sensitive and accurate than serological typing. soluble or recombinant HLA molecules based assay not targeting lymphocytes as lymphocytes can express HLA and non-HLA antigens.
Molecular DNA-based HLA typing techniques It includes different types:
1- sequence specific oligonucleotide probes( PCR-SSOP) which is based on first amplifying genomic DNA using locus- or group specific primers and then detecting the hybridization of specific oligonucleotide probes tagged with enzymatic or fluorescent markers to the amplified product, specific segments of the DNA of different alleles. Unique HLA alleles are then identified using fluorescent tags in commercial kit
2-sequence-specific primer amplification(SSPs)
determined by DNA augmentation using group- or allele-specific primers and
Sensing an amplified product of the correct size by gel electrophoresis
3- Direct DNA sequencing( (SBT) typing determines the precise order of nucleotides in the gene of interest, allowing HLA type comparison, through the amino acid level, to provide insight to the risk monitoring.
SSP and SSOP can easily provide low or intermediate levels of typing of different HLA antigens at allel level ,however Irrespective of the method, molecular typing can detect differences in HLA- Ag between donor and recipient specifics to the amino acid level with no cross-reactivity.
Limitations:
Novel alleles not currently on the HLA sequence database will be not classified.
moving to next-generation sequencing with high resolation
References:
1- Kidney transplant hand book :Gabriel M. Danovitch, MD
2-Human leukocyte antigen typing and crossmatch: A comprehensive review:World J Transplant 2017 December 24; 7(6): 339-348
Excellent
This a molecular method of HLA typing.
The specific molecular method used here is the Sequence-specific oligonucleotide probe (SSOP). This method used single-nucleotide polymorphisms (SNPS) rather than primers.
It allows for testing of large numbers of donors.
The SNPs lie within the probe instead of the 3’termnus position, the probe binds to the denatured amplicon.
Other methods used.
Serological methods; This is being phased out in many transplant centres with emphasis being put on molecular testing methods.
Lymphocytes are tested against a panel of well known HLA- specific alloantibodies. This is incubated and complement added. Cells with the specific HLA will be lysed which allows them to be permeable to fluorochrome and are then identified by microscopy.
This method has very low resolution as it can only identify the HLA gene. Eg HLA-A2.
These are the examples of the molecular methods.
Sequence specific primer (SSP);
Next generation sequencing
RNA sequence based testing
Microarrays
Where are the advantages and disadvantages?
2.Direct DNA sequencing ;
In addition to molecular method there is also serology methods for typing HLA antigens. The principle is similar to CDC & is very limited due to lack of sera with antibody specificites that are capable of identifying the ever growing numbers of HLA alleles.
This HLA typing is a form of molecular typing:
PCR-reverse SSOP
Commercial kits have the process of SSOP reversed, whereby the specific oligoneucleotide probes are attached to microparticles which can be hybridized with labeled PCR products, giving rise to distinct fluorescent beads on hybridization, giving out specific paterns, read by softwares, determining the HLA type.
HLA typing techniques include serological assay, cellular assay and DNA based molecular methods.
a) Serological assay: This method utilizes antibody-dependent cell mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Recipient lymphocyte is added to tray with wells containing serum with antibodies, complement and a dye leading to cell lysis which is observed under a phase contrast microscope. Results are rapidly available leading to a decreased cold ischemia time. These methods are not used nowadays due to limitations like lack of commercially available serum containing specific antibodies against different HLA alleles.
b) Cellular assay: It is a mixed lymphocyte culture (MLC) which is usually utilized for HLA class II typing. It is more sensitive than the serological assay in catching the HLA differences.
c) Molecular methods: These are DNA based methods which are more sensitive, more accurate and have higher resolution, ultimately helping in better HLA typing. These methods include:
i. SSP (Sequence Specific Primer): Extracted DNA from patient is mixed with primers complementary to specific alleles in wells forming an amplification product (using PCR) which, on an agarose gel electrophoresis, forms a band helping in HLA typing. It is a very rapid and sensitive method.
ii. SSOP (Sequence Specific Oligonucleotide Probe hybridization): Amplified DNA is mixed with oligonucleotide probe complementary to specific DNA segment of different alleles which are identified using fluorescent markers leading to HLA typing. It cannot distinguish between alleles, but is useful for typing large number of samples.
iii. SBT (Sequence based typing): Exon specific primers (exon 2 for class II and exon 2 & 3 for class I) are used. It has highest reliability, detecting the nucleotide sequences of HLA alleles directly.
iv. RSCA (Reference Strand based Conformation Analysis): In this, amplified sample is mixed with amplified reference allele and compared on agarose gel electrophoresis.
v. NGS (Next Generation Sequencing): It involves clonal amplification and ability to sequence both introns and exons giving better resolution HLA typing.
vi. STR (Short Tandem Repeat) genotyping: It is a rapid test used for screening sibling donors.
References:
1) Deshpande A. The human leukocyte antigen system … simplified. Glob J Transfus Med 2017;2:77-88.
2) Dunn PP. Human leucocyte antigen typing: techniques and technology, a critical appraisal. Int J Immunogenet. 2011 Dec;38(6):463-73. doi: 10.1111/j.1744-313X.2011.01040.x. PMID: 22059555.
3) Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant. 2017 Dec 24;7(6):339-348. doi: 10.5500/wjt.v7.i6.339. PMID: 29312863; PMCID: PMC5743871.
4) Mahdi BM. A glow of HLA typing in organ transplantation. Clin Transl Med. 2013 Feb 23;2(1):6. doi: 10.1186/2001-1326-2-6. PMID: 23432791; PMCID: PMC3598844.
Where are the advantages and disadvantages?
a) Serological assay:
Advantage: Results are rapidly available leading to a decreased cold ischemia time.
Disadvantage: Not used nowadays due to limitations like lack of commercially available serum containing specific antibodies against different HLA alleles.
b) Cellular assay:
Advantage: More sensitive than the serological assay in catching the HLA differences.
Disadvantage: Less specific.
c) Molecular methods:
Advantage: More sensitive, more accurate and have higher resolution.
Disadvantage: More challenging technically, increased cost. New alleles not currently in the HLA sequence databank will not be recognized.
i. SSP (Sequence Specific Primer):
Advantage: Very rapid and sensitive method.
ii. SSOP (Sequence Specific Oligonucleotide Probe hybridization):
Advantage: Useful for typing large number of samples.
Disadvantage: It cannot distinguish between alleles.
iii. SBT (Sequence based typing):
Advantage: Highest reliability, detecting the nucleotide sequences of HLA alleles directly.
Disadvantage: Time-consuming.
iv. RSCA (Reference Strand based Conformation Analysis):
Advantage: More specific
Disadvantage: High cost, time-consuming
v. NGS (Next Generation Sequencing):
Advantage: Achieves better resolution HLA typing.
vi. STR (Short Tandem Repeat) genotyping:
Advantage: Rapid test for screening sibling donors.
Dear alL, in your responses; respond to the question first and then add theoretical knowledge
HLA typing of deceased donors is done using real-time polymerase chain reaction (RT-PCR) or premade, reverse sequence-specific oligonucleotide probes (rSSO) trays.
High-resolution typing methods can be used in living-donor evaluations.
Serologic methods
Lymphocytes from the donor or recipient are added to several wells of plates containing different sera, incubated then complement is added to wells, the occurrence of reaction leading to dead cells is a positive test. Buy it has some limitations which is inability to recognize HLA large sepicifities.
DNA based molecular methods
Sequence-specific primers (SSP) typing
It recognize a particular HLA sequence (allele) or group of similar alleles.
DNA is extracted and amplified by PCR.
The pattern of amplicons allows for the assignment of the HLA genotype.
The SSP method can be used for either low-resolution typing, by identifying allele groups of a particular antigen, or for high-resolution typing, for a specific allele. Suitable in the typing of deceased donors because it is rapid . However, it is not well suited for typing large numbers of samples
Sequence-specific oligonucleotide probes (SSOP) typing
With SSOP typing, DNA is amplified using a set of primers that recognize a particular HLA locus. As with SSP typing, it amplify the most polymorphic regions of the HLA gene (exons 2 and 3 for class I genes and exon 2 for class II genes).
Primers used in SSOP for DNA amplification are less specific than those used in SSP and cannot distinguish between groups of similar alleles. To discriminate between alleles within a locus, a labeled SSOPs that recognize specific sequences are added . Oligonucleotide probes can also be linked to beads to allow for multiplex analysis of HLA typing using the Luminex platform.
SSOP typing is well suited for typing large numbers of samples.
For less samples, reverse-SSO (rSSO) can be used where oligonucleotide probes are bound to the membranes, with each membrane having all the SSOs bound that are required for typing of a particular HLA locus rSSO typing still has the same drawbacks but is faster
Real-time PCR (RT-PCR)-based typing
It uses allele-specific PCR similar to SSP methods ,amplicons are detected in real time with the use of fluorescent dyes or probes. Fluorescent readings of each well are obtained at different temperatures.and the pattern of reactive wells identifies the HLA typing.
Alternatively, labeled SSOPs can be used instead of the less specific cyanine dye. Probe-based approach allows for different fluorescent reporter molecules to be used in the same reaction well, allowing for a greater degree of multiplexing.
RT PCR is a rapid method
Sequence-based typing (SBT)
Depends on direct amplification and sequencing of the relevant exons using fluorescently labeled dideoxynucleotides , allowing for a high resolution typing.
Next-generation sequencing (NGS)
It permitted high-resolution typing with significantly reduced ambiguity as it allows for base calls to be assigned to the same or different alleles.
Reference
Uptodate May 25 ,2021
Good
Surgical splenectomy has been used as part of desensitization protocol(antibodies removal )and also in ABOI transplantation (evidence is of low quality)but now recently Rituximab(anti-CD20) have taken its place(medical splenectomy).
2- Surgical splenectomy has also been used in refractory AMBR as salvage therapy after failed first line therapy( PLEX,IVIG, rituximab in previously highly sensitized patients).
The major drawback related to surgical splenectomy was that it removes a major source of lymphocytes, including antibody-secreting B cells, B cell precursor cells, and plasma cells and also the effect of splenectomy on the immune system is permanent, which may increase the risk for the development of life-threatening sepsis, especially from encapsulated bacteria.
REFERENCE:
1-Montgomery RA, Locke JE, King KE, et al. ABO incompatible renal transplantation: a paradigm ready for broad implementation. Transplantation 2009; 87:124
2-Morath C., Zieir M., Dohler B.,et al. ABO- Incompatible Kidney Transplantation. Front Immunol :2017 Mar 6;8:234
3-Locke JE, Zachary AA, Haas M, et al. The utility of splenectomy as rescue treatment for severe acute antibody mediated rejection. Am J Transplant 2007; 7:842
This answer doesn’t fit with the scenario, recheck it
Yes, this belongs to another question, it is a mistake.
HLA-TYPING TECHNIQUES.
The Micro cytotoxicity Test:
Its serologic old technique which done by selected antiserum to which lymphocytes from the individual to be typed are added, and incubated then Complement is added, and after another incubation, a vital dye is added to indicate the proportion of dead cells in each well when examined under the microscope, its less specific and sometime can miss several HLA antigens.
DNA Typing Methods:
Three basic methods used in conjunction with polymerase chain reaction (PCR)
1-sequence-specific oligonucleotide probes (SSOPs).
2-sequence-specific primers (SSPs).
3-sequencing-based typing (SBT).
Even with these molecular approaches to HLA typing, it is difficult to produce reagents that uniquely recognize each individual HLA antigen. It is difficult to identify HLA alleles without performing SBT, because the differences between alleles may be determined by single nucleotide differences.
However, SSP and SSOP can easily provide low or intermediate levels of typing, identifying the recognized HLA antigens and major allele groups, respectively.
We still waiting the development of next-generation sequencing will eventually bring higher resolution HLA typing at all loci at decreased costs.
REFERENCE:
Handbook of Kidney Transplantation, 6th Ed, Lippincott Williams & Wilkins (LWW) 2017
HLA-TYPING TECHNIQUES.
The Micro cytotoxicity Test:
Its serologic old technique which done by selected antiserum to which lymphocytes from the individual to be typed are added, and incubated then Complement is added, and after another incubation, a vital dye is added to indicate the proportion of dead cells in each well when examined under the microscope, its less specific and sometime can miss several HLA antigens.
DNA Typing Methods:
Three basic methods used in conjunction with polymerase chain reaction (PCR)
1-sequence-specific oligonucleotide probes (SSOPs).
2-sequence-specific primers (SSPs).
3-sequencing-based typing (SBT).
Even with these molecular approaches to HLA typing, it is difficult to produce reagents that uniquely recognize each individual HLA antigen.
It is difficult to identify HLA alleles without performing SBT, because the differences between alleles may be determined by single nucleotide differences. However, SSP and SSOP can easily provide low or intermediate levels of typing, identifying the recognized HLA antigens and major allele groups, respectively.
We still waiting the development of next-generation sequencing will eventually bring higher resolution HLA typing at all loci at decreased costs.
REFERENCE:
Handbook of Kidney Transplantation, 6th Ed, Lippincott Williams & Wilkins (LWW) 2017
Please describe it in a little more detail. Also, comment on the technique shown in the question asked with more detail and depth.
HLA typing can be done by two methods
1- Serologic method
Serological method –older method, uses serum containing known antibodies to specific HLA antigens. In this method , recipient lymphocytes are added to several wells of plates that contain sera of antibodies to all known HLA antigens. After incubation period, complement is added to produce complement mediated cell lysis in wells where known antibodies are bound to HLA antigens. The presence of lysis indicated the HLA antigen type. But this method would not identify typing at allele specific level or determine the polymorphism of different antigens and also can’t differentiate between HLA protein amino acids which may be antigenic & induce strong immune response.
2- DNA-based HLA typing methods (molecular typing):
Two methods:
A- SSOP sequence specific oligonucleotide probe hybridization (used in our patient)
SSOP is based on first amplifying DNA using specific primers and then detecting the hybridization of specific oligonucleotide probes tagged with fluorescent markers to the amplified product.The microparticles are read on a flow cytometer or Luminex machine.
B- SSP sequence-specific primer amplification
SSP depends on DNA amplification using group- or allele-specific primers and detecting an amplified product of the correct size by gel electrophoresis.
Primers used in SSOP for DNA amplification is less specific than SSP as it can’t differentiate between allele within a locus, or group of similar alleles.
C- RFLP-PCR (restriction fragment length polymorphism polymerase chain reaction)
D- Sequence based typing (high resolution typing)
SBT-considered gold standard ,it may produce uncertain results
E- Next generation sequencing (high resolution typing)
High resolution typing is not required in solid organ transplantation and only low resolution typing is usually sufficient.
REFERENCE:
Handbook of Kidney Transplantation, 6th Ed, Lippincott Williams & Wilkins (LWW) 2017
this is a PCR adjuncted with reverse SSOP using Luminex machine for microparticles reading
HLA-TYPING TECHNIQUES
The Microcytotoxicity Test
This serologic test is performed in small plastic trays with a grid of small flat-bottomed wells, each of which contains a selected antiserum to which lymphocytes from the individual to be typed are added, and incubated. Complement is added, and after another incubation, a vital dye is added to indicate the proportion of dead cells in each well when examined under the microscope. Using the products of an immune response (antibodies) to measure the targets of an immune response (HLA antigens) has a certain inherent logic. If an antigen had provoked an antibody response, its immunologic importance was demonstrated. However, the HLA-typing antisera are seldom monospecific (i.e., they do not recognize a single private specificity), so in most cases it is necessary to examine the patterns of reactivity with several antibodies to determine the HLA type.
DNA Typing Methods
it is now more common to type individuals by DNA-based methods. Three basic methods used in conjunction with polymerase chain reaction (PCR) employ sequence-specific oligonucleotide probes (SSOPs), sequence-specific primers (SSPs), and sequencing-based typing (SBT). SSOP is based on first amplifying genomic DNA using locus- or group-specific primers and then detecting the hybridization of specific oligonucleotide probes tagged with enzymatic or fluorescent markers to the amplified product. The microparticles are read on a flow cytometer or Luminex machine SSP depends on DNA amplification using group- or allele-specific primers and detecting an amplified product of the correct size by gel electrophoresis.
SBT uses gene-specific primers to sequence polymorphic regions of the gene, and alleles can be based on the nucleotides identified at key positions in the sequence. It is difficult to identify HLA alleles without performing SBT, because the differences between alleles may be determined by single nucleotide differences.
SSP and SSOP can easily provide low or intermediate levels of typing, identifying the recognized HLA antigens and major allele groups, respectively.
Reference
Handbook of Kidney Transplantation, 6th Ed,Lippincott Williams & Wilkins (LWW) 2017
This typing is done by sequence based oligonucleotide probe (SSOP)
Other techniques for tissue typing are
I-Serological (microcytotoxicity)
Are the earliest techniques used for HLA typing , not commonly used nowadays. Depends on Mixing the lymphocytes from the tested person with different known antisera then add the complement and a vital dye is added and the percentage of cell lysis is examined under microscopy
Disdvantages ;
1- Can miss several HLA Ags due to lack of antisera
2- Non specific results due to cross reaction between Abs
II-Molecular typing
a- sequence-specific oligonucleotide probe (SSOP) : can produce low or intermediate resolution HLA typing according to number of oligonucleotides used .
b- sequence-specific primer (SSP) : not routemly used. Mainly used for ambiguous results as it can differniate between allele even if they only different in one nucleatide
c- SBT (sequence-based typing) : high-resolution HLA typing
d- NGS (next-generation sequencing )
Good response
HLA TYPING:
Required for transplant, for DS detection and quantification of donor-specific
antibodies are prerequisite investigations which facilitate the assessment of the
recipient’s overall immunological risk.
Automated extraction methods allow for a relatively rapid typing that can be
performed in 3–4hours from the time a sample is received.
Sequence-based typing achieves high-level resolution HLA typing.
1-Sequence-specific primers:
Uses gene-specific primer followed by amplification (DNA polymerase) and
identification by agarose gel electrophoresis.
2-sequence-specific oligonucleotides:SSO
Uses a gene-specific primer with unique fluorescent tags, which are subsequently
identified using a fow cytometer.
HLA Typing techniques are:
A.HLA typing by serology (Based on CDC or microlympocytotoxicity):
Advantages:
– simple
– low cost
Disadvantages:
– low resolution typing of HLA-A & HLA-B (discrimination between groups of related alleles
only)
B.HLA typing DNA-based methods: ( more fast & reliable compared to serology methods)
Methods include:
(i) SSP- (sequence-specific primer)
(ii) SSO- (sequence-specific oligonucleotide); the one used in the case presented here
(iii) RFLP-PCR (restriction fragment length polymorphism polymerase chain reaction)
(iv) SBT (sequence-based typing) (Tait et al., 2009; Bontadini, 2012; Erlich, 2012).
SBT, although considered the gold-standard method for high-resolution HLA genotyping, it may produce uncertain results due to insufficient sequencing and ambiguous haplotype phasing (Erlich, 2012).
(v) NGS (next-generation sequencing ): (Abbott et al., 2006; Bentley et al., 2009; Erlich et al., 2011; Erlich, 2012; Shiina et al., 2012; Hosomichi et al., 2013, 2015; Schöfl et al., 2017).
Methods used in this new approach include:
– amplicon-based HLA sequencing
– target enrichment of HLA genes
– whole exome or genome sequencing data-derived typing
Advantages:
– overcome the usual phase ambiguity of HLA alleles
– enable massive, parallel, & high-resolution HLA-typing.
Thanks Dr Mohamed
-This HLA typing is done by SSOP method.
HLA typing methods:
1. Serologic Typing:
-This serologic test is performed in small plastic trays which contain a selected
antiserum to which lymphocytes from the individual to be typed are added, and incubated. Complement and a vital dye are added.Dye indicates the proportion of dead cells in each well when examined under the microscope.
-The HLA-typing antisera are not monospecific, so it is necessary to examine with several antibodies to determine the HLA type.
2.DNA Typing Methods:
– it is more common to type individuals by DNA-based methods than serological method
-Three basic methods used in conjunction with polymerase chain reaction (PCR) employ sequence-specific oligonucleotide probes (SSOPs), sequence-specific primers (SSPs), and sequencing-based typing (SBT).
– SSOP is based on first amplifying genomic DNA using locus- or group-specific primers and then detecting the hybridization of specific oligonucleotide probes tagged with enzymatic or fluorescent markers to the amplified product.
-The microparticles are read on a flow cytometer or Luminex machine and sophisticated software programs assist in the interpretation of the patterns to determine the HLA type.
–SSP depends on DNA amplification using group- or allele-specific primers and detecting an amplified product of the correct size by gel electrophoresis.
–SBT uses gene-specific primers to sequence polymorphic regions of the gene,
and alleles can be assigned based on the nucleotides identified at key positions in the sequence.
– Even with these molecular approaches to HLA typing, it is difficult to produce reagents that uniquely recognize each HLA antigen.
-SSP and SSOP can easily provide low or intermediate levels of typing, identifying
the recognized HLA antigens and major allele groups, respectively.
-The type of technique used differs between laboratories and depends on experience and cost.
– DNA Typing is more sensitive and precise than serological typing.
Dear All
Please read the handbook I sent to you. HLA typing is well written and well explained.
Reverse_Sequence _specific oligonucleotide probes typing rSSOP:DNA based molecular method, in which DNA is amplified using a set of primer that recognize a particular HLA locus. As with Sequence Specific Primers SSP, primers are designed to amplify thevmist polymorphic regions of the HLA gene (exons 2 and 3 for class I and exon 2 for classvIi gene). Primers used ln
SSOP for DNA amplification isvless specific than SSP. It cant distinguish between allels within a locus, or group of similar allels. SSOP typing is well suited for typing large numbers of samples. For fewer samples reverse_SSO rSSO can be used. rSSO has faster turnaround time than SSP. In comparison, Real_time PCR based typing, shortens turnaround time to approximatly one hour and tequires much less hands on involvement of technicians in addition to automated data interpretation also simplifies the analysis significantly. In Next _generation sequencing(NGS), has enabled high resolution typing with significantly reduced ambiguity however limitations in throughput, scalability, cost, and spees restrict its use in some solid organs transplantation.
Reference:
Melissa Y Yeung, kidney transplantation in adult, overview of HLA sensitization and crossmatch testing. http://Www.uptodate.com
Excellent Dr Wael
I noticed that you use underscore (_) rather than a hyphen (-). This is considered a typo (spelling mistake).
Also, molecular typing achieves standardization and reduces the variability between labs