HLA-C mismatches have been studied in kidney transplant and has been shown to be associated with acute rejection.
Tran et al in their study found that sensitized patients with PRa>10% and HLA-C mismatch had decreased graft survival. (1)
In another study, antibody mediated rejection was seen in 27% of the patients with anti HLA-C antibodies in first year post transplant. (2)
References:
1)Tran Th, Dohler B, Heinold A, t al. Deleterious impact of mismatching for human leukocyte antigen C inpresensitized recipients of kidney transplants. Transplantation 2011;92:419.
2) Aubert O, Bories MC, Suberbielle C, et al. Risk of antibody-mediated rejection in kidney transplant recipients with anti HLA-C donor specific antibodies. Am J Transplant 2014;14:1439-1445.
Studies confirmed the occurrence of antibody mediated rejection and transplant glomerulopathy in patients with HLA-C DSA antibody.1,2,4
A case of kidney transplantation with HLA-C DSA ( MFI= 1000) ended with irreversible ABMR.3
So pretransplant risk stratification of sensitized patients may be accomplished by testing for donor specific HLA-C antibody.4
References:
Bachelet T., Guidicelli G., et al. Anti Cw donor specific alloantibodies can lead to positive flow cytometry crossmatch and irreversible acute antibody mediated rejection. Am J Transplant.2011;11:1543
Rogers N.M., BennetT G.D., Toby Coats B. Transplant nephropathy and rapid allograft loss in the presence of HLA-Cw7 antibody. Transplant Int.2012;25:e38.
Bosch A., Llorente S., Eguia J., Mrowiec A., et al. HLA-C antibodies are associated with irreversible rejection in kidney transplantation: Shared molecular eplets characterization.Human Immunology 75 (2014) 338–341.
Aubert O.,Bories M.C., Suberbielle C., Snanoudj R., et al. Risk of Antibody-Mediated Rejection in Kidney Transplant Recipients With Anti-HLA-C Donor-Specific Antibodies. AmericanJournalofTransplantation2014;14:1439–1445.
Frohn et al. investigated the impact of HLA-C mismatches on rejection in 104 renal transplants (20). They controlled for HLA-B mismatch to eliminate linkage disequilibrium as a confounding factor. They found that patients with one or two mismatches for an HLA-C antigen had a significantly higher incidence of rejection compared to those with no HLA-C mismatch (54 and 100 vs. 0%) but only when there was also one HLA-B mismatch. Patients with one HLA-B and two HLA-C mismatches also had decreased graft survival that approached statistical significance (P = 0.055) (1)
Donor mismatches at each of the HLA-A, -B, and -C loci all contributed to an increasing likelihood of developing sensitization to HLA class I, but HLA-C less than HLA-A and -B
Aubert et al performed case-controlled study of 608 patients of kidney transplantation were screened for the presence of isolated anti-HLA-C DSA at day 0. A total of 22 renal transplant recipients were selected and followed for a period of 1 year.
The 22 patients were compared with 88 immunized patients. Acute AMR was diagnosed in six patients (27.3%). The median level of DSA at day 0 was 1179 (530-17,941). The mean fluorescence intensity in the anti-C group was 4966 (978-17,941) in the AMR group and 981 (530-8012) in the group of patients without AMR. The level of DSA at day 0 was predictive for AMR .
Patients with a high level of pretransplant anti-HLA-C DSAs are likely to develop acute AMR during the first year after transplantation (2).
1 Frohn C, Fricke L, Puchta JC, Kirchner H. The effect of HLA-C matching on acute renal transplant rejection. Nephrol Dial Transplant (2001) 16:355–60. doi:10.1093/ndt/16.2.355
2 Aubert O, Bories MC, Suberbielle C, et al. Risk of antibody-mediated rejection in kidney transplant recipients with anti HLA-C donor specific antibodies. Am J Transplant 2014;14:1439-1445.
Based on the latest observations, I think that pre-transplant evaluation of HLA-C DSA level is reasonable, and if the recipient has high MFI of HLA-C DSA, then it would be better to avoid HLA-C mismatch. Otherwise, if there is no alternative allograft offer, we should intensify the immune suppression considering the higher risk of ABMR.
yes for presensitized candidates and by using the SAB assay with lower cutoff values pf PRA > 10 , based on few studies from single centrs shows the presnce of DSA for this specific class can trigger ABMR in the first year after transplantion ,and offcourse we need more studies to determine the effect of HLA-C ab on graft outcome .
The direct effect of HLA-C mismatch on kidney allograft outcome is still controversial (1). HLA- C was once believed to be of minor immunological importance in the field of kidney transplantation. However, there is a rising interest in its immunological significance due to several observations (1).
A study including 2260 deceased-donor kidney transplant recipients has documented a negative allograft outcome in association with one or both HLA-C antigens mismatch in recipients with panel reactive antibodies (PRA) more than 10%. Surprisingly, the same study showed no significant immunological impact of HLA-C mismatch in recipients who are not sensitized (2).
Another retrospective single-centre study has documented that a high pretransplant HLA-C DSA level was associated with an increased risk of developing ABMR later on while low levels were of minor significance (mean MFI values was 4966 in recipients who developed ABMR versus MFI of 981 those who did not have ABMR) (3).
2) Tran TH, Döhler B, Heinold A, et al. Deleterious impact of mismatching for human leukocyte antigen-C in presensitized recipients of kidney transplants. Transplantation. 2011;92(4):419.
3) Aubert O, Bories MC, Suberbielle C, et al. Risk of antibody-mediated rejection in kidney transplant recipients with anti-HLA-C donor-specific antibodies. Am J Transplant. 2014 Jun;14(6):1439-45.
Presence of HLA-A,B,DR DSA has been proven to increase the risk if acute and/or chronic antibody mediated rejection after transplantation and important in determining graft survival. The association HLA C and anti-DP has not been taken in serious in graft survival. Studies have been done to investigate the role of other HLA in determining graft rejection in fully matched recipients.
Frohn et al showed that HLA-A/-B mismatch was strongly associated with HLA-C mismatch as a result of linkage disequilibrium. HLA -B mismatch was weakly correlated with rejection probability. Univariate analysis showed HLA-C had is strongly influenced on graft survival. It also showed that B cell mismatches when happened together with HLA-C is associated with independent factor in acute rejection.
Albert et al showed increased incidence of AMR in patient with anti-HLA DSA. Moreover the study showed presence of DSA at day 0 in anti-C group more likely to experience AMR. So,the presence of pretransplantation HLA-C DSA appeared to have higher risk of AMR.
References:
1)Tran Th, Dohler B, Heinold A, t al. Deleterious impact of mismatching for human leukocyte antigen C inpresensitized recipients of kidney transplants. Transplantation 2011;92:419.
2) Aubert O, Bories MC, Suberbielle C, et al. Risk of antibody-mediated rejection in kidney transplant recipients with anti HLA-C donor specific antibodies. Am J Transplant 2014;14:1439-1445.
the question what sets HLA-C apart from HLA-A and -B? .
The difference may well be related to the reduced levels of cell-surface expression of HLA-C as compared to HLA-A and -B, possibly due to, among other factors, a limited peptide-binding capacity.
This limited peptide-binding capacity may retain HLA-C in the ER and enhance degradation of the HLA-C protein.
Once degraded, HLA-C-derived peptides can be presented to the immune system via other HLA alleles and are thus available for indirect recognition.
Indeed, such HLA-C-derived peptides have previously been eluted from other HLA alleles.
HLA-C mismatches are clearly associated to alloreactivity after hematopoietic stem-cell transplantation; in a number of large cohorts, HLA-C mismatches are correlated to an increased risk of acute graft-versus-host disease (GVHD) or even impaired survival. While for HLA-A and -B, both antigenic as well as allelic mismatches are associated with an increased risk of acute GVHD, such an increased risk is only observed for antigenic HLA-C mismatches and not for allelic mismatches.
Although HLA-C Ags frequency is lower than HLA-A, & -B loci, but it was found that the presence of pre transplantation anti-HLA-C Abs carry a high risk of AMR, so pre transplant screening is essential & changes in immunosuppression is required for positive cases.
Abuzeineh M., Ziadeh A., Kheradmand T., et al. A curious case of de novo anti-HLA-C antibody-mediated humoral rejection and Fabry-like zebra bodies a renal transplant recipient. Clinical Nephrology- Case Studies. 2020, 8: 12-16.
Aubert O., Bories M., Suberbielle C., et al.Risk of Antibody-Mediated Rejection in Kidney Transplant Recipients With Anti-HLA-C Donor-Specific Antibodies. American Journal of Transplantation. 4014, 14: 1439-1445.
In kidney transplantation, only HLA-A, HLA-B, and HLA-DR antigens are routinely used to identify degree of matching between a donor and recipient, whereas HLA-C and other HLA classes are usually discarded. The introduction of the SAB method allowed, with high sensitivity, the detection of HLA to various classes. This improvement in diagnostic techniques led to the improvement in the identification of DSA and detection of new antibodies not discovered before.
The impact of HLA-C compatibility on renal allograft survival has been investigated in several studies.
Studies on the effect of HLA-C antibodies showed controversial results, as 40-50% of kidney transplant recipients may have preformed anti-HLA-C antibodies. Some studies that addressed the influence of HLA-C mismatches found that mismatching for one or both HLA-C antigens, in recipients who were pretransplant sensitized, was associated with decline in graft survival. Patients with high levels of pretransplant antibodies against HLA-C could be at higher risk to develop ABMR during the first posttransplant year.
Some studies have also suggested that HLA-C antibodies could be associated with acute rejection, while others showed only minimal correlation with graft damage. The use of SAB techniques has allowed better identification of anti-HLA-C DSA and eventually studying their effect on allograft outcome.
For the current clinical practice, HLA-A, HLA-B and HLA-DR antigens are still considered for the basic typing and matching, however in the setting on total match and acurt rejection, anti-HLA-C antibodies and others should be investigated.
References:
1. Tran TH, Döhler B, Heinold A, et al. Deleterious impact of mismatching for human leukocyte antigen-C in presensitized recipients of kidney transplants. Transplantation 2011; 92:419.
2. Ling M, Marfo K, Masiakos P, et al. Pretransplant anti-HLA-Cw and anti-HLA-DP antibodies in sensitized patients. Hum Immunol 2012; 73:879.
3. Chapman JR, Taylor C, Ting A, Morris PJ. Hyperacute rejection of a renal allograft in the presence of anti-HLA-Cw5 antibody. Transplantation 1986; 42:91.
4. Bachelet T, Couzi L, Guidicelli G, et al. Anti-Cw donor-specific alloantibodies can lead to positive flow cytometry crossmatch and irreversible acute antibody-mediated rejection. Am J Transplant 2011; 11:1543.
5. Aubert O, Bories MC, Suberbielle C, et al. Risk of antibody-mediated rejection in kidney transplant recipients with anti-HLA-C donor-specific antibodies. Am J Transplant 2014; 14:1439.
Forty to 50 percent of kidney transplant recipients may have performed anti-HLA-C antibodies that is lower than anti HLA-A or HLA-B. However, the effect of HLA-C antibodies on allograft outcome is controversial. A few reports have suggested that the presence of anti-HLA-C antibodies indicates a deleterious effect caused by severe AMR, while others have suggested that it indicates a low rate of acute rejection.
One study demonstrated, patients with high levels of pretransplant HLA-C DSA may be more likely to develop ABMR during the first posttransplant year.
Another study found that mismatching for one or both HLA-C antigens was associated with decreased graft survival among those who were presensitized (panel reactive antibodies [PRA] >10 percent).
Because of a low expression on renal endothelial cells, HLA-Cw and -DP have been considered to be less immunogenic that other HLA antigens. Recent studies have reported that anti–HLA-Cw and anti–HLA-DP DSA could be associated with an increased risk of acute and chronic antibody-mediated rejection (AMR). Studies suggest that preformed anti–HLA-Cw and anti–HLA-DP DSA are as deleterious as anti–HLA A/B/DR/DQ DSA. It justifies their inclusion in kidney allocation programs and in immunological risk stratification algorithms.
Aubert O, Bories MC, Suberbielle C, et al. Risk of antibody-mediated rejection in kidney transplant recipients with anti-HLA-C donor-specific antibodies. Am J Transplant 2014; 14:1439.
Bachelet T, Martinez C, Del Bello A, Couzi L, Kejji S, Guidicelli G, Lepreux S, Visentin J, Congy-Jolivet N, Rostaing L, Deleterious Impact of Donor-Specific Anti-HLA Antibodies Toward HLA-Cw and HLA-DP in Kidney Transplantation. Transplantation. 2016 Jan;100(1):159-66.
Studies researching the effect of HLA-C mismatching are few. HLA-C matching was found to be an option to decrease probability of acute rejection in the first study by Frohn et al ,2001.Mismatching of one or both HLA-C antigens was found to be associated with lower graft survival in sensitized recipients having deceased -donors .Preformed anti -HLA-C antibodies are generally found in 40-50% of kidney transplant recipients.in spite of this, the effect of that mismatch on allograft outcome is debatable. Some studies suggested association between HLA -C ABs and acute rejection and others found relation to graft loss. SAB assays allowed better HLA-c detection and easier study of HLA-C mismatch. Hence ,it was suggested that antibody mediated rejection is more likely to develop in recipients having high levels of pretransplant HLA-C DSA .
RREFERENCES:
Frohn,Cet al : The effect of HLA-C matching on acute renal transplant rejection, Nephrol Dial Transplant ,2001,Feb,16(2):355-60
Tran,TH,et al :Deleterious impact of mismatching for human leucocyte antigen-c in presensitized recipients of kidney transplants, Transplantion,2011;92(4):419
Ling,M,et al,Pretransplant anti-HLA-Cw and anti-HLA-DPantibodies in sensitized patients,Hum Immunol,2012,Sep;73(9):879-83
Bryan,CF et al, Sharing kidneys across donor-service area boundaries with sensitized candidated can be influenced by HLA C, Clin Transplant,2010,Jan24(1);56-61
Gilbert,Met al, Impact of pretransplant human leucocyte a:1439-45ntigen -c and dp antibodies on kidney graft outcome,Transplant Proc.2011 nov;43(9)3412-4
Aubert,O et al: Risk of antibody -mediated rejection in kidney transplant recipients with anti-HLA-C donor -specific antibodies,2014:Am J Transplant Jun;14(6)
Based on evidence from the large transplant registries which consistently confirm the strong association between HLA matching at the level of HLA-A, -B, DR, DQ loci and both patient, graft survival, however few studies reported that HLA-C miss-match could trigger ABMR with increasing risk of graft failure in presensitized recipients with PRA 10 % and above .
In one single center retrospective study of 608 transplant patients where assessed for DSA by SAB assay with MFI cut vale of 500 and above , they found that 26% of them developed DSA , and another 4% had HLA-C-AB at day0 of the transplantation and upon one year FU they found that 27% had ABMR with significantly higher MFI level= 4966 during the Fu period .
references
up to date , kideny transplataion in adults : HLA matching and outcome .
RELATIVE IMPORTANCE OF HLA-C 40to 50 %of renal Tx recipients may have preformed anti-HLA-C antibodies . The effect of these antibodies on allograft outcome is controversial. Some studies have suggested that they are associated with low levels of acute rejection, while others have shown increased antibody-mediated rejection (AMR) The availability of single-antigen bead assessment of anti-HLA antibodies may add some clarity. In a retrospective, single-center study, 608 transplant patients were assessed for donor-specific antibodies (DSAs) as defined by a mean fluorescent intensity (MFI) of >500 [33]. Of these, 160 (26 percent) had DSA, and 25 (4 percent) had anti-HLA-C antibodies. Among 22 patients who were available for follow-up, the incidence of AMR in the first year was 27 percent in those with anti-HLA-C antibodies.
reference HLA matching and graft survival in kidney transplantation . UpToDate ( accessed on 28/11/2021)
The transplantation barrier is defined by the HLA genes that are responsible for tissue histocompatibility.1-7 Mismatching for HLA-C allotypes between patients and unrelated donors generally leads to very high risks of acute graft-versus-host disease (GVHD) and mortality after hematopoietic cell transplantation, although risks to individual patients may vary.3-7 The success of transplantation for a given patient may depend on the unique features of the HLA-C mismatch itself. Three different models of HLA-C mismatching shed light on the variability of individual risks. Mismatching can occur between allotypes that elicit an antibody (serologic) response (antigen mismatches) or between allotypes that differ for limited nucleotide sequence variation (allele mismatches). The similarity of sequence features between allele mismatches may contribute to their lower immunogenicity.3-7
A second model of HLA-C alloreactivity entails mismatching for amino acid residues that determine the repertoire of peptides presented to T cells. Patient-donor differences at several
residues of the class I molecule might significantly affect the immunogenicity of HLA-C mismatches, and of these residues, residue 116 in the F pocket of the peptide binding groove has a high frequency of patient-donor mismatching and consistently shows an effect on transplant outcome.8-10 HLA-C-mismatched patients who are residue 116 mismatched have higher risks of acute GVHD and mortality than HLA-C-matched patients,10-12 observations that support a critical role for T-cell recognition of class I-peptide complexes.13,14 Most recently, a third model has been proposed in which transplant outcome may depend on the regulation of donor natural killer (NK) cell responses against patient cells.15 Amino acid substitutions at HLA-C residues 77 and 80 define 2 mutually exclusive groups of ligands, each recognized by different killer immunoglobulin-like receptors (KIRs). HLA-C-mismatched patients who are residues 77/80 mismatched may have different transplant outcomes than HLA-C-mismatched patients who are residues 77/80 matched.15-20
Each of the 3 mismatch models suggests that some HLA-C mismatches are less risky than others and therefore represent mismatches that could be considered when matched donors are not available.21 The high overall risks associated with transplantation of HLA-C-mismatched unrelated donors have led some clinicians to abandon the use of such donors altogether. Clinical practice is heterogeneous because the features that define permissive HLA-C mismatches remain ill defined.
Recently, the range of expression across HLA-C allotypes has been elucidated.22 Each serologically defined HLA-C allotype has a characteristic median fluorescence intensity (MFI) of cell surface expression that is reproducible in both healthy and HIV-infected cells in vitro.22 The MFI coefficient is superior to any other marker of expression level, including the previously described single nucleotide polymorphism that resides 35 kb upstream of HLA-C,23 because the MFI provides direct allotype-specific measurement of HLA-C surface expression. Expected levels of HLA-C cell surface expression based on the sum of the 2 allelic MFI coefficients was shown to predict observed HLA-C expression levels among individuals in 2 cohorts, indicating that MFI coefficients can be assigned to each HLA-C allotype in lieu of direct ascertainment of expression.22 Thus, the clinical importance of HLA-C expression can be determined in large-scale retrospective outcome studies where appropriate materials for measuring HLA-C expression directly are not available. Using this approach, higher MFI levels were shown to correlate with better control of HIV viral load and slower progression to HIV-AIDS across ethnic groups, but with increased susceptibility to Crohn disease,22 solidifying the role for HLA-C expression levels in modulating the strength of immune responses. Accordingly, we applied the MFI as a quantitative proxy of HLA-C expression level (simply termed as expression level throughout the manuscript) to assess the clinical significance of the level of HLA-C expression in an exceptionally large international population of patients and unrelated transplant donors whose only HLA mismatch was a single HLA-C allotype.
References:
* Apps R, Qi Y, Carlson JM, et al. Influence of HLA-C expression level on HIV control., Science, 2013, vol. 340 6128(pg. 87-91).
* Fernandez-Viña MA, Wang T, Lee SJ, et al. Identification of a permissible HLA mismatch in hematopoietic stem cell transplantation., Blood, 2014, vol. 123 8(pg. 1270-1278).
* Pidala J, Lee SJ, Ahn KW, et al. Non-permissive -DPB1 mismatch among otherwise HLA-matched donor-recipient pairs results in increased overall mortality after myeloablative unrelated allogeneic hematopoietic cell transplantation for hematologic malignancies., Blood, 2014.
Nearly, all studies have examined outcomes vis-a-vis matching or mismatching of HLA-A, -B, and -DRB1-encoded antigens. However, at the time of this writing, there are limited data on matching at other HLA loci (HLA-C, -DRB3, -DRB4, -DRB5, -DQA, -DQB, -DPA, and -DPB). Frohn et al. investigated the impact of HLA-C mismatches on rejection in 104 renal transplants (20). They controlled for HLA-B mismatch to eliminate linkage disequilibrium as a confounding factor. They found that patients with one or two mismatches for an HLA-C antigen had a significantly higher incidence of rejection compared to those with no HLA-C mismatch (54 and 100 vs. 0%) but only when there was also one HLA-B mismatch. Patients with one HLA-B and two HLA-C mismatches also had decreased graft survival that approached statistical significance (P = 0.055). In an early study of data from the Southeastern Organ Procurement Foundation on 12,050 first deceased donor transplants, no effect of matching for HLA-DQ was found when other factors affecting outcome were taken into account (21). In contrast, in a recent study, Lim et al. found DQ mismatching to incur a significantly increased risk of rejection that was further increased in the presence of DR mismatches (22). Rosenberg et al. (23) found that DP mismatches in patients matched for DR and DQ did not impact graft survival or function. Similarly, data from the Collaborative Transplant Study found no deleterious effect of DP mismatches in first deceased donor transplants but did find significantly reduced graft survival in regraft patients (24). The widespread adoption of DNA-based typing methods has facilitated typing for HLA-DQ and -DP. However, unlike other HLA molecules that have one polypeptide chain that is invariant or has limited variability, both polypeptide chains, the α chain encoded by DQA and DPA and the β chain encoded by DQB and DPB, are polymorphic. Both chains may have immunogenic epitopes and there are epitopes comprised of particular combinations of α and β chains (25, 26). Consequentially, studies that consider matching only for DQB and DPB may incorrectly identify a mismatch as a match between a donor and recipient.
References:
* Broeders N, Racapé J, Hamade A, Massart A, Hoang AD, Mikhalski D, et al. A new HLA allocation procedure of kidneys from deceased donors in the current era of immunosuppression. Transplant Proc (2015) 47:267–74.10.1016/j.transproceed.2014.12.018 .
* Al-Otaibi T, Gheith O, Mosaad A, Nampoory MR, Halim M, Said T, et al. Human leukocyte antigen-DR mismatched pediatric renal transplant: patient and graft outcome with different kidney donor sources. Exp Clin Transplant (2015) 13(Suppl 1):117–23.
Professor Ahmed Halawa
Admin
3 years ago
Dear All This article is directly related to assignment 1. I invite you to read assignment 1. Try to train yourself in writing in your own words. When we allow assignment submission, there is a plagiarism software will be checking if you copied and pasted from other sources.
Akram Abdullah
3 years ago
The HLA system includes complex genes located on chromosome 6. The system is highly polymorphic. HLA classification: ~HLA class I ( present on all nucleated cells of the body ) > 3 majors ( A , B , c ) and 3 minors ( E , F , G)
~HLA class II ( present on the surface of antigen-presenting cells )> 3 major ( DQ , DR , DP) and 2 minor > ( DM , DO).
HLA typing techniques.
Serology technique w is done by known antibodies against specific HLA antigens. Cellular assay in which we use lymphocytes to detect HLA class2 Molecular techniques To detect alleles sequence at DNA level Several methods: 1)SSP :specific primer complementary to specific allele using PCR 2)SSOP :a specific prob used to pair with a specific genetic allele 3)SBT :it detect Exons 4)RSCA 5)NGS (Next Generation Sequencing): which detects both entrons and exons. 6)Short Tandem Repeat. Split antigens: splits mean division of a single antigen into subtypes; an antigen that has a more specific cell surface reaction relative to a broad antigen (Example: HLA-B5 Broad antigen splits to HLA-B51 and HLA-B52 ) HLA matching has a direct effect on graft loss. The effects of HLA-DR mismatches are the most important in the first 6 months after transplantation, the HLA-B effect appears in the first 2 years, and HLA-A mismatches have an unfavorable effect on long-term graft survival. Other types of antibodies that are directed against non-HLA antigens are important like against graft endothelium, MICA and Angiotensin type 1 receptor Techniques of tissue typing HLA typing has been done by two methods: -serologic method using antiserum and mixed lymphocyte culture (MLC): can be made by antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC) -DNA-based HLA typing methods using molecular techniques: DNA based method had more sensitivity, accuracy, and resolving power than serologic typing methods, Sequencing-based typing (SBT) is a high-resolution method for the identification of HLA polymorphisms.
Ahmed Fouad Omar
3 years ago
HLA antigens classification Generally talking The HLA system includes a complex arrangements of genes located on chromosome number 6. The system is highly polymorphic MHC class I proteins present on all nucleated cells of the body: There are 3 major genes: HLA-A,HLA-B and HLA-C and 3 minor genes are HLA-E, HLA-F and HLA-G MHC class II proteins typically present on the surface of antigen-presenting cells: There are 3 major genes :HLA-DP , HLA-DQ and HLA-DR and 2 minor genes :HLA-DM, HLA DO Split antigens: splits means division of a single antigen into subtypes; an antigen that has a more specific cell surface reaction relative to a broad antigen (Example: HLA-B5 Broad antigen splits to HLA-B51 and HLA-B52 ) HLA matching has a direct effect on graft loss. The effects of HLA-DR mismatches are the most important in the first 6 months after transplantation, the HLA-B effect appears in the first 2 years, and HLA-A mismatches have a unfavourable effect on long-term graft survival. Other types of antibodies that directed against non-HLA antigens are important like against graft endothelium, MICA and Angiotensin type 1 receptor Techniques of tissue typing
HLA typing have been done by two methods: -serologic method using antiserum and mixed lymphocyte culture (MLC): can be made by antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC) -DNA-based HLA typing methods using molecular techniques: DNA based method had more sensitivity, accuracy and resolving power than serologic typing methods, Sequencing-based typing (SBT) is a high-resolution method for the identification of HLA polymorphisms.
Mohammed Sobair
3 years ago
HLA System:
Play important role in early recognition of foreign antigen and presenting it to T.cell
which lead to initiation of rejection.
HLA gene present in short arm 6 chromosome which include class one two and three.
Class 1 formed of :
HLA-A, HLA-B and HLA-C, HLA E, HLA-G and HLA-G presents in
nucleated cells.
CLASS 2 composed of :
HLA-DR, HLA-DP and HLA-DQ.HLA –DP HLA-DM and HLA-DO
presented in antigen presenting cells.
HLA mismatch is presence of HLA antigen present in donor but not in the recipient which
increase chance of sensitization and graft loss. Collaboration transplant study show 20%
increase success rate for people with HLA-A, HLA-B AND HLA-DR no mismatch
Compare to those with mismatch.
Now in kidney transplant HLA system most commonly used and compared is HLA-A
,HLA-B and HLA-DR.
Most of comparable studies was examine effect of HLA –A, HLA-B and HLA-DR their
effect on graft survival and outcome , but recent studies show other HLA loci (HLA-C,
DRB3, -DRB4, -DRB5, -DQA, -DQB, -DPA, and -DPB),found her rate of rejection in
patient with mismatch at this loci compare with no mismatch group. Other effect of graft
mismatch is also increase risk of cancer and post-transplant lymphoproliferative
disorder.
What is meant by split antigen:
new technique can identity. Up to 9allele variants.
HLA typing:
Many methods used for HLA typing from old immunological method to recent DNA test
based on polymerase reaction
CR-sequence specific primers (SSP):
In this test PCR primer made to identify HLA allele or group of identical ones, in a way
that t the polymorphism to be identified s located at the 3′ end of the primer.
DNA obtained from a blood sample and amplified by PCR using these primers.
When both primers to bind to the DNA, then amplification occurs and can be detected by
electrophoresis. Its rapid test and can be used when rapid typing is needed as in
deceased donors.
PCR-sequence-specific oligonucleotide (SSO):
IT used asset of primer that recognize specific HLA locus, it’s good to type of large
numbers of samples.
Real-time PCR, Sequencing-Based Typing (SBT):
Typing of real time PCR use of allele- specific PCR similar to SSP typing.
No electrophoresis is used but amplification is identified in real time with fluorescent or
probes.
Sequence-based typing (SBT):
Sequencing and amplification used florescent labeled DNA.
Next-generation sequencing (NGS):
next generation sequencing is common used PCR
test now due to high accuracy.
HLA Matchmaker:
Is recent computer based technology allow more precise detection of eplets, it has better
sensitivity of detection antibodies than other test. (7)
Mohamed Essmat
3 years ago
Types of HLA antigens · Major histocompatibility molecules (MHC) · MHC Class 1, present on all nucleated cells There are 3 major and 3 minor MHC class I genes in HLA: HLA-A, HLA-B and HLA-C; minor genes are HLA-E, HLA-F and HLA-G ·MHC class11 have five loci: HLA-DR, -DQ, -DP, -DM. ·Minor histocompatibility antigens refer to non-MHC antigens trigger rejection, like MHC class 1 related chain (MCIA) antigen its polymorphic and development of anti MCIA AB found to be associated with worse graft survival. · ABO blood group antigens. · Endothelial cell antigens. split antigen The new HLA nomenclature begins with HLA- and the locus name, then number of digits specifying the allele. The first two digits specify a group of alleles, with the new immunogenetic technique they can identify the allele variants up to 9 digits. HLA typing and its influence on organ transplantation: Matching of donor and recipient for MHC antigens showed a significant positive effect on graft acceptance. HLA mismatches may occur at antigenic or allelic level; a better HLA match is associated with significantly better patient and graft survival while HLA mismatches have been significantly associated with increased risk of acute rejection, HLA DR mis-match are associated with higher risk of rejection and poor graft survival especially in the first 6 months post transplantation while the HLA-B mismatch effect emerges in the first 2 years, and HLA-A mismatches have a deleterious effect on long-term graft survival. Techniques of tissue typing ·Serological assay, CDCXM: Used for screening the donor anti HLA-AB in recipients, complement-dependent cytotoxicity (CDCXM for T cells and B cells ·Limitation with this assay is less sensitive for low level of DSA and non-complement HLA AB so can give false negative results due to Non HLA or non complement dependent antibodies, low level of antibodies as well , also false positive results due to inaccurate incubation or dilution methods and in the presence of auto anti bodies Also false positive B-cell CDC-XM following treatment with rituximab. · Antihuman globulin or addition of DTT increases the sensitivity of CDC-XM by excluding auto- immunoglobulin M FC-XM: · Flow cytometry cross match is Sensitive for low titer antibody, detects non-complement binding antibodies and non HLA binding ones . Drawbacks include exclusion patients unnecessarily , falsely positive in patients having previously received monoclonal antibodies like rituximab. ·DNA based typing methods: · more sensitive and accurate compared to serologic assay. · Solid phase immunoassay (SPI), by using enzyme-linked immunosorbent assays (ELISA) ·microbeads, flow cytometry, flow PRA and flow analyzer Luminex with bead base assay (SAB)., CELL based assay for T CELLS, B CELLS (Luminex-SAB) are more sensitive than ELISA by 10%. ·Virtual crossmatch using single antigen beads:
Its more sensitive help in detection of acceptable and unacceptable
Donor antigens
May be performed with stored sera therefore shortening cold
ischemia time
Improves donor allocation for highly sensitized patients, DD allocation program and shorting the waiting list
Improves risk assessment for rejection
Limitation of virtual-FCXM
a false positive result, in the presence of denatured human leucocyte antigens on the beads
Requires more coordination between immunology lab
personnel and transplant team.
Abdullah Raoof
3 years ago
Different types transplantation antigens present like
· themajor histocompatibility molecules,
· minor histocompatibility antigens,
· ABO blood group antigens and
· Endothelialendothelial cell antige
AB produced against these ag has important role in graft rejection .
The sensitization to MHC antigens may be caused by
· transfusions,
· pregnancy, or
· failed previous grafts
leading to development of anti-human leukocyte antigen (HLA) antibodies that are important factor responsible for graft rejection in solid organ transplantation
The HLA system includes a complex array of genes located on chromosome number 6 and their molecular products that are involved in immune regulation and cellular differentiation.
Human leukocyte antigen (HLA) molecules are expressed on almost all nucleated cells, and they are the major molecules that initiate graft rejection.
There are three classical loci at HLA class I:
HLA-A, -B, and -Cw, and
Five loci at class II:
HLA-DR, -DQ, -DP, -DM, and -DO.
The system is highly polymorphic .
The contribution of the allelic diversity of class I and II genes to immune recognition
and alloreactivity can be analyzed by serological
methods and molecular methods at the DNA level by different
methods like
· sequence specific primer (SSP) and
· oligotyping with locus- and
· allele-specific oligonucleotide probes (SSOP) .
HLA class I and II matching is important in organ transplantation especially in kidney and bone marrow transplantation.
Classification
MHC class I proteins form a functional receptor on most nucleated cells of the body.
There are 3 major and 3 minor
MHC class I genes in HLA: HLA-A,HLA-B and HLA-C;
minor genes are HLA-E, HLA-F and HLA-G .
β2- microglobulin binds with major and minor gene subunits to produce a heterodimer.
There are 3 major and 2 minor MHC class II proteins encoded by the HLA. The genes of the class II combine to form heterodimeric (αβ) protein receptors that are typically expressed on the surface of antigen-presenting cells.
Major MHC class II HLA-DP:
α-chain encoded by HLA-DPA1 locus and
β-chain encoded by HLA-DPB1 locus.
HLA-DQ:
α-chain encoded by HLADQA1 locus and
β-chain encoded by HLA-DQB1 locus.
The last one is HLA-DR:
α-chain encoded by HLA-DRA
locus and four β-chains (only 3 possible per person), encoded by HLA-DRB1, DRB3, DRB4, and DRB5 loci.
The other MHC class II proteins, DM and DO, are used in the
internal processing of antigens, loading the antigenic peptides generated from pathogens onto the HLA molecules of antigen-presenting cell.
Variability
the HLA molecules manifested high structural polymorphism.
HLA presents the highest degree of polymorphism of all
human genetic systems.
One mechanism of this polymorphism is due to gene conversion between variable alleles and loci within each HLA gene .
HLA typing and its influence on organ transplantation.
The selection of the optimal donor is based on high resolution HLA typing. The MHC (Major Histocompatibility Complex) contains more than 200 genes which are situated on the short arm of chromosome 6 at 6p21.3
The role of HLA molecules is to present peptides to T cells
(both CD4 and CD8 T cells), enabling them to recognizeand eliminate “foreign” particles and also to prevent the
recognition of “self” as foreign.
HLA mismatches may occur
at antigenic or allelic level; the first are characterized by
amino acid substitutions in both peptide binding and T-cell recognition regions, whereas the latter are characterized by
amino-acid substitution in the peptide binding regions only.
When a human transplant is performed, HLA (human
leukocyte antigens) molecules from a donor are recognized
by the recipient’s immune system by direct and indirect methods of allorecognition triggering an alloimmune response.
Matching of donor and recipient for MHC antigens
showed a significant positive effect on graft acceptance .
In organ transplantation, the adaptive immunity is the main
response exerted to the transplanted tissue, since the main
target of the immune response is the MHC molecules
expressed on the surface of donor cells.
In transplantation immunology, the major impact in graft loss comes from the effects of HLA-B and –DR antigens .
The effects of HLA-DR mismatches are the most important in the first 6 months after transplantation, the HLA-B effect emerges in the first 2 years,
and HLA-A mismatches have a deleterious effect on long-term graft survival .
Techniques of tissue typing
Previously, HLA typing was done by two methods:
1. Serologic method using antiserum and
2. mixed lymphocyte culture (MLC).
But nowadays a more precise DNA-based
HLA typing methods using molecular techniques, is used
· specific oligonucleotide probe hybridization (SSOP),
· sequence-specific primer amplification (SSP),
· sequencing-based typing (SBT), and
· reference strand-based conformation analysis (RSCA).
Ahmed mehlis
3 years ago
●Classify HLA antigens
HLA classified into 3 classes.Class1 includes 3major antigens
(HlA.A
HLA,B
HlA,C .
3minor(E,F,G).
classs 2 include 3 major(DR,DP,DQ) and 2minor (DM,DO).
class 3 .
●What is meant by split antigen?
less specific antigen and may include 2 or more splite antigen
Split antigen is more specific antigen and more reliable in matching than broad antigen.
●How can HLA mismatch influence the outcome of transplantation?
Increased risk of hyperacute/acute rejection class II A/B more than others .
●summery:-
Expansion the field of organ and tissue transplantationhas accelerated amazingly, since the human major histo-
compatibility complex (MHC) was discovered in 1967
The HLA system includes a complex array of genes located on chromosome number 6There are three
classical loci at HLA class I: HLA-A, -B, and -Cw, and five
loci at class II: HLA-DR, -DQ, -DP, -DM, and -DO. The
system is highly polymorphic
●Nomenclature:-
1. serological recognition.
2.Modern HLA nomen-
clature are begin with HLA- and the locus name, then *
and even number of digits specifying the allele
3.Every two years, a nomencla-
ture is put forth to aid researchers in interpreting serotypes
to alleles
●variability:-
HlA system is polymorphic
●HLA typing and its influence on organ transplantation:-
MHC contains more than 200 genes in the short arm of chromosome no 6 .
@●Techniques of tissue typing:-
HLA typing was done by two methods:
1: Cellular method .
Mainly class II ,more sensitive depend on molecular method by dna
2: serologic:
ADCC ..it is antibody mediated .
CDC…complement mediated .
AMAL Anan
3 years ago
Classify HLA antigen:
*HLA class I > 3 majors ( A , B , c ) and 3 minors ( E , F , G)
* HLA class II > 3 major ( DQ , DR , DP) and 2 minor > ( DM , DO).
* HLA class III not related to kidney transplantation.
~ Split antigen: cell surface antigens, an antigen that has a more refined or specific cell surface reaction relative to a broad antigen.(Example: HLA-B51 and HLA-B52 are split antigens of HLA-B5).
* Benefits of HLA matching in organ transplantation:
-better organ function.
– long graft and patient survival.
– decrease risk of sensitisation.
Data from the Collaborative Transplant Study showed that with or without cyclosporine use, the renal transplant success rate was 20% higher when there was no mismatch of HLA-B and -DR than when there was a mismatch.
*Similarly, data from the United Network for Organ Sharing showed that long-term graft survival of deceased donor renal transplants with no HLA-A, -B, and -DR mismatch was nearly 20% better than for fully mismatched grafts with a stepwise reduction in survival with each increased degree of mismatch.
Although there had been reports of the role of HLA-A and/or -B mismatch, it eventually became apparent that of the HLA antigens tested routinely, HLA-DR matching contributed the most to graft survival and function. This is of particular importance since there are fewer antigens encoded by the DRB1 locus than by either the A or B loci making it easier to find zero DR mismatches compared to zero A or B mismatches, particularly when dealing with a very HLA heterogeneous population as in the United States.
~ Increasing numbers of HLA mismatch in renal transplantation were associated with an increased need for antirejection therapy that might account for an increased incidence of death with functioning graft due to infection and cardiovascular disease . It was also shown that among 9,209 pediatric kidney transplants, HLA-A, -B, and -DR mismatches were a risk factor for 5-year graft survival, but two DR mismatches appeared to incur an increased risk for non-Hodgkins lymphoma . There is a differential cost associated with different degrees of HLA match. Schnitzler et al. looked at Medicare payment information in the United States Renal Data System (USRDS) according to the degree of HLA mismatch.
HLA classification:
~HLA class I > 3 majors ( A , B , c ) and 3 minors ( E , F , G)
~HLA class II > 3 major ( DQ , DR , DP) and 2 minor > ( DM , DO).
HLA typing techniques.
Serology technique w is done by known antibodies against specific HLA antigens w extracted from multipara women
Cellular assay in which we use lymphocytes to dectect HLA class2
Molecular techniques
To dectect alleles sequence at DNA level
Several methods:
1)SSP :specific primer complementary to specific allele using PCR
2)SSOP :a specific prob used to pair with specific genetic allele
3)SBT :it detect Exons
4)RSCA
5)NGS (Next Generation Sequencing): which detect both entrons and exons.
6)Short Tandem Repeat.
Ahmed Omran
3 years ago
HLA Antigens Classification :
I,II and (III, not involved in immunogenic process of transplantation)
-HLA I :
3 major and 3 minor. The major are A,B and C. The minor are E and F.HLA -G B2 microglobulin binds to mjor and minor gene subunits producing a heterodimer.
HLA class II;
3 major and 2 minor .The major are DP,DQ and DR.Gene encoding includes HLA-DRB1,3,4 AND 5.Class II genes combine to form aB heterodimer expressed on antigen presenting cells. The minor include DM and DO .They are involved in pathogen related antigenic peptides loading on HLA molecules of antigen presenting cells.
Split HLA antigens :more antigen specificity became possible with improvement of serological methods. Single antigen specificity could be divided into different serological specificities H95)LA -B5,HLA -B51 and HLA-B52.Amodel of writing :HLA-B51(5).
-Influence of HLA mismatch on outcome of kidney transplantation:
Good matching of MHC antigens between both donor and recipients has positive effect on accepting kidney offer. HLA mismatches badly affect long term graft survival. The main influence causing graft loss results from HLA-B and DR antigens.The impact of DR are significant in the first 6 months following Tx. Generally, HLA-B develops in the first 2 years following kidney Tx.
HLA typing techniques:
Serotyping
implementing complement -dependent microlymphocytotoxicity method using multiple small wells in microtiter plate .Lymphocytes to be typed are added to different wells ;each containing certain antiserum with antigen specificity, then incubation and complement which its cascade activation leads to cytotoxicity .Determination of dead cells is done by addition of a vital dye.
DNA Typing:
using standardized probes ,primers or sequencing
DNA probes hyberdize to specific
complementary DNA nucleotide sequence
Mlecular-based HLA typing detect greater degree of polymorphism of single HLA more than serologic based tests
REFERENCES
Mahdi,bm: Aglow of HLA typing in organ transplantation, Clinical and translational medicine ,2013,26
Deshpnde, A:The human leucocytic antigen system simplified,2017Global J Transfusion Med,Nov,2:77-88
Ramy Elshahat
3 years ago
Classify HLA antigens
HLA classified into 3 classes.
Class1 includes 3major antigens (A,B,C)and 3minor(E,F,G)
classs 2 include 3 major(DR,DP,DQ) and 2minor (DM,DO)
class 3 is not important in kidney Transplantation
What is meant by split antigen?
Broad antigen term related to less specific antigen and may include 2 or more splite antigen
Splite antigen is more specific antigen and more reliable in matching than broad antigen.
How can HLA mismatch influence the outcome of transplantation?
UNOS registry showed
1HLA mismatch associated with 16% 10 years graft loss in relation to 64% in 6mismatch.
Hla class 2 (DR)mismatch associated with increase risk of 1y acute rejection.class 1B mismatch associated with increase risk of 1st 2y acute rejection.
Class 2(DR,DQ) mismatch associated with increase risk of post transplant denovo DSA and sensitization
Long term outcomes like PTLPD, PTDM, fracture risk, sensitization, death with functioning graft increase with HLA mismatch
Class1 c matching have linkage to to B.HLA class 1 c matching alone was neglected in studies but when associated with b mismatch it associated with short graft survival.
In your own words, summarise the HLA typing techniques.
Serology technique w is done by known antibodies against specific HLA antigens w extracted from multipara women
Cellular assay in which we use lymphocytes to dectect HLA class2
Molecular techniques
To dectect alleles sequence at DNA level
Includes
1)SSP (Sequence Specific Primer): specific primer complementary to specific allele using PCR
2)SSOP(sequence specific oligonucleotide prob):a specific prob used to pair with specific genetic allele
3)SBT (Sequence based typing):it detect Exons
4)RSCA (Reference Strand based Conformation Analysis)
5)NGS (Next Generation Sequencing): which detect both entrons and exonsSTR
6)Short Tandem Repeat.
Asmaa Khudhur
3 years ago
There are three classical loci at HLA class I: HLA-A, -B, and -Cw, and five loci at class II: HLA-DR, -DQ, -DP, -DM, and -DO.
There are 3 major and 3 minor MHC class I genes in HLA: HLA-A,HLA-B and HLA-C;
minor genes are HLA-E, HLA-F and HLA-G].
There are 3 major and 2 minor MHC class II proteins encoded by the HLA.
Major MHC class II HLA-DP: . HLA-DQ: The last one is HLA-DR:
. The other MHC class II proteins, DM and DO, are used in the internal processing of antigens, loading the antigenic peptides generated from pathogens onto the HLA molecules of antigen presenting cell
Split antigen:
Modern HLA nomen- clature are begin with HLA- and the locus name, then * and even number of digits specifying the allele. The first two digits specify a group of alleles.
The third through fourth digits specify a synonymous allele. Digits five through six denote any synonymous mutations within the coding frame of the gene. The seventh and eighth digits distinguish mutations outside the coding region.
Impact of HLA typing on transplantation outcome
HLA mismatches may occur at antigenic or allelic level; a better HLA match is associated with significantly better patient and graft survival while HLA mismatches have been significantly associated with increased risk of acute rejection, HLA DR mis-match associated with higher risk of rejection and poor graft survival especially in the first 6 months post transplantation
While the HLA-B mismatch effect emerges in the first 2 years, and HLA-A mismatches have a deleterious effect on long-term graft survival.
Classify HLA antigens What is meant by split antigen? How can HLA mismatch influence the outcome of transplantation? In your own words, summarize the HLA typing techniques
1-Types of antigens in relation to organ transplantation
· Major histocompatibility molecules (MHC)
· MHC Class 1, all nucleated cells There are 3 major and 3 minor MHC class I genes in HLA: HLA-A, HLA-B and HLA-C; minor genes are HLA-E, HLA-F and HLA-G
·MHC class11 have five loci: HLA-DR, -DQ, -DP, -DM.
Both MHC antigens class1 ,11 is Highly polymorphic with allelic diversity and HLA matching for the MHC molecules by using serological assay and molecular genetic assay is important in sold organ transplantation including kidney and bone marrow transplantation.
·Minor histocompatibility antigens refer to non-MHC antigens trigger rejection, like MHC class 1 related chain (MCIA) antigen its polymorphic and development of anti MCIA AB found to be associated with worse graft survival.
· ABO blood group antigens.
· Endothelial cell antigens.
2- What is meant by split antigen?
The new HLA nomenclature are begin with HLA- and the locus name, then number of digits specifying the allele. The first two digits specify a group of alleles, with the new immunogenetic technique they can identify the allele variants up to 9 digits.
HLA typing and its influence on organ transplantation:
Matching of donor and recipient for MHC antigens showed a significant positive effect on graft acceptance. HLA mismatches may occur at antigenic or allelic level; a better HLA match is associated with significantly better patient and graft survival while HLA mismatches have been significantly associated with increased risk of acute rejection, HLA DR mis-match associated with higher risk of rejection and poor graft survival especially in the first 6 months post transplantation
While the HLA-B mismatch effect emerges in the first 2 years, and HLA-A mismatches have a deleterious effect on long-term graft survival.
HLA-DR mismatches were a significant risk factor for posttransplant non-Hodgkin lymphoma and hip fracture. Sensitization of the recipient at the B-cell level and development of IgG antibodies still represent a major problem in kidney transplantation. Techniques of tissue typing
·Serological assay, CDCXM:
Used for screening the donor anti HLA-AB in recipients, complement-dependent cytotoxicity (CDCXM for T cells and B cells
·Limitation with this assay is less sensitive for low level of DSA and non-complement HLA AB so can give false negative results, also false positive results due to inaccurate incubation or dilution methods and in the presence of auto anti bodies Also false positive B-cell CDC-XM following treatment with rituximab.
· Antihuman globulin CDC-XM Increased sensitivity over the CDC-XM by detects auto- immunoglobulin M
·DNA based typing methods:
· more sensitive and accurate compared to serologic assay.
· Solid phase immunoassay (SPI), by using enzyme-linked immunosorbent assays (ELISA)
·microbeads, flow cytometry, flow PRA and flow analyzer Luminex with bead base assay (SAB)., CELL based assay for T CELLS, B CELLS (Luminex-SAB) are more sensitive than ELISA by 10%. Advantage of FCXM:
· Flow cytometry crossmatch Sensitive for low titer antibody, detects non-complement binding antibodies
·Both T and B cells can be separated to detect HLA class1,11 specific antibodies
Limitation of FCXM
May exclude patients unnecessarily
Specificity for human leucocyte antigen antibodies is low
May be falsely positive in patients having previously received monoclonal antibodies like rituximab.
·Virtual crossmatch using single antigen beads:
Its more sensitive help in detection of acceptable and unacceptable
Donor antigens
May be performed with stored sera therefore shortening cold
ischemia time
Improves donor allocation for highly sensitized patients, DD allocation program and shorting the waiting list
Improves risk assessment for rejection
Limitation of virtual-FCXM
a false positive result, in the presence of denatured human leucocyte antigens on the beads
Requires more coordination between immunology lab
personnel and transplant team.
CONCLUSION:
The new advances in the DNA molecular immunogenetic crossmatch techniques and human leukocyte antigen (HLA) typing play a crucial role to ensure better organ allocation and provide the recipient with a more favorable match, shortening the waiting list and that improve both the graft and patient survival and allowed for the appropriate use of immunosuppressive therapy with lesser side effects of infection and cancer.
Reem Younis
3 years ago
-HLA system located on the short arm of chromosome 6 at 6p21.
–HLA is classified into 3 major and 3 minor classes:
1. Major HLA class I genes are HLA-A, HLA-B, HLA-C, and minor genes are HLA-E, HLA-F, and HLA-G.
2. Major HLA class II genes are HLA-DP, HLA-DQ, HLA-DR, and minor genes HLA- DM, and HLA-DO.
3. MHC class III is a group of proteins belonging to the class of MHC. They are poorly defined structurally and functionally. It contains many genes for different signaling molecules such as tumor necrosis factor and heat shock protein.
– Split antigen is an antigen that has a more refined or specific cell surface reaction relative to broad antigen. It subgroup (Splits) of broad antigen .examples:
B5 Broad (split B51 and B 52)
B12 Broad (Split B44 and B45)
B15 Broad (Split B62 B63 and B75)
-Split antigens matching give a good transplant outcome
– HLA matching showed a significant positive effect on graft acceptance while HLA mismatch has a critical prognostic effect that affects graft and recipient survival. It is associated with increased risks of overall graft failure, death–censored graft failure, and all-cause mortality. The major graft loss comes from HLA-DR and HLA-B.HLA-DR mismatches are important in the first 6 months after transplantation while the HLA-B effect appears in the first 2 years and HLA –A mismatches have a deleterious effect on long–term graft survival. Techniques of HLA typing:
-Previously, HLA typing was done by serologic method or mixed lymphocyte culture.
-More advanced molecular techniques were developed – DNA –based methods-such as :
1-Sequence-Specific oligonucleotide probe hybridization (SSOP)
2- Sequence-Specific primer amplification (SSP)
3-sequencing-based typing (SBT)
4-Reference strand-based conformation analysis (RSCA).
-In 2013 a new project of 16IHIW demonstrated the benefit of a new project of Next-generation sequencing(NGS).
Ben Lomatayo
3 years ago
HLA antigens are Class I HLA ; Major HLA-A, HLA-B, HLA-C and Minor HLA-E, HLA-F, HLA-G (5) . Class II HLA ; Major HLA-DR, HLA-DP, HLA-DQ and Minor HLA-DO,HLA-DM
Split antigen ; This refined antigen or antigen with specific cell surface reaction relative to broad antigen. e.g. HLA-B51, HLA-B52 are split antigens of HLA-B5
HLA mismatches may take place at the level of antigen or allele. HLA matches between recipient’s and donor’s means better graft outcome(12). The major impact on graft survival comes from the effects of HLA-B and HLA-DR antigens. The effects of HLA-DR mismatch is seen early during the first 6 months, while that of HLA-B appears later in the first two years(16). HLA mismatches of the above antigens leads to allorecognition, stimulation of the immune system and graft loss.
Effects of HLA-C is debatable. The presence of high levels of anti-HLA-C Ab is not innocent and can be associated with adverse graft outcome. HLA-C become more significant in haematopoietic stem cell transplantation.
Hinda Hassan
3 years ago
Classify HLA antigen?
Class I has 3 major genes :A,B and C and 3 minor :E, F and G. Class II has 3 major protins :DP,DQ and DR, which are encoded at multiple loci.minor protin of class II are DM and DO What is meant by split antigen?
in the testing of cell surface antigens, an antigen that has a more refined or specific cell surface reaction relative to a broad antigen eg B51,B52 are split of B5
How can HLA mismatch influence the outcome of transplantation?
HLA molecules present the peptides toT cells so as to differentiate self from non self . the foreign HLA from the doner are recognized as non self by the recipient ending with graft rejection .so in transplant we look for mismatch.this mismatch can occur due to amino acid substitution at the level of antigens or alleles. Mismatch at Dr affect the first 6 month , mismatch at B affect the first 2 years while A mismatch affect the long term survival
In your own words, summarise the HLA typing techniques
1. Serology: with antiserum and mixed lymphocyte culture , antibody-dependent cell-mediated cytotoxicity (ADCC),or complement-dependent cytotoxicity (CDC).
2. Molecular (more sensitive) : assequence-;specific oligonucleotide probe hybridization (SSOP), sequence-specific primer amplification (SSP), sequencing-based typing (SBT), and reference strand-based conformation analysis (RSCA), nextgeneration sequencing (NGS), short tandem repeat (STR) genotyping
Antibodies screening through complement-dependent cytotoxicity (CDC), is important to avoid hyper acute rejection. This was replaced recently with solid-phase immunoassays (SPI):ELISA and bead-based technology (flow cytometry: Flow PRA and Flow Analyzer-Luminex).
Ban Mezher
3 years ago
Split antigen defined as an antigen has more different specific cell surface reaction relative to broad antigen.
The studies proved that any increase in HLA mismatch is strongly associated with very high risk of graft loss, death censored graft failure & all cause mortality.
HLA system is a complex genes located on chromosome 6. It is present in all nucleated cells. HLA classified into class I ( including HLA-A, -B & – Cw) & class II ( including HLA-DR, -DQ, & -DP). Assessment of HLA class I&II matching is very important in transplantation especially in renal & BM transplant.
MHC classified into class I & II. MHC I gene classified into major ( HLA-A, -B, & -C) & minor ( HLA-E, -F & -G) & MHC II also classified into major ( HLA-DR, -DQ & -DP) & minor (HLA-DM & -DO).
The function of HLA is to present an Ag to T cells( CD4 & CD8) to determined & elimination of foreign Ag.
HLA tissue typing done by :
serological methods:
(a) Ab dependent cell-related cytotoxicity.
(b) complement -dependent cytotoxicity .
2 DNA based HLA typing:
(a) sequence -specific oligonucleotide probe hybridization ( SSOP)
(b) Sequence- specific primer amplification ( SSP)
(c) Sequence-based typing ( SBT)
(d) Reference strand based confirmation (RSCA)
(e) Next generation sequencing (NGS)
(f) Short Tandem Repeat (STR).
After typing of HLA , the anti- HLA Abs calculation by CDC, flow cytometry, or Luminex.
Professor Ahmed Halawa
Admin
3 years ago
Dear All Thank you for your answers. HLA C role is highly controversial. HLA C mismatch could play a role on the following conditions:
Double mismatch
High MFI of HLA C DSA (more than 5000)
Retransplant
Bone marrow transplantation.
Mahmoud Hamada
3 years ago
– MHC class I gene has 3 major and 3 minor genes.
– MHC class II has 3 major and 2 minor genes
– HLA CLASS I : A,B,C – then minor E,F ,and G
– HLA class II: DR,DQ.DP
– HLA has the highest polymorphism, hence cross matching has been a mandatory step in pre-transplantation workup
– There are different methods for tissue typing mainly serological (ADCC OR CDC) and mixed lymphocyte culture, with the latter more accurate.
Tahani Hadi
3 years ago
HLA class I present in all nuclear cells and consist of 3 classical loci HLA-A ,HLA -B and HLA-C and 3 minor genes E,F and G while HLA class II present mainly in Ag presenting cells like macrophages and dendritic cells it consists of 3 major and 2 minor loci HLA-DP,DQ AND DR and the minor are DM and DO .
HLA match prior to transplant give better result and long term graft survival the most important Ags that play role in transplant outcome are HLA -DR have high risk of graft failure in the first 6 months in mismatch while HLA -B mismatch effect till 2 years and HLA -A contributes in long term outcomes.
HLA -C mismatch cause acute rejection and alot of cases have show it’s effect on graft survival.
Fatima AlTaher
3 years ago
1- HLA Ag are classified as
a- HLA class I:
Major classes : HLA-A, B,C
Minor classes :HLA E, F, G
b- HLA class II:
Major classes are HLA DR, DQ, DP
Minor classes are DM, DO
2- Split Ag is an antigen that has a more refined or specific cell surface reaction relative to a broad antigen
3- The type and extent of HLA mismatch affects the gragt outcome .poor outcome occurs with mismatch in Dr ( mainly affect short term graft survival )then B and A (maily affect long term graft survival) Also the higher degree of HLA mismatch , the worse graft outcome
As recipient T cells will recognize the mismatched HLA molecule on the graft via direct , indirect and semidierct alloreactivation pathways with subsequent activation of B lymphocytes and production of Ab , enhancing recrtiutment of inflammatory cells and production of various cytokines and chemokines ending in graft injury and rejection . Summary
HLA system is the most biologic system in human being .Itis controlled by genes present on chromosome 6 and is classified into HLA class I including 3 major (HLA A,B,C)|and 3 minor (HLA E,F,G)|and class II including 3 major (HLA DR, DQ,DP) and 2 minor (DM ,DO). HLA system presents the Ags to both CD4 and CD8 T cells to recognize and eliminate forign antiges , they also important for inducing self tolerance.
HLA typing is done by two methods either
1- serological method by mixing recipient serum with donor lymphocytes , but it is less accurate technique , including
a- antibody-dependent cell-mediated cytotoxicity (ADCC),
b- complement-dependent cytotoxicity (CDC)
2- molecular method : a DNA-based technique such as
· SSOP :specific oligonucleotide probe hybridization
· SSP: sequence-specific primer amplification
· SBT : sequencing-based typing
· RSCA :reference strand-based conformation analysis (RSCA)
3- Newer techniques as : nextgeneration sequencing (NGS ) and short tandem repeat (STR) genotyping.
For antibodies screening : either
a- Serological method as CDC crossmatch
b- solid-phase immunoassays (SPI) as ELIZA and bead-based technology such as flow cytometry:
Flow PRA and Flow Analyzer-Luminex).It is better to combie two techniques for better confirmation of of the results .
Nasrin Esfandiar
3 years ago
In a study in UK formation of anti-HLA Abs was studied in candidate patients for retransplantation and its relationship with each donor’s HLA locus mismatches was investigated. Mismatches of all HLAs were effective in forming anti-HLA Abs with different hazard ratios. The order of them was as following: HLA DRB3/4/5→3.9, HLA DRB1→3.5, HLA B→3.4, HLA A→3.2, HLA-DQ→3 and HLA-C→2.5. So, mismatches of HLA-C was associated with the lowest antibody formation.
Kosmoliaptsis, V., Gjorgjimajkoska, O., Sharples, L. D., Chaudhry, A. N., Chatzizacharias, N., Peacock, S., Torpey, N., Bolton, E. M., Taylor, C. J., & Bradley, J. A. (2014). Impact of donor mismatches at individual HLA-A, -B, -C, -DR, and -DQ loci on the development of HLA-specific antibodies in patients listed for repeat renal transplantation. Kidney International, 86(5).
Wessam Moustafa
3 years ago
MHC class I is classified into 3 major and 3 minor genes
Major genes are A,B,C
Minor genes are E,F ,G
MHC class II is classified into 3 major and 2 minor genes
Major are DR DP DQ
Minor genes are DM ,DO
Split Ag is an antigen that has a more refined or specific cell surface reaction relative to a broad antigen.
HLA mismatch triggers immune response as B and T cells recognise foreign donor antigens leading to graft loss .
B and DR mismatch is more important than rest of MHC antigens
DR mismatch results in deleterious effects in 1st 6 months
B mismatch effects appear more than 2years post transplant .
A mismatches affect long term graft outcome.
**Methods of HLA typing **
1) Serological methods is done with either ADCC , CDC
2) cellular assays are done using mixed lymphocyte culture , which is more sensitive than serological tests
That’s because minor differences can be detected by cells more than Abs
3) molecular assays
Done using sequence specific oligonucleotide or sequence specific primer
4) Sequencing-based typing (SBT) is a high-resolution
method for the identification of HLA polymorphisms
Abdulrahman Ishag
3 years ago
1- HLA classification ; There are 3 major MHC class I genes in HLA: HLA-A HLA-B HLA-C There are3 minor MHC class 1 genes in HLA ; HLA-E HLA-F HLA-G. β2- microglobulin binds with major and minor gene subunits to produce a heterodimer. There are 3 major and2 minor MHC class II proteins encoded by the HLA. The genes of the class II combine to form heterodimeric (αβ) protein receptors that are typically expressed on the surface of antigen-presenting cells Major MHC class II HLA-DP: α-chain encoded by HLA-DPA1 locus and β-chain encoded by HLA-DPB1 locus. HLA-DQ: α-chain encoded by HLADQA1 locus and β-chain encoded by HLA-DQB1 locus. Minor MHC class II; DM DO 2- Split antigen; Antigen that has more refined or specific cell surface reaction relatives to broad antigen ,example; HLA-B51and HLA-B52 are split antigen of HLA b5. 3- Influence of HLA mismatch on transplant outcome;
HLA mismatch adversely affect kidney transplant out come and it depends on the degree of mismatch and the gene locus .It is associated with increased incidence of malignancy ,death with censored functioning graft. In transplantation immunology, the major impact in graft loss comes from the effects of HLA-B and -DR antigens .HLA –C was found to be associated with decreased graft survival in sensitized recipient. The effects of HLA-DR mismatches are the most important in the first 6 months after transplantation, the HLA-B effect emerges in the first 2 years, and HLA-A mismatches have a deleterious effect on long-term graft survival . 4- HLA typing techniques; 1- Serological method; using antiserum and mixed lymphocyte culture (MLC). can be made by antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC) 2- Cellular assay; In which lymphocyte culture is used to determine the HLA class II types .It is more sensitive in detection minor HLA disparities which can trigger T-cells. 2- DNA-based HLA typing methods using molecular techniques such as; 1- sequence -specific oligonucleotide probe hybridization(SSOP) 2- sequence-specific primer amplification (SSP) 3- sequencing-based typing (SBT) 4-reference strand-basedconformation analysis (RSCA). DNA based method had more sensitivity, accuracy and resolving power than Sequencing-based typing (SBT) method for the identification of HLA polymorphisms. 3- Next generation sequencing (NGS) . Which combines clonal amplification. 4- short tandem repeat (STR) genotyping provided additional information allowing determination of the extent of HLA identity in families where HLA haplotype inheritance was ambiguous, due to extensive homozygosity or shared parental haplotypes. The HLA STR assay is a reliable and rapid test that used inexpensively screen potential sibling donors for HLA identity .
Mohamed Fouad
3 years ago
HLA antigens classification
Generally talking The HLA system includes a complex arrangements of genes located on chromosome number 6. The system is highly polymorphic
MHC class I proteins present on all nucleated cells of the body: There are 3 major genes: HLA-A,HLA-B and HLA-C and 3 minor genes are HLA-E, HLA-F and HLA-G
MHC class II proteins typically present on the surface of antigen-presenting cells: There are 3 major genes :HLA-DP , HLA-DQ and HLA-DR and 2 minor genes :HLA-DM, HLA DO
Split antigens:splits means division of a single antigen into subtypes; an antigen that has a more specific cell surface reaction relative to a broad antigen (Example: HLA-B5 Broad antigen splits to HLA-B51 and HLA-B52 )
HLA matching has a direct effect on graft loss. The effects of HLA-DR mismatches are the most important in the first 6 months after transplantation, the HLA-B effect appears in the first 2 years, and HLA-A mismatches have a unfavourable effect on long-term graft survival.
Other types of antibodies that directed against non-HLA antigens are important like against graft endothelium, MICA and Angiotensin type 1 receptor
Techniques of tissue typing
HLA typing have been done by two methods: -serologic method using antiserum and mixed lymphocyte culture (MLC): can be made by antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC)
-DNA-based HLA typing methods using molecular techniques: DNA based method had more sensitivity, accuracy and resolving power than serologic typing methods, Sequencing-based typing (SBT) is a high-resolution method for the identification of HLA polymorphisms.
Assafi Mohammed
3 years ago
Classification of HLA antigens
HLA (human leucocyte antigen) are encoded for by genes on chromosome 6 :
Class I antigens (HLA-A, B, C) are expressed on all nucleated cells.
class II antigens HLA-DR, DP, DQ) are expressed on antigen presenting cells like macrophages and dendritic cells.
Vascular endothelial cells of the transplant graft express both these antigens, hence antibody responses to them are of importance in predicting acute antibody mediated rejection.
T cells express only class I antigens while B cells express both.
MHC class I proteins form a functional receptor on most nucleated cells of the body.
There are 3 major and 3 minor MHC class I genes in HLA:
3 major genes are : HLA-A,HLA-B and HLA-C.
3 minor genes are HLA-E, HLA-F and HLA-G
β2- microglobulin binds with major and minor gene subunits to produce a heterodimer.
The genes of the class II combine to form heterodimeric (αβ) protein receptors that are typically expressed on the surface of antigen-presenting cells.
There are 3 major and 2 minor MHC class II proteins encoded by the HLA:
Major MHC class II HLA-DP: α-chain encoded by HLA-DPA1 locus and β-chain encoded by HLA-DPB1 locus.
Major MHC class II HLA-DQ: α-chain encoded by HLA- DQA1 locus and β-chain encoded by HLA-DQB1 locus.
Major MHC class II HLA-DR: α-chain encoded by HLA-DRA locus and four β-chains (only 3 possible per person).
The minor MHC class II proteins, DM and DO, are used in the internal processing of antigens, loading the antigenic peptides generated from pathogens onto the HLA molecules of antigen-presenting cell.
Split Antigen: Ag that has a more refined or specific cell surface reaction relative to abroad antigen. In the testing of cell surface antigens, an antigen that has a more refined or specific cell surface reaction relative to a broad antigen.(Example: HLA-B51 and HLA-B52 are split antigens of HLA-B5)
How can HLA mismatch influence the outcome of transplantation:
In a meta-analysis to evaluate the magnitude effect of HLA mismatching on post-transplant survival outcomes of adult kidney transplantation. The analysis included 23 studies with a large sample of subjects (totally 486,608 recipients). The results indicated that each incremental increase of HLA mismatches was significantly associated with higher risks of overall graft failure, death-censored graft failure and all-cause mortality. The pooled results also indicated that HLA-DR mismatches were significantly associated with a 12% higher risk of overall graft failure. Also observed that HLA-A per mismatch was associated with a 6% higher risk of overall graft failure, but the association was insignificant. There was no significant association between HLA-B mismatching and graft survival. All included studies were in high methodological quality and the heterogeneity between studies was acceptable in each pooling analysis.{What is the impact of human leukocyte antigen mismatching on graft survival and mortality in renal transplantation? A meta-analysis of 23 cohort studies involving 486,608 recipients Xinmiao Shi, Jicheng Lv, Wenke Han, Xuhui Zhong, Xinfang Xie, Baige Su & Jie DingBMC Nephrology volume19, Article number: 116 (2018)}
Primary outcome overall graft failure
HLA per mismatch and overall graft failure Eleven studies (289,987 adult recipients) reported data on HLA mismatching and overall graft failure. The pooled analysis revealed that each incremental increase of HLA-mismatches was significant associated with a higher risk of overall graft failure.
HLA-DR mismatches and overall graft failure Eight studies with 152,105 adult recipients were analyzed to investigate the association between HLA-DR mismatching and overall graft failure.The pooled results revealed an unadjusted HR of 1.44 (95% CI: 0.86–2.41; P = 0.160) with moderate heterogeneity (I2 = 70.0%). After adjustment, each incremental increase of HLA-DR mismatches was significant associated with 12% higher risk of overall graft failure.
HLA-B mismatches and overall graft failure Associations of HLA-B epitope and overall graft failure were reported in 4 studies with 146,019 recipients. The pooled analysis demonstrated that each incremental increase of HLA-B mismatches was not associated with higher risk of overall graft failure (HR: 1.01; 95% CI: 0.90–1.15; P = 0.834; with moderate heterogeneity (I2 = 66.0%). Sensitivity analysis with a fixed-effects model obtained similar effect estimates (HR: 1.01; 95% CI: 0.89–1.14; P = 0.079). In addition, the effect estimates did not changed significantly after stratification for sample size (≥10,000 vs < 10,000) of cohorts.
HLA-A mismatches and overall graft failure Only 3 studies (40,000 recipients) reported data on the association of HLA-A epitope and overall graft failure. The results revealed an insignificant association (HR: 1.06; 95% CI: 0.98–1.14; P = 0.121; with no heterogeneity (I2 = 0%). Sensitivity analysis with a random-effects model showed similar results (HR: 1.06; 95% CI: 0.98–1.15; P = 0.121). The results should be cautiously interpreted because of only three studies included.
Secondary outcomes
Death-censored graft failure
Each incremental increase of HLA mismatches was associated with a higher risk of death-censored graft failure, with summary HR of 1.09 (95% CI: 1.06–1.12; P < 0.001; and moderate heterogeneity (I2 = 70.9%).{What is the impact of human leukocyte antigen mismatching on graft survival and mortality in renal transplantation? A meta-analysis of 23 cohort studies involving 486,608 recipients Xinmiao Shi, Jicheng Lv, Wenke Han, Xuhui Zhong, Xinfang Xie, Baige Su & Jie DingBMC Nephrology volume19, Article number: 116 (2018)}
All-cause mortality
Each incremental increase of HLA mismatches was associated with a higher risk of all-cause mortality rates (HR: 1.04; 95% CI: 1.02–1.07; P = 0.001;The heterogeneity was moderate (I2 = 65.3%). Summary estimates did not changed significantly after analyzing with a fixed-effects model (HR: 1.04; 95% CI: 1.02–1.07; P = 0.001). After stratification for sample size of cohorts (≥10,000 vs < 10,000), the effect estimates were not modified (HR: 1.04; 95% CI: 1.02–1.05; P < 0.001; I2 = 27.8%, Additional file 8: Fig. S4). However, the results should be cautiously interpreted due to small number of included studies (n = 4)..{What is the impact of human leukocyte antigen mismatching on graft survival and mortality in renal transplantation? A meta-analysis of 23 cohort studies involving 486,608 recipients Xinmiao Shi, Jicheng Lv, Wenke Han, Xuhui Zhong, Xinfang Xie, Baige Su & Jie DingBMC Nephrology volume19, Article number: 116 (2018)}
HLA typing techniques:
Old techniques: Previously, HLA typing was done by two methods: serologic method using antiserum and mixed lymphocyte culture (MLC).
Modern DNA-based HLA typing methods: using molecular techniques, such as:
next- generation sequencing (NGS) in the HLA laboratory. NGS may resolve the issue through the combination of clonal amplification, which provides phase information, and the ability to sequence larger regions of genes, including introns, without the additional effort or cost associated with current methods.
short tandem repeat (STR) genotyping provided additional information allowing determination of the extent of HLA identity in families where HLA haplotype inheritance was ambiguous, due to extensive homozygosity or shared parental haplotypes. The HLA STR assay is a reliable and rapid test that used inexpensively screen potential sibling donors for HLA identity.
Tissue typing using serological assaycan be made by:
antibody-dependent cell-mediated cytotoxicity (ADCC): In ADCC, the cytotoxic destruction of antibody-coated target cells by host cells is triggered when an antibody bound to the surface of a cell interacts with Fc receptors on NK cells or macrophages. Serologic method was also used in screening for HLA antibodies in the recipient. These antibodies are important because they are reactive with lymphocytes of a prospective donor (cross matching).
complement-dependent cytotoxicity (CDC): Concerning the cellular assay, this is a mixed lymphocyte culture and used to determine the HLA class II types. The cellular assay is more sensitive in detecting HLA differences than serotyping. This is because minor differences unrecognized by alloantisera can stimulate T cells. This typing is designated as Dw types.
The pioneer method to detect such antibodies is complement-dependent cytotoxicity (CDC).it has been gradually replaced by more-sensitive solid-phase immunoassays (SPI) such as the enzyme-linked immunosorbent assay and the bead-based technology (i.e., flow cytometry: Flow PRA and Flow Analyzer-Luminex).
Tissue typing by molecular method utilizing the followings:
sequence-specific oligonucleotide (SSO).
sequence- specific primer (SSP).
Technologies has been in routine use in many tissue-typing laboratories worldwide for more than 20 years since the
development of the poly- merase chain reaction. Both methods(SSO) & (SSP) are very useful for clinical and
research purposes and can provide generic (low resolution) to allelic (high resolution) typing results.
3. DNA based method had more sensitivity, accuracy and resolving power than serologic typing methods.
Sequencing-based typing (SBT) is a high-resolution method for the identification of HLA polymorphisms.
A comprehensive list of recommendations is provided covering the technical and pretransplantation and posttransplantation monitoring of HLA antibodies in solid organ transplantation:
SPI must be used for the detection of pretransplantation HLA antibodies in solid organ transplant recipients.
In particular the use of the single-antigen bead assay to detect antibodies to HLA loci such as Cw, DQA, DPA, and DPB, which are not readily detected by other methods.
The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive crossmatch (XM).
There must be an awareness of the technical factors that can influence the results and their clinical interpretation when using the Luminex bead technology, such as variation in antigen density and the presence of denatured antigen on the beads.
The recommendations are intended to provide state-of-the-art guidance in the use and clinical application of recently developed methods for HLA antibody detection when used in conjunction with traditional methods.
Mahmud Islam
3 years ago
1. Classify HLA antigens
We have 3 classes of HLA antigens.
I, II, and III
1-MHC class I: we have three major
and three minor MHC class I genes in HLA.
**Major MHC class I: HLA-A, HLA-B,
and HLA-C
*Minor MHC class I: HLA-E, HLA-G,
and HLA-F
2-MHC class II: We have three major
and two minor MHC class II proteins.
**Major MHC class II: HLA-DP,
HLA-DQ, and HLA-DR (DRB1,2,3&5)
*Minor
MHC class II: HLA-DM and HLA DO
3-Class
3: not essential in transplantation. examples(C2, C4A, C4B, PF, TNF- a, b)
2. What is meant by split antigen?
HLA antigen classifications have
evolved. recent technology has allowed the detection of differences
in antigens “split” categories that were previously indistinguishable as broad
categories. For example A203#, A210# are spits
of the original HLA-A2
Here is a list of original broad
and correlated splits http://hla.alleles.org/antigens/broads_splits.html
3. How can HLA mismatch influence the outcome of transplantation?
HLA match is essential for better
transplant outcomes. Conversely, the mismatch is a risk for rejection. The
degree of mismatch means more need for immunosuppression with high
dose-dependency and accordingly higher risk of infections and malignancy.
In renal & bone marrow
transplantation class I&II are more important while in lung and heart
transplantation the DR is of importance.
4. In your own words, summarise the HLA typing techniques
HLA typing was performed by a serologic method using antiserum and cellular mixed lymphocyte culture (MLC). With advanced technology and revolution in PCR technology molecular techniques evolved.
Serological assays can be made by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)
In cellular technique mixed culture of lymphocytes is utilized. This is used to determine class II HLAs.
Molecular methods which are popular today are useful for high-resolution reporting of HLA antigens. Sequence-specific oligonucleotide (SSO) and sequence-specific primer (SSP) are routine in most laboratories. Here both low resolution and high-resolution HLA typing is possible. Another technique is sequencing-based typing (SBT). İn this high-resolution method the polymorphism is detected.
Last edited 3 years ago by Mahmud Islam
Nasrin Esfandiar
3 years ago
HLA molecules are important in kidney transplantation which are contributing in rejection .HLA class I loci are HLA- A ,HLA-B , HLA -C and five HLA class ll are :HLA -DR ,HLA -DP ,HLA-DQ,HLA-DM and HLA-DO .There are two Nomenclature system for HLA .The first one is based on serology that consist ,letters and numbers. The second modern one contains gene name and maximum 8 digits which show alleles. Two first did it’s are Ag or allele from ,Third and fourth for specific HLA allele, fifth and sixth for silent substitute and seventh and eight digits for non-loading substitution molecules and letters like L,N,Q,S. HLA system has high polymorphism and number of their alleles are increasing. Since HLA molecules present foreign antigens to T cells, their mismatches can be recognized by recipient’s immune system and induce alloimmune reaction .So full matching helps graft acceptance. Matching of HLA-DR is important and then HLA-B especially during the first 6 month and 2 years, respectively. HLA-A mismatches may influence long-term graft survival.
2. HLA typing by serological approach has defined the serological subgroup (or broad). By more precise methods such as DNA typing methods HLA is defined as split antigens. Each old broad antigen may have two or more antigens. Split antigens are important in transplantation because of immune system activation.
3. HLA mismatches are important in graft survival. Number of mismatches between donor and recipient’s HLA for six antigens of HLA-A,-B and –DR are usually considered in kidney transplantation.
4. HLA typing methods:
1-Serological typing by antiserum and mixed lymphocyte culture (MĹC) made by ADCC or CDC.
2- DNA typing methods by molecular methods. Different techniques are using like: SSOP, SSP by PCR SSB1, RSCA. SSO and SSP developed based on PCR and can provide both low and high resolution typing results. But SBT only provide high- resolution results. High-resolution results may provide more information especially in sensitized patients.
3. Next- generation sequencing (NGS) through combination of clonal amplification.
4. Short tandem repeat (STR) genotyping allows identification of the extent of HLA in families with unknown HLA haplotype inheritance used for screening of sibling donors.
5. In HLA matched sibling interaction between patients HLA ligand and donor killer–ell immunoglobulin -like receptor (KlR) genes were important in their outcome. So KIR genotyping could be helpful.
Esmat MD
3 years ago
HLA class I proteins are expressed on surface of all nucleated cells. There are 3 major and 3 minor MHC class I genes in HLA: HLA-A, HLA-B and HLA-C; minor genes are HLA-E, HLA-F and HLA-G.
HLA class II proteins are typically expressed on the surface of antigen presenting cells. There are 3 major and 2 minor MHC class II proteins encoded by the HLA.
Major MHC class II HLA-DP: α-chain encoded by HLA-DPA1 locus and β-chain encoded by HLA-DPB1 locus. HLA-DQ: α-chain encoded by HLADQA1 locus and β-chain encoded by HLA-DQB1 locus. The last one is HLA-DR: α-chain encoded by HLA-DRA locus and four β-chains (only 3 possible per person), encoded by HLA-DRB1, DRB3, DRB4, and DRB5 loci. Other HLA class II proteins, DM and DO, are used for internal processing of antigens that are generated from pathogens.
HLA antigen classification has allowed the detection of differences in antigens (split categories) that were previously indistinguishable (broad categories). For example: A9 Broad (splits A23 and A24)
The role of HLA molecules is to present peptides to T cells to recognize and eliminate non self-antigens.
When a transplantation is performed, HLA molecules of the donor are recognized by the recipient’s immune system and provoke an alloimmune response.
The adaptive response is the main response to the HLA molecules expressed on the surface of donor cells. T cell activation leads to the production of cytokines and chemokines and then recruitment of innate immunity.
HLA mismatch correlates with increased probability of graft loss. The effects of HLA-DR mismatches are the most important in the first 6 months after transplantation, the HLA-B effect emerges in the first 2 years, and HLA-A mismatches have a deleterious effect on long-term graft survival.
In addition, an increasing numbers of HLA mismatches of the first transplant, is associated with a significant increase in panel reactive antibody.
Previously, HLA typing was done by serologic method and MLC. Nowadays more precise DNA-based methods have been developed, such as SSPO, SSP, SBP, and RSCA.
Next generation sequencing (NGS) has ability to sequence larger regions of genes, including introns. short tandem repeat (STR) genotyping provided additional information allowing determination of the extent of HLA identity in families where HLA haplotype inheritance was ambiguous, and provides a reliable test for screening of potential sibling donors for HLA identity.
Serological assay, can be made by antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). The cellular assay is more sensitive in detecting HLA differences than serotyping and the concern about it is a mixed lymphocyte culture and determination of the HLA class II.
Sequence specific primer (SSP) and sequence-specific oligonucleotide (SSO) technologies can provide generic (low resolution) to allelic (high resolution) typing results. Sequencing-based typing (SBT) is a high-resolution method for the identification of HLA polymorphisms. Examination of all nucleotides, both at conserved and polymorphic positions, enables the direct identification of new alleles, which may not be possible with techniques such as SSP and SSO typing.
DNA based methods have more sensitivity, accuracy and resolving power than serologic typing methods.
Utilization of solid phase immunoassays makes it possible to detect antibodies to HLA loci such as Cw, DQA, DPA, and DPB which are not readily detected by other methods. The new techniques have been associated with decreased specificity, and some non-HLA antigens with no clinical relevance have been able to give a positive cross match.
The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive crossmatch
Theepa Mariamutu
3 years ago
Glow in HLA typing in organ transplantation
MHC class 1 has been found in all uncleared cells of the body. It has 3 major and 3 minor MHC. HLA -A,B,C are major and HLA-E,F,G are minor. Beta 2 microglobulin binds with major ot minor gene unit to produce heterodimer.
MHC class 2 encoded by 3 major and 2 minor HLA. They combine to form alpha-beta heterodimers, which typical present on APCs.
Nomenclature
Two systems
1. Serological based recognition : antigens assigned letters and numbers ( HLA B-51/B51)
a. Could not do specify alleles more, stop at 2 digits
2. Modern HLA nomenclature: begin with HLA- ,then locus name
a. 1st , 2nd digits – group of alleles
b. 3rd,4th digits – synonymous allele
c. 5th, 6th digits-any synonymous mutations within the coding frame of the gene
d. 7th , 8th digits- distinguishes the mutations outside the coding frame
e. Letter L,N,Q or S – to specify an expression level or other non-genomic a data known about it
Every 2 years – nomenclature is put forth to aid in interpreting serotypes to alleles
Variability
• HLA- highly polymorphism of all human genetic systems
• Polymorphism due to gene conversion between variable alleles and loci within each HLA gene
• HLA allele’s increase in number at about 15% in the high expression loci( A,B,C and DRB1) and 30% in the low expression loci (DRB3/4/5, DQB1,DPB1)
HLA typing and its influences on organ transplantation
HLA molecules present peptides to T cells ( CD4 and CD8 T cells) to enable recognition and eliminate foreign particles
HLA molecules also prevents the recognition if self as non self
HLA mismatch can occur at level of:
• antigenic
o characterised by amino acid substitutions in both peptide binding and T-cell recognition regions
• allelic
o Characterised by amino-acid substitutions in peptide binding region only
Alloimmunity is triggered when a donors kidney is transplanted via direct or indirect methods
Adaptive immunity is the important response exerted to the “foreign” tissue since the main target of immune response is MHC molecules expressed on the surface of donor cells
Activation of T cell stimulate production of cytokines and chemokines then it may recruit components like NK cell or macrophages or complement ( innate immunity) . Defensins and cathelicidin have chemoattractant properties on T lymphocytes
HLA DR mismatches has impact on graft survival during first 6 months
HLA B mismatches has impact on graft survival for first 2 years while HLA A-mismatches has deleterious effect on long term graft survival
Techniques of tissue typing
Classically HLA typing done by serological method using anti-sera and mixed lymphocyte culture (MLC), but now DNA based HLA typing methods using molecular techniques are more accurate
• Sequence specific primer amplification (SSP)
• Assequence-; specific oligonucleotide probe hybridisation
• Sequencing based typing
• Reference strand based conformation analysis (RSCA)
• Next generation sequencing (NGS)
• Short tandem repeat (STR)
ADCC
• cytotoxic destruction of antibody- when an antibody bound to the surface of a cell interacts with Fc receptors on NK cells or macrophages then coated target cells by host cells is triggered
• Also used in screening for HLA antibodies in the recipient – important because they reactive with lymphocytes of a prospective donor (cross matching)
Cellular assay
• used to determine the HLA class II types
• is more sensitive in detecting HLA differences than serotyping- because minor differences unrecognized by alloantisera can trigger T cells
Molecular method
SSO/SSP
• useful for clinical and research purposes and can provide generic to allelic typing results
• more sensitive, accurate and resolving power
SBT
• identification of HLA polymorphisms using high resolution methods
• most of the HLA Class I alleles can be determined by their exon 2 and 3 sequence and for Class II alleles, exon 2 is usually sufficient
• Examination of all nucleotides(at conserved and polymorphic positions) enables the direct identification of new alleles, which may not be possible with techniques such as SSP and SSO typing
Complement-dependent cytotoxicity (CDC)
• very important to identify recipient anti-HLA antibodies to antigens expressed on donor with blood cells
• now it is replaced by more sensitive solid-phase immunoassays (SPI) such as the enzyme-linked immunosorbent assay and the bead-based technology ( flow cytometry: Flow PRA and Flow Analyzer-Luminex)
• The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive XM
• technical factors such as variation in antigen density and the presence of denatured antigen on the beads can alter the results and clinical interpretation when using the Luminex bead technology
New Techniques are associated with decreased specificity, and some non-HLA antigens with no clinical relevance have been able to give a positive cross match
These “false-positive” antibody results have a consequence of decreasing a chance of the patient to receive KT
Study showed that cold ischemia up to 18 hours was found not to be deleterious for graft outcome, and short cold ischemia did not mask effect of HLA matching and HLA-DR mismatches were a significant risk factor for posttransplant non-Hodgkin lymphoma and hip fracture
Another study found that with increasing numbers of HLA mismatches of the first transplant associated with significant increase in PRA. It also showed influence of HLA matching on graft outcome in patients with pretransplant HLA antibodies or high serum levels of the T-cell activation marker sCD30
Even though graft survival rates improved dramatically , sensitization of the recipient at the B-cell level and development of IgG antibodies still a major problem in kidney transplantation .HLA antibodies of the IgG, IgM, or IgA isotypes were detected in almost all patients waiting for transplant
Other antibodies that directed against non-HLA antigens such as against graft endothelium, MICA and Angiotensin type 1 receptor are increasingly detected.
Shereen Yousef
3 years ago
Classification of HLA classes:
HLA system complex it is encoded by genes located on short arm of chromosome 6.
HLA molecules are the major trigger for immune response against foreign oragan .
They are classified into 2 MHC proteins; Class I, Class II
Class I ; is expressed on surface of all nucleated cell it has 3 major HLA types A, B and C, and 3 minor E, F, and G.
Class II; is expressed only on surface of antigen presenting cells it include 3 major types DR, DP and DQ and 2 minor DM and DO.
Split antigens
Spliting antigens into smaller specificities this spliting of HLA antigen matching improved transplant outcome more thsn matching of with broad HLA antigens.
Old serological methods for HLA typing detected HLA genes with allele group but can’t detect specific alleles so broad HLA antigen has a poor specificity.
Recent DNA based techniques allowed better specification and sequencing of the polymorphic regions of genes and identify specific alleles encoding for specific HLA proteins.
similar broad antigen may have different split antigens, and this minor differences may initiate immune response and cause rejection.
There for matching in split antigens has a better transplantation outcomes than matching on broad antigen.
How can HLA mismatch influence the outcome of transplantation?
Transplantation with no mismatch was associated with the best results and the most favourable outcoms not only on patient and graft survival but also on reduction of immunosuppression with all its complications.
Transplantation between homozygous twins showed excellent results
not all HLA antigens have the same effect as mismatch in HLA -DR AND HLA-B have the main effect of graft survival
The effects of HLA-DR mismatches usually appears in first 6 months after transplantation and they were a significant risk factor for posttransplant non-Hodgkin lymphoma and hip fracture .
while HLA-B effect appears in the first 2 years, and HLA-A mismatches effects appears on long-term graft survival
Number of mismatchs in first transplantation lead to significant increase in panel reactive antibody which will affect the next transplantation.
Therefore selection of matched doner is the first step to successfull long term graft survival.
Despite the major effect of HLA antibodies on outcomes of transplantation but also other non HLA antibodies like against graft endothelium , MICA and Angioten-sin type 1 receptor that can affect the graft such effect can be abolished by peritransplant apheresis .
HLA typing techniques
In the past HLA typing was performed using serological methods
Serological method was performed by using known antisera Antibodies to specific HLA antigens to be mixed with lymphocytes culture it can be ADCC OR complement mediated cell lysis.
Cellular method using mixed lymphocyte culture to determine the HLA class II. it is more sensitive in detecting HLA differences than serotyping as minor differences cant be detected by antisera can stimulate T cells.
molecular techniques useing DNA-based for HLA typing include
sequence-specific oligonucleotide probe hybridization (SSOP)
sequence-specific primer amplification(SSP) sequence-based typing (SBT)
reference strand-based conformation analysis (RSCA)
Next-generation sequencing (NGS).
short tandem repeat (STR) genotyping which is reliable test to screen for sibling doner
molecular method, usinf the sequence-specific oligonucleotide (SSO) and sequence-specific primer (SSP) technologies have been used for many years and it can provid high resolution typing (detect specific alleles) or low resolution (generic typing)
While Sequencing-based typing (SBT) is a high-resolution method
In general DNA typing is more sensitive and specific than serotyping
Mujtaba Zuhair
3 years ago
HLA antigens :
Class I antigens : 3 major (classical) Ag : A,B,C , 3 minor (nonclassical) : E,F,G
Class II Ag : 3 major: DR, DQ, DP, 2 minor : DM , DO .
Split antigen :
With the advance of serological methods of HLA typing , they found certain antigen can be divided into many antigen serologically, so they called the parent antigen broad antigen and the splitted antigens split antigen . Nowadays they put the broad antigen between brackets after the split antigen since matching to the split antigen level is associated with better graft outcome.
The HLA mismatch in kidney transplantation is associated with lower graft and patient survival also associated with increased risk of acute rejection and DGF and the use of high doses of immunosuppressive drugs and higher risk of malignancy and serious infections and higher risk of hip fractures.
HLA DR mismatch is most important in the first 6 months after transplantation, HLA B mismatch in the first 2 years post transplantation and HLA A mismatch in the long term graft survival.
HLA typing techniques :
Serological methods : Specific anti HLA antibody is mixed with the lymphocytes of the person , and if the person had that HLA antigen a reaction will occur .
Cellular method using mixed lymphocyte culture : this method is more sensitive in detecting the precise HLA.
Molecular methods : it detect the HLA gene not the protein . multiple methods are available :
PCR sequence specific oligonucleotide probe typing.
PCR sequence specific primers typing.
Sequence based typing : which allows the typing of all the sequence of axon II in HLA class I and axon II III in HLA class II and it is more precise and can discover new alleles.
Next generation sequencing : has the advantage of shorter time and the ability to classify the allele as null allele ( HLA allele which had no expression proteins )
Sherif Yusuf
3 years ago
HLA-C mismatch was found to be associated with reduced graft survival in only those who are presensitized and not in those who are not presensitized.(1)
40 – 50 % of transplant recipient have preformed anti HLA-C antibodies in the serum, Patients with high level of pretransplant HLA-C DSA are more prone to develop ABMR during first post transplant year.(2)
REFERANCES
1- Tran TH, Döhler B, Heinold A, et al. Deleterious impact of mismatching for human leukocyte antigen-C in presensitized recipients of kidney transplants. Transplantation 2011; 92:419.
2- Ling M, Marfo K, Masiakos P, et al. Pretransplant anti-HLA-Cw and anti-HLA-DP antibodies in sensitized patients. Hum Immunol 2012; 73:879.
Last edited 3 years ago by Sherif Yusuf
Ibrahim Omar
3 years ago
1- HLA antigens :
there are 2 major groups of HLA antigens, each one of them include further types and subtypes :
Class I HLA includes 3 major (A,B,CW) and 3 minor (E,F,G).
Class II HLA includes 3 major (DR,DQ,DP) and 2 minor (DM,DO)
2- Split antigens:
split antigen is an antigen having a more refined cell surface markers relateive to the broad antigen e.g HLA-B51 and HLA-B52 are split antigens of HLA-B5
3- HLA mismatch :
no doubt that HLA mismatch will adversely affect the outcome of renal transplantation, regarding rejection, infection, neoplastic disorders, graft survival and patient survival.
before doing transplantation, HLA antigens matching betwen the donor and recepient should be done as a mandatory test pretransplant and below a certain degree of HLA mismatch, transplantation will be allowed. this certain degree is variable according to the experience of the transplant team and the facilities of the the transplant center.
the major impact in gaft loss due to HLA mismatch, is due to HLA-B and HLA-DR mismatch. the 1st will affect it in the 1st 2 years and the 2nd will affect it in the 1st 6 months post-transplantation.
HLA-A mismatch has an adverse effect on long-term survival.
4- HLA typing techniques : 2 main methods.
A- serological :
using anti-serum and mixed lymphocytic culture.
antigens are assigned letters and numbers. e.g HLA-B51
B- molecular :
using PCR technique.
it is more precise, sensitive and accurate.
multiple procedures are available ;
1- SSOP
2- SSP
3- SBT
4- RSCA
5- STR
Doaa Elwasly
3 years ago
1- HLA classification
MajorHistocompitabilityComplex class I genes :
HLA-A
HLA-B
HLA-C
Minor HistocompitabilityComplex class I genes :
HLA-E
HLA-F
HLA-G
Major HistocompitabilityComplex class II :
HLA-DP: α-chain encoded by HLA-DPA1 locus and β-chain encoded by HLA-DPB1 locus.
HLA-DQ: α-chain encoded by HLA-DQA1 locus and β-chain encoded by HLA-DQB1 locus.
HLA-DR: α-chain encoded by HLA-DRA locus and four β-chains encoded by HLA-DRB1, DRB3, DRB4, and DRB5 loci.
Minor HistocompitabilityComplex class II :
DM
DO
2- Split antigen
means division of a single antigen into subtypes according to previously unrecognized differences. Serological identification can be hard if the splits occurs at small numbers .Histocompatibility Committee has established criteria for noticing which splits match themselves and which match the parent antigen too. They depended on the concluded antigens recognised by participating laboratories that should be available during routine donor testing and molecular typing is used to test donors.United Network for Organ Sharing (UNOS )provides a list of antigens that may be entered through UNet and is updated. For example HLA DR Some splits of DR match other splits and some do not.(1)
3- HLA mismatches have a major effect on transplantation outcome as
HLA-DR mismatches are the most crucial drawback in the first 6 months post transplantation,
while HLA-B mismatches are expressed in the first 2 years,
and HLA-A mismatches have a major effect on long-term graft survival which finaly can lead to graft rejection .
4- HLA typing techniques
Serological assay:
Used to screen for recipient’s HLA antibodies By
-Solid-phase immunoassays (SPI) such as the enzyme-linked immunosorbent assay
-Bead-based technology (i.e., flow cytometry: Flow PRA and Flow Analyzer-Luminex).
Cellular assay:
Used to determine types of HLA class II,it is more sensitive in detection of minor HLA disparities which can trigger T cells.
Molecular methodology(DNA based):
It is more sensitive
By using
-sequence-specific oligonucleotide (SSO)
-sequence specific primer (SSP).
-Sequencing-based typing (SBT):a precise technique in determining HLA polymorphism.
-Reference strand-based conformation analysis (RSCA)
Next generation sequencing (NGS) :
Which combines clonal amplification
Short tandem repeat (STR) genotyping:
is an affordable rapid test that can identify HLA
Classification:
The HLA system is highly polymorphic
There are three major (HLA:A, B, C) & three minor MHC class I gene (HLA-E, F, G)
those genes encode for MHC class I proteins that bind to B2 microglobulin to produce heterodimer and form functional receptor on most nucleated cells of the body
Genes of class II encode for 3 major (HLA-DP, DQ, DR) and 2 minor MHC class II proteins (HLA-DM, DO), combine to form heterodimeric protein receptor expressed on surface of APCs.
Split antigens:
Some HLA types are discovered to be multiple splits (broad antigens split into 2 or more antigen), in some occasions the splits may be coded as the broad antigen (1)
A9 was split to A23 and A24
DR2 was split to DR15 and DR16
HLA matching criteria may vary according to consideration of broad or split antigens
increased utilization of broad antigens increase the probability of identifying an HLA matched recipient for a donor (2)
Influence of HLA mismatch on outcome of kidney transplant:
HLA matching of donor and recipient is associated with better graft acceptance
HLA-B and DR antigens have major effect on graft failure
HLA-DR mismatches are most important in the first 6 months, HLA-B effect appear in the first 2 years and HLA-A mismatches affect the long term graft survival
A study showed increased number of HLA mismatches of first transplant is associated with significant increase of PRA.
HLA compatibility significantly affect outcome of transplant in sensitized (second transplant) recipients even at allele level.
HLA typing techniques Serological assay:
made by antibody dependent cell mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC)
antigen level, low resolution
allow discrimination between groups of related HLA alleles Cellular assay:
more sensitive than serotyping in detecting HLA as it detect minor differences that can stimulate T cells
used to determine HLA class II molecular method:
allow HLA genotyping
Sequence specific oligonucleotide (SSO) use oligonucleotide probes
Sequence specific primer (SSP) use arrays of specific primers
(both provide low level of resolution, don’t discriminate between closely related alleles)
Sequencing based typing (SBT): High resolution typing Next generation sequencing:
characterize entire HLA genes including exons other than 2 and 3
provide high resolution typing useful when recipient has allele specific antibodies (3)
help in better compatibility assessment, organ allocation and graft survival. (4)
(1) Takemoto, S., Port, F.K., Claas, F.H. and Duquesnoy, R.J., 2004. HLA matching for kidney transplantation. Human immunology, 65(12), pp.1489-1505.
(2) Takemoto S, Gjertson DW, Cecka JM, Terasaki PI: HLA matching for local pools using fewer HLA factors. Transpl Proc 27:675, 1995
(3) P. Vogiatzi, Some considerations on the current debate about typing resolution in solid organ transplantation, Transplant. Res. 5 (2016) 3.
(4) Bravo-Egana, Valia, Holly Sanders, and Nilesh Chitnis. “New challenges, new opportunities: Next generation sequencing and its place in the advancement of HLA typing.” Human Immunology (2021).
Dalia Ali
3 years ago
*Classification of HLA
class I HLA
3 major
HLA-A,HLA-B and HLA-C
3 minor
HLA-E, HLA-F and HLA-G
Class II HLA
3 major
HLA-DP, HLA-DQ, HLA-DR
2 minor
DM , DO
*Split antigen
antigens are identified using antisera that split HLA antigens into narrower specificities for example HLA-A9 split into HLA-A23 and -A24, and HLA-A10 split into HLA-A25, -A26, -A34, and -A66. splits HLA antigen matching give good transplant outcome if compare to matching of broad HLA antigens
*HLA typing and its influence on organ transplantation
higher degree of human leukocyte antigen (HLA) mismatching is associated with worse transplant outcomes
The initial Collaborative Transplant Study (CTS) analysis showed that the major impact on kidney allograft outcomes came from mismatches for the HLA-DR and -B antigens, with little effect from the HLA-A antigens
Mismatching for HLA-A, -B, and -DR associated with a higher risk of HLA sensitization in both adult and pediatric patients.
HLA-C antigens was associated with decreased graft survival specially in those who are presensitized (panel reactive antibodies [PRA] >10 percent) but not those who are not presensitized. Forty to 50 percent of kidney transplant recipients may have preformed anti-HLA-C antibodies. patients with high levels of pretransplant HLA-C DSA are at high risk for ABMR during the first posttransplant year
major adverse cardiac and cerebrovascular events and high mortality occur in case of Prior sensitization against HLA class II antigens
HLA-DQ — and -DR
Although HLA-DQ is not as polymorphic as HLA-A, -B, and -DR, HLA-DQ mismatches are also associated with an increased risk of rejection and allograft loss in kidney transplant recipients due to development of de novo DSAs
HLA-DP — Anti-HLA-DP antibodies are less common than antibodies to HLA-DR and -DQ, occurring in 5 to 14 percent of transplant recipients but The titer of these antibodies increases in up to 45 percent patients with history of previous transplantation .
exposure to repeated mismatched HLA antigens may trigger reactivation of memory cells and a rebound in DSA immediately posttransplant, leading to earlier graft injury and loss. repeat mismatches alone in the absence of circulating DSA may not need to be categorically avoided, as this practice may further limit access to transplantation.
HLA mismatching is an independent risk factor for the development of posttransplant non-Hodgkin lymphoma and associated with increased risk of posttransplant bone fractures in kidney transplant recipients.
Regarding pediatric age group renal transportation has significant challenges since these patients are more likely to require repeated transplantation. So well-matched organs are a priority for pediatric patients and this may lead to longer wait times. living-donor pediatric kidney transplantation should be done from donors with fewer than four HLA-A+, -B+, -DR mismatches.
Technique of HLA typing
* serological assay
can be made by antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC)
Serologic method used in screening for HLA antibodies in the recipient. These antibodies are important because they are reactive with lymphocytes of a prospective donor (cross matching)
*cellular assay
used to detect the HLA class II types. It is more sensitive in detecting HLA differences than serotyping because minor differences unrecognized by alloantisera can stimulate T cells.
*Tissue typing by molecular method
Using the sequence-specific oligonucleotide (SSO) and sequence-specific primer (SSP) technologies use in many tissue-typing laboratories worldwide for more than 20 years since the development of the polymerase chain reaction.
*DNA based method had more sensitivity, accurate than serologic typing methods
1- Williams RC, Opelz G, McGarvey CJ, et al. The Risk of Transplant Failure With HLA Mismatch in First Adult Kidney Allografts From Deceased Donors. Transplantation 2016; 100:1094.
2- Shi X, Lv J, Han W, et al. What is the impact of human leukocyte antigen mismatching on graft survival and mortality in renal transplantation? A meta-analysis of 23 cohort studies involving 486,608 recipients. BMC Nephrol 2018; 19:116.
3- Malfait T, Emonds MP, Daniëls L, et al. HLA Class II Antibodies at the Time of Kidney Transplantation and Cardiovascular Outcome: A Retrospective Cohort Study. Transplantation 2020; 104:823.
4- Leeaphorn N, Pena JRA, Thamcharoen N, et al. HLA-DQ Mismatching and Kidney Transplant Outcomes. Clin J Am Soc Nephrol 2018; 13:763.
5-Handbook of Kidney Transplantation, SIXTH EDITION.
Thanks Dalia
Yes, splits HLA antigen matching give good transplant outcome if compare to matching of broad HLA antigens. Well done
Weam Elnazer
3 years ago
HLA class I A, B and C antigens are expressed on almost all nucleated cells and platelets.
HLA class II DR, DQ and DP molecules are expressed on a more restricted range of cells, including B cells, activated T-cells, and the monocyte.
split antigen
The antigens were identified using antisera obtained primarily from multiparous women. As the field evolved, new antisera were discovered that could “split” some HLA antigens into narrower specificities. HLA-A9 was split into HLA-A23 and -A24, and HLA-A10 was split into HLA- A25, -A26, -A34, and -A66
only the degree of HLA matching was found to be a significant predictor of delayed graft function, one-year acute rejection, and 10-year graft survival. Using HLA matching for allocation.
a strong association between HLA matching, particularly at the HLA-A, -B, and -DR loci, and patient and graft outcomes. These HLA antigens have the most polymorphisms.
HLA class II mismatches, which include HLA-DQ and -DR, have been found to be a risk factor for the development of de novo DSAs.
*Classification of HLA antigens:
1/ CLASS I :
present on the surface of all nucleated cells
3 major (HLA-A/B/C )
3 minor (HLA E/F/G )
2/ CLASS ll:
present on the surface of APCs
3 major ( HLA-DR/DP/DQ )
2 minor ( HLA-DM/DO )
*Split antigens :
HLA antigens that has a positive reaction with a reaction related to a broad antigen.
for example : HLA B51 & HLA B52 are split antigens to a broad antigen HLA B5.
*Effect of HLA mismatching on TX outcome:
HLA matching improve the graft survival.
HLA mismatching indicate the use of higher doses of immunosuppressive drugs increasing the risk of infection and malignancy and exposure to side effects of these drugs affecting the outcome or the graft.
HLA DR mismatch affects the graft outcome in the first 6 months post transplant, and increase the risk of post TX non Hodgkin lymphoma and hip fracture,
HLA B mismatch affects the graft outcome in the first 2 years post transplant.
HLA A mismatch effect is on long term graft survival.
HLA mismatching in first TX increase the level of PRA leading to lower chances of further TX.
By adding the recipient and donor lymphocytes to reference sera containing specific antibodies then complement is added after incubation period to initiate the complement mediated cell destruction and determination of HLA type.
2/ DNA based HLA typing method :
More sensitive and accurate than serological technique. Includes several types:
a) Sequence specific oligonucleotide probe ( SSOP )
b) Sequence specific primer amplification ( SSP ) both a and b are widely used in most labs worldwide c) Sequence based typing ( SPT ) d) Reference strand based conformation analysis ( RSCA ) e) Next generation sequencing ( NGS ) f ) Short tandem repeat ( STR ) that can determine the possibility of sibling donors.
Huda Al-Taee
3 years ago
Classify HLA antigens
HLA class I: has 3 major & 3 minor genes
major genes: HLA-A, HLA-B, HLA-C
minor genes: HLA-E, HLA-F, HLA-G
HLA class II: has 3 major & 2 minor genes
major genes: HLA-DR, HLA-DP, HLA-DQ
minor genes: HLA-DM, HLA-DO
What is meant by split antigen?
The discovery of new antigens splits the broad antigens into 2 or more antigens named split antigens, for example A9 is a broad antigen split to A23 & A24 split antigens, DR2 broad antigene split into DR15 & DR16 split antigens.
split matching for HLA-A,B,DR appears to be more clinically important.
How can HLA mismatch influence the outcome of transplantation?
HLA mismatch associated with poor graft and patient survival. the major impact on the graft come from HLA-DR & B mismatching. the effect of HLA-DR mismatching is is important in the first 6 months post transplantation, while HLA-B mismatching appear in the first 2 years. HLA mismatching is associated with increased incidence of rejection and delayed graft function. patients my die from infections and other complications of the high level of immunosuppressive medications required for HLA mismatched transplantation.
So better HLA matching is associated with:
lower doses of immunosuppressive medications.
lower incidence of immunosuppressive medications adverse effects including PTLD.
lower incidence of hip fractures.
lower grade of sensitization which is important for second transplantation.
In your own words, summarise the HLA typing techniques
I.Serological method: the first standard method used for HLA typing.
done by using a reference serum combined with donor or recipient lymphocytes and a complement is added to the combination and by the use of viability dye the detection of cell lysis can be done. this method is quick, inexpensive but lack specificity.
II. Cellular assay: a mixed lymphocyte culture
used to determine HLA class II types,
it is more sensitive than serological method in detecting HLA differences.
III. DNA based molecular method: has more sensitivity, accuracy and resolving power than serological methods.
it includes the followings;
a. sequence specific primers typing( SSP): involves the use of primers designated to recognize a particular HLA allele or group of similar alleles
b. sequence specific oligonucleotide probes typing( SSOP): DNA is amplified using a set of primers that recognize a particular HLA locus.
c. real time PCR based typing( RT-PCR): uses allele specific PCR similar to SSP methods
d. sequence based typing( SBT): based on the direct amplification and sequencing of the relevant exons using fluorescently labeled dideoxynucleotides.
e. next generation sequencing( NGS): enabled high resolution typing with significantly reduced ambiguity.
better split matching for HLA-A, HLA-B & HLA-DR is associated with better graft survival
Sherif Yusuf
3 years ago
Classify HLA antigens
HLA antigens are classified into
A- HLA class I which is present in all nucleated cells which are subdivided into
⦁ Major antigens which include HLA -A, HLA- B, HLA-C
⦁ Minor antigens which include HLA-E, HLA-F and HLA-G
B- HLA class II which is present in APCS (macrophages, denderitic cells, vascular endothelial cells and B cells) they are further subdivided into
⦁ Major antigens which include HLA-DR, HLA-DQ, HLA-DP
⦁ Minor antigens which include HLA-DM, HLA DO
What is meant by split antigen?
HLA antigens are either broad or split antigens
Broad antigens are crude measure while split antigens are specific measure of identity of the cells.
Examples
⦁ A9 is broad splits are A23 and A24
⦁ B70 is broad splits B71 and B72
⦁ DR2 is broad splits DR15 and DR1
Matching for split HLA antigens result in better outcome when compared to matching for broad antigens
How can HLA mismatch influence the outcome of transplantation?
⦁ The degree of mismatch between donor and recipient is determined by HLA-A, HLA-B, HLA-DR each contain 2 antigens , so 6 mismatch means that 6 antigens between donor and recipient are not matching , while zero mismatch means that donor and recipient share same antigens meaning that thy are identical.
⦁ Exposure to mismatched antigens leads to production of DSA and increase the risk of antibody medicated rejection, moreover there is increase in the risk of T cell medicated rejection leading to early graft injury and may be loss.
⦁ Patient survival is lower in patients receiving HLA mismatched kidney when compared to those receiving HLA matching kidney
⦁ It was found that only the degree of HLA matching is a strong predictor for delayed graft function, 1 y acute rejection and 10 y graft survival.
⦁ The impact of specific HLA mismatch on graft survival differ, HLA-DR mismatch is associated with the worst outcome ( which is evident in the first 6 m) then HLA-B (which is evident in the first 2 years post transplant) , followed by HLA-A (which is evident on the long term)
⦁ Patients with 2 HLA-DR mismatches had worse graft survival compared to 0 or 1 HLA-DR mismatch
⦁ transplant recipients with 2 HLA-DR mismatch has higher degree of sensitization so they have lower probability of getting 2nd transplant
⦁ HLA- mismatch between donor and recipient especially DR was found to increase the risk of infection, lymphoproliferative diseases, post transplant diabetes, hypertension, hyperlipidemia and cardiovascular events, bone fractures.
⦁ HLA-C mismatch was found to be associated with reduced graft survival in only those who are presensitized and not in those who are not presensitized, moreover patients with high level of pretransplant HLA-C DSA are more prone to develop ABMR
⦁ HLA-DQ mismatch was associated with the development of de novo DSA
⦁ HLA-DP mismatch is not significant in the first transplant but is correlated with reduced graft survival in second transplant
Summarise the HLA typing techniques
1- Serologic method using either antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)
2- Mixed lymphocyte culture (MLC) used for HLA class II, result interpreted in the form of Dw types, more sensitive than serological method
3- DNA-based HLA typing methods which is more sensitive and accurate than both
previous techniques it includes the following:
⦁ SSOP sequence specific oligonucleotide probe hybridization
Yes, well done. “Matching for split HLA antigens result in better outcome when compared to matching for broad antigens”
Ala Ali
Admin
3 years ago
We are looking for more responses
MICHAEL Farag
3 years ago
Classification MHC class I proteins form a functional receptor on most nucleated cells of the body.
There are 3 major and 3 minor MHC class I genes in HLA: HLA-A,HLA-B and HLA-C;
minor genes are HLA-E, HLA-F and HLA-G
There are 3 major (DP, DQ, DR ) and 2 minor ( DM, DO) MHC class II proteins encoded by the HLA. Typically expressed on the surface of antigen-presenting cells.
split antigen
antigen (broad) can be splitter into more antigens and matching for HLA antigen “splits” results in better transplant outcome than matching for “broad” HLA antigens
HLA typing and its influence on organ transplantation
When a human transplant is performed, HLA (human leukocyte antigens) molecules from a donor are recognized by the recipient’s immune system by direct and indirect methods of allorecognition triggering an alloimmune response. Matching of donor and recipient for MHC antigens showed a significant positive effect on graft acceptance
HLA mismatches may occur at antigenic or allelic level; the first are characterized by amino acid substitutions in both peptide binding and T-cell recognition regions, whereas the latter are characterized by amino-acid substitution in the peptide binding regions only.
In transplantation immunology, the major impact in graft loss comes from the effects of HLA-B and -DR antigens.
The effects of HLA-DR mismatches are the most important in the first 6 months after transplantation, the HLA-B effect emerges in the first 2 years, and HLA-A mismatches have a deleterious effect on long-term graft survival.
Techniques of HLA typing
Previously, HLA typing was done by two methods: serologic method using antiserum and mixed lymphocyte culture (MLC). After that a more precise DNA-based
HLA typing methods using molecular techniques, such assequence-;specific oligonucleotide probe hybridization (SSOP), sequence-specific primer amplification (SSP), sequencing-based typing (SBT), and reference strand-based conformation analysis (RSCA), have been developed and are frequently used. In 2013, a new project of the 16IHIW demonstrated the potential benefits of next-generation sequencing (NGS) in the HLA laboratory. Another simplified method using short tandem repeat (STR) genotyping provided additional information allowing determination of the extent of HLA identity in families where HLA haplotype inheritance was
ambiguous
Well done Yes, split antigen match is associated with better outcome, well done
Mohamad Habli
3 years ago
HLA antigen classification
HLA region is located in the short arm of 6th chromosome and in particular 6p21.3. MHC or HLA complex is known to be highly polygenic. Broadly it is divided into three categories: Class I, Class II, and Class III.
Class I include genes that encode for HLA antigens. Three major HLA types A, B and C, and 3 minor E, F, and G.
Class II include 3 major types DR, DP and DQ and 2 minor DM and DO.
Class III is located between class I and II. It is responsible for encoding several complement components, TNF, heat shock protein and others. Split and broad antigens
HLA can be classified into broad and split antigens
Old serological based cellular methods for HLA typing obtain HLA genes with allele group but were not able to identify precisely specific alleles. That’s why broad HLA antigen has a poor specificity. A broad antigen can be divided into 2 or more split antigens.
DNA based molecular techniques allowed for more identification and sequencing of the polymorphic regions of genes and identify specific alleles encoding for specific HLA proteins.
Why split antigens typing is important. In solid organ transplantation, differences in the split antigens, having same broad antigen, could potentially activate immune response and subsequent immune mediated rejection. Amino acid sequencing using DNA based methods is important, because DSA could react against one but not against another in the same broad antigen.
How can HLA mismatch influence the outcome of transplantation?
The better outcomes in transplantation n occur in transplantation between identical twins, which implies that match or mismatch is the predictor of outcomes. Matching for HLA antigens has significant impact on graft survival and overall survival. One mismatch between donor and recipient is associated with an increase risk of rejection, while 2,2,2 mismatch has the poorer outcomes in mismatched groups.
Summarize the HLA typing techniques
Before the widespread availability of DNA based techniques, HLA typing was performed using serological methods.
Serological method uses reference sera containing known antibodies to specific HLA antigens. So donor or recipient lymphocytes are added to several wells of plates that contain different sera and eventually different antibodies. 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. Antibodies used in the reference sera could also be directed against more than HLA molecule causing inconclusive typing.
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.
DNA-based molecular techniques for HLA typing include low to high resolution methods that include sequence-specific oligonucleotide probe typing (SSOP), sequence-specific primer typing(SSP), sequence-based typing (SBT) and Next-generation sequencing (NGS). The type of technique used varies among laboratories based on availability, experience and cost.
SSP involves the use of primers that detect a group of similar alleles or specific HLA sequence.
SSOP uses set of primers that recognize a particular locus. As with SSP, DNA is amplified for better detection of polymorphic regions of HLA.
1. Classify HLA antigens: Human Leukocyte Antigen (HLA) is a gene complex located on short arm of chromosome 6 (6p21.3). It contains many genes and can be classified into class I, II and III. Class I and II have important role in immune regulation.
HLA Class I has 3 major loci (HLA-A, HLA-B and HLA-C) and 3 minor loci (HLA-E, HLA-F, HLA-G)
HLA Class II has 3 major loci (HLA-DP, HLA-DQ, HLA-DR) and 2 minor loci (HLA-DM, HLA-DO) 2. What is meant by split antigen?
HLA can be classified as broad antigen and split antigen.
A broad antigen is HLA molecule with a poor specificity. A broad antigen can be divided into 2 or more split antigens.
A split antigen is HLA molecule with a more refined or specific cell surface reaction relative to a broad antigen.
For example: HLA-A10 is a broad antigen while HLA-A25, HLA-A26, HLA-A34 and HLA-A66 are split antigens.
3. How can HLA mismatch influence the outcome of transplantation? Human Leukocyte Antigen or HLA typing and mismatch determination between the potential transplant recipient and donor pair is an important factor for prognostication in kidney transplantation. Usually the HLA A-, B-, and DR- are typed for both the donor and the recipient and degree of mismatch is looked into. Studies have shown that number of HLA match in the recipient-donor pair is a strong predictor of renal allograft survival. (1) The higher the HLA match, lower the incidence of delayed graft function as well as acute rejection rate in first year and higher the 10 year graft survival. (2,3) HLA mismatches have been shown to be associated with poor transplant outcomes. (4) UNOS registry showed that there was increased risk of allograft failure with number of mismatches (13% in 1/6 mismatch and 64% in 6/6 mismatch). (5) Post-transplant long-term prognosis includes rates of complications like post-transplant lymphoproliferative disease (PTLD), post-transplant bone fractures, development of donor specific antibodies (DSAs) and death with a functioning graft. PTLD risk has been shown to be higher in transplants with HLA DR mismatch (21% increased risk with 1 HLA-DR mismatch and 56% increased risk in 2 HLA-DR mismatch). (6) Post-transplant bone fractures are also shown to be higher in recipients with HLA-DR mismatch (85% increased risk with 1 mismatch and 124% increased risk with 2 mismatch) (7) Increased formation of DSAs is seen in case with Class II HLA mismatch (HLA-DQ, DR) thereby ultimately leading to poor graft survival. (8) Increased risk of death with a functioning kidney graft has been seen in recipients with increased HLA mismatch. (9) Of note is a study by Su et al which deduced that due to availability of better and more potent immunosuppression, HLA mismatch has lost much of its relevance. (10) References:
Yacoub R, gn, Cravedi P et al. Analysis of OPTN/UNOS registry suggests the number of HLA matches and not mismatches is a stronger independent predictor of kidney transplant survival. Kidney Int. 2018;93(2):482.
Takemoto SK, Terasaki PI, Gjertson DW, Cecka JM. Twelve years’ experience with national sharing of HLA-matched cadaveric kidneys for transplantation. N Eng J Med 2000;343(15):1078.
Wissing KM, Fomegne G, Broeders N et al. HLA mismatches remain risk factors for acute kidney allograft rejection in patients receiving quadruple immunosuppression with anti-interleukin-2 receptor antibodies. Transplantation 2008;85(3):411.
Shi X, Lv J, Han W, et al. What is the impact of human leukocyte antigen mismatching on graft survival and mortality in renal transplantation? A metaanalysis of 23 cohort studies involving 486,608 recipients. BMC Nephrology 2018;19:116.
Williams RC, Opelz G, McGarvey CJ et al. The risk of transplant failure with HLA mismatch in first adult kidney allografts from deceased donors. Transplantation 2016;100(5):1094-1102.
Opelz G, Dohler B. Impact of HLA mismatching on incidence of posttransplant non-hodgkin lymphoma after kidney transplantation. Transplantation 2010;89(5):567.
Nikkel LE, Hollenbreak CS, Fox EJ et al. Risk of fractures after renal transplantation in the United States. Transplantation 2009;87(12):1846-51.
Weibe C, Gibson W, Blydt-Hansen TD et al. Evolution and clinical pathologic correlations of de novo donor-specific HLA antibody post kidney transplant. Am J Transplant 2012;12(5):1157-67.
Opelz G, Dohler B. Association of HLA mismatch with death with a functioning graft after kidney transplantation: a collaborative transplant study report. Am J Transplant 2012;12(11):3031.
Su X, Zenios SA, Chakkera H, et al. Diminishing significance of HLA matching in kidney transplantation. Am J Transplant 2004;4:1501-1508.
4. In your own words, summarise the HLA typing techniques
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.
ii. SSOP (Sequence Specific Oligonucleotide Probe hybridization): Amplified DNA is mixed with oligonucleotide probe complementary to specific DNA segment of different alleles which are identified using fluorescent markers leading to HLA typing.
iii. SBT (Sequence based typing): Exon specific primers are used.
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.
class I : A, B. C (major antigens)
E, F, G (minor antigens)
class II DP, DQ, DR (major antigens)
DM, DO (minor antigens)
split antigen: it is antigen split from broad antigen e.g HLA-A2 is broad antigen , HLA-A203, HLA-A210 are split antigens
of course HLA mismatch affect graft survival , more mismatch leads to decrease graft survival, HLA-DR mismatch affects graft survival too early in the first 6 months and HLA-B mismatch affects graft survival in the 1st 2 years , HLA-A mismatch has an effect on long term graft survival.
This mismatch will trigger immune response HLA I and II have protein peptide has its receptor on T cells CD4 has TCR to class II (which present on APC of the donor), and CD8 has TCR to class I (which present in all nucleated cells as donor parenchyma). Activation of T cell will be done when 3 signals stimulated TCR with class I or II, CD28 lock with CD80/86, and IL-2 with CD25.
This will leads acute cellular rejection.
Also endothelial cells have class I and II HLA molecules, this will lead to vascular damage and c4d deposition with antibody formation. ABMR
HLA typing techniques:
1- Serological assay use antiserum and mixed lymphocyte culture
It is made by CDC/ADCC used for screening HLA antibodies in the recipient.
2- Molecular method / DNA based sequence technology which is used widely, also PCR emerging and used.
Sequence based typing technique is more sensitive than serological method
broad antigen used in practice , but any mismatch at the level of split antigen will impact on graft survival as it will induce immune response
molecular method is accurate
molecular typing is accurate than serology, as there is polymorphism of HLA system so, molecular methods can reach to different alleles so more accuracy affecting graft outcome.
All HLA mismatches are associated with some degree of risk of reduced graft function and survival and the risk is proportional to the number of mismatched antigens. HLA C is rarely associated with acute rejection but mostly with chronic rejection.
HLA Mismatching Strategies for Solid Organ Transplantation – A Balancing ActAndrea A. Zachary
Classification
HLA class 1
major-: HLA-A,HLA-B and HLA-C
minor genes are HLA-E, HLA-F and HLA-G
HLA class 2
major-: HLA-DR, -DQ, -DP,
minor- DM, and -DO.
split antigens
HLA-B15 consists of 8 closely related specificities, HLA-B62, B63, B70, B71, B72, B75, B76, and B77.
Serologic supertypes are the broad specificities. Eg: B15
“Splits” or Subtypes are the finer specificities that comprised the supertype. Eg: B62.
As the splits were discovered, they were given number designations that again give no indication as to the supertype to which they belong. Eg: Molecular B*15:12 is equivalent of HLA- B76.
HLA-DR mismatches are the most important in the first 6 months after transplantation.
HLA-B effect emerges in the first 2 years.
HLA-A mismatches have a deleterious effect on long-term graft survival. All HLA mismatches are associated with some degree of risk of reduced graft function and survival and the risk is proportional to the number of mismatched antigens. patients with one or two mismatches for an HLA-C antigen had a significantly higher incidence of rejection compared to those with no HLA-C mismatch (54 and 100 vs. 0%) but only when there was also one HLA-B mismatch.
HLA Mismatching Strategies for Solid Organ Transplantation – A Balancing ActAndrea A Zachary et al .
HLA typing techniques
HLA typing is done by two methods
serologic method using antiserum and mixed lymphocyte culture (MLC).
ADCC or CDC.
DNA-based HLA typing methods
SSOP- can provide low or high resolution
SSP-can provide low or high resolution
SBT- is a high resolution method
RSCA
next gene sequencing-
STR genotyping – rapid and reliable used to screen potential sibling donor.
The difference in survival at three years between grafts with 0 or 6 mismatches for HLA-A, B, DR was 31% when antigen splits were analysed, in contrast to a 6% difference with broad antigens. These results indicate that typing for HLA antigen splits is important in renal transplantation.
Importance of HLA antigen splits for kidney transplant matchingG Opez
molecular typing is more accurate than serological typing since it can identify allelles.
Dear All
We always pay attention to HLA A,B and DR. Antibodies against HLA C may cause troubles
What is the role of HLA C mismatch in transplantation?
There is a reward for the correct answers
Volume 1 and 2 (2020 edition) of this original copy
HLA-C mismatches have been studied in kidney transplant and has been shown to be associated with acute rejection.
Tran et al in their study found that sensitized patients with PRa>10% and HLA-C mismatch had decreased graft survival. (1)
In another study, antibody mediated rejection was seen in 27% of the patients with anti HLA-C antibodies in first year post transplant. (2)
References:
1)Tran Th, Dohler B, Heinold A, t al. Deleterious impact of mismatching for human leukocyte antigen C inpresensitized recipients of kidney transplants. Transplantation 2011;92:419.
2) Aubert O, Bories MC, Suberbielle C, et al. Risk of antibody-mediated rejection in kidney transplant recipients with anti HLA-C donor specific antibodies. Am J Transplant 2014;14:1439-1445.
Thanks Amit
Also in retransplantation.
Studies confirmed the occurrence of antibody mediated rejection and transplant glomerulopathy in patients with HLA-C DSA antibody.1,2,4
A case of kidney transplantation with HLA-C DSA ( MFI= 1000) ended with irreversible ABMR.3
So pretransplant risk stratification of sensitized patients may be accomplished by testing for donor specific HLA-C antibody.4
References:
MFI of more than 5000 is considered significant and also in retransplantation.
Frohn et al. investigated the impact of HLA-C mismatches on rejection in 104 renal transplants (20). They controlled for HLA-B mismatch to eliminate linkage disequilibrium as a confounding factor. They found that patients with one or two mismatches for an HLA-C antigen had a significantly higher incidence of rejection compared to those with no HLA-C mismatch (54 and 100 vs. 0%) but only when there was also one HLA-B mismatch. Patients with one HLA-B and two HLA-C mismatches also had decreased graft survival that approached statistical significance (P = 0.055) (1)
Donor mismatches at each of the HLA-A, -B, and -C loci all contributed to an increasing likelihood of developing sensitization to HLA class I, but HLA-C less than HLA-A and -B
Aubert et al performed case-controlled study of 608 patients of kidney transplantation were screened for the presence of isolated anti-HLA-C DSA at day 0. A total of 22 renal transplant recipients were selected and followed for a period of 1 year.
The 22 patients were compared with 88 immunized patients. Acute AMR was diagnosed in six patients (27.3%). The median level of DSA at day 0 was 1179 (530-17,941). The mean fluorescence intensity in the anti-C group was 4966 (978-17,941) in the AMR group and 981 (530-8012) in the group of patients without AMR. The level of DSA at day 0 was predictive for AMR .
Patients with a high level of pretransplant anti-HLA-C DSAs are likely to develop acute AMR during the first year after transplantation (2).
1 Frohn C, Fricke L, Puchta JC, Kirchner H. The effect of HLA-C matching on acute renal transplant rejection. Nephrol Dial Transplant (2001) 16:355–60. doi:10.1093/ndt/16.2.355
2 Aubert O, Bories MC, Suberbielle C, et al. Risk of antibody-mediated rejection in kidney transplant recipients with anti HLA-C donor specific antibodies. Am J Transplant 2014;14:1439-1445.
Do you think it would be mandatory to test for pretransplant anti-HLA-C DSAs?
Dear Dr Alaa,
Based on the latest observations, I think that pre-transplant evaluation of HLA-C DSA level is reasonable, and if the recipient has high MFI of HLA-C DSA, then it would be better to avoid HLA-C mismatch. Otherwise, if there is no alternative allograft offer, we should intensify the immune suppression considering the higher risk of ABMR.
yes for presensitized candidates and by using the SAB assay with lower cutoff values pf PRA > 10 , based on few studies from single centrs shows the presnce of DSA for this specific class can trigger ABMR in the first year after transplantion ,and offcourse we need more studies to determine the effect of HLA-C ab on graft outcome .
Well done
Winner
Dear Dr Ahmed,
The direct effect of HLA-C mismatch on kidney allograft outcome is still controversial (1). HLA- C was once believed to be of minor immunological importance in the field of kidney transplantation. However, there is a rising interest in its immunological significance due to several observations (1).
A study including 2260 deceased-donor kidney transplant recipients has documented a negative allograft outcome in association with one or both HLA-C antigens mismatch in recipients with panel reactive antibodies (PRA) more than 10%. Surprisingly, the same study showed no significant immunological impact of HLA-C mismatch in recipients who are not sensitized (2).
Another retrospective single-centre study has documented that a high pretransplant HLA-C DSA level was associated with an increased risk of developing ABMR later on while low levels were of minor significance (mean MFI values was 4966 in recipients who developed ABMR versus MFI of 981 those who did not have ABMR) (3).
References:
1) M. C. Philogene and Daniel C Brennan. Kidney transplantation in adults: HLA matching and outcomes. © 2021 UpToDate. (Accessed on 26 November 2021).
2) Tran TH, Döhler B, Heinold A, et al. Deleterious impact of mismatching for human leukocyte antigen-C in presensitized recipients of kidney transplants. Transplantation. 2011;92(4):419.
3) Aubert O, Bories MC, Suberbielle C, et al. Risk of antibody-mediated rejection in kidney transplant recipients with anti-HLA-C donor-specific antibodies. Am J Transplant. 2014 Jun;14(6):1439-45.
HLA-C
Presence of HLA-A,B,DR DSA has been proven to increase the risk if acute and/or chronic antibody mediated rejection after transplantation and important in determining graft survival. The association HLA C and anti-DP has not been taken in serious in graft survival. Studies have been done to investigate the role of other HLA in determining graft rejection in fully matched recipients.
Frohn et al showed that HLA-A/-B mismatch was strongly associated with HLA-C mismatch as a result of linkage disequilibrium. HLA -B mismatch was weakly correlated with rejection probability. Univariate analysis showed HLA-C had is strongly influenced on graft survival. It also showed that B cell mismatches when happened together with HLA-C is associated with independent factor in acute rejection.
Albert et al showed increased incidence of AMR in patient with anti-HLA DSA. Moreover the study showed presence of DSA at day 0 in anti-C group more likely to experience AMR. So,the presence of pretransplantation HLA-C DSA appeared to have higher risk of AMR.
References:
1)Tran Th, Dohler B, Heinold A, t al. Deleterious impact of mismatching for human leukocyte antigen C inpresensitized recipients of kidney transplants. Transplantation 2011;92:419.
2) Aubert O, Bories MC, Suberbielle C, et al. Risk of antibody-mediated rejection in kidney transplant recipients with anti HLA-C donor specific antibodies. Am J Transplant 2014;14:1439-1445.
Winner
Well done, excellent explantation.
the question what sets HLA-C apart from HLA-A and -B? .
The difference may well be related to the reduced levels of cell-surface expression of HLA-C as compared to HLA-A and -B, possibly due to, among other factors, a limited peptide-binding capacity.
This limited peptide-binding capacity may retain HLA-C in the ER and enhance degradation of the HLA-C protein.
HLA-C mismatches are clearly associated to alloreactivity after hematopoietic stem-cell transplantation; in a number of large cohorts, HLA-C mismatches are correlated to an increased risk of acute graft-versus-host disease (GVHD) or even impaired survival. While for HLA-A and -B, both antigenic as well as allelic mismatches are associated with an increased risk of acute GVHD, such an increased risk is only observed for antigenic HLA-C mismatches and not for allelic mismatches.
Yes it is important in hematopiotic stem cell transplantation
Although HLA-C Ags frequency is lower than HLA-A, & -B loci, but it was found that the presence of pre transplantation anti-HLA-C Abs carry a high risk of AMR, so pre transplant screening is essential & changes in immunosuppression is required for positive cases.
Abuzeineh M., Ziadeh A., Kheradmand T., et al. A curious case of de novo anti-HLA-C antibody-mediated humoral rejection and Fabry-like zebra bodies a renal transplant recipient. Clinical Nephrology- Case Studies. 2020, 8: 12-16.
Aubert O., Bories M., Suberbielle C., et al.Risk of Antibody-Mediated Rejection in Kidney Transplant Recipients With Anti-HLA-C Donor-Specific Antibodies. American Journal of Transplantation. 4014, 14: 1439-1445.
In kidney transplantation, only HLA-A, HLA-B, and HLA-DR antigens are routinely used to identify degree of matching between a donor and recipient, whereas HLA-C and other HLA classes are usually discarded. The introduction of the SAB method allowed, with high sensitivity, the detection of HLA to various classes. This improvement in diagnostic techniques led to the improvement in the identification of DSA and detection of new antibodies not discovered before.
The impact of HLA-C compatibility on renal allograft survival has been investigated in several studies.
Studies on the effect of HLA-C antibodies showed controversial results, as 40-50% of kidney transplant recipients may have preformed anti-HLA-C antibodies. Some studies that addressed the influence of HLA-C mismatches found that mismatching for one or both HLA-C antigens, in recipients who were pretransplant sensitized, was associated with decline in graft survival. Patients with high levels of pretransplant antibodies against HLA-C could be at higher risk to develop ABMR during the first posttransplant year.
Some studies have also suggested that HLA-C antibodies could be associated with acute rejection, while others showed only minimal correlation with graft damage. The use of SAB techniques has allowed better identification of anti-HLA-C DSA and eventually studying their effect on allograft outcome.
For the current clinical practice, HLA-A, HLA-B and HLA-DR antigens are still considered for the basic typing and matching, however in the setting on total match and acurt rejection, anti-HLA-C antibodies and others should be investigated.
References:
1. Tran TH, Döhler B, Heinold A, et al. Deleterious impact of mismatching for human leukocyte antigen-C in presensitized recipients of kidney transplants. Transplantation 2011; 92:419.
2. Ling M, Marfo K, Masiakos P, et al. Pretransplant anti-HLA-Cw and anti-HLA-DP antibodies in sensitized patients. Hum Immunol 2012; 73:879.
3. Chapman JR, Taylor C, Ting A, Morris PJ. Hyperacute rejection of a renal allograft in the presence of anti-HLA-Cw5 antibody. Transplantation 1986; 42:91.
4. Bachelet T, Couzi L, Guidicelli G, et al. Anti-Cw donor-specific alloantibodies can lead to positive flow cytometry crossmatch and irreversible acute antibody-mediated rejection. Am J Transplant 2011; 11:1543.
5. Aubert O, Bories MC, Suberbielle C, et al. Risk of antibody-mediated rejection in kidney transplant recipients with anti-HLA-C donor-specific antibodies. Am J Transplant 2014; 14:1439.
Winner
Well done, excellent explantation.
Forty to 50 percent of kidney transplant recipients may have performed anti-HLA-C antibodies that is lower than anti HLA-A or HLA-B. However, the effect of HLA-C antibodies on allograft outcome is controversial. A few reports have suggested that the presence of anti-HLA-C antibodies indicates a deleterious effect caused by severe AMR, while others have suggested that it indicates a low rate of acute rejection.
One study demonstrated, patients with high levels of pretransplant HLA-C DSA may be more likely to develop ABMR during the first posttransplant year.
Another study found that mismatching for one or both HLA-C antigens was associated with decreased graft survival among those who were presensitized (panel reactive antibodies [PRA] >10 percent).
Because of a low expression on renal endothelial cells, HLA-Cw and -DP have been considered to be less immunogenic that other HLA antigens. Recent studies have reported that anti–HLA-Cw and anti–HLA-DP DSA could be associated with an increased risk of acute and chronic antibody-mediated rejection (AMR). Studies suggest that preformed anti–HLA-Cw and anti–HLA-DP DSA are as deleterious as anti–HLA A/B/DR/DQ DSA. It justifies their inclusion in kidney allocation programs and in immunological risk stratification algorithms.
Winner
Well done, excellent explantation.
Refrences:
Aubert O, Bories MC, Suberbielle C, et al. Risk of antibody-mediated rejection in kidney transplant recipients with anti-HLA-C donor-specific antibodies. Am J Transplant 2014; 14:1439.
Bachelet T, Martinez C, Del Bello A, Couzi L, Kejji S, Guidicelli G, Lepreux S, Visentin J, Congy-Jolivet N, Rostaing L, Deleterious Impact of Donor-Specific Anti-HLA Antibodies Toward HLA-Cw and HLA-DP in Kidney Transplantation. Transplantation. 2016 Jan;100(1):159-66.
Role of HLA-C mismatch in transplantation:
Studies researching the effect of HLA-C mismatching are few. HLA-C matching was found to be an option to decrease probability of acute rejection in the first study by Frohn et al ,2001.Mismatching of one or both HLA-C antigens was found to be associated with lower graft survival in sensitized recipients having deceased -donors .Preformed anti -HLA-C antibodies are generally found in 40-50% of kidney transplant recipients.in spite of this, the effect of that mismatch on allograft outcome is debatable. Some studies suggested association between HLA -C ABs and acute rejection and others found relation to graft loss. SAB assays allowed better HLA-c detection and easier study of HLA-C mismatch. Hence ,it was suggested that antibody mediated rejection is more likely to develop in recipients having high levels of pretransplant HLA-C DSA .
RREFERENCES:
Frohn,Cet al : The effect of HLA-C matching on acute renal transplant rejection, Nephrol Dial Transplant ,2001,Feb,16(2):355-60
Tran,TH,et al :Deleterious impact of mismatching for human leucocyte antigen-c in presensitized recipients of kidney transplants, Transplantion,2011;92(4):419
Ling,M,et al,Pretransplant anti-HLA-Cw and anti-HLA-DPantibodies in sensitized patients,Hum Immunol,2012,Sep;73(9):879-83
Bryan,CF et al, Sharing kidneys across donor-service area boundaries with sensitized candidated can be influenced by HLA C, Clin Transplant,2010,Jan24(1);56-61
Gilbert,Met al, Impact of pretransplant human leucocyte a:1439-45ntigen -c and dp antibodies on kidney graft outcome,Transplant Proc.2011 nov;43(9)3412-4
Aubert,O et al: Risk of antibody -mediated rejection in kidney transplant recipients with anti-HLA-C donor -specific antibodies,2014:Am J Transplant Jun;14(6)
Based on evidence from the large transplant registries which consistently confirm the strong association between HLA matching at the level of HLA-A, -B, DR, DQ loci and both patient, graft survival, however few studies reported that HLA-C miss-match could trigger ABMR with increasing risk of graft failure in presensitized recipients with PRA 10 % and above .
In one single center retrospective study of 608 transplant patients where assessed for DSA by SAB assay with MFI cut vale of 500 and above , they found that 26% of them developed DSA , and another 4% had HLA-C-AB at day0 of the transplantation and upon one year FU they found that 27% had ABMR with significantly higher MFI level= 4966 during the Fu period .
references
up to date , kideny transplataion in adults : HLA matching and outcome .
RELATIVE IMPORTANCE OF HLA-C
40to 50 %of renal Tx recipients may have preformed anti-HLA-C antibodies . The effect of these antibodies on allograft outcome is controversial.
Some studies have suggested that they are associated with low levels of acute rejection, while others have shown increased antibody-mediated rejection (AMR)
The availability of single-antigen bead assessment of anti-HLA antibodies may add some clarity.
In a retrospective, single-center study, 608 transplant patients were assessed for donor-specific antibodies (DSAs) as defined by a mean fluorescent intensity (MFI) of >500 [33]. Of these, 160 (26 percent) had DSA, and 25 (4 percent) had anti-HLA-C antibodies. Among 22 patients who were available for follow-up, the incidence of AMR in the first year was 27 percent in those with anti-HLA-C antibodies.
reference
HLA matching and graft survival in kidney transplantation . UpToDate ( accessed on 28/11/2021)
HLA -C mismatch associated with poor graft outcome and increase rate of rejection
The transplantation barrier is defined by the HLA genes that are responsible for tissue histocompatibility.1-7 Mismatching for HLA-C allotypes between patients and unrelated donors generally leads to very high risks of acute graft-versus-host disease (GVHD) and mortality after hematopoietic cell transplantation, although risks to individual patients may vary.3-7 The success of transplantation for a given patient may depend on the unique features of the HLA-C mismatch itself. Three different models of HLA-C mismatching shed light on the variability of individual risks. Mismatching can occur between allotypes that elicit an antibody (serologic) response (antigen mismatches) or between allotypes that differ for limited nucleotide sequence variation (allele mismatches). The similarity of sequence features between allele mismatches may contribute to their lower immunogenicity.3-7
A second model of HLA-C alloreactivity entails mismatching for amino acid residues that determine the repertoire of peptides presented to T cells. Patient-donor differences at several
residues of the class I molecule might significantly affect the immunogenicity of HLA-C mismatches, and of these residues, residue 116 in the F pocket of the peptide binding groove has a high frequency of patient-donor mismatching and consistently shows an effect on transplant outcome.8-10 HLA-C-mismatched patients who are residue 116 mismatched have higher risks of acute GVHD and mortality than HLA-C-matched patients,10-12 observations that support a critical role for T-cell recognition of class I-peptide complexes.13,14 Most recently, a third model has been proposed in which transplant outcome may depend on the regulation of donor natural killer (NK) cell responses against patient cells.15 Amino acid substitutions at HLA-C residues 77 and 80 define 2 mutually exclusive groups of ligands, each recognized by different killer immunoglobulin-like receptors (KIRs). HLA-C-mismatched patients who are residues 77/80 mismatched may have different transplant outcomes than HLA-C-mismatched patients who are residues 77/80 matched.15-20
Each of the 3 mismatch models suggests that some HLA-C mismatches are less risky than others and therefore represent mismatches that could be considered when matched donors are not available.21 The high overall risks associated with transplantation of HLA-C-mismatched unrelated donors have led some clinicians to abandon the use of such donors altogether. Clinical practice is heterogeneous because the features that define permissive HLA-C mismatches remain ill defined.
Recently, the range of expression across HLA-C allotypes has been elucidated.22 Each serologically defined HLA-C allotype has a characteristic median fluorescence intensity (MFI) of cell surface expression that is reproducible in both healthy and HIV-infected cells in vitro.22 The MFI coefficient is superior to any other marker of expression level, including the previously described single nucleotide polymorphism that resides 35 kb upstream of HLA-C,23 because the MFI provides direct allotype-specific measurement of HLA-C surface expression. Expected levels of HLA-C cell surface expression based on the sum of the 2 allelic MFI coefficients was shown to predict observed HLA-C expression levels among individuals in 2 cohorts, indicating that MFI coefficients can be assigned to each HLA-C allotype in lieu of direct ascertainment of expression.22 Thus, the clinical importance of HLA-C expression can be determined in large-scale retrospective outcome studies where appropriate materials for measuring HLA-C expression directly are not available. Using this approach, higher MFI levels were shown to correlate with better control of HIV viral load and slower progression to HIV-AIDS across ethnic groups, but with increased susceptibility to Crohn disease,22 solidifying the role for HLA-C expression levels in modulating the strength of immune responses. Accordingly, we applied the MFI as a quantitative proxy of HLA-C expression level (simply termed as expression level throughout the manuscript) to assess the clinical significance of the level of HLA-C expression in an exceptionally large international population of patients and unrelated transplant donors whose only HLA mismatch was a single HLA-C allotype.
References:
* Apps R, Qi Y, Carlson JM, et al. Influence of HLA-C expression level on HIV control., Science, 2013, vol. 340 6128(pg. 87-91).
* Fernandez-Viña MA, Wang T, Lee SJ, et al. Identification of a permissible HLA mismatch in hematopoietic stem cell transplantation., Blood, 2014, vol. 123 8(pg. 1270-1278).
* Pidala J, Lee SJ, Ahn KW, et al. Non-permissive -DPB1 mismatch among otherwise HLA-matched donor-recipient pairs results in increased overall mortality after myeloablative unrelated allogeneic hematopoietic cell transplantation for hematologic malignancies., Blood, 2014.
Nearly, all studies have examined outcomes vis-a-vis matching or mismatching of HLA-A, -B, and -DRB1-encoded antigens. However, at the time of this writing, there are limited data on matching at other HLA loci (HLA-C, -DRB3, -DRB4, -DRB5, -DQA, -DQB, -DPA, and -DPB). Frohn et al. investigated the impact of HLA-C mismatches on rejection in 104 renal transplants (20). They controlled for HLA-B mismatch to eliminate linkage disequilibrium as a confounding factor. They found that patients with one or two mismatches for an HLA-C antigen had a significantly higher incidence of rejection compared to those with no HLA-C mismatch (54 and 100 vs. 0%) but only when there was also one HLA-B mismatch. Patients with one HLA-B and two HLA-C mismatches also had decreased graft survival that approached statistical significance (P = 0.055). In an early study of data from the Southeastern Organ Procurement Foundation on 12,050 first deceased donor transplants, no effect of matching for HLA-DQ was found when other factors affecting outcome were taken into account (21). In contrast, in a recent study, Lim et al. found DQ mismatching to incur a significantly increased risk of rejection that was further increased in the presence of DR mismatches (22). Rosenberg et al. (23) found that DP mismatches in patients matched for DR and DQ did not impact graft survival or function. Similarly, data from the Collaborative Transplant Study found no deleterious effect of DP mismatches in first deceased donor transplants but did find significantly reduced graft survival in regraft patients (24). The widespread adoption of DNA-based typing methods has facilitated typing for HLA-DQ and -DP. However, unlike other HLA molecules that have one polypeptide chain that is invariant or has limited variability, both polypeptide chains, the α chain encoded by DQA and DPA and the β chain encoded by DQB and DPB, are polymorphic. Both chains may have immunogenic epitopes and there are epitopes comprised of particular combinations of α and β chains (25, 26). Consequentially, studies that consider matching only for DQB and DPB may incorrectly identify a mismatch as a match between a donor and recipient.
References:
* Broeders N, Racapé J, Hamade A, Massart A, Hoang AD, Mikhalski D, et al. A new HLA allocation procedure of kidneys from deceased donors in the current era of immunosuppression. Transplant Proc (2015) 47:267–74.10.1016/j.transproceed.2014.12.018 .
* Al-Otaibi T, Gheith O, Mosaad A, Nampoory MR, Halim M, Said T, et al. Human leukocyte antigen-DR mismatched pediatric renal transplant: patient and graft outcome with different kidney donor sources. Exp Clin Transplant (2015) 13(Suppl 1):117–23.
Dear All
This article is directly related to assignment 1. I invite you to read assignment 1. Try to train yourself in writing in your own words. When we allow assignment submission, there is a plagiarism software will be checking if you copied and pasted from other sources.
The HLA system includes complex genes located on chromosome 6. The system is highly polymorphic.
HLA classification:
~HLA class I ( present on all nucleated cells of the body ) > 3 majors ( A , B , c ) and 3 minors ( E , F , G)
~HLA class II ( present on the surface of antigen-presenting cells )> 3 major ( DQ , DR , DP) and 2 minor > ( DM , DO).
HLA typing techniques.
Serology technique w is done by known antibodies against specific HLA antigens.
Cellular assay in which we use lymphocytes to detect HLA class2
Molecular techniques
To detect alleles sequence at DNA level
Several methods:
1)SSP :specific primer complementary to specific allele using PCR
2)SSOP :a specific prob used to pair with a specific genetic allele
3)SBT :it detect Exons
4)RSCA
5)NGS (Next Generation Sequencing): which detects both entrons and exons.
6)Short Tandem Repeat.
Split antigens: splits mean division of a single antigen into subtypes; an antigen that has a more specific cell surface reaction relative to a broad antigen (Example: HLA-B5 Broad antigen splits to HLA-B51 and HLA-B52 )
HLA matching has a direct effect on graft loss. The effects of HLA-DR mismatches are the most important in the first 6 months after transplantation, the HLA-B effect appears in the first 2 years, and HLA-A mismatches have an unfavorable effect on long-term graft survival.
Other types of antibodies that are directed against non-HLA antigens are important like against graft endothelium, MICA and Angiotensin type 1 receptor
Techniques of tissue typing
HLA typing has been done by two methods:
-serologic method using antiserum and mixed lymphocyte culture (MLC): can be made by antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC)
-DNA-based HLA typing methods using molecular techniques: DNA based method had more sensitivity, accuracy, and resolving power than serologic typing methods, Sequencing-based typing (SBT) is a high-resolution method for the identification of HLA polymorphisms.
HLA antigens classification
Generally talking The HLA system includes a complex arrangements of genes located on chromosome number 6. The system is highly polymorphic
MHC class I proteins present on all nucleated cells of the body: There are 3 major genes: HLA-A,HLA-B and HLA-C and 3 minor genes are HLA-E, HLA-F and HLA-G
MHC class II proteins typically present on the surface of antigen-presenting cells: There are 3 major genes :HLA-DP , HLA-DQ and HLA-DR and 2 minor genes :HLA-DM, HLA DO
Split antigens: splits means division of a single antigen into subtypes; an antigen that has a more specific cell surface reaction relative to a broad antigen (Example: HLA-B5 Broad antigen splits to HLA-B51 and HLA-B52 )
HLA matching has a direct effect on graft loss. The effects of HLA-DR mismatches are the most important in the first 6 months after transplantation, the HLA-B effect appears in the first 2 years, and HLA-A mismatches have a unfavourable effect on long-term graft survival.
Other types of antibodies that directed against non-HLA antigens are important like against graft endothelium, MICA and Angiotensin type 1 receptor
Techniques of tissue typing
HLA typing have been done by two methods:
-serologic method using antiserum and mixed lymphocyte culture (MLC): can be made by antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC)
-DNA-based HLA typing methods using molecular techniques: DNA based method had more sensitivity, accuracy and resolving power than serologic typing methods, Sequencing-based typing (SBT) is a high-resolution method for the identification of HLA polymorphisms.
HLA System:
Play important role in early recognition of foreign antigen and presenting it to T.cell
which lead to initiation of rejection.
HLA gene present in short arm 6 chromosome which include class one two and three.
Class 1 formed of :
HLA-A, HLA-B and HLA-C, HLA E, HLA-G and HLA-G presents in
nucleated cells.
CLASS 2 composed of :
HLA-DR, HLA-DP and HLA-DQ.HLA –DP HLA-DM and HLA-DO
presented in antigen presenting cells.
HLA mismatch is presence of HLA antigen present in donor but not in the recipient which
increase chance of sensitization and graft loss. Collaboration transplant study show 20%
increase success rate for people with HLA-A, HLA-B AND HLA-DR no mismatch
Compare to those with mismatch.
Now in kidney transplant HLA system most commonly used and compared is HLA-A
,HLA-B and HLA-DR.
Most of comparable studies was examine effect of HLA –A, HLA-B and HLA-DR their
effect on graft survival and outcome , but recent studies show other HLA loci (HLA-C,
DRB3, -DRB4, -DRB5, -DQA, -DQB, -DPA, and -DPB),found her rate of rejection in
patient with mismatch at this loci compare with no mismatch group. Other effect of graft
mismatch is also increase risk of cancer and post-transplant lymphoproliferative
disorder.
What is meant by split antigen:
new technique can identity. Up to 9allele variants.
HLA typing:
Many methods used for HLA typing from old immunological method to recent DNA test
based on polymerase reaction
CR-sequence specific primers (SSP):
In this test PCR primer made to identify HLA allele or group of identical ones, in a way
that t the polymorphism to be identified s located at the 3′ end of the primer.
DNA obtained from a blood sample and amplified by PCR using these primers.
When both primers to bind to the DNA, then amplification occurs and can be detected by
electrophoresis. Its rapid test and can be used when rapid typing is needed as in
deceased donors.
PCR-sequence-specific oligonucleotide (SSO):
IT used asset of primer that recognize specific HLA locus, it’s good to type of large
numbers of samples.
Real-time PCR, Sequencing-Based Typing (SBT):
Typing of real time PCR use of allele- specific PCR similar to SSP typing.
No electrophoresis is used but amplification is identified in real time with fluorescent or
probes.
Sequence-based typing (SBT):
Sequencing and amplification used florescent labeled DNA.
Next-generation sequencing (NGS):
next generation sequencing is common used PCR
test now due to high accuracy.
HLA Matchmaker:
Is recent computer based technology allow more precise detection of eplets, it has better
sensitivity of detection antibodies than other test. (7)
Types of HLA antigens
· Major histocompatibility molecules (MHC)
· MHC Class 1, present on all nucleated cells There are 3 major and 3 minor MHC class I genes in HLA: HLA-A, HLA-B and HLA-C; minor genes are HLA-E, HLA-F and HLA-G
·MHC class11 have five loci: HLA-DR, -DQ, -DP, -DM.
·Minor histocompatibility antigens refer to non-MHC antigens trigger rejection, like MHC class 1 related chain (MCIA) antigen its polymorphic and development of anti MCIA AB found to be associated with worse graft survival.
· ABO blood group antigens.
· Endothelial cell antigens.
split antigen
The new HLA nomenclature begins with HLA- and the locus name, then number of digits specifying the allele. The first two digits specify a group of alleles, with the new immunogenetic technique they can identify the allele variants up to 9 digits.
HLA typing and its influence on organ transplantation:
Matching of donor and recipient for MHC antigens showed a significant positive effect on graft acceptance. HLA mismatches may occur at antigenic or allelic level; a better HLA match is associated with significantly better patient and graft survival while HLA mismatches have been significantly associated with increased risk of acute rejection, HLA DR mis-match are associated with higher risk of rejection and poor graft survival especially in the first 6 months post transplantation while the HLA-B mismatch effect emerges in the first 2 years, and HLA-A mismatches have a deleterious effect on long-term graft survival.
Techniques of tissue typing
·Serological assay, CDCXM:
Used for screening the donor anti HLA-AB in recipients, complement-dependent cytotoxicity (CDCXM for T cells and B cells
·Limitation with this assay is less sensitive for low level of DSA and non-complement HLA AB so can give false negative results due to Non HLA or non complement dependent antibodies, low level of antibodies as well , also false positive results due to inaccurate incubation or dilution methods and in the presence of auto anti bodies Also false positive B-cell CDC-XM following treatment with rituximab.
· Antihuman globulin or addition of DTT increases the sensitivity of CDC-XM by excluding auto- immunoglobulin M
FC-XM:
· Flow cytometry cross match is Sensitive for low titer antibody, detects non-complement binding antibodies and non HLA binding ones .
Drawbacks include exclusion patients unnecessarily , falsely positive in patients having previously received monoclonal antibodies like rituximab.
·DNA based typing methods:
· more sensitive and accurate compared to serologic assay.
· Solid phase immunoassay (SPI), by using enzyme-linked immunosorbent assays (ELISA)
·microbeads, flow cytometry, flow PRA and flow analyzer Luminex with bead base assay (SAB)., CELL based assay for T CELLS, B CELLS (Luminex-SAB) are more sensitive than ELISA by 10%.
·Virtual crossmatch using single antigen beads:
Its more sensitive help in detection of acceptable and unacceptable
Donor antigens
May be performed with stored sera therefore shortening cold
ischemia time
Improves donor allocation for highly sensitized patients, DD allocation program and shorting the waiting list
Improves risk assessment for rejection
Limitation of virtual-FCXM
a false positive result, in the presence of denatured human leucocyte antigens on the beads
Requires more coordination between immunology lab
personnel and transplant team.
Different types transplantation antigens present like
· themajor histocompatibility molecules,
· minor histocompatibility antigens,
· ABO blood group antigens and
· Endothelialendothelial cell antige
AB produced against these ag has important role in graft rejection .
The sensitization to MHC antigens may be caused by
· transfusions,
· pregnancy, or
· failed previous grafts
leading to development of anti-human leukocyte antigen (HLA) antibodies that are important factor responsible for graft rejection in solid organ transplantation
The HLA system includes a complex array of genes located on chromosome number 6 and their molecular products that are involved in immune regulation and cellular differentiation.
Human leukocyte antigen (HLA) molecules are expressed on almost all nucleated cells, and they are the major molecules that initiate graft rejection.
There are three classical loci at HLA class I:
HLA-A, -B, and -Cw, and
Five loci at class II:
HLA-DR, -DQ, -DP, -DM, and -DO.
The system is highly polymorphic .
The contribution of the allelic diversity of class I and II genes to immune recognition
and alloreactivity can be analyzed by serological
methods and molecular methods at the DNA level by different
methods like
· sequence specific primer (SSP) and
· oligotyping with locus- and
· allele-specific oligonucleotide probes (SSOP) .
HLA class I and II matching is important in organ transplantation especially in kidney and bone marrow transplantation.
Classification
MHC class I proteins form a functional receptor on most nucleated cells of the body.
There are 3 major and 3 minor
MHC class I genes in HLA: HLA-A,HLA-B and HLA-C;
minor genes are HLA-E, HLA-F and HLA-G .
β2- microglobulin binds with major and minor gene subunits to produce a heterodimer.
There are 3 major and 2 minor MHC class II proteins encoded by the HLA. The genes of the class II combine to form heterodimeric (αβ) protein receptors that are typically expressed on the surface of antigen-presenting cells.
Major MHC class II HLA-DP:
α-chain encoded by HLA-DPA1 locus and
β-chain encoded by HLA-DPB1 locus.
HLA-DQ:
α-chain encoded by HLADQA1 locus and
β-chain encoded by HLA-DQB1 locus.
The last one is HLA-DR:
α-chain encoded by HLA-DRA
locus and four β-chains (only 3 possible per person), encoded by HLA-DRB1, DRB3, DRB4, and DRB5 loci.
The other MHC class II proteins, DM and DO, are used in the
internal processing of antigens, loading the antigenic peptides generated from pathogens onto the HLA molecules of antigen-presenting cell.
Variability
the HLA molecules manifested high structural polymorphism.
HLA presents the highest degree of polymorphism of all
human genetic systems.
One mechanism of this polymorphism is due to gene conversion between variable alleles and loci within each HLA gene .
HLA typing and its influence on organ transplantation.
The selection of the optimal donor is based on high resolution HLA typing. The MHC (Major Histocompatibility Complex) contains more than 200 genes which are situated on the short arm of chromosome 6 at 6p21.3
The role of HLA molecules is to present peptides to T cells
(both CD4 and CD8 T cells), enabling them to recognizeand eliminate “foreign” particles and also to prevent the
recognition of “self” as foreign.
HLA mismatches may occur
at antigenic or allelic level; the first are characterized by
amino acid substitutions in both peptide binding and T-cell
recognition regions, whereas the latter are characterized by
amino-acid substitution in the peptide binding regions only.
When a human transplant is performed, HLA (human
leukocyte antigens) molecules from a donor are recognized
by the recipient’s immune system by direct and indirect
methods of allorecognition triggering an alloimmune response.
Matching of donor and recipient for MHC antigens
showed a significant positive effect on graft acceptance .
In organ transplantation, the adaptive immunity is the main
response exerted to the transplanted tissue, since the main
target of the immune response is the MHC molecules
expressed on the surface of donor cells.
In transplantation immunology, the major impact in graft loss comes from the effects of HLA-B and –DR antigens .
The effects of HLA-DR mismatches are the most important in the first 6 months after transplantation, the HLA-B effect emerges in the first 2 years,
and HLA-A mismatches have a deleterious effect on long-term graft survival .
Techniques of tissue typing
Previously, HLA typing was done by two methods:
1. Serologic method using antiserum and
2. mixed lymphocyte culture (MLC).
But nowadays a more precise DNA-based
HLA typing methods using molecular techniques, is used
· specific oligonucleotide probe hybridization (SSOP),
· sequence-specific primer amplification (SSP),
· sequencing-based typing (SBT), and
· reference strand-based conformation analysis (RSCA).
●Classify HLA antigens
HLA classified into 3 classes.Class1 includes 3major antigens
(HlA.A
HLA,B
HlA,C .
3minor(E,F,G).
classs 2 include 3 major(DR,DP,DQ) and 2minor (DM,DO).
class 3 .
●What is meant by split antigen?
less specific antigen and may include 2 or more splite antigen
Split antigen is more specific antigen and more reliable in matching than broad antigen.
●How can HLA mismatch influence the outcome of transplantation?
Increased risk of hyperacute/acute rejection class II A/B more than others .
●summery:-
Expansion the field of organ and tissue transplantationhas accelerated amazingly, since the human major histo-
compatibility complex (MHC) was discovered in 1967
The HLA system includes a complex array of genes located on chromosome number 6There are three
classical loci at HLA class I: HLA-A, -B, and -Cw, and five
loci at class II: HLA-DR, -DQ, -DP, -DM, and -DO. The
system is highly polymorphic
●Nomenclature:-
1. serological recognition.
2.Modern HLA nomen-
clature are begin with HLA- and the locus name, then *
and even number of digits specifying the allele
3.Every two years, a nomencla-
ture is put forth to aid researchers in interpreting serotypes
to alleles
●variability:-
HlA system is polymorphic
●HLA typing and its influence on organ transplantation:-
MHC contains more than 200 genes in the short arm of chromosome no 6 .
@●Techniques of tissue typing:-
HLA typing was done by two methods:
1: Cellular method .
Mainly class II ,more sensitive depend on molecular method by dna
2: serologic:
ADCC ..it is antibody mediated .
CDC…complement mediated .
Classify HLA antigen:
*HLA class I > 3 majors ( A , B , c ) and 3 minors ( E , F , G)
* HLA class II > 3 major ( DQ , DR , DP) and 2 minor > ( DM , DO).
* HLA class III not related to kidney transplantation.
~ Split antigen: cell surface antigens, an antigen that has a more refined or specific cell surface reaction relative to a broad antigen.(Example: HLA-B51 and HLA-B52 are split antigens of HLA-B5).
* Benefits of HLA matching in organ transplantation:
-better organ function.
– long graft and patient survival.
– decrease risk of sensitisation.
Data from the Collaborative Transplant Study showed that with or without cyclosporine use, the renal transplant success rate was 20% higher when there was no mismatch of HLA-B and -DR than when there was a mismatch.
*Similarly, data from the United Network for Organ Sharing showed that long-term graft survival of deceased donor renal transplants with no HLA-A, -B, and -DR mismatch was nearly 20% better than for fully mismatched grafts with a stepwise reduction in survival with each increased degree of mismatch.
Although there had been reports of the role of HLA-A and/or -B mismatch, it eventually became apparent that of the HLA antigens tested routinely, HLA-DR matching contributed the most to graft survival and function. This is of particular importance since there are fewer antigens encoded by the DRB1 locus than by either the A or B loci making it easier to find zero DR mismatches compared to zero A or B mismatches, particularly when dealing with a very HLA heterogeneous population as in the United States.
~ Increasing numbers of HLA mismatch in renal transplantation were associated with an increased need for antirejection therapy that might account for an increased incidence of death with functioning graft due to infection and cardiovascular disease . It was also shown that among 9,209 pediatric kidney transplants, HLA-A, -B, and -DR mismatches were a risk factor for 5-year graft survival, but two DR mismatches appeared to incur an increased risk for non-Hodgkins lymphoma . There is a differential cost associated with different degrees of HLA match. Schnitzler et al. looked at Medicare payment information in the United States Renal Data System (USRDS) according to the degree of HLA mismatch.
HLA classification:
~HLA class I > 3 majors ( A , B , c ) and 3 minors ( E , F , G)
~HLA class II > 3 major ( DQ , DR , DP) and 2 minor > ( DM , DO).
HLA typing techniques.
Serology technique w is done by known antibodies against specific HLA antigens w extracted from multipara women
Cellular assay in which we use lymphocytes to dectect HLA class2
Molecular techniques
To dectect alleles sequence at DNA level
Several methods:
1)SSP :specific primer complementary to specific allele using PCR
2)SSOP :a specific prob used to pair with specific genetic allele
3)SBT :it detect Exons
4)RSCA
5)NGS (Next Generation Sequencing): which detect both entrons and exons.
6)Short Tandem Repeat.
HLA Antigens Classification :
I,II and (III, not involved in immunogenic process of transplantation)
-HLA I :
3 major and 3 minor. The major are A,B and C. The minor are E and F.HLA -G B2 microglobulin binds to mjor and minor gene subunits producing a heterodimer.
HLA class II;
3 major and 2 minor .The major are DP,DQ and DR.Gene encoding includes HLA-DRB1,3,4 AND 5.Class II genes combine to form aB heterodimer expressed on antigen presenting cells. The minor include DM and DO .They are involved in pathogen related antigenic peptides loading on HLA molecules of antigen presenting cells.
Split HLA antigens :more antigen specificity became possible with improvement of serological methods. Single antigen specificity could be divided into different serological specificities H95)LA -B5,HLA -B51 and HLA-B52.Amodel of writing :HLA-B51(5).
-Influence of HLA mismatch on outcome of kidney transplantation:
Good matching of MHC antigens between both donor and recipients has positive effect on accepting kidney offer. HLA mismatches badly affect long term graft survival. The main influence causing graft loss results from HLA-B and DR antigens.The impact of DR are significant in the first 6 months following Tx. Generally, HLA-B develops in the first 2 years following kidney Tx.
HLA typing techniques:
Serotyping
implementing complement -dependent microlymphocytotoxicity method using multiple small wells in microtiter plate .Lymphocytes to be typed are added to different wells ;each containing certain antiserum with antigen specificity, then incubation and complement which its cascade activation leads to cytotoxicity .Determination of dead cells is done by addition of a vital dye.
DNA Typing:
using standardized probes ,primers or sequencing
DNA probes hyberdize to specific
complementary DNA nucleotide sequence
Mlecular-based HLA typing detect greater degree of polymorphism of single HLA more than serologic based tests
REFERENCES
Mahdi,bm: Aglow of HLA typing in organ transplantation, Clinical and translational medicine ,2013,26
Deshpnde, A:The human leucocytic antigen system simplified,2017Global J Transfusion Med,Nov,2:77-88
Classify HLA antigens
HLA classified into 3 classes.
Class1 includes 3major antigens (A,B,C)and 3minor(E,F,G)
classs 2 include 3 major(DR,DP,DQ) and 2minor (DM,DO)
class 3 is not important in kidney Transplantation
What is meant by split antigen?
Broad antigen term related to less specific antigen and may include 2 or more splite antigen
Splite antigen is more specific antigen and more reliable in matching than broad antigen.
How can HLA mismatch influence the outcome of transplantation?
UNOS registry showed
1HLA mismatch associated with 16% 10 years graft loss in relation to 64% in 6mismatch.
Hla class 2 (DR)mismatch associated with increase risk of 1y acute rejection.class 1B mismatch associated with increase risk of 1st 2y acute rejection.
Class 2(DR,DQ) mismatch associated with increase risk of post transplant denovo DSA and sensitization
Long term outcomes like PTLPD, PTDM, fracture risk, sensitization, death with functioning graft increase with HLA mismatch
Class1 c matching have linkage to to B.HLA class 1 c matching alone was neglected in studies but when associated with b mismatch it associated with short graft survival.
In your own words, summarise the HLA typing techniques.
Serology technique w is done by known antibodies against specific HLA antigens w extracted from multipara women
Cellular assay in which we use lymphocytes to dectect HLA class2
Molecular techniques
To dectect alleles sequence at DNA level
Includes
1)SSP (Sequence Specific Primer): specific primer complementary to specific allele using PCR
2)SSOP(sequence specific oligonucleotide prob):a specific prob used to pair with specific genetic allele
3)SBT (Sequence based typing):it detect Exons
4)RSCA (Reference Strand based Conformation Analysis)
5)NGS (Next Generation Sequencing): which detect both entrons and exonsSTR
6)Short Tandem Repeat.
There are three classical loci at HLA class I: HLA-A, -B, and -Cw, and five loci at class II: HLA-DR, -DQ, -DP, -DM, and -DO.
There are 3 major and 3 minor MHC class I genes in HLA: HLA-A,HLA-B and HLA-C;
minor genes are HLA-E, HLA-F and HLA-G].
There are 3 major and 2 minor MHC class II proteins encoded by the HLA.
Major MHC class II HLA-DP: . HLA-DQ: The last one is HLA-DR:
. The other MHC class II proteins, DM and DO, are used in the internal processing of antigens, loading the antigenic peptides generated from pathogens onto the HLA molecules of antigen presenting cell
Split antigen:
Modern HLA nomen- clature are begin with HLA- and the locus name, then * and even number of digits specifying the allele. The first two digits specify a group of alleles.
The third through fourth digits specify a synonymous allele. Digits five through six denote any synonymous mutations within the coding frame of the gene. The seventh and eighth digits distinguish mutations outside the coding region.
Impact of HLA typing on transplantation outcome
HLA mismatches may occur at antigenic or allelic level; a better HLA match is associated with significantly better patient and graft survival while HLA mismatches have been significantly associated with increased risk of acute rejection, HLA DR mis-match associated with higher risk of rejection and poor graft survival especially in the first 6 months post transplantation
While the HLA-B mismatch effect emerges in the first 2 years, and HLA-A mismatches have a deleterious effect on long-term graft survival.
Techniques:
1- serological
2-molecular like:
SSP
SSOP
SBT
RSCA
Classify HLA antigens
What is meant by split antigen?
How can HLA mismatch influence the outcome of transplantation?
In your own words, summarize the HLA typing techniques
1-Types of antigens in relation to organ transplantation
· Major histocompatibility molecules (MHC)
· MHC Class 1, all nucleated cells There are 3 major and 3 minor MHC class I genes in HLA: HLA-A, HLA-B and HLA-C; minor genes are HLA-E, HLA-F and HLA-G
·MHC class11 have five loci: HLA-DR, -DQ, -DP, -DM.
Both MHC antigens class1 ,11 is Highly polymorphic with allelic diversity and HLA matching for the MHC molecules by using serological assay and molecular genetic assay is important in sold organ transplantation including kidney and bone marrow transplantation.
·Minor histocompatibility antigens refer to non-MHC antigens trigger rejection, like MHC class 1 related chain (MCIA) antigen its polymorphic and development of anti MCIA AB found to be associated with worse graft survival.
· ABO blood group antigens.
· Endothelial cell antigens.
2- What is meant by split antigen?
The new HLA nomenclature are begin with HLA- and the locus name, then number of digits specifying the allele. The first two digits specify a group of alleles, with the new immunogenetic technique they can identify the allele variants up to 9 digits.
HLA typing and its influence on organ transplantation:
Matching of donor and recipient for MHC antigens showed a significant positive effect on graft acceptance. HLA mismatches may occur at antigenic or allelic level; a better HLA match is associated with significantly better patient and graft survival while HLA mismatches have been significantly associated with increased risk of acute rejection, HLA DR mis-match associated with higher risk of rejection and poor graft survival especially in the first 6 months post transplantation
While the HLA-B mismatch effect emerges in the first 2 years, and HLA-A mismatches have a deleterious effect on long-term graft survival.
HLA-DR mismatches were a significant risk factor for posttransplant non-Hodgkin lymphoma and hip fracture. Sensitization of the recipient at the B-cell level and development of IgG antibodies still represent a major problem in kidney transplantation.
Techniques of tissue typing
·Serological assay, CDCXM:
Used for screening the donor anti HLA-AB in recipients, complement-dependent cytotoxicity (CDCXM for T cells and B cells
·Limitation with this assay is less sensitive for low level of DSA and non-complement HLA AB so can give false negative results, also false positive results due to inaccurate incubation or dilution methods and in the presence of auto anti bodies Also false positive B-cell CDC-XM following treatment with rituximab.
· Antihuman globulin CDC-XM Increased sensitivity over the CDC-XM by detects auto- immunoglobulin M
·DNA based typing methods:
· more sensitive and accurate compared to serologic assay.
· Solid phase immunoassay (SPI), by using enzyme-linked immunosorbent assays (ELISA)
·microbeads, flow cytometry, flow PRA and flow analyzer Luminex with bead base assay (SAB)., CELL based assay for T CELLS, B CELLS (Luminex-SAB) are more sensitive than ELISA by 10%.
Advantage of FCXM:
· Flow cytometry crossmatch Sensitive for low titer antibody, detects non-complement binding antibodies
·Both T and B cells can be separated to detect HLA class1,11 specific antibodies
Limitation of FCXM
May exclude patients unnecessarily
Specificity for human leucocyte antigen antibodies is low
May be falsely positive in patients having previously received monoclonal antibodies like rituximab.
·Virtual crossmatch using single antigen beads:
Its more sensitive help in detection of acceptable and unacceptable
Donor antigens
May be performed with stored sera therefore shortening cold
ischemia time
Improves donor allocation for highly sensitized patients, DD allocation program and shorting the waiting list
Improves risk assessment for rejection
Limitation of virtual-FCXM
a false positive result, in the presence of denatured human leucocyte antigens on the beads
Requires more coordination between immunology lab
personnel and transplant team.
CONCLUSION:
The new advances in the DNA molecular immunogenetic crossmatch techniques and human leukocyte antigen (HLA) typing play a crucial role to ensure better organ allocation and provide the recipient with a more favorable match, shortening the waiting list and that improve both the graft and patient survival and allowed for the appropriate use of immunosuppressive therapy with lesser side effects of infection and cancer.
-HLA system located on the short arm of chromosome 6 at 6p21.
–HLA is classified into 3 major and 3 minor classes:
1. Major HLA class I genes are HLA-A, HLA-B, HLA-C, and minor genes are HLA-E, HLA-F, and HLA-G.
2. Major HLA class II genes are HLA-DP, HLA-DQ, HLA-DR, and minor genes HLA- DM, and HLA-DO.
3. MHC class III is a group of proteins belonging to the class of MHC. They are poorly defined structurally and functionally. It contains many genes for different signaling molecules such as tumor necrosis factor and heat shock protein.
– Split antigen is an antigen that has a more refined or specific cell surface reaction relative to broad antigen. It subgroup (Splits) of broad antigen .examples:
B5 Broad (split B51 and B 52)
B12 Broad (Split B44 and B45)
B15 Broad (Split B62 B63 and B75)
-Split antigens matching give a good transplant outcome
– HLA matching showed a significant positive effect on graft acceptance while HLA mismatch has a critical prognostic effect that affects graft and recipient survival. It is associated with increased risks of overall graft failure, death–censored graft failure, and all-cause mortality. The major graft loss comes from HLA-DR and HLA-B.HLA-DR mismatches are important in the first 6 months after transplantation while the HLA-B effect appears in the first 2 years and HLA –A mismatches have a deleterious effect on long–term graft survival.
Techniques of HLA typing:
-Previously, HLA typing was done by serologic method or mixed lymphocyte culture.
-More advanced molecular techniques were developed – DNA –based methods-such as :
1-Sequence-Specific oligonucleotide probe hybridization (SSOP)
2- Sequence-Specific primer amplification (SSP)
3-sequencing-based typing (SBT)
4-Reference strand-based conformation analysis (RSCA).
-In 2013 a new project of 16IHIW demonstrated the benefit of a new project of Next-generation sequencing(NGS).
Effects of HLA-C is debatable. The presence of high levels of anti-HLA-C Ab is not innocent and can be associated with adverse graft outcome. HLA-C become more significant in haematopoietic stem cell transplantation.
Classify HLA antigen?
Class I has 3 major genes :A,B and C and 3 minor :E, F and G. Class II has 3 major protins :DP,DQ and DR, which are encoded at multiple loci.minor protin of class II are DM and DO
What is meant by split antigen?
in the testing of cell surface antigens, an antigen that has a more refined or specific cell surface reaction relative to a broad antigen eg B51,B52 are split of B5
How can HLA mismatch influence the outcome of transplantation?
HLA molecules present the peptides toT cells so as to differentiate self from non self . the foreign HLA from the doner are recognized as non self by the recipient ending with graft rejection .so in transplant we look for mismatch.this mismatch can occur due to amino acid substitution at the level of antigens or alleles. Mismatch at Dr affect the first 6 month , mismatch at B affect the first 2 years while A mismatch affect the long term survival
In your own words, summarise the HLA typing techniques
1. Serology: with antiserum and mixed lymphocyte culture , antibody-dependent cell-mediated cytotoxicity (ADCC),or complement-dependent cytotoxicity (CDC).
2. Molecular (more sensitive) : assequence-;specific oligonucleotide probe hybridization (SSOP), sequence-specific primer amplification (SSP), sequencing-based typing (SBT), and reference strand-based conformation analysis (RSCA), nextgeneration sequencing (NGS), short tandem repeat (STR) genotyping
Antibodies screening through complement-dependent cytotoxicity (CDC), is important to avoid hyper acute rejection. This was replaced recently with solid-phase immunoassays (SPI):ELISA and bead-based technology (flow cytometry: Flow PRA and Flow Analyzer-Luminex).
Split antigen defined as an antigen has more different specific cell surface reaction relative to broad antigen.
The studies proved that any increase in HLA mismatch is strongly associated with very high risk of graft loss, death censored graft failure & all cause mortality.
HLA system is a complex genes located on chromosome 6. It is present in all nucleated cells. HLA classified into class I ( including HLA-A, -B & – Cw) & class II ( including HLA-DR, -DQ, & -DP). Assessment of HLA class I&II matching is very important in transplantation especially in renal & BM transplant.
MHC classified into class I & II. MHC I gene classified into major ( HLA-A, -B, & -C) & minor ( HLA-E, -F & -G) & MHC II also classified into major ( HLA-DR, -DQ & -DP) & minor (HLA-DM & -DO).
The function of HLA is to present an Ag to T cells( CD4 & CD8) to determined & elimination of foreign Ag.
HLA tissue typing done by :
(a) Ab dependent cell-related cytotoxicity.
(b) complement -dependent cytotoxicity .
2 DNA based HLA typing:
(a) sequence -specific oligonucleotide probe hybridization ( SSOP)
(b) Sequence- specific primer amplification ( SSP)
(c) Sequence-based typing ( SBT)
(d) Reference strand based confirmation (RSCA)
(e) Next generation sequencing (NGS)
(f) Short Tandem Repeat (STR).
After typing of HLA , the anti- HLA Abs calculation by CDC, flow cytometry, or Luminex.
Dear All
Thank you for your answers. HLA C role is highly controversial. HLA C mismatch could play a role on the following conditions:
– MHC class I gene has 3 major and 3 minor genes.
– MHC class II has 3 major and 2 minor genes
– HLA CLASS I : A,B,C – then minor E,F ,and G
– HLA class II: DR,DQ.DP
– HLA has the highest polymorphism, hence cross matching has been a mandatory step in pre-transplantation workup
– There are different methods for tissue typing mainly serological (ADCC OR CDC) and mixed lymphocyte culture, with the latter more accurate.
HLA class I present in all nuclear cells and consist of 3 classical loci HLA-A ,HLA -B and HLA-C and 3 minor genes E,F and G while HLA class II present mainly in Ag presenting cells like macrophages and dendritic cells it consists of 3 major and 2 minor loci HLA-DP,DQ AND DR and the minor are DM and DO .
HLA match prior to transplant give better result and long term graft survival the most important Ags that play role in transplant outcome are HLA -DR have high risk of graft failure in the first 6 months in mismatch while HLA -B mismatch effect till 2 years and HLA -A contributes in long term outcomes.
HLA -C mismatch cause acute rejection and alot of cases have show it’s effect on graft survival.
1- HLA Ag are classified as
a- HLA class I:
Major classes : HLA-A, B,C
Minor classes :HLA E, F, G
b- HLA class II:
Major classes are HLA DR, DQ, DP
Minor classes are DM, DO
2- Split Ag is an antigen that has a more refined or specific cell surface reaction relative to a broad antigen
3- The type and extent of HLA mismatch affects the gragt outcome .poor outcome occurs with mismatch in Dr ( mainly affect short term graft survival )then B and A (maily affect long term graft survival) Also the higher degree of HLA mismatch , the worse graft outcome
As recipient T cells will recognize the mismatched HLA molecule on the graft via direct , indirect and semidierct alloreactivation pathways with subsequent activation of B lymphocytes and production of Ab , enhancing recrtiutment of inflammatory cells and production of various cytokines and chemokines ending in graft injury and rejection .
Summary
HLA system is the most biologic system in human being .Itis controlled by genes present on chromosome 6 and is classified into HLA class I including 3 major (HLA A,B,C)|and 3 minor (HLA E,F,G)|and class II including 3 major (HLA DR, DQ,DP) and 2 minor (DM ,DO). HLA system presents the Ags to both CD4 and CD8 T cells to recognize and eliminate forign antiges , they also important for inducing self tolerance.
HLA typing is done by two methods either
1- serological method by mixing recipient serum with donor lymphocytes , but it is less accurate technique , including
a- antibody-dependent cell-mediated cytotoxicity (ADCC),
b- complement-dependent cytotoxicity (CDC)
2- molecular method : a DNA-based technique such as
· SSOP :specific oligonucleotide probe hybridization
· SSP: sequence-specific primer amplification
· SBT : sequencing-based typing
· RSCA :reference strand-based conformation analysis (RSCA)
3- Newer techniques as : nextgeneration sequencing (NGS ) and short tandem repeat (STR) genotyping.
For antibodies screening : either
a- Serological method as CDC crossmatch
b- solid-phase immunoassays (SPI) as ELIZA and bead-based technology such as flow cytometry:
Flow PRA and Flow Analyzer-Luminex).It is better to combie two techniques for better confirmation of of the results .
In a study in UK formation of anti-HLA Abs was studied in candidate patients for retransplantation and its relationship with each donor’s HLA locus mismatches was investigated. Mismatches of all HLAs were effective in forming anti-HLA Abs with different hazard ratios. The order of them was as following: HLA DRB3/4/5→3.9, HLA DRB1→3.5, HLA B→3.4, HLA A→3.2, HLA-DQ→3 and HLA-C→2.5. So, mismatches of HLA-C was associated with the lowest antibody formation.
Kosmoliaptsis, V., Gjorgjimajkoska, O., Sharples, L. D., Chaudhry, A. N., Chatzizacharias, N., Peacock, S., Torpey, N., Bolton, E. M., Taylor, C. J., & Bradley, J. A. (2014). Impact of donor mismatches at individual HLA-A, -B, -C, -DR, and -DQ loci on the development of HLA-specific antibodies in patients listed for repeat renal transplantation. Kidney International, 86(5).
MHC class I is classified into 3 major and 3 minor genes
Major genes are A,B,C
Minor genes are E,F ,G
MHC class II is classified into 3 major and 2 minor genes
Major are DR DP DQ
Minor genes are DM ,DO
Split Ag is an antigen that has a more refined or specific cell surface reaction relative to a broad antigen.
HLA mismatch triggers immune response as B and T cells recognise foreign donor antigens leading to graft loss .
B and DR mismatch is more important than rest of MHC antigens
DR mismatch results in deleterious effects in 1st 6 months
B mismatch effects appear more than 2years post transplant .
A mismatches affect long term graft outcome.
**Methods of HLA typing **
1) Serological methods is done with either ADCC , CDC
2) cellular assays are done using mixed lymphocyte culture , which is more sensitive than serological tests
That’s because minor differences can be detected by cells more than Abs
3) molecular assays
Done using sequence specific oligonucleotide or sequence specific primer
4) Sequencing-based typing (SBT) is a high-resolution
method for the identification of HLA polymorphisms
1- HLA classification ;
There are 3 major MHC class I genes in HLA:
HLA-A
HLA-B
HLA-C
There are3 minor MHC class 1 genes in HLA ;
HLA-E
HLA-F
HLA-G.
β2- microglobulin binds with major and minor gene subunits to produce a heterodimer.
There are 3 major and2 minor MHC class II proteins encoded by the HLA.
The genes of the class II combine to form heterodimeric (αβ)
protein receptors that are typically expressed on the surface of antigen-presenting cells
Major MHC class II HLA-DP: α-chain encoded by HLA-DPA1 locus and β-chain encoded by HLA-DPB1 locus. HLA-DQ: α-chain encoded by HLADQA1 locus and β-chain encoded by HLA-DQB1 locus.
Minor MHC class II;
DM
DO
2- Split antigen;
Antigen that has more refined or specific cell surface reaction relatives to broad antigen ,example; HLA-B51and HLA-B52 are split antigen of HLA b5.
3- Influence of HLA mismatch on transplant outcome;
HLA mismatch adversely affect kidney transplant out come and it depends on the degree of mismatch and the gene locus .It is associated with increased incidence of malignancy ,death with censored functioning graft. In transplantation immunology, the major impact in graft loss comes from the effects of HLA-B and -DR antigens .HLA –C was found to be associated with decreased graft survival in sensitized recipient.
The effects of HLA-DR mismatches are the most important in the first 6 months after transplantation, the HLA-B effect emerges in the first 2 years, and HLA-A mismatches have a deleterious effect on long-term graft survival .
4- HLA typing techniques;
1- Serological method;
using antiserum and mixed lymphocyte culture (MLC). can be made by antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC)
2- Cellular assay;
In which lymphocyte culture is used to determine the HLA class II types .It is more sensitive in detection minor HLA disparities which can trigger T-cells.
2- DNA-based HLA typing methods using molecular techniques such as;
1- sequence -specific oligonucleotide probe hybridization(SSOP)
2- sequence-specific primer amplification (SSP)
3- sequencing-based typing (SBT)
4-reference strand-basedconformation analysis (RSCA).
DNA based method had more sensitivity, accuracy and resolving power than Sequencing-based typing (SBT) method for the identification of HLA polymorphisms.
3- Next generation sequencing (NGS) .
Which combines clonal amplification.
4- short tandem repeat (STR) genotyping provided additional information allowing determination of the extent of HLA identity in families where HLA haplotype inheritance was ambiguous, due to extensive homozygosity or shared parental haplotypes. The HLA STR assay is a reliable and rapid test that used inexpensively screen potential sibling donors for HLA identity .
HLA antigens classification
Generally talking The HLA system includes a complex arrangements of genes located on chromosome number 6. The system is highly polymorphic
MHC class I proteins present on all nucleated cells of the body: There are 3 major genes: HLA-A,HLA-B and HLA-C and 3 minor genes are HLA-E, HLA-F and HLA-G
MHC class II proteins typically present on the surface of antigen-presenting cells: There are 3 major genes :HLA-DP , HLA-DQ and HLA-DR and 2 minor genes :HLA-DM, HLA DO
Split antigens: splits means division of a single antigen into subtypes; an antigen that has a more specific cell surface reaction relative to a broad antigen (Example: HLA-B5 Broad antigen splits to HLA-B51 and HLA-B52 )
HLA matching has a direct effect on graft loss. The effects of HLA-DR mismatches are the most important in the first 6 months after transplantation, the HLA-B effect appears in the first 2 years, and HLA-A mismatches have a unfavourable effect on long-term graft survival.
Other types of antibodies that directed against non-HLA antigens are important like against graft endothelium, MICA and Angiotensin type 1 receptor
Techniques of tissue typing
HLA typing have been done by two methods:
-serologic method using antiserum and mixed lymphocyte culture (MLC): can be made by antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC)
-DNA-based HLA typing methods using molecular techniques: DNA based method had more sensitivity, accuracy and resolving power than serologic typing methods, Sequencing-based typing (SBT) is a high-resolution method for the identification of HLA polymorphisms.
Classification of HLA antigens
HLA (human leucocyte antigen) are encoded for by genes on chromosome 6 :
MHC class I proteins form a functional receptor on most nucleated cells of the body.
There are 3 major and 3 minor MHC class I genes in HLA:
The genes of the class II combine to form heterodimeric (αβ) protein receptors that are typically expressed on the surface of antigen-presenting cells.
There are 3 major and 2 minor MHC class II proteins encoded by the HLA:
Split Antigen:
Ag that has a more refined or specific cell surface reaction relative to abroad antigen.
In the testing of cell surface antigens, an antigen that has a more refined or specific cell surface reaction relative to a broad antigen.(Example: HLA-B51 and HLA-B52 are split antigens of HLA-B5)
How can HLA mismatch influence the outcome of transplantation:
In a meta-analysis to evaluate the magnitude effect of HLA mismatching on post-transplant survival outcomes of adult kidney transplantation. The analysis included 23 studies with a large sample of subjects (totally 486,608 recipients). The results indicated that each incremental increase of HLA mismatches was significantly associated with higher risks of overall graft failure, death-censored graft failure and all-cause mortality. The pooled results also indicated that HLA-DR mismatches were significantly associated with a 12% higher risk of overall graft failure. Also observed that HLA-A per mismatch was associated with a 6% higher risk of overall graft failure, but the association was insignificant. There was no significant association between HLA-B mismatching and graft survival. All included studies were in high methodological quality and the heterogeneity between studies was acceptable in each pooling analysis.{What is the impact of human leukocyte antigen mismatching on graft survival and mortality in renal transplantation? A meta-analysis of 23 cohort studies involving 486,608 recipients Xinmiao Shi, Jicheng Lv, Wenke Han, Xuhui Zhong, Xinfang Xie, Baige Su & Jie Ding BMC Nephrology volume19, Article number: 116 (2018)}
Primary outcome overall graft failure
Secondary outcomes
Each incremental increase of HLA mismatches was associated with a higher risk of death-censored graft failure, with summary HR of 1.09 (95% CI: 1.06–1.12; P < 0.001; and moderate heterogeneity (I2 = 70.9%).{What is the impact of human leukocyte antigen mismatching on graft survival and mortality in renal transplantation? A meta-analysis of 23 cohort studies involving 486,608 recipients Xinmiao Shi, Jicheng Lv, Wenke Han, Xuhui Zhong, Xinfang Xie, Baige Su & Jie Ding BMC Nephrology volume19, Article number: 116 (2018)}
Each incremental increase of HLA mismatches was associated with a higher risk of all-cause mortality rates (HR: 1.04; 95% CI: 1.02–1.07; P = 0.001;The heterogeneity was moderate (I2 = 65.3%). Summary estimates did not changed significantly after analyzing with a fixed-effects model (HR: 1.04; 95% CI: 1.02–1.07; P = 0.001). After stratification for sample size of cohorts (≥10,000 vs < 10,000), the effect estimates were not modified (HR: 1.04; 95% CI: 1.02–1.05; P < 0.001; I2 = 27.8%, Additional file 8: Fig. S4). However, the results should be cautiously interpreted due to small number of included studies (n = 4)..{What is the impact of human leukocyte antigen mismatching on graft survival and mortality in renal transplantation? A meta-analysis of 23 cohort studies involving 486,608 recipients Xinmiao Shi, Jicheng Lv, Wenke Han, Xuhui Zhong, Xinfang Xie, Baige Su & Jie Ding BMC Nephrology volume19, Article number: 116 (2018)}
HLA typing techniques:
Tissue typing using serological assay can be made by:
The pioneer method to detect such antibodies is complement-dependent cytotoxicity (CDC).it has been gradually replaced by more-sensitive solid-phase immunoassays (SPI) such as the enzyme-linked immunosorbent assay and the bead-based technology (i.e., flow cytometry: Flow PRA and Flow Analyzer-Luminex).
Tissue typing by molecular method utilizing the followings:
Technologies has been in routine use in many tissue-typing laboratories worldwide for more than 20 years since the
development of the poly- merase chain reaction. Both methods(SSO) & (SSP) are very useful for clinical and
research purposes and can provide generic (low resolution) to allelic (high resolution) typing results.
3. DNA based method had more sensitivity, accuracy and resolving power than serologic typing methods.
A comprehensive list of recommendations is provided covering the technical and pretransplantation and posttransplantation monitoring of HLA antibodies in solid organ transplantation:
The recommendations are intended to provide state-of-the-art guidance in the use and clinical application of recently developed methods for HLA antibody detection when used in conjunction with traditional methods.
1. Classify HLA antigens
We have 3 classes of HLA antigens.
I, II, and III
1-MHC class I: we have three major
and three minor MHC class I genes in HLA.
**Major MHC class I: HLA-A, HLA-B,
and HLA-C
*Minor MHC class I: HLA-E, HLA-G,
and HLA-F
2-MHC class II: We have three major
and two minor MHC class II proteins.
**Major MHC class II: HLA-DP,
HLA-DQ, and HLA-DR (DRB1,2,3&5)
*Minor
MHC class II: HLA-DM and HLA DO
3-Class
3: not essential in transplantation. examples(C2, C4A, C4B, PF, TNF- a, b)
2. What is meant by split antigen?
HLA antigen classifications have
evolved. recent technology has allowed the detection of differences
in antigens “split” categories that were previously indistinguishable as broad
categories. For example A203#, A210# are spits
of the original HLA-A2
Here is a list of original broad
and correlated splits
http://hla.alleles.org/antigens/broads_splits.html
3. How can HLA mismatch influence the outcome of
transplantation?
HLA match is essential for better
transplant outcomes. Conversely, the mismatch is a risk for rejection. The
degree of mismatch means more need for immunosuppression with high
dose-dependency and accordingly higher risk of infections and malignancy.
In renal & bone marrow
transplantation class I&II are more important while in lung and heart
transplantation the DR is of importance.
4. In your own words, summarise the HLA typing techniques
HLA typing was performed by a serologic method using antiserum and cellular mixed lymphocyte culture (MLC). With advanced technology and revolution in PCR technology molecular techniques evolved.
Serological assays can be made by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)
In cellular technique mixed culture of lymphocytes is utilized. This is used to determine class II HLAs.
Molecular methods which are popular today are useful for high-resolution reporting of HLA antigens. Sequence-specific oligonucleotide (SSO) and sequence-specific primer (SSP) are routine in most laboratories. Here both low resolution and high-resolution HLA typing is possible. Another technique is sequencing-based typing (SBT). İn this high-resolution method the polymorphism is detected.
HLA molecules are important in kidney transplantation which are contributing in rejection .HLA class I loci are HLA- A ,HLA-B , HLA -C and five HLA class ll are :HLA -DR ,HLA -DP ,HLA-DQ,HLA-DM and HLA-DO .There are two Nomenclature system for HLA .The first one is based on serology that consist ,letters and numbers. The second modern one contains gene name and maximum 8 digits which show alleles. Two first did it’s are Ag or allele from ,Third and fourth for specific HLA allele, fifth and sixth for silent substitute and seventh and eight digits for non-loading substitution molecules and letters like L,N,Q,S. HLA system has high polymorphism and number of their alleles are increasing. Since HLA molecules present foreign antigens to T cells, their mismatches can be recognized by recipient’s immune system and induce alloimmune reaction .So full matching helps graft acceptance. Matching of HLA-DR is important and then HLA-B especially during the first 6 month and 2 years, respectively. HLA-A mismatches may influence long-term graft survival.
2. HLA typing by serological approach has defined the serological subgroup (or broad). By more precise methods such as DNA typing methods HLA is defined as split antigens. Each old broad antigen may have two or more antigens. Split antigens are important in transplantation because of immune system activation.
3. HLA mismatches are important in graft survival. Number of mismatches between donor and recipient’s HLA for six antigens of HLA-A,-B and –DR are usually considered in kidney transplantation.
4. HLA typing methods:
1-Serological typing by antiserum and mixed lymphocyte culture (MĹC) made by ADCC or CDC.
2- DNA typing methods by molecular methods. Different techniques are using like: SSOP, SSP by PCR SSB1, RSCA. SSO and SSP developed based on PCR and can provide both low and high resolution typing results. But SBT only provide high- resolution results. High-resolution results may provide more information especially in sensitized patients.
3. Next- generation sequencing (NGS) through combination of clonal amplification.
4. Short tandem repeat (STR) genotyping allows identification of the extent of HLA in families with unknown HLA haplotype inheritance used for screening of sibling donors.
5. In HLA matched sibling interaction between patients HLA ligand and donor killer–ell immunoglobulin -like receptor (KlR) genes were important in their outcome. So KIR genotyping could be helpful.
HLA class I proteins are expressed on surface of all nucleated cells. There are 3 major and 3 minor MHC class I genes in HLA: HLA-A, HLA-B and HLA-C; minor genes are HLA-E, HLA-F and HLA-G.
HLA class II proteins are typically expressed on the surface of antigen presenting cells. There are 3 major and 2 minor MHC class II proteins encoded by the HLA.
Major MHC class II HLA-DP: α-chain encoded by HLA-DPA1 locus and β-chain encoded by HLA-DPB1 locus. HLA-DQ: α-chain encoded by HLADQA1 locus and β-chain encoded by HLA-DQB1 locus. The last one is HLA-DR: α-chain encoded by HLA-DRA locus and four β-chains (only 3 possible per person), encoded by HLA-DRB1, DRB3, DRB4, and DRB5 loci. Other HLA class II proteins, DM and DO, are used for internal processing of antigens that are generated from pathogens.
HLA antigen classification has allowed the detection of differences in antigens (split categories) that were previously indistinguishable (broad categories). For example: A9 Broad (splits A23 and A24)
The role of HLA molecules is to present peptides to T cells to recognize and eliminate non self-antigens.
When a transplantation is performed, HLA molecules of the donor are recognized by the recipient’s immune system and provoke an alloimmune response.
The adaptive response is the main response to the HLA molecules expressed on the surface of donor cells. T cell activation leads to the production of cytokines and chemokines and then recruitment of innate immunity.
HLA mismatch correlates with increased probability of graft loss. The effects of HLA-DR mismatches are the most important in the first 6 months after transplantation, the HLA-B effect emerges in the first 2 years, and HLA-A mismatches have a deleterious effect on long-term graft survival.
In addition, an increasing numbers of HLA mismatches of the first transplant, is associated with a significant increase in panel reactive antibody.
Previously, HLA typing was done by serologic method and MLC. Nowadays more precise DNA-based methods have been developed, such as SSPO, SSP, SBP, and RSCA.
Next generation sequencing (NGS) has ability to sequence larger regions of genes, including introns. short tandem repeat (STR) genotyping provided additional information allowing determination of the extent of HLA identity in families where HLA haplotype inheritance was ambiguous, and provides a reliable test for screening of potential sibling donors for HLA identity.
Serological assay, can be made by antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). The cellular assay is more sensitive in detecting HLA differences than serotyping and the concern about it is a mixed lymphocyte culture and determination of the HLA class II.
Sequence specific primer (SSP) and sequence-specific oligonucleotide (SSO) technologies can provide generic (low resolution) to allelic (high resolution) typing results. Sequencing-based typing (SBT) is a high-resolution method for the identification of HLA polymorphisms. Examination of all nucleotides, both at conserved and polymorphic positions, enables the direct identification of new alleles, which may not be possible with techniques such as SSP and SSO typing.
DNA based methods have more sensitivity, accuracy and resolving power than serologic typing methods.
Utilization of solid phase immunoassays makes it possible to detect antibodies to HLA loci such as Cw, DQA, DPA, and DPB which are not readily detected by other methods. The new techniques have been associated with decreased specificity, and some non-HLA antigens with no clinical relevance have been able to give a positive cross match.
The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive crossmatch
Glow in HLA typing in organ transplantation
MHC class 1 has been found in all uncleared cells of the body. It has 3 major and 3 minor MHC. HLA -A,B,C are major and HLA-E,F,G are minor. Beta 2 microglobulin binds with major ot minor gene unit to produce heterodimer.
MHC class 2 encoded by 3 major and 2 minor HLA. They combine to form alpha-beta heterodimers, which typical present on APCs.
Nomenclature
Two systems
1. Serological based recognition : antigens assigned letters and numbers ( HLA B-51/B51)
a. Could not do specify alleles more, stop at 2 digits
2. Modern HLA nomenclature: begin with HLA- ,then locus name
a. 1st , 2nd digits – group of alleles
b. 3rd,4th digits – synonymous allele
c. 5th, 6th digits-any synonymous mutations within the coding frame of the gene
d. 7th , 8th digits- distinguishes the mutations outside the coding frame
e. Letter L,N,Q or S – to specify an expression level or other non-genomic a data known about it
Every 2 years – nomenclature is put forth to aid in interpreting serotypes to alleles
Variability
• HLA- highly polymorphism of all human genetic systems
• Polymorphism due to gene conversion between variable alleles and loci within each HLA gene
• HLA allele’s increase in number at about 15% in the high expression loci( A,B,C and DRB1) and 30% in the low expression loci (DRB3/4/5, DQB1,DPB1)
HLA typing and its influences on organ transplantation
HLA molecules present peptides to T cells ( CD4 and CD8 T cells) to enable recognition and eliminate foreign particles
HLA molecules also prevents the recognition if self as non self
HLA mismatch can occur at level of:
• antigenic
o characterised by amino acid substitutions in both peptide binding and T-cell recognition regions
• allelic
o Characterised by amino-acid substitutions in peptide binding region only
Alloimmunity is triggered when a donors kidney is transplanted via direct or indirect methods
Adaptive immunity is the important response exerted to the “foreign” tissue since the main target of immune response is MHC molecules expressed on the surface of donor cells
Activation of T cell stimulate production of cytokines and chemokines then it may recruit components like NK cell or macrophages or complement ( innate immunity) . Defensins and cathelicidin have chemoattractant properties on T lymphocytes
HLA DR mismatches has impact on graft survival during first 6 months
HLA B mismatches has impact on graft survival for first 2 years while HLA A-mismatches has deleterious effect on long term graft survival
Techniques of tissue typing
Classically HLA typing done by serological method using anti-sera and mixed lymphocyte culture (MLC), but now DNA based HLA typing methods using molecular techniques are more accurate
• Sequence specific primer amplification (SSP)
• Assequence-; specific oligonucleotide probe hybridisation
• Sequencing based typing
• Reference strand based conformation analysis (RSCA)
• Next generation sequencing (NGS)
• Short tandem repeat (STR)
Serological method
Antibody-dependent cell-mediated cytotoxicity (ADCC)/complement-dependent cytotoxicity (CDC)
ADCC
• cytotoxic destruction of antibody- when an antibody bound to the surface of a cell interacts with Fc receptors on NK cells or macrophages then coated target cells by host cells is triggered
• Also used in screening for HLA antibodies in the recipient – important because they reactive with lymphocytes of a prospective donor (cross matching)
Cellular assay
• used to determine the HLA class II types
• is more sensitive in detecting HLA differences than serotyping- because minor differences unrecognized by alloantisera can trigger T cells
Molecular method
SSO/SSP
• useful for clinical and research purposes and can provide generic to allelic typing results
• more sensitive, accurate and resolving power
SBT
• identification of HLA polymorphisms using high resolution methods
• most of the HLA Class I alleles can be determined by their exon 2 and 3 sequence and for Class II alleles, exon 2 is usually sufficient
• Examination of all nucleotides(at conserved and polymorphic positions) enables the direct identification of new alleles, which may not be possible with techniques such as SSP and SSO typing
Complement-dependent cytotoxicity (CDC)
• very important to identify recipient anti-HLA antibodies to antigens expressed on donor with blood cells
• now it is replaced by more sensitive solid-phase immunoassays (SPI) such as the enzyme-linked immunosorbent assay and the bead-based technology ( flow cytometry: Flow PRA and Flow Analyzer-Luminex)
• The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive XM
• technical factors such as variation in antigen density and the presence of denatured antigen on the beads can alter the results and clinical interpretation when using the Luminex bead technology
New Techniques are associated with decreased specificity, and some non-HLA antigens with no clinical relevance have been able to give a positive cross match
These “false-positive” antibody results have a consequence of decreasing a chance of the patient to receive KT
Study showed that cold ischemia up to 18 hours was found not to be deleterious for graft outcome, and short cold ischemia did not mask effect of HLA matching and HLA-DR mismatches were a significant risk factor for posttransplant non-Hodgkin lymphoma and hip fracture
Another study found that with increasing numbers of HLA mismatches of the first transplant associated with significant increase in PRA. It also showed influence of HLA matching on graft outcome in patients with pretransplant HLA antibodies or high serum levels of the T-cell activation marker sCD30
Even though graft survival rates improved dramatically , sensitization of the recipient at the B-cell level and development of IgG antibodies still a major problem in kidney transplantation .HLA antibodies of the IgG, IgM, or IgA isotypes were detected in almost all patients waiting for transplant
Other antibodies that directed against non-HLA antigens such as against graft endothelium, MICA and Angiotensin type 1 receptor are increasingly detected.
Classification of HLA classes:
HLA system complex it is encoded by genes located on short arm of chromosome 6.
HLA molecules are the major trigger for immune response against foreign oragan .
They are classified into 2 MHC proteins; Class I, Class II
Class I ; is expressed on surface of all nucleated cell it has 3 major HLA types A, B and C, and 3 minor E, F, and G.
Class II; is expressed only on surface of antigen presenting cells it include 3 major types DR, DP and DQ and 2 minor DM and DO.
Split antigens
Spliting antigens into smaller specificities this spliting of HLA antigen matching improved transplant outcome more thsn matching of with broad HLA antigens.
Old serological methods for HLA typing detected HLA genes with allele group but can’t detect specific alleles so broad HLA antigen has a poor specificity.
Recent DNA based techniques allowed better specification and sequencing of the polymorphic regions of genes and identify specific alleles encoding for specific HLA proteins.
similar broad antigen may have different split antigens, and this minor differences may initiate immune response and cause rejection.
There for matching in split antigens has a better transplantation outcomes than matching on broad antigen.
How can HLA mismatch influence the outcome of transplantation?
Transplantation with no mismatch was associated with the best results and the most favourable outcoms not only on patient and graft survival but also on reduction of immunosuppression with all its complications.
Transplantation between homozygous twins showed excellent results
not all HLA antigens have the same effect as mismatch in HLA -DR AND HLA-B have the main effect of graft survival
The effects of HLA-DR mismatches usually appears in first 6 months after transplantation and they were a significant risk factor for posttransplant non-Hodgkin lymphoma and hip fracture .
while HLA-B effect appears in the first 2 years, and HLA-A mismatches effects appears on long-term graft survival
Number of mismatchs in first transplantation lead to significant increase in panel reactive antibody which will affect the next transplantation.
Therefore selection of matched doner is the first step to successfull long term graft survival.
Despite the major effect of HLA antibodies on outcomes of transplantation but also other non HLA antibodies like against graft endothelium , MICA and Angioten-sin type 1 receptor that can affect the graft such effect can be abolished by peritransplant apheresis .
HLA typing techniques
In the past HLA typing was performed using serological methods
Serological method was performed by using known antisera Antibodies to specific HLA antigens to be mixed with lymphocytes culture it can be ADCC OR complement mediated cell lysis.
Cellular method using mixed lymphocyte culture to determine the HLA class II. it is more sensitive in detecting HLA differences than serotyping as minor differences cant be detected by antisera can stimulate T cells.
molecular techniques useing DNA-based for HLA typing include
sequence-specific oligonucleotide probe hybridization (SSOP)
sequence-specific primer amplification(SSP) sequence-based typing (SBT)
reference strand-based conformation analysis (RSCA)
Next-generation sequencing (NGS).
short tandem repeat (STR) genotyping which is reliable test to screen for sibling doner
molecular method, usinf the sequence-specific oligonucleotide (SSO) and sequence-specific primer (SSP) technologies have been used for many years and it can provid high resolution typing (detect specific alleles) or low resolution (generic typing)
While Sequencing-based typing (SBT) is a high-resolution method
In general DNA typing is more sensitive and specific than serotyping
HLA antigens :
Class I antigens : 3 major (classical) Ag : A,B,C , 3 minor (nonclassical) : E,F,G
Class II Ag : 3 major: DR, DQ, DP, 2 minor : DM , DO .
Split antigen :
With the advance of serological methods of HLA typing , they found certain antigen can be divided into many antigen serologically, so they called the parent antigen broad antigen and the splitted antigens split antigen . Nowadays they put the broad antigen between brackets after the split antigen since matching to the split antigen level is associated with better graft outcome.
The HLA mismatch in kidney transplantation is associated with lower graft and patient survival also associated with increased risk of acute rejection and DGF and the use of high doses of immunosuppressive drugs and higher risk of malignancy and serious infections and higher risk of hip fractures.
HLA DR mismatch is most important in the first 6 months after transplantation, HLA B mismatch in the first 2 years post transplantation and HLA A mismatch in the long term graft survival.
HLA typing techniques :
HLA-C mismatch was found to be associated with reduced graft survival in only those who are presensitized and not in those who are not presensitized.(1)
40 – 50 % of transplant recipient have preformed anti HLA-C antibodies in the serum, Patients with high level of pretransplant HLA-C DSA are more prone to develop ABMR during first post transplant year.(2)
REFERANCES
1- Tran TH, Döhler B, Heinold A, et al. Deleterious impact of mismatching for human leukocyte antigen-C in presensitized recipients of kidney transplants. Transplantation 2011; 92:419.
2- Ling M, Marfo K, Masiakos P, et al. Pretransplant anti-HLA-Cw and anti-HLA-DP antibodies in sensitized patients. Hum Immunol 2012; 73:879.
1- HLA antigens :
there are 2 major groups of HLA antigens, each one of them include further types and subtypes :
2- Split antigens:
split antigen is an antigen having a more refined cell surface markers relateive to the broad antigen e.g HLA-B51 and HLA-B52 are split antigens of HLA-B5
3- HLA mismatch :
4- HLA typing techniques : 2 main methods.
A- serological :
B- molecular :
1- SSOP
2- SSP
3- SBT
4- RSCA
5- STR
1- HLA classification
MajorHistocompitabilityComplex class I genes :
HLA-A
HLA-B
HLA-C
Minor HistocompitabilityComplex class I genes :
HLA-E
HLA-F
HLA-G
Major HistocompitabilityComplex class II :
HLA-DP: α-chain encoded by HLA-DPA1 locus and β-chain encoded by HLA-DPB1 locus.
HLA-DQ: α-chain encoded by HLA-DQA1 locus and β-chain encoded by HLA-DQB1 locus.
HLA-DR: α-chain encoded by HLA-DRA locus and four β-chains encoded by HLA-DRB1, DRB3, DRB4, and DRB5 loci.
Minor HistocompitabilityComplex class II :
DM
DO
2- Split antigen
means division of a single antigen into subtypes according to previously unrecognized differences. Serological identification can be hard if the splits occurs at small numbers . Histocompatibility Committee has established criteria for noticing which splits match themselves and which match the parent antigen too. They depended on the concluded antigens recognised by participating laboratories that should be available during routine donor testing and molecular typing is used to test donors. United Network for Organ Sharing (UNOS )provides a list of antigens that may be entered through UNet and is updated. For example HLA DR Some splits of DR match other splits and some do not.(1)
3- HLA mismatches have a major effect on transplantation outcome as
HLA-DR mismatches are the most crucial drawback in the first 6 months post transplantation,
while HLA-B mismatches are expressed in the first 2 years,
and HLA-A mismatches have a major effect on long-term graft survival which finaly can lead to graft rejection .
4- HLA typing techniques
Serological assay:
Used to screen for recipient’s HLA antibodies By
– Antibody-dependent cell-mediated cytotoxicity (ADCC)
-Complement-dependent cytotoxicity (CDC).
Then updated to more sensitive and specific tests
-Solid-phase immunoassays (SPI) such as the enzyme-linked immunosorbent assay
-Bead-based technology (i.e., flow cytometry: Flow PRA and Flow Analyzer-Luminex).
Cellular assay:
Used to determine types of HLA class II,it is more sensitive in detection of minor HLA disparities which can trigger T cells.
Molecular methodology(DNA based):
It is more sensitive
By using
-sequence-specific oligonucleotide (SSO)
-sequence specific primer (SSP).
-Sequencing-based typing (SBT):a precise technique in determining HLA polymorphism.
-Reference strand-based conformation analysis (RSCA)
Next generation sequencing (NGS) :
Which combines clonal amplification
Short tandem repeat (STR) genotyping:
is an affordable rapid test that can identify HLA
1-Helman S W.etal, Interpretation of HLA Typing Results for Entry into UNet. Organ Procurement and Transplantation Network,Jan 2003.
Classification:
The HLA system is highly polymorphic
There are three major (HLA:A, B, C) & three minor MHC class I gene (HLA-E, F, G)
those genes encode for MHC class I proteins that bind to B2 microglobulin to produce heterodimer and form functional receptor on most nucleated cells of the body
Genes of class II encode for 3 major (HLA-DP, DQ, DR) and 2 minor MHC class II proteins (HLA-DM, DO), combine to form heterodimeric protein receptor expressed on surface of APCs.
Split antigens:
Some HLA types are discovered to be multiple splits (broad antigens split into 2 or more antigen), in some occasions the splits may be coded as the broad antigen (1)
A9 was split to A23 and A24
DR2 was split to DR15 and DR16
HLA matching criteria may vary according to consideration of broad or split antigens
increased utilization of broad antigens increase the probability of identifying an HLA matched recipient for a donor (2)
Influence of HLA mismatch on outcome of kidney transplant:
HLA matching of donor and recipient is associated with better graft acceptance
HLA-B and DR antigens have major effect on graft failure
HLA-DR mismatches are most important in the first 6 months, HLA-B effect appear in the first 2 years and HLA-A mismatches affect the long term graft survival
A study showed increased number of HLA mismatches of first transplant is associated with significant increase of PRA.
HLA compatibility significantly affect outcome of transplant in sensitized (second transplant) recipients even at allele level.
HLA typing techniques
Serological assay:
made by antibody dependent cell mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC)
antigen level, low resolution
allow discrimination between groups of related HLA alleles
Cellular assay:
more sensitive than serotyping in detecting HLA as it detect minor differences that can stimulate T cells
used to determine HLA class II
molecular method:
allow HLA genotyping
Sequence specific oligonucleotide (SSO) use oligonucleotide probes
Sequence specific primer (SSP) use arrays of specific primers
(both provide low level of resolution, don’t discriminate between closely related alleles)
Sequencing based typing (SBT): High resolution typing
Next generation sequencing:
characterize entire HLA genes including exons other than 2 and 3
provide high resolution typing useful when recipient has allele specific antibodies (3)
help in better compatibility assessment, organ allocation and graft survival. (4)
(1) Takemoto, S., Port, F.K., Claas, F.H. and Duquesnoy, R.J., 2004. HLA matching for kidney transplantation. Human immunology, 65(12), pp.1489-1505.
(2) Takemoto S, Gjertson DW, Cecka JM, Terasaki PI: HLA matching for local pools using fewer HLA factors. Transpl Proc 27:675, 1995
(3) P. Vogiatzi, Some considerations on the current debate about typing resolution in solid organ transplantation, Transplant. Res. 5 (2016) 3.
(4) Bravo-Egana, Valia, Holly Sanders, and Nilesh Chitnis. “New challenges, new opportunities: Next generation sequencing and its place in the advancement of HLA typing.” Human Immunology (2021).
*Classification of HLA
class I HLA
3 major
HLA-A,HLA-B and HLA-C
3 minor
HLA-E, HLA-F and HLA-G
Class II HLA
3 major
HLA-DP, HLA-DQ, HLA-DR
2 minor
DM , DO
*Split antigen
antigens are identified using antisera that split HLA antigens into narrower specificities for example HLA-A9 split into HLA-A23 and -A24, and HLA-A10 split into HLA-A25, -A26, -A34, and -A66. splits HLA antigen matching give good transplant outcome if compare to matching of broad HLA antigens
*HLA typing and its influence on organ transplantation
higher degree of human leukocyte antigen (HLA) mismatching is associated with worse transplant outcomes
The initial Collaborative Transplant Study (CTS) analysis showed that the major impact on kidney allograft outcomes came from mismatches for the HLA-DR and -B antigens, with little effect from the HLA-A antigens
Mismatching for HLA-A, -B, and -DR associated with a higher risk of HLA sensitization in both adult and pediatric patients.
HLA-C antigens was associated with decreased graft survival specially in those who are presensitized (panel reactive antibodies [PRA] >10 percent) but not those who are not presensitized. Forty to 50 percent of kidney transplant recipients may have preformed anti-HLA-C antibodies. patients with high levels of pretransplant HLA-C DSA are at high risk for ABMR during the first posttransplant year
major adverse cardiac and cerebrovascular events and high mortality occur in case of Prior sensitization against HLA class II antigens
HLA-DQ — and -DR
Although HLA-DQ is not as polymorphic as HLA-A, -B, and -DR, HLA-DQ mismatches are also associated with an increased risk of rejection and allograft loss in kidney transplant recipients due to development of de novo DSAs
HLA-DP — Anti-HLA-DP antibodies are less common than antibodies to HLA-DR and -DQ, occurring in 5 to 14 percent of transplant recipients but The titer of these antibodies increases in up to 45 percent patients with history of previous transplantation .
exposure to repeated mismatched HLA antigens may trigger reactivation of memory cells and a rebound in DSA immediately posttransplant, leading to earlier graft injury and loss. repeat mismatches alone in the absence of circulating DSA may not need to be categorically avoided, as this practice may further limit access to transplantation.
HLA mismatching is an independent risk factor for the development of posttransplant non-Hodgkin lymphoma and associated with increased risk of posttransplant bone fractures in kidney transplant recipients.
Regarding pediatric age group renal transportation has significant challenges since these patients are more likely to require repeated transplantation. So well-matched organs are a priority for pediatric patients and this may lead to longer wait times. living-donor pediatric kidney transplantation should be done from donors with fewer than four HLA-A+, -B+, -DR mismatches.
Technique of HLA typing
* serological assay
can be made by antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC)
Serologic method used in screening for HLA antibodies in the recipient. These antibodies are important because they are reactive with lymphocytes of a prospective donor (cross matching)
*cellular assay
used to detect the HLA class II types. It is more sensitive in detecting HLA differences than serotyping because minor differences unrecognized by alloantisera can stimulate T cells.
*Tissue typing by molecular method
Using the sequence-specific oligonucleotide (SSO) and sequence-specific primer (SSP) technologies use in many tissue-typing laboratories worldwide for more than 20 years since the development of the polymerase chain reaction.
*DNA based method had more sensitivity, accurate than serologic typing methods
SSOP
assequence-;specific oligonucleotide probe hybridization
SSP
sequence-specific primer amplification
SBT
sequencing-based typing
RSCA
reference strand-based conformation analysis
1- Williams RC, Opelz G, McGarvey CJ, et al. The Risk of Transplant Failure With HLA Mismatch in First Adult Kidney Allografts From Deceased Donors. Transplantation 2016; 100:1094.
2- Shi X, Lv J, Han W, et al. What is the impact of human leukocyte antigen mismatching on graft survival and mortality in renal transplantation? A meta-analysis of 23 cohort studies involving 486,608 recipients. BMC Nephrol 2018; 19:116.
3- Malfait T, Emonds MP, Daniëls L, et al. HLA Class II Antibodies at the Time of Kidney Transplantation and Cardiovascular Outcome: A Retrospective Cohort Study. Transplantation 2020; 104:823.
4- Leeaphorn N, Pena JRA, Thamcharoen N, et al. HLA-DQ Mismatching and Kidney Transplant Outcomes. Clin J Am Soc Nephrol 2018; 13:763.
5-Handbook of Kidney Transplantation, SIXTH EDITION.
Thanks Dalia
Yes, splits HLA antigen matching give good transplant outcome if compare to matching of broad HLA antigens. Well done
HLA class I A, B and C antigens are expressed on almost all nucleated cells and platelets.
HLA class II DR, DQ and DP molecules are expressed on a more restricted range of cells, including B cells, activated T-cells, and the monocyte.
split antigen
The antigens were identified using antisera obtained primarily from multiparous women. As the field evolved, new antisera were discovered that could “split” some HLA antigens into narrower specificities. HLA-A9 was split into HLA-A23 and -A24, and HLA-A10 was split into HLA- A25, -A26, -A34, and -A66
only the degree of HLA matching was found to be a significant predictor of delayed graft function, one-year acute rejection, and 10-year graft survival. Using HLA matching for allocation.
a strong association between HLA matching, particularly at the HLA-A, -B, and -DR loci, and patient and graft outcomes. These HLA antigens have the most polymorphisms.
HLA class II mismatches, which include HLA-DQ and -DR, have been found to be a risk factor for the development of de novo DSAs.
HLA Typing technique:
Serologic methods
DNA-based molecular methods :
HLA typing techniques
Sequence-specific primers (SSP) typing
Sequence-specific oligonucleotide probes (SSOP) typing
Real-time PCR (RT-PCR)-based typing
Sequence-based typing (SBT)
Next-generation sequencing (NGS)
*Classification of HLA antigens:
1/ CLASS I :
present on the surface of all nucleated cells
3 major (HLA-A/B/C )
3 minor (HLA E/F/G )
2/ CLASS ll:
present on the surface of APCs
3 major ( HLA-DR/DP/DQ )
2 minor ( HLA-DM/DO )
*Split antigens :
HLA antigens that has a positive reaction with a reaction related to a broad antigen.
for example : HLA B51 & HLA B52 are split antigens to a broad antigen HLA B5.
*Effect of HLA mismatching on TX outcome:
* HLA typing techniques :
1/ Serological technique :
By adding the recipient and donor lymphocytes to reference sera containing specific antibodies then complement is added after incubation period to initiate the complement mediated cell destruction and determination of HLA type.
2/ DNA based HLA typing method :
More sensitive and accurate than serological technique. Includes several types:
a) Sequence specific oligonucleotide probe ( SSOP )
b) Sequence specific primer amplification ( SSP )
both a and b are widely used in most labs worldwide
c) Sequence based typing ( SPT )
d) Reference strand based conformation analysis ( RSCA )
e) Next generation sequencing ( NGS )
f ) Short tandem repeat ( STR ) that can determine the possibility of sibling donors.
Classify HLA antigens
HLA class I: has 3 major & 3 minor genes
major genes: HLA-A, HLA-B, HLA-C
minor genes: HLA-E, HLA-F, HLA-G
HLA class II: has 3 major & 2 minor genes
major genes: HLA-DR, HLA-DP, HLA-DQ
minor genes: HLA-DM, HLA-DO
What is meant by split antigen?
The discovery of new antigens splits the broad antigens into 2 or more antigens named split antigens, for example A9 is a broad antigen split to A23 & A24 split antigens, DR2 broad antigene split into DR15 & DR16 split antigens.
split matching for HLA-A,B,DR appears to be more clinically important.
How can HLA mismatch influence the outcome of transplantation?
HLA mismatch associated with poor graft and patient survival. the major impact on the graft come from HLA-DR & B mismatching. the effect of HLA-DR mismatching is is important in the first 6 months post transplantation, while HLA-B mismatching appear in the first 2 years. HLA mismatching is associated with increased incidence of rejection and delayed graft function. patients my die from infections and other complications of the high level of immunosuppressive medications required for HLA mismatched transplantation.
So better HLA matching is associated with:
lower doses of immunosuppressive medications.
lower incidence of immunosuppressive medications adverse effects including PTLD.
lower incidence of hip fractures.
lower grade of sensitization which is important for second transplantation.
References:
In your own words, summarise the HLA typing techniques
I. Serological method: the first standard method used for HLA typing.
done by using a reference serum combined with donor or recipient lymphocytes and a complement is added to the combination and by the use of viability dye the detection of cell lysis can be done. this method is quick, inexpensive but lack specificity.
II. Cellular assay: a mixed lymphocyte culture
used to determine HLA class II types,
it is more sensitive than serological method in detecting HLA differences.
III. DNA based molecular method: has more sensitivity, accuracy and resolving power than serological methods.
it includes the followings;
a. sequence specific primers typing( SSP): involves the use of primers designated to recognize a particular HLA allele or group of similar alleles
b. sequence specific oligonucleotide probes typing( SSOP): DNA is amplified using a set of primers that recognize a particular HLA locus.
c. real time PCR based typing( RT-PCR): uses allele specific PCR similar to SSP methods
d. sequence based typing( SBT): based on the direct amplification and sequencing of the relevant exons using fluorescently labeled dideoxynucleotides.
e. next generation sequencing( NGS): enabled high resolution typing with significantly reduced ambiguity.
Dear Huda
“Split matching for HLA-A,B,DR appears to be more clinically important”.
Please explain
better split matching for HLA-A, HLA-B & HLA-DR is associated with better graft survival
Classify HLA antigens
HLA antigens are classified into
A- HLA class I which is present in all nucleated cells which are subdivided into
⦁ Major antigens which include HLA -A, HLA- B, HLA-C
⦁ Minor antigens which include HLA-E, HLA-F and HLA-G
B- HLA class II which is present in APCS (macrophages, denderitic cells, vascular endothelial cells and B cells) they are further subdivided into
⦁ Major antigens which include HLA-DR, HLA-DQ, HLA-DP
⦁ Minor antigens which include HLA-DM, HLA DO
What is meant by split antigen?
HLA antigens are either broad or split antigens
Broad antigens are crude measure while split antigens are specific measure of identity of the cells.
Examples
⦁ A9 is broad splits are A23 and A24
⦁ B70 is broad splits B71 and B72
⦁ DR2 is broad splits DR15 and DR1
Matching for split HLA antigens result in better outcome when compared to matching for broad antigens
How can HLA mismatch influence the outcome of transplantation?
⦁ The degree of mismatch between donor and recipient is determined by HLA-A, HLA-B, HLA-DR each contain 2 antigens , so 6 mismatch means that 6 antigens between donor and recipient are not matching , while zero mismatch means that donor and recipient share same antigens meaning that thy are identical.
⦁ Exposure to mismatched antigens leads to production of DSA and increase the risk of antibody medicated rejection, moreover there is increase in the risk of T cell medicated rejection leading to early graft injury and may be loss.
⦁ Patient survival is lower in patients receiving HLA mismatched kidney when compared to those receiving HLA matching kidney
⦁ It was found that only the degree of HLA matching is a strong predictor for delayed graft function, 1 y acute rejection and 10 y graft survival.
⦁ The impact of specific HLA mismatch on graft survival differ, HLA-DR mismatch is associated with the worst outcome ( which is evident in the first 6 m) then HLA-B (which is evident in the first 2 years post transplant) , followed by HLA-A (which is evident on the long term)
⦁ Patients with 2 HLA-DR mismatches had worse graft survival compared to 0 or 1 HLA-DR mismatch
⦁ transplant recipients with 2 HLA-DR mismatch has higher degree of sensitization so they have lower probability of getting 2nd transplant
⦁ HLA- mismatch between donor and recipient especially DR was found to increase the risk of infection, lymphoproliferative diseases, post transplant diabetes, hypertension, hyperlipidemia and cardiovascular events, bone fractures.
⦁ HLA-C mismatch was found to be associated with reduced graft survival in only those who are presensitized and not in those who are not presensitized, moreover patients with high level of pretransplant HLA-C DSA are more prone to develop ABMR
⦁ HLA-DQ mismatch was associated with the development of de novo DSA
⦁ HLA-DP mismatch is not significant in the first transplant but is correlated with reduced graft survival in second transplant
Summarise the HLA typing techniques
1- Serologic method using either antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)
2- Mixed lymphocyte culture (MLC) used for HLA class II, result interpreted in the form of Dw types, more sensitive than serological method
3- DNA-based HLA typing methods which is more sensitive and accurate than both
previous techniques it includes the following:
⦁ SSOP sequence specific oligonucleotide probe hybridization
⦁ SSP sequence-specific primer amplification
⦁ SBT sequencing-based typing
⦁ RSCA reference strand-based conformation analysis
⦁ NGS next generation sequencing
Yes, well done. “Matching for split HLA antigens result in better outcome when compared to matching for broad antigens”
We are looking for more responses
Classification
MHC class I proteins form a functional receptor on most nucleated cells of the body.
There are 3 major and 3 minor MHC class I genes in HLA: HLA-A,HLA-B and HLA-C;
minor genes are HLA-E, HLA-F and HLA-G
There are 3 major (DP, DQ, DR ) and 2 minor ( DM, DO) MHC class II proteins encoded by the HLA. Typically expressed on the surface of antigen-presenting cells.
split antigen
antigen (broad) can be splitter into more antigens and matching for HLA antigen “splits” results in better transplant outcome than matching for “broad” HLA antigens
HLA typing and its influence on organ transplantation
When a human transplant is performed, HLA (human leukocyte antigens) molecules from a donor are recognized by the recipient’s immune system by direct and indirect methods of allorecognition triggering an alloimmune response. Matching of donor and recipient for MHC antigens showed a significant positive effect on graft acceptance
HLA mismatches may occur at antigenic or allelic level; the first are characterized by amino acid substitutions in both peptide binding and T-cell recognition regions, whereas the latter are characterized by amino-acid substitution in the peptide binding regions only.
In transplantation immunology, the major impact in graft loss comes from the effects of HLA-B and -DR antigens.
The effects of HLA-DR mismatches are the most important in the first 6 months after transplantation, the HLA-B effect emerges in the first 2 years, and HLA-A mismatches have a deleterious effect on long-term graft survival.
Techniques of HLA typing
Previously, HLA typing was done by two methods: serologic method using antiserum and mixed lymphocyte culture (MLC). After that a more precise DNA-based
HLA typing methods using molecular techniques, such assequence-;specific oligonucleotide probe hybridization (SSOP), sequence-specific primer amplification (SSP), sequencing-based typing (SBT), and reference strand-based conformation analysis (RSCA), have been developed and are frequently used. In 2013, a new project of the 16IHIW demonstrated the potential benefits of next-generation sequencing (NGS) in the HLA laboratory. Another simplified method using short tandem repeat (STR) genotyping provided additional information allowing determination of the extent of HLA identity in families where HLA haplotype inheritance was
ambiguous
Well done
Yes, split antigen match is associated with better outcome, well done
HLA antigen classification
HLA region is located in the short arm of 6th chromosome and in particular 6p21.3. MHC or HLA complex is known to be highly polygenic. Broadly it is divided into three categories: Class I, Class II, and Class III.
Class I include genes that encode for HLA antigens. Three major HLA types A, B and C, and 3 minor E, F, and G.
Class II include 3 major types DR, DP and DQ and 2 minor DM and DO.
Class III is located between class I and II. It is responsible for encoding several complement components, TNF, heat shock protein and others. Split and broad antigens
HLA can be classified into broad and split antigens
Old serological based cellular methods for HLA typing obtain HLA genes with allele group but were not able to identify precisely specific alleles. That’s why broad HLA antigen has a poor specificity. A broad antigen can be divided into 2 or more split antigens.
DNA based molecular techniques allowed for more identification and sequencing of the polymorphic regions of genes and identify specific alleles encoding for specific HLA proteins.
Why split antigens typing is important. In solid organ transplantation, differences in the split antigens, having same broad antigen, could potentially activate immune response and subsequent immune mediated rejection. Amino acid sequencing using DNA based methods is important, because DSA could react against one but not against another in the same broad antigen.
How can HLA mismatch influence the outcome of transplantation?
The better outcomes in transplantation n occur in transplantation between identical twins, which implies that match or mismatch is the predictor of outcomes. Matching for HLA antigens has significant impact on graft survival and overall survival. One mismatch between donor and recipient is associated with an increase risk of rejection, while 2,2,2 mismatch has the poorer outcomes in mismatched groups.
Summarize the HLA typing techniques
Before the widespread availability of DNA based techniques, HLA typing was performed using serological methods.
Serological method uses reference sera containing known antibodies to specific HLA antigens. So donor or recipient lymphocytes are added to several wells of plates that contain different sera and eventually different antibodies. 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. Antibodies used in the reference sera could also be directed against more than HLA molecule causing inconclusive typing.
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.
DNA-based molecular techniques for HLA typing include low to high resolution methods that include sequence-specific oligonucleotide probe typing (SSOP), sequence-specific primer typing(SSP), sequence-based typing (SBT) and Next-generation sequencing (NGS). The type of technique used varies among laboratories based on availability, experience and cost.
SSP involves the use of primers that detect a group of similar alleles or specific HLA sequence.
SSOP uses set of primers that recognize a particular locus. As with SSP, DNA is amplified for better detection of polymorphic regions of HLA.
Excellent Mohamed
Excellent summary
1. Classify HLA antigens:
Human Leukocyte Antigen (HLA) is a gene complex located on short arm of chromosome 6 (6p21.3). It contains many genes and can be classified into class I, II and III. Class I and II have important role in immune regulation.
HLA Class I has 3 major loci (HLA-A, HLA-B and HLA-C) and 3 minor loci (HLA-E, HLA-F, HLA-G)
HLA Class II has 3 major loci (HLA-DP, HLA-DQ, HLA-DR) and 2 minor loci (HLA-DM, HLA-DO)
2. What is meant by split antigen?
HLA can be classified as broad antigen and split antigen.
A broad antigen is HLA molecule with a poor specificity. A broad antigen can be divided into 2 or more split antigens.
A split antigen is HLA molecule with a more refined or specific cell surface reaction relative to a broad antigen.
For example: HLA-A10 is a broad antigen while HLA-A25, HLA-A26, HLA-A34 and HLA-A66 are split antigens.
3. How can HLA mismatch influence the outcome of transplantation?
Human Leukocyte Antigen or HLA typing and mismatch determination between the potential transplant recipient and donor pair is an important factor for prognostication in kidney transplantation.
Usually the HLA A-, B-, and DR- are typed for both the donor and the recipient and degree of mismatch is looked into.
Studies have shown that number of HLA match in the recipient-donor pair is a strong predictor of renal allograft survival. (1) The higher the HLA match, lower the incidence of delayed graft function as well as acute rejection rate in first year and higher the 10 year graft survival. (2,3)
HLA mismatches have been shown to be associated with poor transplant outcomes. (4) UNOS registry showed that there was increased risk of allograft failure with number of mismatches (13% in 1/6 mismatch and 64% in 6/6 mismatch). (5)
Post-transplant long-term prognosis includes rates of complications like post-transplant lymphoproliferative disease (PTLD), post-transplant bone fractures, development of donor specific antibodies (DSAs) and death with a functioning graft.
PTLD risk has been shown to be higher in transplants with HLA DR mismatch (21% increased risk with 1 HLA-DR mismatch and 56% increased risk in 2 HLA-DR mismatch). (6)
Post-transplant bone fractures are also shown to be higher in recipients with HLA-DR mismatch (85% increased risk with 1 mismatch and 124% increased risk with 2 mismatch) (7)
Increased formation of DSAs is seen in case with Class II HLA mismatch (HLA-DQ, DR) thereby ultimately leading to poor graft survival. (8)
Increased risk of death with a functioning kidney graft has been seen in recipients with increased HLA mismatch. (9)
Of note is a study by Su et al which deduced that due to availability of better and more potent immunosuppression, HLA mismatch has lost much of its relevance. (10)
References:
4. In your own words, summarise the HLA typing techniques
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.
ii. SSOP (Sequence Specific Oligonucleotide Probe hybridization): Amplified DNA is mixed with oligonucleotide probe complementary to specific DNA segment of different alleles which are identified using fluorescent markers leading to HLA typing.
iii. SBT (Sequence based typing): Exon specific primers are used.
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.
Excellent
Very impressive summary Amit
classification of HLA system :
class I : A, B. C (major antigens)
E, F, G (minor antigens)
class II DP, DQ, DR (major antigens)
DM, DO (minor antigens)
split antigen: it is antigen split from broad antigen e.g HLA-A2 is broad antigen , HLA-A203, HLA-A210 are split antigens
of course HLA mismatch affect graft survival , more mismatch leads to decrease graft survival, HLA-DR mismatch affects graft survival too early in the first 6 months and HLA-B mismatch affects graft survival in the 1st 2 years , HLA-A mismatch has an effect on long term graft survival.
This mismatch will trigger immune response HLA I and II have protein peptide has its receptor on T cells CD4 has TCR to class II (which present on APC of the donor), and CD8 has TCR to class I (which present in all nucleated cells as donor parenchyma). Activation of T cell will be done when 3 signals stimulated TCR with class I or II, CD28 lock with CD80/86, and IL-2 with CD25.
This will leads acute cellular rejection.
Also endothelial cells have class I and II HLA molecules, this will lead to vascular damage and c4d deposition with antibody formation. ABMR
HLA typing techniques:
1- Serological assay use antiserum and mixed lymphocyte culture
It is made by CDC/ADCC used for screening HLA antibodies in the recipient.
2- Molecular method / DNA based sequence technology which is used widely, also PCR emerging and used.
Sequence based typing technique is more sensitive than serological method
Thank you Riham
broad antigen used in practice , but any mismatch at the level of split antigen will impact on graft survival as it will induce immune response
molecular method is accurate
molecular typing is accurate than serology, as there is polymorphism of HLA system so, molecular methods can reach to different alleles so more accuracy affecting graft outcome.
molecular method used in workplace.
All HLA mismatches are associated with some degree of risk of reduced graft function and survival and the risk is proportional to the number of mismatched antigens.
HLA C is rarely associated with acute rejection but mostly with chronic rejection.
HLA Mismatching Strategies for Solid Organ Transplantation – A Balancing ActAndrea A. Zachary
Classification
HLA class 1
major-: HLA-A,HLA-B and HLA-C
minor genes are HLA-E, HLA-F and HLA-G
HLA class 2
major-: HLA-DR, -DQ, -DP,
minor- DM, and -DO.
split antigens
HLA-B15 consists of 8 closely related specificities, HLA-B62, B63, B70, B71, B72, B75, B76, and B77.
Serologic supertypes are the broad specificities. Eg: B15
“Splits” or Subtypes are the finer specificities that comprised the supertype. Eg: B62.
As the splits were discovered, they were given number designations that again give no indication as to the supertype to which they belong. Eg: Molecular B*15:12 is equivalent of HLA- B76.
HLA-DR mismatches are the most important in the first 6 months after transplantation.
HLA-B effect emerges in the first 2 years.
HLA-A mismatches have a deleterious effect on long-term graft survival.
All HLA mismatches are associated with some degree of risk of reduced graft function and survival and the risk is proportional to the number of mismatched antigens.
patients with one or two mismatches for an HLA-C antigen had a significantly higher incidence of rejection compared to those with no HLA-C mismatch (54 and 100 vs. 0%) but only when there was also one HLA-B mismatch.
HLA Mismatching Strategies for Solid Organ Transplantation – A Balancing ActAndrea A Zachary et al .
HLA typing techniques
HLA typing is done by two methods
serologic method using antiserum and mixed lymphocyte culture (MLC).
ADCC or CDC.
DNA-based HLA typing methods
SSOP- can provide low or high resolution
SSP-can provide low or high resolution
SBT- is a high resolution method
RSCA
next gene sequencing-
STR genotyping – rapid and reliable used to screen potential sibling donor.
The difference in survival at three years between grafts with 0 or 6 mismatches for HLA-A, B, DR was 31% when antigen splits were analysed, in contrast to a 6% difference with broad antigens. These results indicate that typing for HLA antigen splits is important in renal transplantation.
Importance of HLA antigen splits for kidney transplant matchingG Opez
molecular typing is more accurate than serological typing since it can identify allelles.