Study on HLA matching in sensitized recipients of renal allografts and its clinical application
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Objective To evaluate the clinical application of HLA matching in highly sensitized recipients of renal allografts.Methods Recipient's panel reactive antibody (PRA) was detected by using ELISA test with Lambda antigen tray (LAT).Donor and recipient HLA classⅠtyping was performed with special monoclonal tray,and HLA classⅡgene typing with micro-sequence specific primers (Micro-SSP).Results There were 104 recipients with anti-HLA class-ⅠIgG antibody,76 with anti-HLA class-ⅡIgG antibody,and 44 with both anti-HLA class-1 and anti HLA class-ⅡIgG antibody respectively in 136 sensitized recipients.HLA class-ⅠIgG antibody positive rate was 11%-97 %,with an average of 49.6%±23.8%;The common public epitopes antibody was not found in each recipient of 13 cases with PRA＜20%,but was found in I2 recipients in 44 cases with PRA be- tween 20%-50%,and 39 recipients in 47 cases with PRA＞50%.HLA class-ⅡIgG antibody posi- tive rate was 17%-100%,with an average of 28.2%±63.8%.The number of cases of 0,1,2,3, 4 MM was 7 (5.1%),26 (19.1%),47 (34.6%),39 (28.7%) and 17 (12.5%) respectively by the standard of conventional HLA antigen matching;however the number of the recipients with 0,1, 2,3 MM was 31 (22.8%),53 (39.0%),36 (26.5%) and 16 (11.7%) respectively according to the rule of HLA CREGs matching and none with 4 MM.Rates of acute rejection in sensitized recipi- ents with 2MM and 3MM HLA-CREGs were 25.0% and 37.5% respectively and were significantly higher than those with 0MM (P＜0.05,＜0.05 respectively).Kidney year-survival was decreased when the number of MM of HLA CREGs matching increased.Conclusion The HLA CREGs matching can improve the ratio of well-matched significantly.Good HLA matching can reduce the incidence of acute rejection in sensitized recipients and increase the survival rate of grafts.Keywords:
Panel reactive antibody
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Donor-Specific Antibodies
Complement-dependent cytotoxicity
Histocompatibility Testing
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Objective
To interrogate the detection of anti-HLA antibodies using two methods of Luminex xMAP, and to compare their detection capacity and to analyze their misdetection rate for initial screening, providing more accurate results in clinical practice.
Methods
214 serum samples from recipients with a history of sensitization before renal transplantation were collected and detected by LM (LABScreen Mixed) and LSA (LABScreen Single Antigen) respectively on the Luminex xMAP platform.
Results
For the LM detection, the positive rates of anti-HLA class I and II were 50.9% and 23.4% respectively, which were lower than those used by the LSA detection (58.9% and 46.7% respectively). The difference had statistical significance (P<0.05). The sensitivity, specificity, miss rate and mistake rate of anti-HLA class Ⅰ and Ⅱ detection were 80.2%, 90.9%, 19.8%, 9.1% and 49.0%, 99.1%, 51.0%, 0.9% respectively. The missed detection gene with the highest rate was Cw*17: 01, B*15: 12, B*45: 01 for anti-HLA class I and DPA1*01: 03, DPB1*06: 01, DPA1*01: 03, DPB1*01: 01 for anti-HLA class II. The highest MFI value was 10603 and 3659. For the recipients with only blood transfusion history or pregnancy history, LM and LSA detection showed no statistically significant difference when detecting anti-HLA class I antibodies, but had statistically significant difference when testing anti-HLA class II antibodies. For the patients with history of both blood transfusion and pregnancy history, LM and LSA showed no significant difference in the detection of anti-HLA class Ⅰ antibodies and anti-HLA class Ⅱ antibodies. The miss rate of anti-HLA class I antibody detection was lower than that of anti-HLA class Ⅱ antibody detection.
Conclusion
LSA detection has the higher sensitivity and lower miss rate than LM detection. In the light of the disadvantage of LM detection as diagnostic preliminary screening, it is suggested that LSA detection should be used directly for the recipients with a history of sensitization. By this process optimization, it is more likely to cause missed inspection and the occurrence of rejection, as well as a poor long-term survival rate.
Key words:
Kidney transplantation; Anti-HLA antibodies; Luminex xMAP
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Background: Introduction of the Luminex panel reactive antibody (PRA)-single antigen (SA) assay has increased the detection rates of unacceptable antigens in sensitized patients; the calculated PRA (CPRA) level represents the percentage of actual organ donors that express 1 or more of these unacceptable antigens.We developed a CPRA calculator based on the HLA frequencies in Koreans to measure sensitization levels in Korean patients.Methods: To develop the calculator, we obtained the HLA-A, HLA-B, and HLA-DR phenotypes of 1,622 Koreans, and compared these with previously reported frequencies in Koreans.Sera from patients awaiting kidney transplantation were tested for HLA antibodies by Luminex PRA-screen, PRA-identification (ID), and PRA-SA assays.The measured %PRA from the PRA-screen (N = 55) and PRA-ID (N = 71) were compared to the %CPRA for the unacceptable antigens obtained from PRA-SA.Results: Phenotype frequencies used for the CPRA calculator agreed with previously reported data.The concordance rates among the 3 PRA methods for the detection of class I and class II antibodies were 76.1-81.8%(kappa, 0.519-0.636)and 72.7-83.6%(0.463-0.650), respectively.For the detection of broadly sensitized sera ( >50% or > 80%), the concordance rates were over 80%.In sera with 80-100% CPRA, 91.7% and 94.4% of the samples had concordant results (80-100% PRA) in the PRA-screen and PRA-ID assay, respectively.Conclusions: Although further clinical studies are required to confirm the benefits of CPRA values, adoption of CPRA analysis based on HLA frequencies in Koreans may be useful for sensitization measurements and organ-allocation algorithms.
Concordance
Panel reactive antibody
Kappa
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Aim: Panel Reactive Antibodies (PRA) analysis is important not only in virtual cross-match of donors and recipients but also in follow-up of recipients immune response after transplantation. Although bead based tests give faster and more reliable, it may have false positive and negative results. We investigated HLA groups of patients with false negative results and the frequencies of anti-HLA antibodies in all three false negative situations.
Materials and Methods: PRA results of all patients were divided into 3 groups according to negative controls and samples results. First one is negative controls were false negative and samples were false negative (1st Situation); second one is negative controls were false negative but samples were true negative (2nd Situation); third one is negative controls were true negative but samples were false negative (3rd Situation). All serum samples were tested by bead based Luminex assay.
Results: While anti-HLA-A68, A24, A32 were the most frequent antibodies for the first situation, anti-HLA-A03,24,32 were the most frequent three antibodies for the second situation. In case of anti-HLA-B antibodies; anti-HLA-B38, B44, B48 were detected for the first situation, and anti-HLA-B44, B58, B49 were detected for the second situation. In case of class II antibodies; we detected anti-HLA-DRB110, DRB115 and DRB151 antibodies as the most frequent three antibodies at 1st situation. Frequencies of anti-HLA antibodies of 120 patients with false negative results were determined in serum samples. In case of 3rd situation, while anti-A23, A68, A24; anti-B06, B04, B39 and anti-Cw07 were the most frequent anti-HLA Class I antibodies, anti-HLA DQ07, DQ05, DQ04 and anti-HLA-DRB152, DRB113, DRB153 were the most frequent HLA-Class II antibodies detected in serum samples.
Conclusion: Samples with false negative PRA results should be repeated and these samples should be recorded in the laboratory for internal control system.
Identification
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The clinical relevance of the detection of human leucocyte antigen (HLA) antibodies in sera of renal transplant recipients by highly sensitive methods such as Luminex alone is uncertain and a matter of debate. The choice of output thresholds affects antibody detection and thus organ allocation, yet there are no internationally agreed threshold levels. This study aims at evaluating our current practice of using an MFI threshold of 1000 in antibody detection. We carried out a case–control study by looking at 761 renal transplant recipients at one unit between 2000 and 2010. Of these, there were 93 cases of graft loss within 1 year and stored serum samples of 40 cases were available for testing. Controls were selected (graft function >2 years) and individually matched according to age, sex, number of transplants and date of transplant. All 40 cases and 40 controls had negative crossmatch by complement-dependent cytotoxicity (CDC) at the time of transplant, and pre-transplant sera were re-analysed for the presence of detectable HLA and donor-specific antibodies (DSAs) using Luminex screen and single-antigen beads and MFI threshold values of 1000, 2000 and 4000. In nearly 48% of cases with graft loss within a year, HLA antibodies were detectable by Luminex when using a 1000 MFI threshold. This was 25% greater than in controls (P = 0.017). There was also a 15% increase in detected DSAs; however, statistical significance depends on the inclusion or exclusion of one specific case. Using MFI thresholds of 2000 and 4000, no DSAs were found in any long-term surviving grafts. Selection of appropriate MFI cut-off values influences the detection of DSAs and, thus, organ allocation. Using a threshold of 1000 led to the detection of DSAs in 5% of long-term graft survivors in our population and should be considered too sensitive. Using a detection threshold of 2000 is sufficiently sensitive and leads to clinically relevant detection of DSA.
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Plasmapheresis
Peritubular capillaries
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Because of the inherent difficulties in allele assignment with HLA-DR serological typing, in 1993 our organ procurement organization-based HLA laboratory replaced serology with the molecular method of polymerase chain reaction using sequence-specific primer mixes (PCR-SSP) to type for DR and DQ at a resolution level equivalent to that of serologically defined antigens. In this study, we compared the incidence of DR blanks, where allocative homozygosity occurred, and graft outcome during our serology epoch (1987-1993) with that of our molecular epoch (1993-1996). The incidence of DR blanks by PCR-SSP (17.0%; 138/1101) was significantly lower (P<0.005) than in the serology epoch (21.5%; 569/2647). Although DQ is not a component of the allocation algorithm, the incidence of blanks in the molecular era (21.9%; 196/895) was 46% lower (P<0.001) than in the serology epoch (40.8%; 931/2277). Graft survival in 163 cadaveric renal transplant recipients for whom molecular DR allocation occurred (patient and donor were molecularly typed) showed that PCR-SSP typing had no significant effect on 2.5-year graft survival for patients mismatched for 0 (97%), 1 (90%), or 2 (94%) HLA-DR antigens (P=0.4; log-rank). In conclusion, molecular typing lowered the rate of DR and DQ blanks, but molecular matching for HLA DR and DQ did not influence graft outcome at 2.5 years.
Primer (cosmetics)
HLA-DR
Histocompatibility Testing
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Donor-Specific Antibodies
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Background : When organ transplantation or HLA-matched platelet transfusion is considered, accurate identification of HLA antibody specificity in the recipient’s serum is very important. In this study, we report our experience in an international quality control program. Methods : For external quality control in a HLA antibody test, the International Serum Exchange Program distributes serum samples, generally showing polyspecific reactivity for cross-reactive epitope groups (CREGs), to participating laboratories: 4 samples per survey, 10 surveys per year. Participating in the program from May 1998 to August 2000 (24 surveys), we performed HLA antibody identification of 96 serum samples by the AHG-CDC (anti-human globulin-complement dependent cytotoxicity) method using frozen lymphocyte trays (36 lymphocyte panels). We compared the results of our laboratory with those of the total participants (all methods combined, 72 to 92 laboratories per survey) using the analyzed survey results distributed by the program organizer. Results : We analyzed the survey results for the antibodies to relatively common HLA antigens in Koreans (antigen frequency >1%). For the HLA antibodies detected in 20% of participants, our detection rate was higher by 10-15% than that of all laboratories (HLA-A, 76% vs 65%; HLA-B, 73% vs 57%). And for the HLA antibodies detected in 50% of the participants, our detection rate was as high as 88% for HLA-A and 87% for HLA-B. Our detection rate for a few antibody specificities was lower than that of all laboratories, namely HLA-A1, A3, B35, and B55. Among these, A1, A3, and B55 were of lower incidence antigens in Koreans (antigen frequency 3-4%), indicating that the low detection rate was due to a limitation in the composition of lymphocyte panels. Conclusions : In general, our detection rate of HLA antibodies was superior to the average detection rate of the total participant laboratories. We would be able to improve the low detection rate for a few antibody specificities to lower incidence antigens by refining the composition of lymphocyte panels.
Panel reactive antibody
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[Objective]To set up the flow cytometry of complement-dependent experiment me thod to improve the accuracy of distinguishing the IgG-HLA antibody. [Methods]Sixty-two patients were used Flow-CDC and NIH-CDC . The correlatio n between different techniques for detection of donor specific anti-HLA antibod ies was evaluated. The effect of both methods on clinical transplantation outcom e was observed. [Results]NIH-CDC and Flow-CDC were negative for PRA negative group. In PRA p ositive group , there was significant difference between two methods (χ~2=5.1 4,P= 0.016 ). Five patients received transplantation. One of them with ne gative NIH-CDC and positive Flow-CDC suffered from acute rejection after trans plantation and lost the graft, and the other patients with negative NIH-CDC and Flow-CDC had good outcome. [Conclusion]Flow-CDC can detect specifically complement-fixing IgG alloantib odies against donor HLA and is more sensitive than NIH-CDC. Additionally, the c omputer printouts represent a permanent record of the crossmatch for retrospecti ve review. Flow-CDC may become the standard crossmatch method as a possible alt ernative to conventional NIH-CDC testing.
Complement
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