False-Positive T-Cell Cytotoxicity Crossmatch Results Due to Autoantibodies in Korean Network for Organ Sharing Crossmatch Tests / 대한이식학회지

BACKGROUND: Basic National Institute of Health (NIH) and sensitive antihuman globulin (AHG) methods are widely used for T-cell complement-dependent cytotoxicity crossmatch (XM) tests. Whereas NIH-negative, AHG-positive (NIH⁻/AHG⁺) results are caused by weak antibodies, NIH⁺/AHG⁻ results are usually due to autoantibodies. We found that solid organ transplantation candidates with NIH⁺/AHG⁻ XM results are repeatedly excluded from allocation of deceased donor organs by the Korean Network for Organ Sharing (KONOS) allocation system. Here, we attempted to demonstrate that these patients do not have donor-specific HLA antibodies (DSAs). METHODS: Sera showing NIH⁺/AHG⁻ results in the analysis of 1,668 KONOS T-cell XM tests were screened for panel reactive antibody (PRA) using a Luminex test. For screen-positive samples, antibody identification was conducted using a Luminex single antigen assay and the presence or absence of class I DSAs was determined. For positive controls, 42 KONOS XM tests showing probable true-positive (NIH⁻/AHG⁺ or NIH⁺/AHG⁺) results were reviewed for PRA results based on electronic medical records and the presence or absence of DSAs was determined. RESULTS: NIH⁺/AHG⁻ results were observed in 1.3% (21/1,668) of KONOS XM tests analyzed. Most of these (18/21, 85.7%) were negative for PRA or DSAs. All probable true-positive cases were either positive for DSAs (24/42, 57.1%) or had high PRA (mean, 92% [range; 42%~100%]), complicating accurate identification of antibody specificities. CONCLUSIONS: NIH⁺/AHG⁻ results are not rare (1.3%) in KONOS XM tests. Most of these results are not due to DSAs, and these patients should not be excluded from organ allocation.

A Questionnaire Survey of HLA Crossmatch Tests in Korea (2015)

BACKGROUND: We carried out a questionnaire survey for laboratories performing human leukocyte antigen-crossmatch (HLA-XM) to provide a basis for laboratory standardization of HLA-XM tests in Korea. METHODS: The questionnaires were distributed to 51 HLA laboratories participating in the HLA-XM part of the HLA proficiency survey program organized by the Korean Society for Laboratory Medicine and replies from 50 laboratories were analyzed. The questionnaires included following items: 1) HLA-XM methods performed and annual number of tests, 2) types of the specimen and lymphocyte separation methods, 3) test procedures and reagents for complement-dependent cytotoxicity crossmatch (CDC-XM) and flow cytometry crossmatch (FCXM). RESULTS: The number of laboratories performing anti-human globulin (AHG) CDC-XM (47/49, 96%) and FCXM (30/50, 60%) was considerably increased compared to the 2005 survey (AHG CDC-XM, 35/43, 81%; FCXM, 7/44, 16%). As for the annual number of XM tests, more than 50% of the laboratories were low volume laboratories performing ≤50 tests, and only 10% of the laboratories were performing >500 tests. For cell isolation methods, negative selection was used by 43% (21/49) of laboratories performing CDC-XM. Number of cells reacted per 1 µL of serum varied among different laboratories in both CDC-XM (1,000–8,000) and FCXM tests (1,300-20,000). For the interpretation of FCXM, log fluorescence ratio (26/30, 87%) was more commonly used than channel shift values (5/30, 17%). CONCLUSIONS: Considerable variation is noted in both CDC-XM and FCXM methods performed by different laboratories. A continuous effort for laboratory standardization is needed to reduce inter-laboratory variation in the HLA-XM test results.

Flow Cytometric AHG-CDC for HLA Crossmatch: A Pilot Study / 대한수혈학회지

BACKGROUND: For HLA crossmatch in organ transplantation, complement-dependent cytotoxicity (CDC) is a very useful methodology to detect donor-specific HLA antibodies and their complement fixing ability. In this preliminary pilot study, we investigated whether dead cells induced in anti-human globulin (AHG)-augmented CDC (AHG-CDC) reaction could be measured by flow cytometry ('FC AHG-CDC'). METHODS: This FC AHG-CDC measured the percentage of dead cells (% dead cells) after 7-aminoactinomycin D staining using 3 color flow cytometry. A total 45 flow cytometry crossmatch (FCXM) cases of 12 positives and 33 negatives were further tested using this FC AHG-CDC. RESULTS: The % dead cells of FC AHG-CDC was significantly correlated with the mean fluorescent intensity ratio of FCXM (T cells: r=0.613, P=7.45x10(-6); B cells: r=0.404, P=0.006). The positivity rate of FC AHG-CDC among FCXM positive cases was relatively high: 80% (8/10) for T cells and 75% (9/12) for B cells. The negativity rate of FC AHG-CDC among FCXM negative cases was 100% (35/35) for T cells and 91% (30/33) for B cells. CONCLUSION: In this pilot study, FC AHG-CDC could yield quantitative values, % dead cells, which was proportional to the level of complement-fixing cytotoxic antibodies against T and B cells, respectively, even without physical separation of cells or serial dilution of serum.

An Evaluation of Platelet Transfusion Response Using HLA Crossmatch-compatible Donors in Patients with Platelet Refractoriness / 대한진단검사의학회지

BACKGROUND: Majority of immune-mediated platelet refractoriness is caused by HLA alloimmunization and can be effectively managed by HLA-matched platelet transfusions. However, HLA class I-typed large-sized donor registry has not been well established in Korea. We evaluated the effectiveness of platelet transfusion using HLA crossmatch-compatible donors without HLA typing. METHODS: Sixteen patients showing platelet refractoriness to random donor platelets (1 hr corrected count increment [CCI] 60%) were crossmatched with 78 platelet apheresis-eligible donors using National Institute of Health (NIH) and anti-human globulin (AHG) lymphocytotoxicity methods. NIH negative/AHG negative and NIH negative/AHG positive donors were selected as best and second choice donors, respectively. RESULTS: Eleven patients (11/16, 69%) could find NIH-crossmatch negative donors and 27 donors (27/78, 35%) belonged to the best donors. To 8 patients, 32 apheresis platelet products from 19 donors were transfused. The mean 1 hr and 24 hr CCI values from the best donors were significantly higher than those from random donors (17,893 vs 2,358, P=0.003; 8,292 vs -614, P<0.001), whereas such differences were not observed for those from the second choice donors. Platelet storage time was inversely correlated with CCI values and platelets stored < or =10 hr after collection gave significantly higher CCI values. Neither ABO match nor donor status (related vs unrelated) affected the transfusion effectiveness. CONCLUSIONS: Effective post-transfusion platelet increment using HLA crossmatch-compatible donors was attained in patients with platelet refractoriness due to HLA antibodies, and this method can be used effectively where HLA-typed platelet donor registry is not available.

Human Leukocyte Antigen Typing Proficiency Surveys in Korea, 2005-2006 / 대한진단검사의학회지

BACKGROUND: To monitor the performance of histocompatibility testing laboratories, HLA proficiency survey in Korea has been conducted biannually since 1996. In this report, we summarized the results of the surveys performed in recent two years (2005-2006). METHODS: A total of four proficiency surveys were performed, in which 59-61 laboratories participated. Each survey included three tests for HLA class I (serology and DNA) and class II (DNA) typing and six tests for HLA crossmatch. RESULTS: The overall concordance of serologic typing was 98.9% (355/359) for HLA-A, 97.5% (350/ 359) for HLA-B, and 94.7% (337/356) for HLA-C. The antigens assigned correctly by less than 95% of the participating laboratories were A26 (93.8%), B38 (94.2%), Cw3/Cw10 (90.9%), Cw6 (94.4%), and Cw8 (74.3%). The overall concordance rates of DNA typing were 99.6% (533/535) for HLA-A, 99.8% (539/540) for HLA-B, and 100% (392/392) for HLA-C. Correct assignment of HLA-DRB1 and -DQB1 was reported by 99.2% (98.1-100%) and 96.7% (88.9-100%) for the generic level and 100% and 95.8% (75-100%) for the allelic level, respectively. On the average 3.8% (0-7.7%) of the total laboratories showed unacceptable results in the crossmatch tests. CONCLUSIONS: The rates of correct antigen identification and of unacceptable crossmatch were similar to those of previous surveys, which were considered satisfactory. The Korean proficiency survey program may have contributed to a high quality of HLA tests today and should be continued for further improvements of the tests tomorrow.

A Living Donor Liver Transplantation after Therapeutic Plasmapheresis in a Patient with Positive HLA Crossmatch / 대한수혈학회지

A positive HLA crossmatch in cadevaric liver transplantation is relatively acceptable, but in living donor liver transplantation (LDLT) using relatively small sized grafts, the rejection rates were higher in positive crossmatchcases than in negative cases, as described in several previous reports. We report a case of LDLT performed with therapeutic plasmapheresis, in a recipient with a positive HLA crossmatch to donor before transplantation. The patient was a 56-year-old male patient with liver cirrhosis (UNOS status IIA, MELD score 28) caused by chronic hepatitis B. The HLA crossmatch results were 1:2 and 1:8 positive for NIH-CDC (complement dependent cytotoxicity) and AHG-CDC, respectively. The flow cytometric crossmatch (FCXM) was also positive (T-MFI ratio 9.0 and B-MFI ratio 3.4). With 5 cycles of preoperative therapeutic plasmapheresis, the HLA crossmatch converted to negative and liver transplantation was performed. The liver function of the patient was well maintained for 5 months, without any sign of hyperacute or acute rejection. However, the patient eventually died from suddenly occurred infection-associated hemophagocytic syndrome at 5 months after surgery. Therapeutic plasmapheresis can be considered as one of therapeutic options for LDLT patients with a positive HLA crossmatch to donor.

Experimental Application of Whole Blood Flow Cytometry to HLA Crossmatch for Renal Transplantation / 대한진단검사의학회지

BACKGROUND: The lymphocytes separated from whole blood are used in HLA flow cytometry crossmatch (FCXM) for renal transplantation. In this study, the methodology of whole blood flow cytometry was applied to FCXM, omitting lymphocyte separation step. METHODS: In the 20 cases (including positive 5 cases) of T cell FCXM for renal transplantation, the standard assay using the separated mononuclear cells (MNC) was compared with the two variant assays using whole blood. In the latter assay, the donor whole blood was incubated with the excessive recipient serum. The red cells were lysed (lysed whole blood, LWB). Otherwise, instead of red cell lysis, the signals of T cells among whole blood (WB) were acquired using fluorescence triggering. The sample/negative control mean fluorescence intensity (MFI) ratio was calculated for the interpretation. RESULTS: The MFI ratio of the 20 cases by MNC, LWB and WB assay were 4.9+/-8.1, 5.4+/-9.7 and 4.8+/-7.8, respectively. Both LWB and WB assay were not significantly different from MNC assay (P= 0.313, 0.831, respectively, paired t-test). The qualitative determinations were concordant in all cases, except for one case which was weakly positive with MFI ratio 2.2 by LWB assay. CONCLUSIONS: The assays using whole blood were comparable to the standard assay in FCXM for renal transplantation. This study indirectly supports that the variant methods can be used reliably in the case of the MNC preparation erroneously mixed with other blood cells.

HLA Typing Proficiency Survey in Korea, 2003-2004 / 대한진단검사의학회지

BACKGROUND: HLA proficiency survey in Korea started in 1996 and the results of the survey were last reported in 1999. In this report, we summarized the results of the survey performed in recent 2 years. METHODS: A total of four proficiency surveys were performed, in which 54-59 laboratories participated. Each survey included 3 tests for HLA class I (serology and DNA) and class II (DNA) typing and 6 for HLA crossmatch test (3 cells x 2 sera). RESULTS: Overall concordance of serologic typing was 99.5% (436/438) for HLA-A, 95.7% (419/438) for HLA-B, and 94.8% (199/210) for HLA-C. The antigens assigned incorrectly by more than 5% of the participating laboratories were B54 (10.3%), B55 (10.3%), B27 (5.4%), Cw6 (22.9%), and C-blank (5.7%). Overall concordance rates of DNA typing were 99.7% (393/394) for HLA-A, 99.8% (415/416) for HLA-B, 100% (156/156) for HLA-C. Correct assignment of HLA-DRB1 and -DQB1 was reportred by 99.7% (98.1-100%) and 99.2% (88.9-100%) for generic and 99.2% and 98.1% (80-100%) for allelic level, respectively. Most laboratories (93.5-97.9%) were using sensitive methods of crossmatch such as T-long, T-AHG, and flowcytometry. The proportion of laboratories evaluated as unacceptable was on the average 3.1% of total laboratories. CONCLUSIONS: The rate of correct identification of HLA antigens was higher this time than in the previous survey in 1999. The rate of unacceptable crossmatch was also low enough to be satisfactory. It is thought that the proficiency survey has contributed to the high quality of HLA tests in the participating laboratories and should be continued to maintain the proficiency in Korea.

Results of the HLA Typing Proficiency Survey in Korea, 2000-2002 / 대한진단검사의학회지

BACKGROUND: HLA proficiency survey was started in 1996 in Korea, and the results of the 1996-1998 surveys were reported previously. Here, we report the results of the surveys performed in recent three years (2000-2002). METHODS: Six surveys were carried out with the participation of 52-54 laboratories. For each survey, 3 peripheral blood samples and 2 sera were distributed for 3 HLA class I serology, 3 HLA class I DNA, 3 HLA class II DNA, 6 HLA crossmatch, and 3 PRA tests. RESULTS: Overall consensus of serologic typing was similar to the results of the previous survey: HLA-A 93.5%, HLA-B 88.3%, and HLA-A, B 82.7%. There were an increasing number of the laboratories that were using DNA typing for HLA-DR (51 laboratories, 94%) and HLA-A and B (26 laboratories, 48%). Overall consensus of DNA typing was very high: HLA-A 100%, HLA-B 99.1%, HLAC 97.9%, HLA-DRB1 low/high resolution 99.2/99.0%, HLA-DQB1 low/high resolution 99.3/97.5%. HLA crossmatch (T cells) was reported by 44-49 laboratories, and the use of sensitive methods was increased: AHG 33 laboratories and flow cytometry 7 laboratories. For incompatible (positive) crossmatches, 4.9% (0-14.3%) of cytotoxicity tests and 7.1% (0-16.7%) of flow tests were reported as negative. PRA was reported by 5 laboratories only. CONCLUSIONS: The use of DNA tests for HLA typing and AHG or flow cytometry methods for HLA crossmatch tests has much increased compared to the previous report. A continuous survey program would play an important role in the standardization and maintenance of laboratory proficiency in histocompatibility testing in Korea.

An Evaluation of HLA-matched Platelet Transfusion Effect in Patients with Platelet Refractoriness / 대한진단검사의학회지

BACKGROUND: Patients with platelet refractoriness as a result of human leukocyte antigen (HLA) alloimmunization can be effectively managed by transfusion of HLA-matched platelets. In this study, we have retrospectively evaluated the effect of HLA-matched platelet transfusion using a hospital based donor pool of 450 HLA typed donors. METHODS: For 17 patients showing platelet refractoriness to random donor platelets [1 hr corrected count increment (CCI) or =7, 500/microliter/m2) was obtained. HLA crossmatch (NIH method) negative patients showed a significantly higher platelet increment compared with crossmatch positive patients (23, 877 vs 10, 823; P=0.000). Although better transfusion effect was obtained in higher grade HLA match of A-B2U by selection of HLA compatible donors according to patients' HLA antibody specificities, an effective platelet increment was obtained in lower grade matches as well. Platelets transfused 24 hours (20, 325 vs 11, 417; P=0.029). CONCLUSIONS: Although many low grade matched donors were selected due to a relatively small size of HLA typed donor pool, effective platelet increments were obtained by selecting platelet donors on the basis of HLA antibody specificity.

Plasmapheresis in a Renal Transplant Patient with Positive Crossmatch only Detected by Flow Cytometry / 대한수혈학회지

Complement-dependent cytotoxicity (CDC) has been established as a crossmatch (XM) technique that can effectively prevent hyperacute transplantation rejetion by detecting preformed complement-fixing antibodies. The anti-human globulin crossmatch (AHGXM) has been performed in an effort to improve sensitivity for detecting anti-donor antibodies. Flow cytometry crossmatch (FCXM) was introduced as a more sensitive technique than the traditional CDCXM or AHGXM. A positive pre-transplant FCXM in recipients with a negative CDCXM or AHGXM has been found to be associated with a poor graft survival in several studies. Many clinical studies have focused on suppressing or eliminating anti-donor antibodies through the use of immunosuppresive drugs, immunoadsorption, intravenous immunoglobulin, or plasmapheresis. We performed plasmapheresis with intravenous immunoglobulin and FK 506 for a 38-year old female patient with chronic renal failure to remove anti-donor antibody which was only detected by FCXM. After transplant, no evidence of hyperacute or acute rejection was found. After 6 month, the recipient was surviving with well-functioning graft.

HLA Cross-match Negative Conversion by Therapeutic Plasmapheresis in a Patient with Positive HLA Cross-match / 대한진단검사의학회지

It is well accepted that kidney transplantation cannot be done if the recipient has antibodies showing a positive HLA cross-match to the donor. Recently, Schweitzer and his associates used a combination therapy with plasmapheresis, IV gamma globulin, and potent immunosuppression to induce HLA cross-match negative conversion in patients with a positive HLA cross-match to living donors and they reported good results after the trials. Therefore, we treated a patient with combination therapy who had persistent-positive HLA cross-match to multiple living donors. The patient was a 38-year-old female suffering from chronic renal failure and she showed persistent positive HLA cross-match to multiple living donors. Using a combination therapy with plasmapheresis, IV gamma globulin and immunosuppression, we have successfully achieved a HLA cross-match negative conversion in a patient and we did kidney transplantation without any sign of hyperacute or acute rejection. Although we present possibility of a HLA cross-match negative conversion by combination therapy, especially in a recipient with a low titer cross-match positive to a family donor, further long-term study with more patients is needed for evaluation of the efficacy of this trial.

A Questionnaire Survey of HLA Laboratories in Korea (1997) / 대한임상병리학회지

BACKGROUND: We have performed questionnaire surveys of HLA laboratories in 1993 and 1995 and here we report the results of a survey performed in 1997. METHODS: The questionnaires were distributed to 39 HLA laboratories enrolled in the HLA quality assessment (QA) program (started in 1996) in Korea. The questionnaire items were slightly modified from those of the previous survey. RESULTS: Most of the HLA laboratories (31/39, 80%) belonged to the specialties of clinical pathology. Most of the HLA laboratories were of small scale in the number of HLA technicians and the annual number of HLA tests. The methods used for HLA crossmatch were quite improved compared to those of the previous survey (1995). The number of laboratories using sensitive methods such as T-AHG and/or T-long methods has markedly increased (31/34 laboratories, 91%) compared to that of the previous survey (5/29 laboratories, 17%). DNA typing methods for HLA-DR were used in 27 (69%) laboratories, among which 25 laboratories used commercial kits. Some laboratories stored complement at inappropriate temperature, which could adversely affect the test results. As for external QA programs for HLA tests, 7 laboratories were participating in international programs. Most of the laboratories responded that the domestic HLA QA program was of much help for HLA tests, especially for HLA crossmatch tests, and 21 laboratories changed the HLA crossmatch test methods after participating in the QA program. CONCLUSIONS: In recent 2 years, the most prominent changes in domestic HLA laboratories were increased use of HLA-DR DNA typing methods and improvement and standardization of HLA crossmatch test methods. The domestic HLA QA program was considered to be very helpful for quality improvement and standardization of HLA test.