Sezai Yilmaz, Veysel Ersan, Zeynal Mete Karaca, Basak Kayhan

False negative results in luminex panel reactive antibodyidentification assay: Should we be alert on them?

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 antiHLA 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.

PDF

___

1. Terasaki PI, McClelland JD. Microdroplet Assay of Human Serum Cytotoxins. Nature 1964;204:998- 1000.
2. Picascia A, Infante T, Napoli C. Luminex and antibody detection in kidney transplantation. Clin Exp Nephrol 2012;16:373-81.
3. Kim JJ, Fuggle SV, Marks SD. Does HLA matching matter in the modern era of renal transplantation? Pediatric Nephrology 2019.
4. Vo AA, Aubert O, Haas M, et al. Clinical Relevance of Posttransplant DSAs in Patients Receiving Desensitization for HLA-incompatible Kidney Transplantation. Transplantation 2019;103:2666-74.
5. Huh KH, Kim MS, Kim HJ, et al. Renal transplantation in sensitized recipients with positive luminex and negative CDC (complement-dependent cytotoxicity) crossmatches. Transpl Int 2012;25:1131-7.
6. Fuggle SV, Martin S. Tools for human leukocyte antigen antibody detection and their application to transplanting sensitized patients. Transplantation 2008;86:384-90.
7. Khishigsuren B, Demir E, Akgul SU, et al. Panel Reactive Antibody Responses Against Influenza Vaccination in Kidney Transplant Recipients. Transplant Proc 2019;51:1115-7.
8. Jang JY, Kim YJ, Kim Y, et al. Application of calculated panel reactive antibody using HLA frequencies in Koreans. Ann Lab Med 2012;32:66-72.
9. Cecka JM. Calculated PRA (CPRA): the new measure of sensitization for transplant candidates. Am J Transplant 2010;10:26-9.
10. Inal A, Ozcelik U, Ogan Uyanik E, et al. Analysis of Panel Reactive Antibodies in Renal Transplant Recipients Detected by Luminex: A Single-Center Experience. Exp Clin Transplant 2016;14:401-4.
11. Gebel HM, Bray RA. Sensitization and sensitivity: defining the unsensitized patient. Transplantation 2000;69:1370-4.
12. Murphey CL, Forsthuber TG. Trends in HLA antibody screening and identification and their role in transplantation. Expert Rev Clin Immunol 2008;4:391- 9.
13. In JW, Rho EY, Shin S, et al. False-positive reactions against HLA class II molecules detected in Luminex single-antigen bead assays. Ann Lab Med 2014;34:408-10.
14. Jain D, Choudhuri J, Chauhan R, et al. False negative single antigen bead assay: Is it always an effect of prozone? J Clin Lab Anal 2018;32.
15. Phanish MK. Immunological risk assessment and human leukocyte antigen antibody testing in kidney transplantation. Indian J Nephrol 2016;26:80-5.
16. Kurtoglu EL, Sahin I, Taskapan H, et al. Diyaliz hastalarında panel reaktif antikor düzeyinin tespiti: iki yöntem ve iki analizin karșılaștırılması. İnönü Üniversitesi Sağlık Bilimleri Dergisi 2015;4:22-5.
17. Valenzuela NM, Reed EF. Antibody-mediated rejection across solid organ transplants: manifestations, mechanisms, and therapies. J Clin Invest 2017;127:2492-504.
18. Martinez-Mier G, Vazquez-Crespo LV, AngelesHernández F, et al. Effect of Panel-Reactive Antibody on Graft Survival in Living Kidney Donor Transplantation: Analysis of 10 Years in a Transplant Center in Veracruz, Mexico. Transplantation Proceedings 2020;52:1140- 2.
19. Barocci S, Valente U, Nocera A. Detection and analysis of HLA class I and class II specific alloantibodies in the sera of dialysis recipients waiting for a renal retransplantation. Clin Transplant 2007;21:47-56.
20. Colombo MB, Haworth SE, Poli F, et al. Luminex technology for anti-HLA antibody screening: evaluation of performance and of impact on laboratory routine. Cytometry B Clin Cytom 2007;72:465-71.
21. Jung S, Oh EJ, Yang CW, et al. [Comparative evaluation of ELISA and Luminex panel reactive antibody assays for HLA alloantibody screening]. Korean J Lab Med 2009;29:473-80.
22. Weinstock C, Schnaidt M. The complement-mediated prozone effect in the Luminex single-antigen bead assay and its impact on HLA antibody determination in patient sera. Int J Immunogenet 2013;40:171-7.
23. Konvalinka A, Tinckam K. Utility of HLA Antibody Testing in Kidney Transplantation. J Am Soc Nephrol 2015;26:1489-502.
24. Jacob EK, De Goey SR, Gandhi MJ. Positive virtual crossmatch with negative flow crossmatch results in two cases. Transpl Immunol 2011;25:77-81.
25. Carey BS, Boswijk K, Mabrok M, et al. A reliable method for avoiding false negative results with Luminex single antigen beads; evidence of the prozone effect. Transpl Immunol 2016;37:23-7