J Korean Soc Transplant.  2012 Jun;26(2):92-100.

Evaluation of Flow Cytometric Crossmatch Results in Comparison with Donor-specific Antibodies Detected by Luminex-PRA Tests in Organ Transplantation Patients

Affiliations
  • 1Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea. parkmhee@snu.ac.kr
  • 2Department of Laboratory Medicine, Hallym University Sacred Heart Hospital, Anyang, Korea.
  • 3Department of Laboratory Medicine, Seoul Paik Hospital, Inje University College of Medicine, Seoul, Korea.

Abstract

BACKGROUND
Two of the most sensitive methods for detecting donor-specific HLA antibodies (DSAs) are solid phase panel reactive antibody (PRA) assay using Luminex platform (Luminex-PRA), and a cell-based flow cytometric crossmatch (FCXM) test. We evaluated FCXM results in relation to DSAs detected by the Luminex-PRA method in solid organ transplantation candidates or post-transplant follow-up patients.
METHODS
A total of 171 donor-recipient pairs were evaluated by Luminex-PRA (LIFECODES Class I and Class II ID kits; Gen-Probe, USA) and FCXM (T- and B-cells) tests. DSA levels were analyzed using a sum of median fluorescence intensity (MFI) values, and FCXM results were analyzed using MFI ratios.
RESULTS
Class I and II DSAs were detected in 11.7% (20/171) and 11.1% (19/171) of tested sera, respectively. T-FCXM was negative in 97.4% (147/151) of Class I DSA negative sera, and B-FCXM was negative in 99.3% (137/138) of Class I and II DSA negative sera. T-FCXM was positive in 91.7% (11/12) of sera with moderate to strong Class I DSAs and B-FCXM was positive in 88.9% (16/18) of sera with moderate to strong Class II and/or Class I DSAs in the evaluation of sensitivities of FCXM in relation to DSA. There were significant correlations between FCXM ratios and DSA levels for both T-FCXM (P=0.008) and B-FCXM (P<0.001).
CONCLUSIONS
The FCXM results correlated well with the DSAs detected by the Luminex-PRA method. The specificities of T- and B-FCXM in relation to DSAs were high (>97%) and the sensitivities of T- and B-FCXM were satisfactory (>88%) in detecting moderate to strong DSAs.

Keyword

HLA antigens; Panel reactive antibody; Histocompatibility testing; Flow cytometry; Crossmatching

MeSH Terms

Antibodies
Flow Cytometry
Fluorescence
Follow-Up Studies
Histocompatibility Testing
HLA Antigens
Humans
Organ Transplantation
Transplants
Antibodies
HLA Antigens

Figure

  • Fig. 1 Relationship between DSA intensity (MFI sum) and FCXM strength (MFI ratio). (A) T-FCXM results in patients with Class I DSA (n=20), and (B) B-FCXM results in patients with Class I or Class II DSA (n=33). Abbreviations: DSA, donor-specific antibody; FCXM, flow cytometric crossmatch; MFI, median fluorescence intensity.

  • Fig. 2 Relationship between DSA intensity and flow cytometric crossmatch (FCXM) results. Sensitivities of FCXM tests detecting DSA-positive samples of different intensities are shown. Abbreviations: DSA, donor-specific antibody; MFI, median fluorescence intensity.

  • Fig. 3 Receiver operating characteristics (ROC) curve analysis of DSA intensity (MFI sum) for FCXM positivity. MFI sum values of Class I DSAs were used for T-FCXM (A), and those of Class I and Class II were used for B-FCXM (B). Abbreviations: AUC, area under the curve; DSA, donor-specific antibody; FCXM, flow cytometric crossmatch; MFI, median fluorescence intensity.


Reference

1. Patel R, Terasaki PI. Significance of the positive crossmatch test in kidney transplantation. N Engl J Med. 1969. 280:735–739.
Article
2. Süsal C, Döhler B, Sadeghi M, Ovens J, Opelz G. HLA antibodies and the occurrence of early adverse events in the modern era of transplantation: a collaborative transplant study report. Transplantation. 2009. 87:1367–1371. Erratum in Transplantation. 2009;88(2):293.
Article
3. Fernández-Fresnedo G, Pastor JM, López-Hoyos M, Ruiz JC, Zubimendi JA, Gonzalez-Cotorruelo J, et al. ELISA after kidney transplantation on the development of acute rejection and graft survival. Nephrol Dial Transplant. 2003. 18:990–995.
4. Gebel HM, Bray RA, Nickerson P. Pre-transplant assessment of donor-reactive, HLA-specific antibodies in renal transplantation: contraindication vs. risk. Am J Transplant. 2003. 3:1488–1500.
Article
5. Worthington JE, Martin S, Al-Husseini DM, Dyer PA, Johnson RW. Posttransplantation production of donor HLA-specific antibodies as a predictor of renal transplant outcome. Transplantation. 2003. 75:1034–1040.
Article
6. Cardarelli F, Pascual M, Tolkoff-Rubin N, Delmonico FL, Wong W, Schoenfeld DA, et al. Prevalence and significance of anti-HLA and donor-specific antibodies long-term after renal transplantation. Transpl Int. 2005. 18:532–540.
Article
7. Leffell MS, Zachary AA. Antiallograft antibodies: relevance, detection, and monitoring. Curr Opin Organ Transplant. 2010. 15:2–7.
Article
8. Eng HS, Bennett G, Bardy P, Coghlan P, Russ GR, Coates PT. Clinical significance of anti-HLA antibodies detected by Luminex: enhancing the interpretation of CDC-BXM and important post-transplantation monitoring tools. Hum Immunol. 2009. 70:595–599.
Article
9. Zeevi A, Lunz JG 3rd, Shapiro R, Randhawa P, Mazariegos G, Webber S, et al. Emerging role of donor-specific anti-human leukocyte antigen antibody determination for clinical management after solid organ transplantation. Hum Immunol. 2009. 70:645–650.
Article
10. Terasaki PI, McClelland JD. Microdroplet assay of human serum cytotoxins. Nature. 1964. 204:998–1000.
Article
11. Zachary AA, Houp JA, et al. Detrick B, Hamilton RG, editors. Evaluation of the humoral response in transplantation. Manuals of molecular and clinical laboratory immunology. 2006. 7th ed. Washington, D.C.: ASM Press;1215–1227.
Article
12. Colombo MB, Haworth SE, Poli F, Nocco A, Puglisi G, Innocente A, et al. Luminex technology for anti-HLA antibody screening: evaluation of performance and of impact on laboratory routine. Cytometry B Clin Cytom. 2007. 72:465–471.
Article
13. Vaidya S, Cooper TY, Avandsalehi J, Barnes T, Brooks K, Hymel P, et al. Improved flow cytometric detection of HLA alloantibodies using pronase: potential implications in renal transplantation. Transplantation. 2001. 71:422–428.
Article
14. Rodey GE, Neylan JF, Whelchel JD, Revels KW, Bray RA. Epitope specificity of HLA Class. I. alloantibodies I Frequency analysis of antibodies to private versus public specificities in potential transplant recipients. Hum Immunol. 1994. 39:272–280.
Article
15. Rodey GE, Revels K, Fuller TC. Epitope specificity of HLA Class I alloantibodies: II. Stability of cross-reactive group antibody patterns over extended time periods. Transplantation. 1997. 63:885–893.
16. Reinsmoen NL, Lai CH, Vo A, Cao K, Ong G, Naim M, et al. Acceptable donor-specific antibody levels allowing for successful deceased and living donor kidney transplantation after desensitization therapy. Transplantation. 2008. 86:820–825.
Article
17. Zachary AA, Leffell MS. Detecting and monitoring human leukocyte antigen-specific antibodies. Hum Immunol. 2008. 69:591–604.
Article
18. Gebel HM, Moussa O, Eckels DD, Bray RA. Donor-reactive HLA antibodies in renal allograft recipients: considerations, complications, and conundrums. Hum Immunol. 2009. 70:610–617.
Article
19. Tait BD, Hudson F, Brewin G, Cantwell L, Holdsworth R. Solid phase HLA antibody detection technology - challenges in interpretation. Tissue Antigens. 2010. 76:87–95.
Article
20. Muro M, Llorente S, Marín L, Moya-Quiles MR, Gonzalez-Soriano MJ, Prieto A, et al. Acute vascular rejection mediated by HLA antibodies in a cadaveric kidney recipient: discrepancies between FlowPRA, ELISA and CDC vs luminex screening. Nephrol Dial Transplant. 2005. 20:223–226.
Article
21. Lee PC, Ozawa M, Hung CJ, Lin YJ, Chang SS, Chou TC. Reappraisal of HLA antibody analysis and cross-matching in kidney transplantation. Transplant Proc. 2009. 41:95–98.
Article
22. Morris GP, Phelan DL, Jendrisak MD, Mohanakumar T. Virtual crossmatch by identification of donor-specific anti-human leukocyte antigen antibodies by solid-phase immunoassay: a 30-month analysis in living donor kidney transplantation. Hum Immunol. 2010. 71:268–273.
Article
23. Lee YS, Won DI. Analysis of positive flow cytometric crossmatch in organ transplantation. Lab Med Online. 2011. 1:43–50.
Article
24. Christiaans MH, Overhof-de Roos R, Nieman F, van Hooff JP, van den Berg-Loonen EM. Donor-specific antibodies after transplantation by flow cytometry: relative change in fluorescence ratio most sensitive risk factor for graft survival. Transplantation. 1998. 65:427–433.
Article
25. Riethmüller S, Ferrari-Lacraz S, Müller MK, Raptis DA, Hadaya K, Rüsi B, et al. Donor-specific antibody levels and three generations of crossmatches to predict antibody-mediated rejection in kidney transplantation. Transplantation. 2010. 90:160–167.
Article
26. Aubert V, Venetz JP, Pantaleo G, Pascual M. Low levels of human leukocyte antigen donor-specific antibodies detected by solid phase assay before transplantation are frequently clinically irrelevant. Hum Immunol. 2009. 70:580–583.
Article
27. Vlad G, Ho EK, Vasilescu ER, Colovai AI, Stokes MB, Markowitz GS, et al. Relevance of different antibody detection methods for the prediction of antibody-mediated rejection and deceased-donor kidney allograft survival. Hum Immunol. 2009. 70:589–594.
Article
Full Text Links
  • JKSTN
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr