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Table of Contents
CASE REPORT
Year : 2021  |  Volume : 15  |  Issue : 3  |  Page : 279-281

Pretransplant compatibility tests in kidney transplants: Significance of adding HLA bead-based assay to direct cell-based cross-match assays - A case report


Department of Transfusion Medicine, Molecular and Transplant Immunology Laboratory, Medanta-The Medicity, Gurgaon, Haryana, India

Date of Submission10-Jun-2020
Date of Decision12-Dec-2020
Date of Acceptance28-Apr-2021
Date of Web Publication30-Sep-2021

Correspondence Address:
Ms. Chhavi Rajvanshi
Department of Transfusion Medicine, Molecular and Transplant Immunology Laboratory, Medanta-The Medicity, Gurgaon, Haryana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijot.ijot_54_20

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  Abstract 

Pre-transplant compatibility work-up has evolved tremendously from conventional CDC only to CDC with more sensitive FCXM testing with bead-based assays (LMX and SAB). Detection of alloantibodies is one of the major objective in work-up algorithm. Conventionally cell-based tests were used, if both are negative, hospital usually proceeds for transplantation. If one of the screening tests (CDC/FCXM) is or both (CDC and FCXM) screening tests are positive, antibody identification is performed on Luminex platform to determine DSA by virtual cross-match. However, cell-based tests have their limitations. Here, we described a case which could have gone unnoticed if HLA bead-based assay was not there and could have triggered AMR (Antibody mediated rejection) as detected antibody (DRB1*07:01; BCM is 16416) was DSA.

Keywords: Bead-based assay, cell-based assay, prozone effect, renal transplant


How to cite this article:
Rajvanshi C, Tiwari AK, Pabbi S, Aggarwal G, Saini A, Chauhan R, Mehra S, Kumari P. Pretransplant compatibility tests in kidney transplants: Significance of adding HLA bead-based assay to direct cell-based cross-match assays - A case report. Indian J Transplant 2021;15:279-81

How to cite this URL:
Rajvanshi C, Tiwari AK, Pabbi S, Aggarwal G, Saini A, Chauhan R, Mehra S, Kumari P. Pretransplant compatibility tests in kidney transplants: Significance of adding HLA bead-based assay to direct cell-based cross-match assays - A case report. Indian J Transplant [serial online] 2021 [cited 2021 Nov 29];15:279-81. Available from: https://www.ijtonline.in/text.asp?2021/15/3/279/327393


  Introduction Top


Compatibility of donor with the recipient is one of the main objectives of compatibility workup before transplantation, and this has a direct bearing of graft and patient outcome. Common screening test strategies employed in India are to perform complement-dependent cytotoxicity cross-match (CDC) alone or CDC cross-match with flow cytometry-based cross-match (FCXM) tests. If both are negative, the hospital usually proceeds for transplantation.[1] If one of the screening tests (CDC/FCXM) is or both (CDC and FCXM) screening tests are positive, antibody identification is performed on Luminex platform and is matched with donor human leukocyte antigen (HLA) typing test results to determine donor-specific antibody (DSA). DSA may lead to donor deferral.[2]

However, both (CDC and FCXM) these assays are cell-based assays and have their limitations.[3] Recently, few laboratories, including ours, have added HLA bead-based assays to the conventional screening algorithm. We would like to present a case which could have gone unnoticed if HLA bead-based assay was not there and could have resulted in possible antibody-mediated rejection (AMR).


  Case Report Top


Recipient serum and lymphocytes of the donor were collected. CDC-XM and FCXM were performed. Luminex-based bead assay (LIFECODES Life Screen Deluxe- LMX, Immucor Inc, Atlanta, Georgia, USA) was also done with the serum of recipient to screen for anti-HLA antibodies. HLA typing for class I (A and B) and class II (DR) antigens was performed using polymerase chain reaction–sequence-specific oligonucleotide probes. HLA typing is usually done for assessing kinship between organ recipient and donor as a prerequisite in accordance with the Human Organ Transplant Act, India.[4] Single antigen bead assay (SAB) (LIFECODES LSATM Kit, Immucor Transplant Diagnostics, Inc. USA) was done on serum to identify the specificity of antibody (gold standard) and for virtual cross-match. In virtual cross-match, anti-HLA antibodies against donor's HLA antigen were mapped and compatibility was monitored virtually. BD FACSuite software was used to analyze FCXM files (cutoff; MCS for T-cell is 26; B-cell is 110) and Luminex software (MatchIT antibody) was used for LMX and SAB assay analysis (cutoff; background corrected MFI: positive by machine).

As described in [Table 1], we present a case where the patient was male and prospective donor (paternal aunt) was female, having no history of sensitization in either of them. All three tests (CDCXM, FCXM, and LMX) were performed for pretransplant workup. In this case, the CDC cross-match was negative. There was a discordance between FCXM and LMX results. While FCXM was negative for both T-cell and B-cell, LMX came out positive for class II. To resolve this discordance, an SAB assay was performed and DSA was identified in class II. Out of scientific curiosity, FCXM was repeated with serum dilutions and this confirmed our initial suspicion of negative FCXM because of pro-zone.
Table 1: Summary of initial complement-dependent cytotoxicity, flow cytometric cross-match, LMX and supplemental repeat flow cytometric cross-match, and single antigen bead assay results in the reported case

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In this case, LMX revealed that antibody against DRB1 locus could have been missed if we were not employing LMX assay. More importantly, the identified antibody (DRB1 * 07:01) was DSA and could have triggered AMR, if remained undetected.

[Table 2] describes FCXM between recipient neat serum and donor cells plus diluted serum of patient and donor cells. Neat serum indicates a negative FCXM reaction for T- and B-cells. However, positive B–cells' reactions were found in diluted serum reactions at 1:2 (MCS = 544), 1:4 (MCS = 775), and at 1:8 (MCS = 391) dilutions. [Table 2] clearly is an illustration of the prozone effect in FCXM.
Table 2: Flow cytometry-based cross-match results with neat and serially diluted serum confirming prozone phenomenon

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  Discussion Top


Prozone effect is defined as false-negative antigen–antibody reaction mainly due to high titer of immunoglobulin G (IgG) antibodies or other causes like interference of IgM antibodies (IgM are not clinically relevant) or excess of C1q antibodies which hinder binding of labeled anti-IgG antibodies (secondary antibodies) to antigen–antibody complex.[5] Hence, this effect sometimes gives unexpected negative results in FCXM or bead-based assays. In case of CDC testing, serum dilution is an essential part to avoid prozone effect. However, CDC has the limitation of low sensitivity. In FCXM test, though DSA is usually identified with higher sensitivity,[6],[7] dilutions are not done routinely in most of the laboratories. Likewise in LMX, dilutions are not done routinely.

SAB is more sensitive than all the three (CDC, FCXM, LMX) screening tests and is usually employed as a confirmatory test. However, even SAB is not performed in dilutions as part of the routine test procedure.[8],[9],[10] However, in this report though there was no routine use of diluted serum tube in FCXM, bead-based LMX test alerted us for the possibility of prozone phenomenon, which was confirmed with repeated FCXM test [performed in dilutions, [Table 2]] supplemented with SAB assay.

This patient had DSA in class II (DRB1 * 07:01) with significant MFI (16416), which lead to donor deferral. However, what we would like the readers to know that if we had done only cell-based (CDCXM and FCXM) assays and transplant would have occurred, and it would have possibly led to AMR. It was the HLA bead-based (LMX) test that drew our attention to retest FCXM and eventually deferring the donor since the identified antibody was DSA.

Since a single test is never 100% sensitive (and specific), the testing strategy should always combine two or more tests as part of pretransplant compatibility testing.[11]

For example, we usually adopt CDC, FCXM, and LMX for screening and the test results give concordant results (commonly concordant negative) and therefore allow clear and unambiguous interpretation. Whenever one of the screening tests or more than one test are positive, it calls for supplementary testing by SAB for confirmation.

Whenever there is discordance in the results of screening assays, one should consider possibility of prozone, autoantibody, non-HLA antibodies, etc.[12] In this case, since the initial suspicion was of prozone, FCXM was repeated in dilutions to confirm the results.

The authors strongly recommend the addition of HLA bead-based test to the existing armamentarium of cell-based cross-match test in compatibility testing algorithm to enhance the sensitivity of test strategy.

Declaration of patient consent

Patient consent was obtained for diagnosis and treatment to the hospital. This was an observational study and no additional sample was drawn for this study. All investigation, treatment, and monitoring were according to the current “Standard-of-Care.”

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Rajvanshi C, Tiwari AK, Choudhuri J, Mehra S, Chauhan R. Pre-transplant compatibility tests in kidney transplants: case report on significance of epitope-based analysis in donor selection. Indian J Nephrol 2020;30:57-8.  Back to cited text no. 1
[PUBMED]  [Full text]  
2.
Zachary AA, Sholander JT, Houp JA, Leffell MS. Using real data for a virtual crossmatch. Hum Immunol 2009;70:574-9.  Back to cited text no. 2
    
3.
Altermann WW, Seliger B, Sel S, Wendt D, Schlaf G. Comparison of the established standard complement-dependent cytotoxicity and flow cytometric crossmatch assays with a novel ELISA-based HLA crossmatch procedure. Histol Histopathol 2006;21:1115-24.  Back to cited text no. 3
    
4.
Sahay M. Transplantation of human organs and tissues act. Indian J Transplant 2020;14:188.  Back to cited text no. 4
  [Full text]  
5.
Jain D, Choudhuri J, Chauhan R, Dorwal P, Sharma D, Tiwari AK, et al. False negative single antigen bead assay: Is it always an effect of prozone? J Clin Lab Anal 2018;32:e22237.  Back to cited text no. 5
    
6.
Schnaidt M, Weinstock C, Jurisic M, Schmid-Horch B, Ender A, Wernet D. HLA antibody specification using single-antigen beads–A technical solution for the prozone effect. Transplantation 2011;92:510-5.  Back to cited text no. 6
    
7.
Zhang R. Donor-specific antibodies in kidney transplant recipients. Clin J Am Soc Nephrol 2018;13:182-92.  Back to cited text no. 7
    
8.
Gebel HM, Harris SB, Zibari G, Bray RA. Conundrums with FlowPRA™ beads. Clin Transplant 2002;16:24-9.  Back to cited text no. 8
    
9.
Morris AB, Sullivan HC, Krummey SM, Gebel HM, Bray RA. Out with the old, in with the new: Virtual versus physical crossmatching in the modern era. HLA 2019;94:471-81.  Back to cited text no. 9
    
10.
Sağıroğlu T, Tozkır H, Kılıçarslan-Ayna T, Yağcı MA, Sezer A, Carin M. Is flow cytometry crossmatch analysis using sera with different dilutions important for pretransplant analysis? A case report. Transplant Proc 2012;44:1767-9.  Back to cited text no. 10
    
11.
Dorwal P, Jain D, Tyagi N, Mehra S, Pande A, Chauhan R, et al. Pre-transplant anti-HLA antibody work-up of prospective renal transplant patients in a developing country: A flexible, efficient, yet cost-effective approach: P2. Int J Immunogenet 2015;42:375-6.  Back to cited text no. 11
    
12.
Schinstock CA, Gandhi MJ, Stegall MD. Interpreting Anti-HLA antibody testing data: A practical guide for physicians. Transplantation 2016;100:1619-28.  Back to cited text no. 12
    



 
 
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