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Year : 2021  |  Volume : 15  |  Issue : 2  |  Page : 99-103

Evaluation of screening tests for pre-transplant compatibility testing in live-related kidney transplants: Single-center report from India - A prospective observational study

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

Date of Submission11-Jul-2020
Date of Acceptance15-Aug-2020
Date of Web Publication30-Jun-2021

Correspondence Address:
Ms. Rajni Chauhan
Department of Transfusion Medicine, Molecular and Transplant Immunology Laboratory, Medanta-The Medicity, Sector-38, Gurgaon - 122 001, Haryana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijot.ijot_76_20

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Introduction: Pre-transplant compatibility testing involves the use of different methodologies (cell-based and solid phase based) for the determination of anti-human-leukocyte antigen (HLA) antibodies. Implementation of these donor-recipient methods in the screening of patients awaiting transplantation increased their chance of successful graft and patient outcomes. Materials and Methods: A total of 1054 patients visiting tertiary care hospitals for pretransplant compatibility testing were screened with cell-based tests; complement-dependent cytotoxicity crossmatch (CDC-XM) and flow cytometric crossmatch (FC-XM). The patients positive for either or both screening tests were suspected to have anti-HLA antibodies. Luminex single-antigen bead (SAB) tests were performed in such patients to determine and identify antibody specificity and establish donor-specific antibody (DSA). Results: The study showed a significantly higher sensitivity of the FCXM (94.6%) method when compared with CDC-XM (35.7%), considering the SAB assay as the gold standard technique. The specificity of CDC-XM (100%) was slightly higher than the FC-XM (76.3%). Combination of FC-XM with CDC-XM (17 cases) was 100% sensitive and specific to identify DSA (s). The graft-survival was 94.77% using the proposed algorithm. Conclusions: The combination of CDC-XM and FC-XM, along with SAB assay, could be used as a screening algorithm as it is a useful technique in identifying the specificities of alloantibodies, assessment of DSAs. Hence, the presented algorithm can become a new standard for the identification of potential recipients awaiting kidney transplantation in India.

Keywords: Compatibility, live-related, pre-transplant, renal transplant, single-antigen bead

How to cite this article:
Chauhan R, Tiwari AK, Rajvanshi C, Mehra S, Aggarwal G, Bansal SB, Kher V. Evaluation of screening tests for pre-transplant compatibility testing in live-related kidney transplants: Single-center report from India - A prospective observational study. Indian J Transplant 2021;15:99-103

How to cite this URL:
Chauhan R, Tiwari AK, Rajvanshi C, Mehra S, Aggarwal G, Bansal SB, Kher V. Evaluation of screening tests for pre-transplant compatibility testing in live-related kidney transplants: Single-center report from India - A prospective observational study. Indian J Transplant [serial online] 2021 [cited 2021 Oct 20];15:99-103. Available from: https://www.ijtonline.in/text.asp?2021/15/2/99/319891

  Introduction Top

Preformed anti-HLA antibodies pose a high risk of rejection and remain a significant barrier in renal transplantation.[1] The incidence of acute humoral rejection and chronic rejection increases even with a low level of donor-specific antibodies (DSAs) and leads to organ failure.[2],[3] The complement-dependent cytotoxicity cross-match (CDC-XM) has been the standard assay for the detection of cytotoxic DSA before transplantation.[4] However, CDC-XM is less sensitive in detecting all clinically significant antibodies and thus may result in antibody-mediated rejection (AMR) in some patients even with negative CDC-XM.[5],[6]

With the advent of time, improved methodologies for cross-matching and antibody detection have evolved with increased sensitivity and specificity. Flow cytometric cross-match (FC-XM) is a sensitive technique that enables the detection of the antibody of all immunoglobulin (IgG) isotypes, including both complement-fixing and noncomplement-fixing.[7],[8],[9],[10] Luminex single-antigen bead (SAB) technology allows the determination of anti-HLA antibody specificity in patients. This helps to resolve much of the uncertainty associated with the interpretation of CDC-XM and FC-XM.[11]

Different laboratories follow different pretransplant testing algorithms based on a combination of various assays that are available CDC-XM, FC-XM, panel reacting antibodies (PRA) and SAB. Some transplant centers have even abandoned the use of CDC-XM and rely on a combination of FC-XM and SAB. However, the use of very sensitive techniques like SAB universally reduces the chances of sensitized patients to undergo timely transplantation though the clinical significance of very-low-level DSA detected only by Luminex assay (SAB) is uncertain.[12],[13],[14],[15]

This study discusses the authors' experience with different testing methods such as CDC-XM, FC-XM, and Luminex (SAB) assay in the detection of DSA and co-relates results of these methods with actual graft and patient survival. The objective of the study was to evaluate pretransplant testing algorithm that would be feasible and cost-effective for a developing country like India.

  Materials and Methods Top

Study design

This prospective observational study comprised pre-transplant workup data (and analysis) from 1054 potential kidney recipients at a tertiary care center in the national capital region (India) during 2012–2017. As per institutional guidelines, all recipients underwent kinship testing (human-leukocyte antigen [HLA] typing) with the prospective donor (s) in accordance with the Human Organ Transplantation Act, India.[16] According to the Gazette of India. Extraordinary Transplantation of Human Organs and Tissues Rules, 2014, “Donors are near relative which includes spouse, son, daughter, father, mother, brother, or sister, grandparents, grandchildren.”[17] Compatibility testing was performed by using CDC-XM and FC-XM for all potential recipients. Luminex SAB testing was performed as a reflex test when either CDC-XM or FC-XM positive or both were positive. SAB was done to determine the specificity of the anti-HLA antibody and confirm DSA.

Technique, and equipment

HLA typing was performed using sequence-specific oligonucleotide probes method (HLA-A, B, DR Immucor, Inc., GA, US). In addition, HLA-C, DQ (HLA-C, DR Immucor, Inc., GA, US) and HLA-DP (HLA-DP Immucor, Inc., GA, US) were performed in SAB positive cases to rule-out/rule-in DSA.

Anti-human globulin (AHG)-CDC-XM was performed using lymphocytes[18] isolated from peripheral blood. Cells were incubated with recipient's serum in serial dilution (1:1–1:64) for 75 min at 37°C and room temperature, which was then followed by incubation with AHG and complement for another 75 min. Eosin dye was added to differentiate live and dead cells under an inverted microscope and based on the percent of dead cells present; scoring was done.

FC-XM was performed using three-color flow cytometer (BD FACS Verse) and anti-human IgG (Jackson Immuo Research Laboratories, USA) after discriminating T- and B-cells from the isolated donor mononuclear cells using CD3 and CD22 (BD Biosciences, US, as per the protocols derived from Current Protocols in Cytometry 2004).[19] Our laboratory (median channel shift [MCS]) cutoff for T-cells was 34 and B-cell was 110 for FC-XM positive samples. To minimize the probability of any technical error creeping in, each assay is performed in duplicate, and the mean value of MCS is considered for result evaluation. If the results of replicate testing are grossly discordant, the entire assay is repeated with the same sample or fresh sample, if required.

SAB assay was performed on the Luminex platform using Lifecodes® SAB Class I and Class II kits (Immucor, Inc., GA, US). The strength of the antibody was measured as median fluorescence intensity (MFI); interpretation as “more the MFI,” “more the strength” of the antibody. If the specificity of the anti-HLA antibody detected by SAB was against one of the donors' HLA antigen (s), then they were called DSA.

Statistical analysis

The analysis included demographic profiling of all the patients on different parameters. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for CDC-XM and FC-XM in comparison with SAB assay with the presence of DSA. Additional 100 concordant negative samples (i.e., negative on both CDC-XM and FC-XM) were also tested with SAB assay for appropriate statistical evaluation. P < 0.05 was considered statistically significant.

Declaration of patient consent

The patient consent has been taken for participation in the study and for publication of clinical details and images. Patients understand that the names, initials would not be published, and all standard protocols will be followed to conceal their identity.

Ethics statement

Patients' written informed consent was obtained for diagnosis and treatment at the hospital. This was an observational study; no personal identifiers such as name, addresses were used, and no additional sample was drawn for this study and EC approval was not needed. All investigation, treatment, and monitoring were according to the current hospital's “standard-of-care” and institutional standard operating procedure. The study was carried out as per declaration of Helsinki.

  Results Top

Out of the total, 1054 patients, 834 were male and 220 were female with a median age of 40 years. Among them, 967 patients were negative by both CDC-XM and FC-XM. Therefore, no reflex testing was performed in these cases. Out of 87 positive samples, 17 samples were positive by both CDC-XM and FC-XM, 67 samples were positive by FC-XM only, three samples were positive by CDC-XM only. In all these 87 positive cases, SAB assay was performed to confirm the presence and determine the specificity of anti-HLA antibody. [Table 1] and [Table 2] compare the results of CDC-XM and FC-XM with DSA (determined by SAB assay). Of 17 samples positive by both techniques, all were positive for DSA. All three cases positive by CDC-XM only were also positive for DSA. Of 67 samples positive by FC-XM only, 57 were positive for SAB (36 were positive for DSA and 21 were negative for DSA) and 10 were negative for SAB.
Table 1: Two by two table of complement-dependent cytotoxicity-crossmatch and donor-specific antibody (single antigen bead assay)

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Table 2: Two by two table of flow cytometric-crossmatch and donor-specific antibody (single antigen bead assay)

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SAB assay was negative for DSA in all additional 100 concordant negative samples performed for statistical analysis. The sensitivity, specificity, PPV, and NPV of CDC-XM and FC-XM in comparison to DSA is shown in [Table 3]. Out of 1054 patients, 1034 patients underwent a transplant and mean serum creatinine of these patients at 6 months of post-transplant follow up was 1.4. At the mean 2-year follow-up, overall patient survival was 97.08% and the graft survival was 94.77%.
Table 3: Sensitivity, specificity, positive predictive value, negative predictive value of complement-dependent cytotoxicity-crossmatch and flow cytometric-crossmatch in comparison with donor-specific antibody (determined by single antigen bead assay)

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  Discussion Top

Several methods such as CDC-XM, FC-XM, PRA, and SAB assays are used alone or in combination (algorithm) with an objective to detect any preexisting DSA before transplant. Different laboratories follow different testing algorithms for the detection of anti-HLA antibodies.[20]

CDC-XM alone as a screening technique is widely used but has various limitations in terms of low sensitivity, subjectivity, and inability to detect noncomplement-fixing antibodies.[21] Modifications of conventional CDC-XM, such as AHG addition (AHG-CDC-XM) and Dithiothreitol (DTT) addition (DTT-CDC-XM) are more sensitive than the conventional method. However, these modifications are, in turn, is less sensitive and Luminex for the detection of DSA. Therefore, we used AHG-CDC-XM in this study.[22],[23],[24],[25] FC-XM is a prevalent technique at many transplant centers as it is a very sensitive cross-match method available.[15] Luminex solid phase (SAB) assay is the most sensitive method to detect low levels of anti HLA antibodies.[26],[27],[28],[29] Hence, SAB is used as a gold standard to confirm and detect anti-HLA antibodies in the present study. However, SAB was not performed in all patients due to cost constraints.

The algorithm used in this study [Figure 1] involved the use of CDC-XM and FC-XM as screening techniques and SAB assay as reflex confirmation tests in patients positive by either or both techniques.
Figure 1: Algorithm for pretransplant compatibility testing followed in the study

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In the present study, the sensitivity of FC-XM (94.6%) was found to be much higher than CDC-XM (35.7%), whereas the specificity of CDC-XM (100%) was slightly higher than the FC-XM (76.3%). Lack of sensitivity of CDC-XM in detecting clinically significant antibodies leads to a high incidence of AMR occurring in patients with a negative CDC-XM.[5],[6] Therefore, the authors recommend that no laboratory should have CDC-XM as the sole screening test for pretransplant compatibility testing. However, even FC-XM (sensitivity of 94.6%), though far superior to CDC-XM, may also miss a few patients with clinically relevant antibodies. A combination of FC-XM with CDC-XM or PRA (not tested in the present study) would possibly be a better screening algorithm. Guidelines for testing of HLA antibodies confirm the use of both cell-based and solid-phase assays to assess the antibody status of the intended donor.[30] In the current study, even positive FC-XM (specificity of 76.3%) assay seems like “absolute contraindication” for transplant. Ten cases, which were FC-XM positive and were SAB negative, could be due to the underrepresentation of indigenous HLA antigen in the SAB kit.[31] Twenty-one FC-XM positive cases, where DSAs were not identified, could be due to the non-HLA antibodies (e.g. MHC class 1 chain-related) alloantibodies.[32] However, non-HLA antibodies were not tested in the present study. This also suggests the use of PRA as a screening test for differentiating HLA-antibodies from non-HLA antibodies. Three cases, which were CDC-XM positive and FC-XM negative, cannot be explained and these could have possibly happened due to some technical error (s). Seventeen cases positive by both CDC-XM and FC-XM were all positive for DSA and therefore validated the proposed algorithm.

The algorithm is for risk stratification, or in simple words, means that if both screening tests are negative, the transplant can be safely performed, and likewise, if one or both screening tests are positive, it is best to look for another prospective matched donor.

The solid-phase Luminex (SAB) assays demonstrate high sensitivity and specificity in detecting anti-HLA antibodies.[15] Transplant after identification of DSA in SAB is associated with a higher incidence of graft rejection and in several studies.[20] The risk may vary from high risk to almost no risk. This variation is partly due to technical aspects such as MFI, antibody class (complement fixing vs. noncomplement-fixing antibodies), antibody titer, and type of antibodies (native antibodies or antibodies to denatured HLA molecules on the beads), which have extensively been reviewed in a recent consensus paper.[6] Therefore, in the present study, SAB assay was used as a gold standard in determining DSA in positive cases. Our patient (97.08%) and graft (94.77%) survival rates are at par with Indian and international published literature.[33],[34]

The average cost of performing the screening (CDC-XM and FC-XM) and reflex SAB in positive cases worked out as 100 US$ per patient, while an algorithm comprising CDCXM and SAB in all patients would have worked out approximately 320 US$. The algorithm proposed in the present study is three times less expensive.

Limitations of the study

The study had a few limitations; SAB was performed in only 100 concordant negative samples and not all 967, and therefore, 867 were assumed to be negative for DSA. In our screening algorithm, we used both cell-based tests (CDC-XM and FC-XM), and no solid-phase test (PRA) was part of the screening tests.

  Conclusions Top

The algorithm comprising a combination of CDC-XM and FC-XM along with SAB assay (used as a reflex test when either or both are positive) could be used as testing algorithm as it is a useful cost-effective technique in identifying the specificities of alloantibodies, and assessment of DSAs. This proposed algorithm can become a new standard for pretransplant testing in potential recipients awaiting kidney transplantation in India.


We would like to thank the team members of the Molecular and Transplant Immunology Laboratory for their technical support.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Patel R, Terasaki PI. Significance of the positive cross-match test in kidney transplantation. N Engl J Med 1969;280:735-9.  Back to cited text no. 1
Süsal C, Döhler B, Opelz G. Presensitized kidney graft recipients with HLA Class I and II antibodies are at increased risk for graft failure: A collaborative transplant study report. Hum Immunol 2009;70:569-73.  Back to cited text no. 2
Gebel HM, Bray RA, Nickerson P. Pre-transplant assessment of donor-reactive, HLA-specific antibodies in renal transplantation: Contraindication vs. risk. Am J Trans 2003;12:1488-500.  Back to cited text no. 3
Mulley WR, Kanellis J. Understanding crossmatch testing in organ transplantation: A case-based guide for the general nephrologist. Nephrol 2011;16:125-33.  Back to cited text no. 4
Banner B, Makowka L, Demetris J, Tzakis A, Griffin M, Starzl TE. Hyperacute rejection of the kidney in patients with a negative cross-match. Trans Proceed 1988;20 Suppl 1:453.  Back to cited text no. 5
Iwaki Y. Primary nonfunction in human cadaver kidney transplantation: Evidence for hidden hyperacute rejection. Clin Trans 1989;1:125-31.  Back to cited text no. 6
Cho YW, Cecka JM. Crossmatch tests-an analysis of UNOS data from 1991-2000. Clin Transpl 2001:237-46.  Back to cited text no. 7
Iwaki Y, Cook DJ, Terasaki PI, Lau M, Terashita GY, Danovitch G, et al. Flow cytometry cross-matching in human cadaver kidney transplantation. Trans Proceed 1987;19 Pt 1:764.  Back to cited text no. 8
Talbot D, Givan AL, Shenton BK, Stratton A, Proud G, Taylor RM. The relevance of a more sensitive cross-match assay to renal transplantation. Transplant 1989;47:552-5.  Back to cited text no. 9
Mahoney RJ, Ault KA, Given SR, Adams RJ, Breggia AC, Paris PA, et al. The flow cytometric cross-match and early renal transplant loss. Transplant 1990;49:527-35.  Back to cited text no. 10
Taylor CJ, Kosmoliaptsis V, Summers DM, Bradley JA. Back to the future: Application of contemporary technology to long-standing questions about the clinical relevance of human leukocyte antigen-specific alloantibodies in renal transplantation. Hum Immunol 2009;70:563-8.  Back to cited text no. 11
Jin YP, Fishbein MC, Said JW, Jindra PT, Rajalingam R, Rozengurt E, et al. Anti-HLA class I antibody-mediated activation of the PI3K/Akt signaling pathway and induction of Bcl-2 and Bcl-xL expression in endothelial cells. Hum immunol 2004;65:291-302.  Back to cited text no. 12
Salama AD, Delikouras A, Pusey CD, Cook HT, Bhangal G, Lechler RI, et al. Transplant accommodation in highly sensitized patients: A potential role for Bcl-xL and alloantibody. Am J Transplant 2001;1:260-9.  Back to cited text no. 13
Eckels DD. Solid phase testing in the HLA laboratory: Implications for organ allocation. Int J Immunogenet 2008;35:265-74.  Back to cited text no. 14
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;70:595-9.  Back to cited text no. 15
Shroff S. Legal and ethical aspects of organ donation and transplantation. Indian J Urol 2009;25:348-55.  Back to cited text no. 16
[PUBMED]  [Full text]  
Sahay M. Transplantation of human organs and tissues Act-”Simplified”. Indian J Transplant 2018;12:84.  Back to cited text no. 17
Feucht HE, Schneeberger H, Hillebrand G, Burkhardt K, Weiss M, Riethmüller G, et al. Capillary deposition of C4d complement fragment and early renal graft loss. Kidney Int 1993;43:1333-8.  Back to cited text no. 18
Cook, Daniel J. Detection of HLA specific alloantibody by flow Cytometry and solid phase assays: Approved guideline. Clin Lab Stand Institute 2008:I-LA, 29-A, 28.  Back to cited text no. 19
Süsal C, Roelen DL, Fischer G, Campos EF, Gerbase-DeLima M, Hönger G, et al. Algorithms for the determination of unacceptable HLA antigen mismatches in kidney transplant recipients. Tissue Antigens 2013;82:83-92.  Back to cited text no. 20
Süsal C, Pelzl S, Simon T, Opelz G. Advances in pre- and posttransplant immunologic testing in kidney transplantation. Transplant Proc 2004;36:29-34.  Back to cited text no. 21
Karpinski M, Rush D, Jeffery J, Exner M, Regele H, Dancea S, et al. Flow cytometric cross-matching in primary renal transplant recipients with a negative anti-human globulin enhanced cytotoxicity cross-match. Clin J Am Soc Nephrol 2001;12:2807-14.  Back to cited text no. 22
O'Rourke RW, Osorio RW, Freise CE, Lou CD, Garovoy MR, Bacchetti P, et al. Flow cytometry crossmatching as a predictor of acute rejection in sensitized recipients of cadaveric renal transplants. Clin Transplant 2000;14:167-73.  Back to cited text no. 23
Wen R, Wu V, Dmitrienko S, Yu A, Balshaw R, Keown PA, et al. Biomarkers in transplantation: Prospective, blinded measurement of predictive value for the flow cytometry cross-match after negative antiglobulin cross-match in kidney transplantation. Kidney Int 2006;0:1474-81.  Back to cited text no. 24
Herczyk W. Luminex. ASHI Quarterly. 3rd ed. Quarter; 2003. p. 104.  Back to cited text no. 25
Moszkowska G, Zielińska H, Zieliński M, Dukat-Mazurek A, Dębska-Ślizień A, Rutkowski B, et al. Identification of patients with increased immunological risk among potential kidney recipients in the Polish population. Hum immunol 2014;75:650-5.  Back to cited text no. 26
Gupta A, Sinnott P. Clinical relevance of pretransplant human leukocyte antigen donor-specific antibodies in renal patients waiting for a transplant: A risk factor. Hum Immunol 2009;70:618-22.  Back to cited text no. 27
Mathur A, Thapa S, Jagannathan L. Luminex-based donor-specific antibody cross-matching for renal transplant: A 3-year experience in South India. Global J Trans Med 2018;3:34.  Back to cited text no. 28
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.  Back to cited text no. 29
Tait BD, Süsal C, Gebel HM, Nickerson PW, Zachary AA, Claas FH, et al. Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation. Transplant 2013;95:19-47.  Back to cited text no. 30
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. 31
Chowdhry M, Makroo RN, Singh M, Kumar M, Thakur Y, Sharma V. Role of anti-MICA antibodies in Graft Survival of Renal Transplant Recipients of India. J Immunol Res 2018;2018:3434050.  Back to cited text no. 32
Wang JH, Skeans MA, Israni AK. Current status of kidney transplant outcomes: Dying to survive. Adv Chronic Kidney Dis 2016;23:281-6.  Back to cited text no. 33
Srivastava A, Prabhakaran S, Sureka SK, Kapoor R, Kumar A, Sharma RK, et al. The challenges and outcomes of living donor kidney transplantation in pediatric and adolescent age group in a developing country: A critical analysis from a single center of North India. Indian J Urol 2015;31:33.  Back to cited text no. 34


  [Figure 1]

  [Table 1], [Table 2], [Table 3]


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