Journal of The Egyptian Society of Nephrology and Transplantation

REVIEW ARTICLE
Year
: 2017  |  Volume : 17  |  Issue : 4  |  Page : 107--114

Case-based review of cross-match techniques in kidney transplantation


Obadiah Moyo1, Ajay K Sharma2, Ahmed Halawa3,  
1 Department of Nephrology, Chitungwiza Central Hospital, Harare, Zimbabwe
2 Royal Liverpool University Hospital, Liverpool, UK
3 Sheffield Teaching Hospitals, Sheffield Kidney Institute, University of Sheffield, Sheffield, UK

Correspondence Address:
Dr. Ahmed Halawa
Sheffield Teaching Hospital, Herries Road, Sheffield S5 7AU
UK

Abstract

To avoid renal allograft rejection and loss, human leucocyte antigen compatibility and lymphocytotoxic cross-matching of renal donors against prospective renal allograft recipients are obligatory and fundamental steps in pretransplant evaluation. The complement-dependent cytotoxicity assay has been the mainstay technique for ascertaining the presence of donor-specific antibodies since the 1970s. Improvement in the performance of cross-matching has seen the introduction of more sensitive and specific techniques such as flow cytometry, and highly sensitive enzyme-linked immunosorbent assay, Luminex platforms and virtual cross-match. This review article is an in-depth comparison of the advantages and disadvantages of each of the cross-match techniques. We demonstrate the application of these techniques through a clinical case scenario. A step-by-step analysis and interpretation of cross-match modalities of varying levels of sensitivity and specificity is needed to arrive at a pragmatic decision, which will lead to better transplantation outcome. In some patients with immunologically complex issues, a spectrum of cross-match techniques may be required for a safe outcome of transplant.



How to cite this article:
Moyo O, Sharma AK, Halawa A. Case-based review of cross-match techniques in kidney transplantation.J Egypt Soc Nephrol Transplant 2017;17:107-114


How to cite this URL:
Moyo O, Sharma AK, Halawa A. Case-based review of cross-match techniques in kidney transplantation. J Egypt Soc Nephrol Transplant [serial online] 2017 [cited 2018 Jul 18 ];17:107-114
Available from: http://www.jesnt.eg.net/text.asp?2017/17/4/107/223413


Full Text



 Introduction



Antibody-mediated rejection presents in three forms with hyperacute rejection occurring within minutes to hours following transplantation owing to an amnestic response to alloantigen exposure. Acute rejection occurs within weeks, whereas chronic rejection occurs several years post-transplantation. During the initial stages of transplantation, it was observed that a high number of kidney transplants performed in patients with donor-specific antibodies (DSAs) resulted in hyperacute rejection [1],[2]. The major histocompatibility complex in humans is called human leucocyte antigen (HLA) and is located on the short arm of chromosome 6. Mismatch of HLA between donor and recipient would determine the frequency and magnitude of allograft acute rejection.

There are two types of HLA molecules called HLA class I and HLA class II. The genes coding for HLA class I are called HLA A, B and C and are expressed on all nucleated cells and activate CD8 T cells. HLA class II are HLA DR, DQ and DP, which encode antigens responsible for presenting peptide to CD4 T-helper cells for an immune response, and also expressed on B cells and T lymphocytes.

Many patients are transplanted with an HLA mismatched organ, and mismatch in clinical setting ranges from zero mismatch to six mismatches. Long-term graft survival is possible provided the patient does not have preformed antibodies to the donor. Pregnancy, blood transfusions and previous transplants can induce such antibodies [3]. Panel reactive antibodies (PRA) are used as a measure of the level of sensitization in recipients of allografts [4]. PRA levels are the percentage of incompatible donors that would have positive cross-match. A high PRA suggests that a candidate will immunologically react against a large section of the population. The recipient sera are tested against cells from a panel of HLA-typed donors or solid-phase-based assays with HLA antigens.

The use of calculated panel reactive antibodies (cPRA) is a standardized method of estimating the possibility of the recipient having DSA through measuring the difference between antibody specificities and the prevalence of HLA alleles in a target population [5]. The basis of cPRA values is the list of HLA antigens considered as unacceptable for a prospective kidney transplant patient against a panel of 10 000 newly assigned deceased donors, and thereby giving an estimation of being allocated a suitable donor from a pool [6].

To prevent hyperacute rejection, a lymphocytotoxic cross-match has to be performed to allow for the elimination of adverse recipient donor combination [7]. Cytotoxicity-dependent cross-match (CDC) was introduced in the 1970s that was followed by the flow cytometry version in the 1980s. Currently, solid-phase assays based on enzyme-linked immunosorbent assay (ELISA), and Luminex platforms, which are very sensitive, have also been developed for the detection of DSAs. HLA antibody specificities would help the transplant immunologists to perform virtual cross-match without really doing that in the laboratory [8].

 Clinical case scenario



A 30-year-old male patient with chronic kidney disease stage 5 secondary to lupus nephritis who has been on haemodialysis for 5 years was offered a kidney from a deceased donor. This is his first kidney transplantation and he had no blood transfusions in the past. Laboratory results at admission for transplantation were as follows:1–0–0 mismatch.Complement-dependent cytotoxicity (CDC) cross-match positive for B cell but flow cytometry cross-match reported negative for both B and T cells.Luminex single-antigen bead (SAB) did not identify any DSAs.

 End-stage renal disease in lupus nephritis



Lupus nephritis is more prevalent in younger individuals [9],[10]. Lupus nephritis patients who progress to end-stage renal disease (ESRD) are currently commenced on haemodialysis, the logical basis being to inhibit any remaining lupus activity and create a ‘quiescent’ and ‘burnt-out’ state of the disease before transplantation [11]. The patients with ESRD due to lupus nephritis show remarkable outcomes following renal transplantation, which are comparable to outcomes of transplant recipients with ESRD secondary to other causes [12],[13]. Lupus nephritis patients who receive cadaveric graft transplants have been shown to have a poor allograft survival than those who have been transplanted with a living donor allograft [14],[15].

 Interpretation of laboratory results in our index case



A1–0–0 mismatch

It has been shown that in first cadaveric transplants there is an increased chance of allograft acute rejection if the HLA mismatch is anything other than a beneficial mismatch such as 0–0–0, 1–0–0, 0–1–0 or 1–1–0 [16]. Class I HLA-A, B and C antigens matching are key in survival, whereas class II HLA DR was also shown to have a greater influence on outcome [17]. HLA DQ matching is considered to be of less significance on outcomes in transplantation [18]. There are reports that suggest that the essence of matching lies in matching for HLA epitopes and amino acid sequencing [19]. HLA mismatch has been shown to have a linear relationship with allograft failure ([Figure 1]). With a 1–0–0 mismatch, this patient therefore has a low hazard ratio of 1.2, which translates to a low allograft failure risk [16].{Figure 1}

Interpretation of complement-dependent cytotoxicity cross-match, flow cytometry cross-match and Luminex single-antigen bead results

Flow cytometry and Luminex T-cell results show that there are no antibodies to HLA class 1, and the B-cell results also indicate the absence of antibodies targeting HLA class I and II individually or both. However, in view of the positive CDC cross-match result, before proceeding with the transplant, there is a need to verify whether the result is not a false positive owing to laboratory error or immunoglobulin (Ig) M autoantibodies in the patient. An auto-cross-match, where the recipient’s lymphocytes and serum are cross-matched, would need to be done to confirm whether the result is due to autoantibodies.

A cytotoxic cross-match between the serum of the recipient and the donor cells, which tests positive, is linked with hyperacute rejection and predicts loss of graft with certainty [20]. To confirm IgM as a cause of false positive result, a repeat assay is relevant with dithiothreitol (DTT) to reduce disulphide bonds in IgM antibodies and, thereby, blocks IgM antibodies from giving rise to false positive results. Controls using PBS are included in the test assay to control for DTT dilution effect on antibody level [21],[22]. It is acceptable to go ahead with the transplant if the DTT assay is negative. The fact that our index patient has lupus nephritis gives a high possibility that this is a false positive CDC cross-match caused by autoantibodies in the recipient. Schlaf et al. [22] highlighted the possibility of artificially positive cross-matches in prospective kidney transplant recipients as a result of autoantibodies that develop in autoimmune diseases such as systemic lupus erythematosus (SLE).

Complement-dependent cytotoxicity cross-match

IgG and IgM class HLA-specific and non-HLA-specific antibodies are detected by the standard CDC cross-match, which is a cell-based assay [23]. Aliquoted T and B cells from donor lymphocytes are incorporated with recipient serum in a microtiter plate followed by addition of rabbit-derived complement if present; DSA will bind and cause lysis to the donor lymphocytes via the classical pathway. Through a two-colour fluorescence microscopy, the percentage of dead cells that are stained red by ethidium bromide versus the live cells stained green through uptake of acridine orange are verified ([Figure 2]). The scoring ranges from 0 to 8 depending upon the rate of lysis of cells. A score of 2 (20%) is considered as a low and cutoff point of a positive result with a score of 8 (with 80% cell lysis) being regarded as a strong positive.{Figure 2}

Modifications were made to the assay to enhance the specificity by using DTT, which is a disulphide bond reducing agent in IgM. To increase the sensitivity of the cross-match, antihuman globulin is added to bind to DSAs by making Fc receptors accessible to the complement and a subsequently improved activation of the complement and lysis of the cells [21].

Other approaches to improving the responsiveness and quality of results of the CDC test include lengthening the incubation time, and addition of wash steps (Amos modified) to remove any anticomplimentary factors that may cause false negative [24].

CDC cross-match has been the gold standard for over 40 years. It gives a good prediction of clinical outcomes and detects clinically important antibodies before and after transplantation. The assay does not differentiate complement-fixing antibodies and allows for a subjective analysis of cross-match results [25].

Hyperacute graft rejection does happen in some patients who were negative in the CDC cross-match before kidney transplantation [4]. These false negatives are associated with laboratory error and low-titre antibody. CDC has also been linked to false positives owing to autoantibodies and contamination by carryover of a negative test with positive antiserum.

Basiliximab and rituximab, which are IgG isotypes, can cause complement activation, which will result in a positive CDC result [26]. This assay does not detect DSAs without complement-activating function, which are deemed to be detrimental to the donated organ. The donor cells used in the assay are hard to come by and have to be of high degree of vitality if the results are to be of high quality and accurate [22].

Flow cytometry cross-match

It uses polystyrene microbeads containing fluorochromes. The incubated recipient serum and lymphocytes from the donor are stained with the fluorochrome-labelled antibodies against IgG. The result is considered negative if there are no antibodies detected. If DSAs are present, they adhere to the fluorochrome-labelled anti-human IgG antibody, which is read in the flow cytometer ([Figure 3]); the quantified results are reported as ‘channel shifts’ ([Figure 4]).{Figure 3}{Figure 4}

The sensitivity of the flow cytometry cross-match is three times more than the antihuman globulin–CDC assay [20],[21],[28]. Flow cytometry detects IgG antibodies independent of complement fixation. Antibody reaction on donor T and B lymphocytes can be measured at the same time but as separate entities [20].

There have been situations in which CDC is negative but flow cytometry cross-match is positive, which might be due to non-complement-fixing antibody, low-level antibody, non-HLA antibody or reduced cutoff value of anti-HLA antibody. False positive flow cytometry cross-match in nonsensitized patients is not predictive of increased graft rejection [21].

Enzyme-specific immunosorbent assay

This assay uses microtiter plates with antigen bound to flat-bottom wells. The HLA-specific antibodies are identified after addition of test serum to the wells, an enzyme conjugate antiglobulin antibody and a chromogenic substrate, which results in a colour change in wells containing HLA-specific antibody [29] ([Figure 5]).{Figure 5}

The ELISA assay has several advantages over lymphocytotoxicity tests, which include the elimination of the need for lymphocytes and complement that are capable of normal growth. The assay has the capability of identifying HLA-specific antibodies and identifying the difference between anti-HLA class I and II. ELISA has an increased sensitivity than CDC or flow cytometry. ELISA cross-matching is used to verify invalid CDC results [26]. ELISA allows for retrospective de-facto cross-match using frozen sample, especially in cadaver donations [26].

In ELISA, non-complement-binding HLA antibodies can cause positive results. ELISA excludes non-HLA alloantibodies [30]. There is a risk of HLA molecules on the solid surface changing configuration during the processing, which is likely to cause a false positive owing to exposure of epitopes or a negative result due to loss of epitopes [31].

Luminex single-antigen bead assay

Luminex is a solid-phase immunoassay that is more sensitive than other forms of cross-matching and uses recombinant HLA antigens instead of lymphocytes [8]. The platform consists of a dedicated instrument and a fluorescent-bead-based array. The fluorescent beads acts as a solid substrate for an immunocaptive assay similar to polystyrene plates in a traditional ELISA. The bead types are of different colours and intensity. Each unique bead type is labelled by a particular antibody for detecting specific proteins of interest ([Figure 6]).{Figure 6}

For screening purposes, beads may be multi-HLA antigen coated, and for specific, precise antibody definition, single HLA antigen coated beads can be used. The result is determined by using a dual beam laser that measures the fluorescent intensity of the complement detection antibody reporter dye on the beads. Different beads can be combined together in a single well. When beads pass through the two lasers, the reporter laser reports occurrence of antigen–antibody reaction, whereas the classification laser recognizes the beads as per colour coding [28] ([Figure 7]).{Figure 7}

The fluorescence-based readout gives the Luminex assay greater sensitivity and autoantibodies, which are antagonistic towards immune complexes and non-HLA do not interfere in the assay [8]. Because of its improved separation efficiency, it is at the moment the assay of choice for determining HLA antibody specificities [8]. Luminex allows for pretransplant and post-transplant detection of HLA class II antibodies, which are linked to chronic rejection. Delineation of antibodies with linkage disequilibrium from HLA-DRB1 activity is possible with this assay, as well as detection of antibodies targeted at specific differences in alleles contained within a recipient’s self-antigen group. Luminex has created awareness on the role of HLA-DP antibodies in allograft rejection through the use of HLA-DPB1-molecule-loaded beads. Before any successive transplant, Luminex is able to detect sensitization to previous mismatches [32]. Luminex is affected by the prozone phenomenon where neat serum, which is known to contain high levels of DSAs but does test falsely negative, and yet, if diluted, the same serum test would test positive for these antibodies. This ‘phenomenon’ happens because by the C1 part of the complement system binds to the Fc component of IgG1 and IgG3 and, thereby, does not allow antihuman antibodies (detecting second antibodies) to bind the antibodies it is supposed to detect [33]. EDTA or complement inhibitor has been used to overcome the prozone phenomenon by chelating calcium that would prevent binding C1 component of complement to the target antibodies [34].

False negative results occurring because of the prozone effect lead to underestimation of some higher-titre anti-HLA antibodies [35]. The strength of HLA antibodies read as median fluorescence intensity needs to be correlated with other cross-match methods to avoid discrepant results. Besides the expensiveness of the Luminex assay, its limitation is that it can only detect antigens that are incorporated on the beads [7].

Comparison of Luminex (single-antigen bead) and enzyme-linked immunosorbent assay

ELISA is less sensitive in detecting HLA class I antibodies than Luminex as its panel is composed of a pool of platelet antigens from various ethnological backgrounds in contrast to Luminex, which uses recombinant HLA antigens [36]. Serum dilution of 1 : 4 in ELISA in comparison with neat serum in Luminex renders it less sensitive. Luminex has a better capability of identifying anti-DQ and anti-DP antibodies than ELISA owing to linkage equilibrium between these alleles. Luminex can show specificity at low levels of antibody and has reproducibility of specific epitopes targeted to antibodies [36].

Virtual cross-match

Virtual cross-match is not an assay where you physically mix test cells and serum. It is an application of collated antibody data on a prospective transplant patient to make a prediction of the outcome of the final cross-match. It, therefore, requires a broad knowledge of the detected antibody’s specificity and its reaction with a donor of a particular HLA type. If antibody specificity of the recipient matches one or more of the HLA antigens of the donor, then DSA is present. With this database history, pretransplant laboratory-based cross-match can therefore be avoided [23]. Virtual cross-match should be carried out using all available results that should be as latest as 3 months before transplant. In a study by Bielman et al. [37], there was 86% concordance between the virtual cross-match and flow cytometry cross-match.

The virtual cross-match adds precision to actual cross-match and it eliminates the physical cross-match in that no samples are required from the donor. It reduces laboratory workload, and thereby improves allocation efficiency and increases the rate of transplantation for sensitized patients. Furthermore, it permits national paired exchange and risk of memory response can be accounted for.

The reliability of the virtual cross-match is variable because changes inpatient’s antibody status including specificity and levels. A previously recorded absent patient’s antibody specificity may become detectable due to blood transfusion pregnancy or transplantations. Donors can be mistakenly excluded because of virtual cross-match false positives, which are known to occur when the titres or non-complement-binding antibody are very low or if the cross-match is not as sensitive as the assay for antibody detecting. As solid-phase tests cannot necessarily accommodate all potential HLA antigens, this could result in a false negative virtual cross-match.

There is variable correlation between virtual cross-match and an actual cross-match. The virtual cross-match is overall not fully predictive of positive or negative results, thus supporting the need for an actual cross-match [38].

Discussion in regard to index case

In this particular case after a thorough interpretation of all the results, it is revealed that the CDC positive results are because of autoantibodies in SLE. The positive CDC cross-match in this patient is due to autoantibodies. The patient has SLE and therefore autoantibodies are expected. The initial CDC cross-match test should also be redone with the addition of DTT. If the test becomes negative, then the autoantibodies are IgM and transplantation can proceed. If it is still positive, then IgG alloantibodies are likely to be the cause of false positive CDC cross-match. SAB, therefore, we infer that these IgG alloantibodies would be non-HLA. To rule out laboratory error, the quality of cells and other test conditions should be checked. In this situation, it is logical to recommend for the transplantation to proceed because there is no history of sensitization, there is only one HLA mismatch and that too at HLA-A, flow cytometry is negative for both T and B cells and Luminex SAB is negative for DSA.

This CDC is false positive owing to autoimmune disease, that is, lupus nephritis, thereby demonstrating the importance of carefully interpreting available reports understanding its limitations. Further testing to negate IgM and autoantibodies has been suggested for the given case. In some patients with complex immunology, a careful and judicious use of a battery of immunology tests is of utmost importance to come up with a meaningful conclusion.

 Conclusion



It is relevant to note the progress that has been made to date in the development of techniques for the detection of DSAs before transplantation. The current solid-phase cross-matching techniques such as Luminex SAB have higher sensitivities and specificities over the standard CDC, flow cytometry and ELISA assays. The pitfalls, advantages and comparative protocols have been clearly defined and, therefore, no single cross-matching technique would be good enough in isolation when faced with a dilemma of managing immunologically complex prospective recipient. For a successful transplant and to avoid exclusion of a good donor, a careful and comprehensive immunological assessment of a prospective recipient requires a careful discussion between transplant immunologist, transplant surgeon and nephrologist to interpret clinical background, range of cross-match tests and cPRA. Each of these tests do have inherent strengths and limitations.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Claas FH. Clinical relevance of circulating donor-specific HLA antibodies. Curr Opin Organ Transplant 2010; 15:462–466.
2Salvalaggio PR, Graff RJ, Pinsky B, Schnitzler MA, Takemoto SK, Burroughs TE et al. Crossmatch testing in kidney transplantation: patterns of practice and associations with rejection and graft survival. Saudi J Kidney Dis Transpl 2009; 20:577–589.
3Puttarajappa C, Shapiro R, Tan HP. Antibody mediated rejection in kidney transplantation: a review. J Transplant 2012; 2012:193724.
4Jang J, Kim YJ, Kim Y, Park Y, Han K, Oh E. Applications of calculated panel reactive antibody using HLA frequencies in Koreans. Ann Lab Med 2012; 32:66–72.
5Tinckman KJ. Basic histocompatibility testing methods. In: Core concepts in renal transplantation. Springer; 2012. pp. 21–42.
6Delordson K. A collection of brief revision notes. In: Histocompatibility and immunogenetics. Word Press; 2011.
7Patel R, Terasaki PI. Significance of the positive crossmatch test in kidney transplantation. N Engl J Med 1969; 280:735–739.
8Susal C, Dohler B, Ruhenstroth A, Morath C, Slavcev A, Fehr T et al. Donor specific antibodies require preactived immune system to harm renal transplant. A Collaborative Transplant Study Report. EBiomedicine 2016; 19:366–371.
9Costenbader KH, Desai A, Alarcón GS, Hiraki L, Shaykevich T, Brookhart MA et al. Trends in the incidence, demographics, and outcomes of end stage renal disease due to lupus nephritis in the US from1995 to 2006. Arthritis Rheum 2011; 63:1681–1688.
10Katalinić L, Eliasson E, Bubić-Filipi L, Kes P, Anić B, Bašić-Jukić N. Renal transplantation in patients with lupus nephritis. Lijec Vjesn 2014; 136:219–223.
11Fries JF, Powers R, Kempson RL. Late stage lupus nephropathy. J Rheumatol 1974; 1:166–175.
12Ponticelli C, Moroni G. Renal transplantation in lupus nephritis. Lupus 2005; 14:95–98.
13Ward MM. Outcomes of renal transplantation among patients with end stage renal disease caused by lupus nephritis, Kidney Int 2000; 57:2136–2143.
14Chelamcharla M, Javaid B, Baird BC, Goldfarb-Rumyantzev AS. The outcome of renal transplantation among systemic lupus erythematosus patients. Nephrol Dial Transplant 2007; 22:3623–3630.
15Cairoli E, Sanchez-Marcos C, Espinosa G, Glucksmann C, Ercilla G, Oppenheimer F, Cervera R. Renal transplantation in systemic lupus erythematosus: outcome and prognostic factors in 50 cases from a single centre. Biomed Res Int 2014;2014:746192.
16Williams RC, Opelz G, McGarvey CJ, Weil EJ, Chakkera HA. The risk of transplant failure with HLA mismatch in first adult kidney allografts from deceased donors. Transplant J 2016; 100:1094–1102.
17Ting A. Morris powerful effect of HLA-DR matching on survival of cadaveric renal allografts. Lancet 1980; 2:282–285.
18Bushell A, Higgins RM, Wood KJ, Morris PJ. HLA-DQ mismatches between donor and recipient in the presence of HLA-DR compatibility do not influence the outcome of renal transplants. Hum Immunol 1989; 26:179–189.
19Duquesnoy RJ, Takemoto S, de Lange P, Doxiadis IIN, Schreuder GMT, Claas FHJ. HLA matchmaker: a molecularly based algorithm for histocompatibility determination III: effect of matching at the HLA-A, B amino acid triplet level on kidney transplant survival. Transplantation 2003; 75:884.
20Gebel HM, Bray RA, Nickerson P. Pre-transplant assessment of donor-reactive, HLA-specific antibodies in renal transplantation: contraindication vs risk. Am J Tansplant 2003; 3:1488–1500.
21Mulley WR, Kanellis J. Understanding crossmatch testing in organ transplantation: a case-based guide for the general nephrologist. Nephrology 2011; 16:125–133.
22Schlaf G, Pollok-Kopp B, Schabel E, Altermann W. Artificially positive crossmatches not leading to the refusal of kidney donations due to the usage of adequate diagnostic tools. Case Rep Transplant 2013; 2013:746395.
23British Transplantation Society. Guidelines for the detection and characterisation of clinically relevant antibodies in allotransplantation. 2016; version 4:39.
24Amos DB, Cohen I, Klein WJ Jr. Mechanisms of immunologic enhancement. Transplant Proc 1970; 2:68–75.
25Grimaldi V, Cesario E, Casamassimi A, Infante T, Mapoli C. Flow cytometry analysis and crossmatch detection techniques in transplantation. Immunol Endocr Metab Agents Med Chem 2012; 12:34–39.
26Schlaf G, Apel S, Wahle A, Altermann WW. Solid phase-based cross matching as solution for kidney allograft recipients pretreated with therapeutic antibodies. Biomed Res Int 2015; 2015:587158.
27Fuggle SV, Taylor CJ. Chapter 10: histocompatibility in renal transplantation. In: Morris P, editor. Kidney transplantation: principles and practice. Philadelphia, PA: Saunders Elsevier; 2008. pp. 143–160.
28Garovoy MR, Rheinshmilt MA, Bigos M, Perkins H, Colombe B, Feduska N, Salvatierra O. Flow cytometry analysis: a high technology crossmatch technique facilitating transplantation. Transplant Proc 1983; 15:1939–1944.
29Biorad clinical diagnostics. HLA antibody ELISA manual; 2017.
30Susal C, Opelz G. Options for immunologic support of renal transplantation through the HLA and immunology laboratories. Am J Transplant 2007; 7:1450–1456.
31Chacko MP, Mathan A, Daniel D, Basu G, Varughese S. Significance of pre-transplant anti HLA antibodies detected on an ELISA mixed antigen tray platform. Indian J Nephrol 2013; 23:351–353.
32Tait BD, Hudson F, Cantwell L, Brewin G, Holdsworth R, Bennett G, Jose M. Review article: Luminex technology for HLA antibody detection in organ transplantation. Nephrology (Carlton) 2009; 14:247–254.
33Weinstock 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 2012; 40:171–177.
34Vittoraki A, Iannou SI, Vallindra HM, Siorenta AA, Milonas AG, Seimenis NM et al. Treating sera with ethylene diamine tetraacetic acid (EDTA): a promising technical solution for the complement-mediated prozone effect in anti-HLA antibody detection by single antigen bead assay. Hum Immunol J 2015; 76:65.
35Jain D, Chouddhuri J, Chauhan R, Raina V. False negative single antigen bead assay: is it always an effect of prozone. J Clin Lab Anal 2017. [Epub ahead of print].
36Minucci PB, Grimaldi V, Casamassimi A, Cacciatore F, Sommese L, Picascia A et al. Methodologies for anti-HLA antibody screening in patients awaiting kidney transplant: a comparative study. Exp Clin Transplant 2011; 9:381–386.
37Bielman D, Honger G, Lutz D, Mihatsch MJ, Steiger J, Schaub S. Pretransplant risk assessment in renal allograft recipients using virtual crossmatching. Am J Transplant 2007; 7:626–632.
38Bray RA, Nolen JD, Larsen C, Pearson T, Newell KA, Kokko K et al. Transplanting a highly sensitised patient: the emory algorithm. Am J Transplant 2006; 6:2307–2315.