The number of solid organ transplantation (such as liver, kidney, pancreas, heart, and lung) is on the increasing trend in the past few years due to the rise in the affordability, accessibility, and availability of the good patient and donor workup protocols (both pre- and posttransplant). Despite this, a huge number of patients awaiting renal transplants are on the waiting list. Published data suggested that about 150,000 people in India are waiting for renal transplantation due to a shortage of organs. Hence, an accurate and timely investigation of donor matching to obtain the best outcome of a transplant using various techniques is required. The detection of preformed HLA donor-specific antibodies (DSAs) is primarily the main target of all these investigations which plays a role in graft rejection. Commonly, several cell-based assays ([complement-dependent cytotoxicity crossmatch (CDC-XM) and flow cytometry crossmatch (FCXM]) are available for the detection of HLA antibodies and now upgrading to virtual crossmatch, in which single-antigen bead (SAB) assay is widely used for characterization preformed HLA DSA. Results of SAB and FCXM generally match up if proper cutoffs are maintained in SAB interpretation, but there are a few instances when the reports of these tests do not show any correlation. In such cases of discordance interpretation, further workup needs to be done before making a final decision about the transplant. Here, we report one such case of positive SAB (the presence of DSA) and with negative CDC and flow crossmatch.
A case of 49-year-old male patient with chronic kidney disease-V and on maintenance hemodialysis was planned for renal transplant with his daughter as a prospective donor. He was hypertensive, nondiabetic, and history of one unit of blood transfusion. His pretransplant histocompatibility workup came to our laboratory which is ISO15189: 2012 accredited, and the required internal and external quality control and assurance are routinely undertaken. The tests requisitioned from the treating physician were CDC, FCXM, HLA (A, B, and DRB1 typing of both patient and donor),a SAB assay for HLA class I and II DSA identification. HLA-A, B, and DRB1 testing results are shown in Table 1 and they were 3/6 matched at the antigenic level. CDC using antihuman globulin (AHG) and FCXM were performed and it turned out to be negative. SAB testing using LIFECODES, LSA Class I and II, Immucor, USA, kit to detect the presence of DSA for both HLA Class I and Class II was performed. SAB testing showed various HLA Class I and Class II antibodies. There was no DSA identified against HLA Class II antigens. However, DSA was detected against HLA Class I, A *33:01 (MFI: 3360). The cutoff value for a positive antibody was an MFI of ≥1000 in our laboratory.
To rule out the possibility of error, as results of cell-based and virtual crossmatch were not correlated, a repeat CDC (AHG) and FCXM (serum dilution up to 1:4) were repeated on a fresh sample and again both were negative. SAB testing was also repeated with another kit (One Lambda LABScreen Kit, USA) and there was no variation in the result. After a literature search and discussion, it was hypothesized that these contradictions may result from denatured HLA antigens and hence needed further workup to come up to a conclusion. To prove this hypothesis, acid treatment of the beads to denature the antigens was performed with the patient's serum. To find out the nature of the antibody found against anti-HLA-A*33:01 in our case, the beads used in the SAB assay were treated with 0.1 M NaAc, pH 2.7 (10 × volume, by mixing 10 μl of beads + 100 μl of 0.1 M NaAc), and incubated at room temperature for 30 min. The mixture was then washed with basic wash buffer (×2) and resuspended in phosphate buffer solution.
The MFIs of the anti-HLA antibodies from the patient sera with acid-treated beads increased significantly in contrast to the routinely untreated SAB indicating that additional antigen epitopes became available by the denaturation (acid treatment) process as shown in [Figure 1]. These findings support the presence of antibodies against a denatured antigen present on the beads.
After a discussion with the treating physician, a consensus was made to go ahead with the transplant and the patient underwent a transplant. The patient had two double-filtration plasmapheresis sessions before the transplant. A repeat SAB assay for Class I was repeated after 1-week posttransplant and the results were similar with a slight decrease in MFI. In addition, a flow crossmatch was also repeated with this sample and reported negative. A Luminex donor lysate crossmatch and flow crossmatch (serum dilution up to 1:4) were repeated after 6 months of the transplant and the results were negative.
After 9 months of transplant, the creatinine value continues to be within the normal range and no further complications.
Virtual crossmatch (SAB assay) is an evaluation of immunological compatibility based on the comparison between the anti-HLA antibodies profile of the recipient with the HLA of the prospective donor. The HLA antigens of the donor against which these DSAs are identified are considered unacceptable to proceed for a transplant and hence to assess pretransplant immunological risk SAB assay should be performed in recipients. However, there is a limitation in SAB that it detects only anti-HLA DSAs and not non-HLA antibodies. False-positive or high titers may be reported due to the presence of antibodies to denatured HLA antigens. A report published by Jacob et al. also supports the above-mentioned hypothesis of acid treatment. A study published by Schinstock et al. also suggested if after acid treatment, SAB is still positive then the alloantibody may be toward denatured HLA and as per the risk stratification of antibody-mediated rejection based on the combination of SABs and flow cytometric crossmatch it will come under “Low” as a risk for AMR. Acid treatment does increase the further unmasking of the denatured antigens and an increase in MFI values after acid treatment favors the fact that the antibodies are not against intact antigens but against immunologically insignificant denatured HLA antigens. Similarly, a couple of more reports published by Nishida et al. and Ferris et al. also stated that the human immune system can react to denatured antigens. Whereas Morales-Buenrostro et al. has stated that this may signify the specificity of some “natural” HLA antibodies. Despite all these reports and theories, the implication of such antibodies is not very clear as this has been explained in the literature to a lesser amount. A new generation of SABs known as iBeads:which is largely devoid of denatured HLA class I/clean beads is now available to overcome this issue. They concluded that DSAs against denatured HLA are clinically irrelevant and only antibodies against intact HLA determine the risk for graft loss. Further, this theory was also supported by Visentin et al. The transplanted patient reported above was doing well until the latest follow-up with creatinine value continuing to be within the normal range supporting the above conclusion.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the legal guardian has given her consent for images and other clinical information to be reported in the journal. The guardian understands that her names and initials will not be published and due efforts will be made to conceal the patient's identity, but anonymity cannot be guaranteed.
The results of SAB should be correlated with the patient clinical history and results of another compatibility testing (such as flow crossmatch) especially in cases of discordance between cell-based assay and virtual crossmatch before “ruling in” or “ruling out” the patient for transplant.
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Conflicts of interest
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