Association of prior sensitizing events with anti-human leukocyte antigen antibodies: An analysis of renal transplant recipients in a tertiary care centre in South India.

Traditionally, sensitizing events such as previous pregnancies, previous transfusions and prior transplants result in the production of anti-Human Leukocyte Antigen (HLA) antibodies. However, it has been observed that, anti-HLA antibodies have been detected in many patients with no prior history of sensitizing events. This retrospective study analysed the most recent 100 consecutive Single Antigen Bead (SAB) assay results performed on 100 patients. The SAB assay is used routinely to detect anti-HLA antibodies in transplant recipients. Results of the SAB assay were analyzed and subsequently studied to see if a correlation existed between sensitizing events, the type of events and presence of antibody. Analysis showed that 77% (77/100) had anti-HLA antibodies. 61 out of 100 patients had prior sensitizing events while the remaining 39 had none. Both these groups showed an almost equal percent of patients with anti-HLA antibodies 77% (47/61) and 76.9% (30/39) respectively. A single sensitizing event was seen in 54.1% (33/61) patients including previous transfusions in 29.5% (18/61), pregnancies in 11.4% (7/61) and prior transplant in 13.1% (8/61). Our study suggests that irrespective of whether patients have prior sensitizing events or not, patients run the risks of alloimmunization, and therefore appropriate screening tests should be included in the pre-transplant compatibility algorithm.

Fallacies of a purely virtual platform: Virtual plus reality versus virtual reality - A case study.

Pretransplant immunological assessment of a transplant donor has evolved significantly over the last few decades with the advent of testing platforms with enhanced sensitivity and varying formats. The single antigen bead assay (SAB) assay, a virtual crossmatch (vXM) is used extensively and considered the gold standard for defining donor-specific antibodies (DSA) in many parts of the World. A country like India, is however challenged by the lack of adequate representation of locally frequent HLA alleles and hence in our institution, we continue to perform a physical crossmatch (pXM) on the Complement Dependent Cytotoxicity and flow cytometry platforms alongside the SAB. We report here a case report where the discrepancy between platforms of testing have raised certain pertinent questions in our interpretation of the vXM.

Impact of rituximab on the T-cell flow cytometric crossmatch.

Rituximab is frequently used in the setting of ABO-incompatible renal transplants, and highly sensitized patients. Its interference with B-cell flow cytometric crossmatch (B-FCXM) is well known. However, its effect on the T-cell flow cytometric crossmatch (T-FCXM) has not been described. We aimed to evaluate the effect of rituximab on the T-FCXM using non-pronase and pronase treated donor lymphocytes and compare results with the single antigen bead (SAB) assay. In this retrospective study, 28 patients on rituximab therapy were evaluated against 30 donors. Using non-pronase treated donor lymphocytes, all 30 FCXMs showed strong B-cell positivity {median (IQR) B-cell ratio: 184.65 (253.17)} which significantly reduced {1.0 (1.18); p < 0.00001} with pronase treatment. 'T-cell tailing' phenomenon was observed in 17/30 FCXMs in the non-pronase group as a 'tail of T-cells', indicating a rare sub-population. However, it disappeared in the pronase-treated group. SAB assay did not show donor-specific antibodies (DSA) in all 17 patients with 'T-cell tailing' phenomenon. Although, rituximab is described to impact only B-FCXM, we have consistently found 'T-cell tailing' in 57% of T-FCXMs, which clears with pronase treatment. The 'T-cell tailing' led to weak positive T-FCMX ratios due to increased MFI in the FL1 channel. However, the absence of DSA in all recipients reinforces the fact that this is a false positive finding and should not be misconstrued as a possible class I DSA. Structural homology of Fc receptors on activated T-cells to CD20 could be a possible explanation of the same and provide insight into a novel mechanism of action of rituximab.

The challenge of using the virtual crossmatch as a singular tool for the detection of Anti-HLA antibodies- A study from a tertiary care institute from South India.

INTRODUCTION: Detection of donor specific antibodies (DSA) is critical in both solid organ and mismatched haematopoietic stem cell transplants. The single antigen bead assay (SAB) is widely used as a virtual crossmatch in these settings. However, HLA allele variation across ethnicities and differing genetic backgrounds is a well-known and acknowledged fact and representation of alleles prevalent in a population is key while using a virtual crossmatch as a sole decision making tool. Against this background, this study was performed to assess the feasibility of using the SAB as a single tool to identify DSA in our population. MATERIALS AND METHODS: The HLA alleles identified in the study population were analysed to assess their representation on SAB panels from two different vendors. RESULTS: The study population comprised of a total of 966 subjects for whom 6 loci high resolution HLA typing was done. A total of 241 different alleles were assigned in the population. Among the 241 alleles identified in our study population, 48.55% (n = 117) alleles were represented in the SAB A panel and 48.13% (n = 116) represented in the SAB B panel. Unrepresented alleles were 51.45% (n = 124) in panel A and 51.87% (n = 125) in panel B. All the twelve alleles were represented for 16.05% (n = 155) and 16.25% (n = 157) of study population in panel A and in panel B respectively. The remaining individuals (83.95%, (n = 811) in panel A and 83.75%, (n = 809) in panel B) had at least one allele unrepresented. CONCLUSION: Our study addresses an important limitation in utilizing the SAB as a single tool to identify DSA, owing to non-representation of locally prevalent / unique alleles in our population. More than 50% of alleles were unrepresented in both the SAB assays we studied, which included alleles from both Class I and Class II. We recommend therefore that, until a comprehensive coverage of alleles is provided, or epitope matching becomes robust, that the SAB be combined with a physical crossmatch when mismatched alleles are not represented.