Positive flow cytometry crossmatch with discrepant antibody testing results following COVID-19 vaccination.

The impact of COVID-19 vaccination on the alloimmunity of transplant candidates is unknown. We report a case of positive B cell flow cytometry crossmatch in a patient waiting for second kidney transplantation, 37 days after receiving the COVID-19 vaccine. The preliminary crossmatch, using sample collected before COVID-19 vaccination, was negative. The antibodies to mismatched donor HLA-DR7 were detected only with multi-antigen beads but not with single-antigen beads, excluding possible prozone effects in solid-phase antibody assays. The crossmatches were positive with HLA-DR7-positive surrogates (n = 2) while negative with HLA-DR7-negative surrogates (n = 3), which confirms the HLA-DR7 alloreactivity. The antigen configurations on B lymphocytes are similar to that on the multi-antigen beads while distinct from the single-antigen beads. HLA-DR7 was the repeating mismatched antigen with the failing first kidney allograft. The newly emerged antibody to HLA-DR7 probably is the consequence of bystander activation of memory response by the COVID-19 vaccination. This case highlights the importance of verifying allo-sensitization history and utilizing multiple assays, including cell-based crossmatch and solid-phase assays with multi-antigens. COVID-19 immunization may deserve special attention when assessing the immunological risk before and after organ transplantation.

The big picture: A case report of antibody mediated rejection and treatment after lung transplantation illustrating the need to correlate laboratory findings with clinical status.

We report the case of a patient who developed respiratory failure in the presence of de novo donor-specific antibody (DSA) two years after lung transplantation, following recurrent acute cellular rejection. The patient underwent salvage therapy with plasma exchange, intravenous immunoglobulins, and proteasome inhibitor carfilzomib (CFZ). DSA was detected prior to admission for antibody-mediated rejection by single antigen bead Luminex (SAB) testing and indicated the presence of a DQ3 pattern (DQ7, DQ8, and DQ9). The patient initially responded to CFZ-based treatment with a decline in DQ3-DSA strengths, but DQ7-DSA persisted at low-levels. However, the DQ7-C1q reactivity that was absent after therapy recovered, indicating a potential "prozone" effect on SAB testing. Treating sera with dithiothreitol/ heat and ethylenediaminetetraacetic acid was able to relieve the "prozone" effect, resulting in an increased DQ7 immunoglobulin G (IgG) mean fluorescence intensity. HLAMatchmaker eplet analysis suggested reactivity towards the DQB1* eplet 55PPP expressed on all DQ3 antigens and DQB1* eplet 45EV(DQ7). In this case, we illustrate the functional diversity of DQ3/DQ7-specific DSA reactivity patterns obtained by IgG-SAB and C1q-SAB assays and determined the eplet repertoire using HLAMatchmaker. DSA analysis should include tests to evaluate DSA strength, titer, and function, along with communications with clinical colleagues to correlate laboratory findings with clinical parameters.

How did a patient who types for HLA-B*4403 develop antibodies that react with HLA-B*4402?

This case report shows that the sensitization of a HLA-B*4403 patient by a kidney transplant with a HLA-Cw*0704 mismatch led to antibodies reacting with the 156DA eplet shared with B*4402 and other HLA-B antigens including B*0801, B*3701, B*4101, B*4201, B*4501, and B*8201. It demonstrates that antibodies induced by an HLA-C mismatch can render certain HLA-B antigens unacceptable mismatches although the patient has never been exposed to them. This finding illustrates the importance of analyzing antibody specificities against HLA epitopes in the determination of mismatch acceptability for sensitized patients.

Anti-human leukocyte antigen antibodies, vascular C4d deposition and increased soluble c4d in broncho-alveolar lavage of lung allografts.

BACKGROUND: The hallmark of humoral rejection is the presence of subendothelial C4d in the allograft. A simultaneous determination of vascular C4d with soluble C4d in broncho-alveolar lavage fluid (BAL) and circulating anti-human leukocyte antigen (HLA) antibodies (HLA-Ab) has not been reported in lung transplantation. METHODS: Forty-two consecutive lung-transplant patients were included in this cross-sectional study. The presence and specificity of HLA-Ab was determined at the same frequency with transbronchial biopsies. Soluble C4d levels were measured by enzyme-linked immunosorbent assay in all 42 patients. In a subgroup of 32 patients with available timely matched paraffin-embedded tissue sections, the vascular C4d deposition was also assessed. RESULTS: The presence of HLA-Ab in 16 patients was associated with biopsy-proven acute rejection (10/16 vs. 3/16, P<0.01) and increased immunosuppression (13/16 vs. 4/16, P<0.005). Pulmonary function was also decreased in patients with HLA-Ab (mean forced expiratory volume in 1 second=49%) when compared with the control group (mean forced expiratory volume in 1 second=66%, P<0.05). Nine patients exhibited specific vascular C4d deposition and in eight of nine (89%) cases HLA-Ab were detected, versus 8 of 23 (35%) in C4d-negative patients (P<0.05). Soluble C4d in BAL was highly (>0.5 microg/mL) elevated in patients with HLA-Ab and vascular C4d and was moderately (0.2 microg/mL) increased in patients with antibodies but C4d-negative. In contrast, only a slight elevation of soluble C4d (<0.1 microg/mL) was detected in patients without HLA-specific antibodies. CONCLUSIONS: The association of HLA-specific antibodies with vascular C4d deposition and soluble C4d in BAL, in addition to the reduced pulmonary function, might constitute a diagnostic triad for antibody-mediated rejection in lung transplant patients.

Retransplant candidates have donor-specific antibodies that react with structurally defined HLA-DR,DQ,DP epitopes.

This report describes a detailed analysis how donor-specific HLA class II epitope mismatching affects antibody reactivity patterns in 75 solid organ transplant recipients with an in situ allograft and who were considered for retransplantation. Sera were tested for antibodies in a sensitive antigen-binding assay (Luminex) with single class II alleles. Their reactivity was analyzed with HLAMatchmaker, a structural matching algorithm that considers so-called eplets to define epitopes recognized by antibodies. Only 24% of the patients showed donor-specific anti-DRB1 antibodies and there was a significant correlation with a low number of mismatched DRB1 eplets. This low detection rate of anti-DRB1 antibodies may also be due to allograft absorption. In contrast, antibodies to DRB3/4/5 mismatches were more common. Especially, 83% of the DRB4 (DR53) mismatches resulted in detectable antibodies against an eplet uniquely found on DR53 antigens. Donor-specific DQB mismatches led to detectable anti-DQB antibodies with a frequency of 87%. Their specificity correlated with eplets uniquely found on DQ1-4. The incidence of antibodies induced by 2-digit DQA mismatches was 64% and several eplets appeared to play a dominant role. These findings suggest that both alpha and beta chains of HLA-DQ heterodimers have immunogenic epitopes that can elicit specific antibodies. About one-third of the sera had anti-DP antibodies; they reacted primarily with two DPB eplets and an allelic pair of DPA eplets. These data demonstrate that HLA class II reactive sera display distinct specificity patterns associated with structurally defined epitopes on different HLA-D alleles.