Jane Kearns

Detection of anti-HLA antibody by flow cytometry in patients with a left ventricular assist device is associated with early rejection following heart transplantation.

BACKGROUND Patients with a left ventricular assist device (LVAD) as a bridge to heart transplantation (HT) often have elevated levels of panel reactive antibodies (PRA). The clinical significance of anti-human histocompatibility leukocyte antigen (HLA) antibodies detected by flow cytometry in PRA negative patients remains unclear. METHODS Eighteen patients who underwent LVAD placement as a successful bridge to HT had standard anti-human globulin complement-dependent cytotoxicity and retrospective flow cytometry assays performed to detect class I anti-HLA antibodies. A positive flow result was defined as a fluorescent ratio of 23:1 versus a negative control. RESULTS Six patients had anti-HLA antibodies detected by flow cytometry. Univariate analysis demonstrated more moderate-severe rejection episodes (ISHLT > or = IIIA) at 2 months (0.83+/-0.75 vs. 0; P=0.04) and a trend toward decreased time to first rejection (61+/-17 vs. 225+/-62 days; P=0.06) in these patients. No differences were observed in donor-recipient HLA mismatch or 1 year Kaplan-Meier survival between patients with or without anti-HLA antibodies. CONCLUSION Despite a negative PRA, LVAD patients with class I anti-HLA antibodies detected by flow cytometry have a greater incidence of moderate-severe rejection in the first 2 months after HT. Flow cytometry may be a useful clinical tool in screening PRA negative LVAD patients before transplantation. Patients with positive anti-HLA antibody screening by flow cytometry may require more intensive immunosuppression in the early post-HT period.

Renal transplantation despite a positive antiglobulin crossmatch with and without prophylactic OKT3.

The antiglobulin crossmatch (AGXM) is a sensitive technique employed by many transplant centers to enhance detection of preformed antibody to donor antigens that may cause hyperacute rejection. However, positive AGXM may detect irrelevant or very low titers of anti-HLA antibody precluding transplantation in suitable recipients. To investigate the significance of a positive AGXM, cadaveric renal transplantation was carried out despite a weakly positive AGXM (defined as cell killing above background but not greater than 20%) in 48 recipients. In an initial group (n = 10), maintained on triple therapy (cyclosporine, azathioprine, and prednisone), accelerated acute rejection occurred in 4 recipients and 3 grafts were lost. A subsequent group (n = 38) was treated with a prophylactic course of OKT3 then triple therapy. There were no episodes of accelerated acute rejection (P less than 0.01) although clinical hyperacute rejection claimed one graft and the incidence of delayed graft function was high (75%). The prophylactic OKT3 group had a reduced incidence of acute rejection (0.5 versus 1.0) per recipient and the onset of first episodes was delayed (mean onset: 13 versus 35 days after transplantation). One year actuarial primary graft survival was 88% in the prophylactic OKT3 group as compared with only 50% in the initial group. The outcome in the positive AGXM group was similar to a concurrent group (n = 32) with a negative AGXM and immediate graft function. On the other hand, the subset of positive AGXM regraft recipients treated with prophylactic OKT3 fared poorly, with a 36% (4/11) incidence of primary nonfunction. In summary, a positive AGXM, as defined in this report, is not a contraindication to primary renal transplantation--in fact, the use of the AGXM will identify recipients that would benefit from prophylactic OKT3.

HLA-A amino acid polymorphism and delayed kidney allograft function

Delayed allograft function (DGF) is a common adverse event in postrenal transplantation. The etiology of DGF is thought to include both nonimmunologic (donor age, cold ischemia time, and recipient race) and immunologic factors. We examined the association of DGF with amino acid mismatches at 66 variable sites of the HLA-A molecule in a prospective cohort study of 697 renal transplant recipients of deceased donors. Using a multivariate logistic regression model adjusted for nonimmunologic risk factors, we show that combinations of a few amino acid mismatches at crucial sites of HLA-A molecules were associated with DGF. In Caucasian recipients, a mismatch at position 62, 95, or 163, all known to be functionally important within the antigen recognition site, was associated with an increased risk for DGF. Furthermore, a decreased risk for DGF was associated with a mismatch at HLA-A family-specific sites (149, 184, 193, or 246), indicating that evolutionary features of HLA-A polymorphism separating HLA-A families and lineages among donor-recipient pairs may correlate with the magnitude of alloreactivity influencing the development of DGF. These findings suggest that amino acid polymorphisms at functionally important positions at the antigen recognition site of the HLA-A molecule have a significant influence on DGF.

Correlation of Circulating Complement-Fixing Donor-Specific Antibodies Identified by the C1q Assay and Presence of C4d in Endomyocardial Biopsy Specimens

OBJECTIVES Donor-specific antibodies (DSAs) are associated with increased cardiac graft loss. We applied a C1q solid-phase assay in parallel with the standard immunoglobulin G (IgG) single antigen bead (SAB) assay to examine the correlation of circulating complement-fixing donor-specific antibodies and the presence of C4d in endomyocardial biopsy (EMB) specimens. METHODS We retrospectively studied the relationship of C1q+ DSAs and C4d immunofluorescence (IF) in 49 EMB specimens from 44 heart transplant recipients who had concurrent EMB, C4d IF, and DSA measurements. We applied a C1q SAB in parallel with the standard IgG SAB assay to examine the DSA profiles in heart transplant patients posttransplant. RESULTS A better concordance is observed between C1q+ DSAs with C4d IF+ compared with IgG DSAs with C4d IF + (40% vs 24%, P = .02). However, the correlation of C1q DSAs with C4d IF is not statistically significant (P = .24). Importantly, C1q+ DSAs were observed in 16 of 17 cases with C4d IF+; 24 cases had circulating C1q+ DSAs without detectable C4d staining, suggesting that that the presence of C1q+ DSAs may precede the detection of C4d deposition in EMB specimens and/or the development of antibody-mediated rejection. CONCLUSIONS In this cohort of 44 patients, no significant correlation was observed between circulating C1q DSAs and C4d IF in EMB specimens. Additional studies are needed to further evaluate the association of C1q DSAs with EMB specimens and C4d staining.

Renal Transplantation in Patients With Pretransplant Donor‐Specific Antibodies and Negative Flow Cytometry Crossmatches

Patel et al. (1) performed a retrospective cohort study of 60 living donor renal transplant recipients; the authors observed that despite negative pretransplant cytotoxicity and flow cytometry crossmatches (FCXM), renal transplant recipients with pretransplant donor-specific antibodies (DSA) detected by solid-phase methods appear to have an increased incidence of acute humoral rejection (AHR). While these observations are interesting, only a small number (20) of DSA-positive patients were evaluated. Moreover, a relative high cutoff value was used for determination of a positive B-cell FCXM; weak DSA may not be detected by the type of crossmatch used in this study (1). The decision threshold used for the interpretation of the flow cytomtery crossmatch results in pretransplant risk assessment in sensitized kidney transplant candidates is an important parameter to consider. The sensitivity of the FCXM compared to DSA detected by bead assays can vary significantly with the cutoff value used for these assays. Transplant centers usually set their own criteria for decision threshold for antiHLA antibody and flow crossmatching (2). A low cutoff value may be used in particular when B cells are treated with Pronase in the B-cell FCXM. A low cutoff value in the B-cell FCXM can detect weak DSA including antibodies to HLA class I antigens that can go undetected by the T-cell FCXM. In addition, the inclusion of historical sera in the analysis of anti-HLA antibody specificity and pretransplant PRA is critical in assessing donor compatibility and risk factors for the development of AHR due to a memory alloresponse. Unfortunately, this information can be difficult to obtain like the situation in three of the four patients who had AHR (1).

Assessing a single targeted next generation sequencing for human leukocyte antigen typing protocol for interoperability, as performed by users with variable experience

BACKGROUND A simplified protocol for HLA-typing -by NGS, developed for use with the Illumina MiSeq, was performed by technologists with varying NGS experience to assess accuracy and reproducibility. METHODS Technologists from six laboratories typed the same 16 samples at HLA-A, B, C, DRB1, and DQB1. The protocol includes long range PCR, library preparation, and paired-end 250bp sequencing. Two indexing strategies were employed: locus-specific indexing whereby each locus was tagged uniquely and sample-specific indexing whereby all 5 loci for a sample were pooled prior to library preparation. Sequence analysis was performed with two software packages, Target HLA (Omixon) and NGSengine (GenDx). RESULTS The average number of sequence reads per library was 387,813; however, analysis was limited to 40,000 reads for locus-indexed libraries and 200,000 reads for sample-indexed libraries resulting in an average depth of coverage of 1444 reads per locus. Sufficient reads for genotype analysis were obtained for 98.4% of libraries. Genotype accuracy was >97% in pooled amplicons and >99% in individual amplicons by both software analysis. Inter-laboratory reproducibility was 99.7% and only cause of discordance was cross-contamination of a single amplicon. CONCLUSIONS This NGS HLA-typing protocol is simple, reproducible, scalable, highly accurate and amenable to clinical testing.

Transplant Nephrectomy: Histologic Findings - A Single Center Study

Aims: To identify the histopathological features of transplant nephrectomy (TN) specimens. Methods: We performed retrospective analysis of 73 nephrectomies to review the histopathology in detail and correlate the Banff grading characteristics of TN specimens with time post engraftment and clinical features. Retrospective data on donor-specific antibodies (DSA) were also collected. Results: The majority of patients who had TN in less than 3 months posttransplant (n = 20; median time to TN: 4 days) had hemorrhagic infarction; 7 patients (35%) had grade 3 acute rejection (AR). Patients who had TN later than 3 months posttransplant (n = 53; median time to TN: 67 months) had AR, grade 2B (21%) and 3 (43%), coexisting with advanced vascular injury in the form of interstitial hemorrhage, extensive interstitial fibrosis and tubular atrophy (IF/TA) as well as the presence of DSAs. Overall, the majority of patients without DSA pre-TN developed DSA post-TN. Conclusions: Our data revealed extensive inflammation and ongoing immunologic activity in a subset of patients with a failed graft. Careful and individualized approach based on clinical and laboratory data should guide the decision for transplant nephrectomy. i 2014 S. Karger AG, Basel