Solid phase multiplex-bead arrays for the detection and characterization of HLA antibodies provide increased sensitivity and specificity compared to conventional lymphocyte-based assays. Assay variability due to inconsistencies in commercial kits and differences in standard operating procedures (SOP) hamper comparison of results between laboratories. The Clinical Trials in Organ Transplantation Antibody Core Laboratories investigated sources of assay variation and determined if reproducibility improved through utilization of SOP, common reagents and normalization algorithms. Ten commercial kits from two manufacturers were assessed in each of seven laboratories using 20 HLA reference sera. Implementation of a standardized (vs. a nonstandardized) operating procedure greatly reduced MFI variation from 62% to 25%. Although laboratory agreements exceeded 90% (R2), small systematic differences were observed suggesting center specific factors still contribute to variation. MFI varied according to manufacturer, kit, bead type and lot. ROC analyses showed excellent consistency in antibody assignments between manufacturers (AUC > 0.9) and suggested optimal cutoffs from 1000 to 1500 MFI. Global normalization further reduced MFI variation to levels near 20%. Standardization and normalization of solid phase HLA antibody tests will enable comparison of data across laboratories for clinical trials and diagnostic testing.

Letter to the Editor.

Although rare, infection and vaccination can result in antibodies to human leukocyte antigens (HLA). We analyzed the effect of SARS-CoV-2 infection or vaccination on HLA antibodies in waitlisted renal transplant candidates. Specificities were collected and adjudicated if the calculated panel reactive antibodies (cPRA) changed after exposure. Of 409 patients, 285 (69.7 %) had an initial cPRA of 0 %, and 56 (13.7 %) had an initial cPRA > 80 %. The cPRA changed in 26 patients (6.4 %), 16 (3.9 %) increased, and 10 (2.4 %) decreased. Based on cPRA adjudication, cPRA differences generally resulted from a small number of specificities with subtle fluctuations around the borderline of the participating centers’ cutoff for unacceptable antigen listing. All five COVID recovered patients with an increased cPRA were female (p = 0.02). In summary, exposure to this virus or vaccine does not increase HLA antibody specificities and their MFI in approximately 99 % of cases and 97 % of sensitized patients. These results have implications for virtual crossmatching at the time of organ offer after SARS-CoV-2 infection or vaccination, and these events of unclear clinical significance should not influence vaccination programs.

The requirement for immunosuppression in organ transplant recipients, particularly suppression of the T cell response, has made this population especially vulnerable to prolonged and severe infections during the ongoing SARS-CoV-2 pandemic.1 Acknowledgment of this increased risk led to the reduction of deceased-donor organ transplantation, and for many programs, complete cessation of living-donor operations during the early periods of the COVID-19 pandemic.2,3 Nevertheless, for transplant recipients, the risk of contracting viral disease does not exist in a vacuum. Solid organ transplant candidates have a number of characteristics that increase their morbidity and mortality risk above that of the general population. Delineating the presentation and effects of SARS-CoV-2 in this cohort is critical to understanding the new landscape of organ transplantation in the context of an ongoing pandemic.4, 5, 6

Technological advances in HLA laboratory testing undoubtedly improved the sensitivity and specificity of HLA antibody assessment but not without introducing a set of challenges regarding data interpretation. In particular, the introduction of solid-phase single-antigen bead (SAB) antibody assessment brought the belief that mean fluorescence intensity (MFI) was a quantifiable value. As such, MFI levels heavily influenced HLA antibody reporting, monitoring, and clinical practice. However, given that SAB testing was neither intended for nor approved to be quantifiable, is the use of MFI in current clinical and laboratory practice valid? What, if anything, does this numerical value actually reveal about the pathogenic potential of the antibody? What are the pitfalls and caveats associated with reporting MFI? Herein, we travel the road to HLA antibody assessment and explore the reliability of MFI values to make clinical decisions.

Background African American (AA) recipients of deceased-donor (DD) kidney transplants (KT) have shorter allograft survival than recipients of other ethnic groups. Reasons for this disparity encompass complex interactions between donors and recipients characteristics. Methods Outcomes from 3872 AA and 19,719 European American (EA) DDs who had one kidney transplanted in an AA recipient and one in an EA recipient were analyzed. Four donor/recipient pair groups (DRP) were studied, AA/AA, AA/EA, EA/AA, and EA/EA. Survival random forests and Cox proportional hazard models were fitted to rank and evaluate modifying effects of DRP on variables associated with allograft survival. These analyses sought to identify factors contributing to the observed disparities in transplant outcomes among AA and EA DDKT recipients. Results Transplant era, discharge serum creatinine, delayed graft function, and DRP were among the top predictors of allograft survival and mortality among DDKT recipients. Interaction effects between DRP with the kidney donor risk index and transplant era showed significant improvement in allograft survival over time in EA recipients. However, AA recipients appeared to have similar or poorer outcomes for DDKT performed after 2010 versus before 2001; allograft survival hazard ratios (95% CI) were 1.15 (0.74, 1.76) and 1.07 (0.8, 1.45) for AA/AA and EA/AA, compared to 0.62 (0.54, 0.71) and 0.5 (0.41, 0.62) for EA/EA and AA/EA DRP, respectively. Recipient mortality improved over time among all DRP, except unemployed AA/AAs. Relative to DDKT performed pre-2001, employed AA/AAs had HR = 0.37 (0.2, 0.69) versus 0.59 (0.31, 1.11) for unemployed AA/AA after 2010. Conclusion Relative to DDKT performed before 2001, similar or worse overall DCAS was observed among AA/AAs, while EA/EAs experienced considerable improvement regardless of employment status, KDRI, and EPTS. AA recipients of an AA DDKT, especially if unemployed, had worse allograft survival and mortality and did not appear to benefit from advances in care over the past 20 years.

The virtual crossmatch (VXM) is gaining acceptance as an alternative approach to assess donor:recipient compatibility prior to transplantation. In contrast to a physical crossmatch, the virtual crossmatch does not require viable donor cells but rather relies on complete HLA typing of the donor and current antibody assessment of the recipient. Thus, the VXM can be performed in minutes which allows for faster transplant decisions thereby increasing the likelihood that organs can be shipped across significant distances yet safely transplanted. Here, we present a brief review of the past 50 years of histocompatibility testing; from the original complement-dependent cytotoxicity crossmatch in 1969 to the new era of molecular HLA typing, solid-phase antibody testing and virtual crossmatching. These advancements have shaped a paradigm shift in our approach to transplantation. That is, foregoing a prospective physical crossmatch in favor of a VXM. In this review, we undertake an in-depth analysis of the pros- and cons- of physical and virtual crossmatching.Finally, we provide objective data on the selected use of the VXM which demonstrate the value of a VXM in lieu of the traditional physical crossmatch for safe and efficient organ transplantation.

Accurate deceased donor HLA typing assumes that the blood sample tested contains only DNA from the organ donor. Prior to procurement, many organ donors are transfused at least one unit of red blood cells (RBC). Non-organ donor DNA acquired from transfusions may result in incorrect and/or ambiguous HLA typing. To address this question, we investigated the impact of RBC transfusion on organ donor HLA typing by using different in vitro transfusion models: leukoreduced (LR) and non-LR RBCs. Various quantities of LR and non-LR RBCs were added to normal peripheral blood and HLA typing was performed by real time PCR. Our results show that HLA typing of deceased donors can be impacted dependent upon the type and quantity of transfused RBCs. Importantly, if LR RBCs are given, HLA typing is unlikely to be affected, precluding the need to delay typing and obtain an alternative source of donor DNA.

Background. Alloimmunization to minor red blood cell (RBC) antigens occurs commonly in sickle cell disease (SCD). Patients with alloimmunization demonstrate increased risk for new alloantibody formation with subsequent transfusion. Alloimmunization to human leukocyte antigens (HLA) can occur with RBC transfusion and may result in graft rejection during stem cell or organ transplantation. The prevalence and risk factors for HLA alloimmunization in multiply transfused pediatric SCD patients are unknown. Procedure. A cross-sectional study of HLA alloimmunization in SCD patients aged 3-21 years with a history of ≥3 RBC transfusions was performed to test the hypothesis that HLA alloimmunization is associated with RBC alloimmunization. Antibodies to class I and class II HLA were measured by Flow Panel Reactive Antibody (FlowPRA®). Results. Seventy-three SCD patients (30 with RBC antibodies) were tested. HLA antibodies were detected in 25/73 (34%) patients; class I HLA antibodies occurred in 24/73 (33%) and class II HLA antibodies occurred in 3 (4%). Among patients with RBC antibodies, 16/30 (53%) had HLA antibodies, while 9/43 (21%) patients without RBC antibodies had HLA antibodies (OR 4.32 [1.6-12.1]). In a multivariate analysis, antibodies to RBC antigens were an independent predictor of HLA alloimmunization (P=0.041). The association of RBC and HLA immunization was strongest among patients with no history of chronic transfusion therapy. Conclusions. This analysis is the first description of HLA alloimmunization in pediatric SCD patients who receive primarily leukoreduced RBC transfusions and demonstrates that HLA alloimmunization tendency is associated with antibodies to RBC antigens.

Background. Two apolipoprotein L1 gene (APOL1) renal-risk variants in donors and African American (AA) recipient race are associated with worse allograft survival in deceased-donor kidney transplantation (DDKT) from AA donors. To detect other factors impacting allograft survival from deceased AA kidney donors, APOL1 renal-risk variants were genotyped in additional AA kidney donors. Methods. The APOL1 genotypes were linked to outcomes in 478 newly analyzed DDKTs in the Scientific Registry of Transplant Recipients. Multivariate analyses accounting for recipient age, sex, race, panel-reactive antibody level, HLAmatch, cold ischemia time, donor age, and expanded criteria donation were performed. These 478 transplantations and 675 DDKTs from a prior report were jointly analyzed. Results. Fully adjusted analyses limited to the new 478 DDKTs replicated shorter renal allograft survival in recipients of APOL1 2-renal-risk-variant kidneys (hazard ratio [HR], 2.00; P = 0.03). Combined analysis of 1153 DDKTs from AA donors revealed donor APOL1 high-risk genotype (HR, 2.05; P = 3 - 10-4), older donor age (HR, 1.18; P = 0.05), and younger recipient age (HR, 0.70; P = 0.001) adversely impacted allograft survival. Although prolonged allograft survival was seen in many recipients of APOL1 2-renal-risk-variant kidneys, follow-up serum creatinine concentrations were higher than that in recipients of 0/1 APOL1 renal-risk-variant kidneys. A competing risk analysis revealed that APOL1 impacted renal allograft survival, but not recipient survival. Interactions between donor age and APOL1 genotype on renal allograft survival were nonsignificant. Conclusions. Shorter renal allograft survival is reproducibly observed after DDKT from APOL1 2-renal-risk-variant donors. Younger recipient age and older donor age have independent adverse effects on renal allograft survival.