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Antimicrobial stewardship programs (ASPs) have made immense strides in optimizing antibiotic, antifungal, and antiviral use in clinical settings. However, although ASPs are required institutionally by regulatory agencies in the United States and Canada, they are not mandated for transplant centers or programs specifically. Despite the fact that solid organ transplant recipients in particular are at increased risk of infections from multidrug-resistant organisms, due to host and donor factors and immunosuppressive therapy, there currently are little rigorous data regarding stewardship practices in solid organ transplant populations, and thus, no transplant-specific requirements currently exist. Further complicating matters, transplant patients have a wide range of variability regarding their susceptibility to infection, as factors such as surgery of transplant, intensity of immunosuppression, and presence of drains or catheters in situ may modify the risk of infection. As such, it is not feasible to have a “one-size-fits-all” style of stewardship for this patient population. The objective of this white paper is to identify opportunities, risk factors, and ASP strategies that should be assessed with solid organ transplant recipients to optimize antimicrobial use, while producing an overall improvement in patient outcomes. We hope it may serve as a springboard for development of future guidance and identification of research opportunities.

The COVID‐19 pandemic has brought unprecedented challenges to the transplant community. The reduction in transplantation volume during this time is partly due to concerns over potentially increased susceptibility and worsened outcomes of COVID‐19 in immunosuppressed recipients. The consequences of COVID‐19 on patients waitlisted for kidney transplantation, however, have not previously been characterized. We studied 56 waitlisted patients and 80 kidney transplant recipients diagnosed with COVID‐19 between March 13 and May 20, 2020. Despite similar demographics and burden of comorbidities between waitlisted and transplant patients, waitlisted patients were more likely to require hospitalization (82% vs. 65%, P = .03) and were at a higher risk of mortality (34% vs. 16%, P = .02). Intubation was required in one third of hospitalized patients in each group, and portended a very poor prognosis. The vast majority of patients who died were male (84% waitlist, 100% transplant). Multivariate analysis demonstrated waitlist status, age, and male sex were independently associated with mortality. COVID‐19 has had a dramatic impact on waitlisted patients, decreasing their opportunities for transplantation and posing significant mortality risk. Understanding the impact of COVID‐19 on waitlist patients in comparison to transplant recipients may aid centers in weighing the risks and benefits of transplantation in the setting of ongoing COVID‐19.

A consensus conference on frailty in kidney, liver, heart and lung transplantation, sponsored by the American Society of Transplantation (AST) and endorsed by the American Society of Nephrology (ASN), the American Society of Transplant Surgeons (ASTS) and the Canadian Society of Transplantation (CST), took place on February 11, 2018 in Phoenix, Arizona. Input from the transplant community through scheduled conference calls enabled wide discussion of current concepts in frailty, exploration of best practices for frailty risk assessment of transplant candidates and for management after transplant, and development of ideas for future research. A current understanding of frailty was compiled by each of the solid organ groups and is presented in this paper. Frailty is a common entity in patients with end-stage organ disease who are awaiting organ transplantation, and affects mortality on the wait-list and in the post-transplant period. The optimal methods by which frailty should be measured in each organ group are yet to be determined, but studies are underway. Interventions to reverse frailty vary among organ groups and appear promising. This conference achieved its intent to highlight the importance of frailty in organ transplantation and to plant the seeds for further discussion and research in this field.

BK virus nephropathy (BKVN) is a frequent and serious post-transplant complication and undermines realization of the full benefits of kidney transplantation. We developed a Bak amplicon-based standard curve for absolute quantification of BKV VP1 mRNA copy number in the real time quantitative PCR (RT-qPCR) assay and investigated the performance characteristics of this novel assay.We determined analytical specificity, sensitivity, and precision of our 73 bp mouse Bak amplicon based standard curve for absolute quantification of BKV VP1 mRNA in RT-qPCR assays. The diagnostic accuracy of the Bak standard curve in the RT-qPCR assay for the noninvasive diagnosis of BKVN in human kidney allograft recipients was investigated by quantification of BKV VP1 mRNA copy number in 192 urine samples matched to 192 kidney allograft biopsies from 155 unique kidney allograft recipients. Intraclass correlation coefficients (ICC) were calculated for the threshold cycles (Ct) and BKV VP1 mRNA copy number observed in the RT-qPCR assay with the Bak standard curve or the BKV standard curve.Performance characteristics of the Bak amplicon-based RT-qPCR assay were exceptional with a slope of -3.291, Y-intercept of 38.60, Rsup2/supvalue of 1.00, efficiency of 101% and error of 0.014. Amplification was specific for the Bak amplicon. Intra assay standard deviation (SD) was 0.08 or less and inter assay SD was 0.11 or less for 31 cycles or less of amplification of the Bak amplicon. Receiver operating characteristic (ROC) curve analysis of BKV VP1 mRNA copy number in 192 biopsy matched urines yielded an area under the ROC of 0.982 (95% CI, 0.964 to 0.999, P lt; 0.0001) for discriminating patients with BKVN biopsies from patients without BKVN biopsies. The striking identity in the measurement of BKV VP1 mRNA copy numbers in the Bak amplicon-based RT-qPCR assay and in the BKV amplicon-based RT-qPCR assay was shown by an ICC of 1.00 when the Cts were compared, and an ICC of 0.99 when the logsub10/subBKV VP1 mRNA copy numbers were compared.Bak standard curve for absolute quantification of BKV VP1 mRNA copy number in the RT-qPCR assay demonstrated high efficiency, short and long-term precision and analytical specificity. BKVN was diagnosed with high accuracy. Our new findings, viewed in the light of our earlier demonstration that absolute quantification of a panel of mRNAs encoding immunoregulatory proteins is feasible with the Bak amplicon-based RT-qPCR assays, suggest that the Bak standard curve could serve as a universal calibrator for absolute quantification of transcripts in RT-qPCR assays and help reduce the workload, costs and eliminate contamination of genes of interest by repeated amplification of gene specific standard curves.

Posttransplant diarrhea is associated with kidney allograft failure and death, but its etiology remains unknown in the majority of cases. Because altered gut microbial ecology is a potential basis for diarrhea, we investigated whether posttransplant diarrhea is associated with gut dysbiosis. We enrolled 71 kidney allograft recipients for serial fecal specimen collections in the first 3 months of transplantation and profiled the gut microbiota using 16S ribosomal RNA (rRNA) gene V4-V5 deep sequencing. The Shannon diversity index was significantly lower in 28 diarrheal fecal specimens from 25 recipients with posttransplant diarrhea than in 112 fecal specimens from 46 recipients without posttransplant diarrhea. We found a lower relative abundance of 13 commensal genera (Benjamini-Hochberg adjusted P ≤ .15) in the diarrheal fecal specimens including the same 4 genera identified in our prior study. The 28 diarrheal fecal specimens were also evaluated by a multiplexed polymerase chain reaction (PCR) assay for 22 bacterial, viral, and protozoan gastrointestinal pathogens, and 26 specimens were negative for infectious etiologies. Using PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) to predict metagenomic functions, we found that diarrheal fecal specimens had a lower abundance of metabolic genes. Our findings suggest that posttransplant diarrhea is not associated with common infectious diarrheal pathogens but with a gut dysbiosis.

Advances in bioinformatics allow identification of single nucleotide polymorphisms (variants) from RNA sequence data. In an allograft biopsy, 2 genomes contribute to the RNA pool, 1 from the donor organ and the other from the infiltrating recipient's cells. We hypothesize that imbalances in genetic variants of RNA sequence data of kidney allograft biopsies provide an objective measure of cellular infiltration of the allograft. We performed mRNA sequencing of 40 kidney allograft biopsies, selected to represent a comprehensive range of diagnostic categories. We analyzed the sequencing reads of these biopsies and of 462 lymphoblastoid cell lines from the 1000 Genomes Project, for RNA variants. The ratio of heterozygous to nonreference genome homozygous variants (Het/Hom ratio) on all autosomes was determined for each sample, and the estimation of stromal and immune cells in malignant tumors using expression data (ESTIMATE) score was computed as a complementary estimate of the degree of cellular infiltration into biopsies. The Het/Hom ratios (P = .02) and the ESTIMATE scores (P < .001) were associated with the biopsy diagnosis. Both measures correlated significantly (r = .67, P < .0001), even though the Het/Hom ratio is based on mRNA sequence variation, while the ESTIMATE score uses mRNA expression. Het/Hom ratio and the ESTIMATE score may offer unbiased and quantitative parameters for characterizing cellular traffic into human kidney allografts.

In order to evaluate whether distinct cell types and states may be identified in human kidney allograft rejection compared to normal kidney, we sequenced 3 kidney biopsies using the 10x Chromium platform and generated 7,217 high-quality single-cell transcriptomes. Taking advantage of the recipient-donor sex mismatches in the allograft biopsies, we unexpectedly observed that in the biopsy with fibrosis, more than half of the kidney allograft fibroblasts were recipient-derived and therefore likely migratory and graft infiltrative, whereas in the biopsy without fibrosis, all the fibroblasts were donor-derived. Furthermore, tubular progenitor cells that overexpress profibrotic extracellular matrix genes potentially contributing to fibrosis, were enriched in the biopsy with fibrosis. In addition, our analysis revealed unique cell types and states in the kidney. Single-cell transcriptomics complemented histopathology and yielded promising mechanistic insights for individualizing the care of transplant recipients and revealed a new approach to study cell migratory contributions in allograft fibrosis.

Introduction HLA sensitization presents a major barrier to transplantation yet sensitive techniques such as the Luminex Single Antigen Bead (SAB) assay, when used in isolation, may inappropriately limit the donor pool. We report a case of apparent robust HLA sensitization following HBV vaccination and describe our approach to the assessment of HLA reactivity leading to successful heart transplantation (HTx). Case Report A 50 year old Caucasian male with long standing ARVD developed recurrent VT and worsening RV dysfunction leading to HTx evaluation. There was no history of transfusion, surgery, or mechanical circulatory support. The Luminex SAB assay at listing and serially thereafter was negative. Unexpectedly, broad class II HLA sensitization (cPRA 98%) was suddenly detected by SAB assay after HBV vaccination concerning for either a heterologous immune response or false reactivity to the beads. Multiple antibodies (Abs) persisted at 1:8 dilution and included Abs against the recipients own HLA (autologous flow XM negative). At the time when a donor became available, 4 DSA were identified on the most recent Luminex SAB. Given his high cPRA in the absence of a strong allosensitizing event, we combined assessment with an alternative solid phase assay (LABScreen PRA), 4-digit donor typing, and prospective crossmatches by Flow Cytometry (FC) and Complement Dependent Cytotoxicity (CDC) to determine the safety of proceeding with HTx (Figure 1). Notably, no HLA Abs were detected using the LABScreen PRA and both FC and CDC crossmatches were negative. The patient underwent successful HTx with no rejection and normal graft function. Summary HLA allosensitization portends worse outcomes after transplant. However, the high sensitivity of the Luminex SAB and potential for false positivity may result in inappropriately limiting access to HTx. This case highlights the importance of comprehensive assessment of HLA sensitization in situations of uncertainty to help identify pathogenic HLA Abs and allow for successful HTx.

To compare the capability of ultrasound strain and Doppler parameters in the assessment of renal allograft interstitial fibrosis/tubular atrophy (IF/TA), we prospectively measured ultrasound corticomedullary strain (strain) and intra-renal artery Doppler end-diastolic velocity (EDV), peak systolic velocity (PSV) and resistive index (RI) in 45 renal transplant recipients before their kidney biopsies. We used 2-D speckle tracking to estimate strain, the deformation ratio of renal cortex to medulla produced by external compression using the ultrasound transducer. We also measured Doppler EDV, PSV and RI at the renal allograft inter-lobar artery. Using the Banff scoring system for renal allograft IF/TA, 45 patients were divided into the following groups: group 1 with ≤5% (n = 12) cortical IF/TA; group 2 with 6%-25% (n = 12); group 3 with 26%-50% (n = 11); and group 4 with50% (n = 10). We performed receiver operating characteristic curve analysis to test the accuracy of these ultrasound parameters and duration of transplantation in determining26% cortical IF/TA. In our results, strain was statistically significant in all paired groups (all p0.005) and inversely correlated with the grade of cortical IF/TA (p0.001). However, the difference in PSV and EDV was significant only between high-grade (26%, including 26%-50% and50%) and low-grade (≤25%, including5% and 6%-25%) cortical IF/TA (p0.001). RI did not significantly differ in any paired group (all p0.05). The areas under the receiver operating characteristic curve for strain, EDV, PSV, RI and duration of transplantation in determining26% cortical IF/TA were 0.99, 0.94, 0.88, 0.52 and 0.92, respectively. Our results suggest that corticomedullary strain seems to be superior to Doppler parameters and duration of transplantation in assessment of renal allograft cortical IF/TA.

Use of organs from donors testing positive for hepatitis B virus (HBV) may safely expand the donor pool. The American Society of Transplantation convened a multidisciplinary expert panel that reviewed the existing literature and developed consensus recommendations for recipient management following the use of organs from HBV positive donors. Transmission risk is highest with liver donors and significantly lower with non-liver (kidney and thoracic) donors. Antiviral prophylaxis significantly reduces the rate of transmission to liver recipients from isolated HBV core antibody positive (anti-HBc+) donors. Organs from anti-HBc+ donors should be considered for all adult transplant candidates after an individualized assessment of the risks and benefits and appropriate patient consent. Indefinite antiviral prophylaxis is recommended in liver recipients with no immunity or vaccine immunity but not in liver recipients with natural immunity. Antiviral prophylaxis may be considered for up to 1 year in susceptible non-liver recipients but is not recommended in immune non-liver recipients. Although no longer the treatment of choice in patients with chronic HBV, lamivudine remains the most cost-effective choice for prophylaxis in this setting. Hepatitis B immunoglobulin is not recommended.

Because the kidney allograft has the potential to function as an in-vivo flow cytometer and facilitate the access of immune cells and kidney parenchymal cells in to the urinary space, we hypothesized that mRNA profiling of urinary cells offers a noninvasive means of assessing the kidney allograft status. We overcame the inherent challenges of urinary cell mRNA profiling by developing pre-amplification protocols to compensate for low RNA yield from urinary cells and by developing robust protocols for absolute quantification mRNAs using RT-PCR assays. Armed with these tools, we undertook first single-center studies urinary cell mRNA profiling and then embarked on the multicenter Clinical Trials in Organ Transplantation-04 study of kidney transplant recipients. We report here our discovery and validation of diagnostic and prognostic biomarkers of acute cellular rejection and of interstitial fibrosis and tubular atrophy (IF/TA). Our urinary cell mRNA profiling studies, in addition to demonstrating the feasibility of accurate diagnosis of acute cellular rejection and IF/TA in the kidney allograft, advance mechanistic and potentially targetable biomarkers. Interventional trials, guided by urinary cell mRNA profiles, may lead to personalized immunosuppression in recipients of kidney allografts.

Aim Emerging data suggest that DSA development is a frequent complication post BKV replication, and most DSA are directed against donor HLA-DQ. In the current investigation, we quantified DQ epitope mismatches between the recipient and donor in individuals with BKVN diagnosis and investigated the association between DQ epitope load and development of DSA. Methods In this pilot study, using the most probable 4-digit HLA typings, we investigated epitope mismatches in individuals who developed DSA post BKVN diagnosis compared to those who did not and correlated them to DSA development and graft dysfunction defined as an increase in serum creatinine by > 0.5 mg/dL at 12 months post-BKVN diagnosis. We had SSOP based intermediate HLA typing available for recipients and donors in 13 recipients who developed BKVN confirmed by renal allograft biopsy. Of the 13 recipients, 2 were excluded: 1 for early graft loss and 1 who had zero DQ mismatches. None of the 11 recipients with BKVN diagnosis had a positive XM or DSA at time of transplantation. Results Time from kidney transplantation to BKVN diagnosis was 8.5 ± 5.1 months. Serum creatinine at time of diagnosis was 1.78 ± 0.56. Of the 11 with BKVN, 36% developed de novo DSA. All DSAs were directed against DQ mismatches. Time from BKVN diagnosis to DSA development was 5.5 ± 2.3 months. Table below lists the epitope mismatch number at each locus. Of the 4 patients with de novo DSA, 75% experienced graft dysfunction during the 12 months post BKVN and of the 7 patients without de novo DSA, 29% experienced graft dysfunction by 12 months. Our data demonstrated that the most frequent DQ epitope mismatches in those with de novo DSA were 52PL3, 45EV, 52PQ2, and 52PR. Conclusions In our pilot study, the epitope load was not different between BKVN patients who developed or did not develop de novo DSA. The most frequent target of de novo antibodies were DQ locus mismatched epitopes. Knowledge of epitope targets of de novo DSA in patients with BKVN may facilitate targeted therapies and improve kidney allograft outcomes. Download : Download high-res image (148KB) Download : Download full-size image

Aim Luminex Single Antigen bead assay (LSAB) derived mean fluorescence intensity (MFI) values of anti-HLA antibodies directed at the potential donor’s HLA are the primary parameter used in the virtual crossmatch to predict physical crossmatch (XM) outcome. We aimed to develop statistically validated LSAB MFI cutpoints for predicting CDC XM and FCXM results. Methods We leveraged the ASHI proficiency testing (PT) 80% consensus results of 7156 T FCXM, 6758 B FCXM, 2917 T CDC XM and 2233 B CDC XM as the reference results to investigate whether LSAB MFI of IgG anti-HLA antibodies predict validated physical XM results. LSAB MFI was determined in our laboratory using One Lambda Single Antigen HLA Class I and Class II beads. The 80% consensus results are from 8 consecutive challenges during 2013 to 2016, and 107 laboratories across USA tested sera and HLA typed cells distributed by ASHI, and reported the results to ASHI for assessment of the laboratory’s proficiency. Data analysis included: summing of MFI of IgG antibodies directed at HLA-A, B and C for investigating their association with T FCXM and T CDC XM results; summing of MFI of antibodies directed at HLA-A, B, C, DR, DQ and DP for investigating their association with B FCXM and B CDC XM results; assigning alternate ASHI challenges to the Discovery set and the Validation set; investigating the association between LSAB MFI and physical XM results by logistic regression analysis corrected for overdispersion; and identification of LSAB MFI cutpoint for maximizing the sum of sensitivity and specificity. LSAB MFI cutpoints derived from the Discovery set to predict FCXM and CDC XM results were investigated in an independent validation set. Results Table 1 demonstrates that LSAB MFI cutpoints from the Discovery set predicts XM outcomes in the Validation set (ROC AUC range from 0.974 to 0.999). Conclusions We have developed and validated LSAB MFI cutpoints for use in the virtual crossmatch to accurately predict physical T FCXM, B FCXM, T CDC XM and B CDC XM results.

Aim Interfering factors in sera may confound crossmatch (XM) results. Heat inactivation (HI), DTT and dialysis treatment of sera have been utilized to minimize these confounders. We investigated the impact of HI on donor T cell complement dependent cytotoxicity (CDC) XM (T Cell AHG XM) and donor B cell CDC XM (B Cell NIH XM). Methods We queried our electronic database for all donor CDC T cell and B cell XM performed during 2011–2017 in which potential kidney transplant recipient’s serum was tested in parallel with or without HI and using potential donor’s T cells and B cells as target cells. Results A total of 6295 living donor XMs and 15880 deceased donor XMs performed in our laboratory were reviewed. Table 1 shows the impact of HI on the 22175 consecutive XM results. 394 of the 6295 living donor T Cell AHG XMs were positive using neat sera and 303 were positive following HI at 63C for 6 min, a 23% conversion from positive to negative results. 698 of the 15880 deceased donor T Cell AHG XMs were positive using neat sera and 540 XMs were positive following HI, a 23% reduction. 1372 of the 6295 living donor B Cell NIH XMs were positive using neat sera and 539 were positive following HI, a 61% reduction. 3015 of the 15880 deceased donor B Cell NIH XMs were positive using neat sera and 842 XMs were positive following HI, a 72% reduction from a positive result to negative result. Conclusions A substantial percentage of XMs are converted from a positive CDC result to negative result and such conversion is more frequent with B cell XM compared to T cell XM. Transplant centers seldom transplant across a positive T cell CDC XM even when HI converts a positive result to a negative result. On the other hand, it is not uncommon to proceed with a kidney transplant when HI converts a positive B cell CDC to a negative CDC provided there are no other immune contraindications. There is an urgent need to establish the clinical significance of XMs converted from a positive to a negative result by heat inactivation.

Aim Luminex Single Antigen bead assays (LSAB) are robust for detecting and identifying IgG anti-HLA antibodies, and the LSAB mean fluorescence intensity (MFI) is currently used to list unacceptable antigens and to predict physical crossmatch (XM) results. Concerns exist regarding the use of MFI as the criterion because MFI thresholds for accurately predicting physical XM results are far from established. We aimed to address this unmet need. Methods We leveraged the XM results from ASHI Proficiency Testing (PTs) to investigate whether LSAB MFI cutpoints are predictive of physical XM results. ASHI graded (⩾80% consensus) XM results from 8 consecutive ASHI PTs in which up to 30 labs tested 40 sera and 16 cells distributed by ASHI were used to examine whether LSAB MFI data we generated in the same PTs predict physical XM results. Cumulative MFIs of DSA directed at HLA-A, B, and C were tested for their ability to predict T cell Flow XM and T cell AHG XM results; cumulative MFIs of DSA directed at HLA-A, B, C, DRB1, DRB3/4/5, DQB and DP were tested for their ability to predict B cell Flow XM and B cell CDC XM results. Results Our data analysis identified that cumulative MFI of DSA less than 6000 directed at HLA-class I antigens predicted T cell Flow XM results with a 100% negative predictive value (NPV) and MFI > 7000 predicted T cell Flow XM results with a 100% positive predictive value (PPV) (Chi-Square for trend, X 2 = 70, df = 1, P 8000 predicted B Flow XM result with a 100% PPV (X 2 ; =; 67, df = 1, P Download high-res image (343KB) Download full-size image Conclusions Our data demonstrating the feasibility of developing LSAB MFI cutpoints for the accurate prediction of T cell Flow XM, B cell Flow XM, T cell AHG XM, and B cell CDC XM results advance a strategy for the interpretation of virtual crossmatches and the prediction of physical XM results.

Aim LSAB mean fluorescence intensity (MFI) is often used to predict physical FCXM results, and LSAB MFI cutpoints are used to list unacceptable antigens. We therefore determined the accuracy of LSAB assay MFI cutpoint used to score the LSAB assay as positive or negative in foretelling FCXM results. Methods One hundred and forty-six pre-transplant sera from 146 patients with end stage renal disease were tested against potential kidney donor’s T cells and B cells using a 3-color flow cytometry clinical protocol; the same 146 sera were tested using LSAB assay and the cumulative MFI directed at donor HLA class I and/or II antigens were determined. A T cell FCXM with a median channel shift (MCS) ≥ 40 with the patients serum vs. negative control serum was classified as a positive test; a B cell FCXM with MCS ≥ 50 with the patients serum vs. negative control serum was classified as a positive test. In the LSAB assay, MFI > 2000 directed at donor HLA antigens was scored as positive DSA. These cutpoints are our laboratory’s cutpoints for both ASHI proficiency testing and for clinical use. Results Statistical analysis demonstrated a significant association between LSAB results and FXCM results (Table 1). LSAB assay results predicted T Cell FCXM results with a sensitivity of 70% and a specificity of 100% (Fischers Exact Test, P 0.0001, Positive Predictive Value (PPV = 100%, Negative Predictive Value (NPV) = 91%). LSAB assay results also predicted B Cell FCXM results with a sensitivity of 86% and a specificity of 72% (Fischers Exact Test, P 0.0001, PPV = 62%, NPV = 91%) (Table 1). In a subset of 31 patients, both donor FCXM and auto FCXM results were available and 17 of 31 donor positive FCXM were also auto B cell FCXM positive (17/17) and auto T cell FCXM positive (1/17). Conclusions LSAB assay results are significantly associated with FCXM results. Auto B cell FCXM and high resolution HLA typing should further refine the relationship between these two high sensitive assays. Download high-res image (138KB) Download full-size image

Detection of donor specific HLA antibodies (DSA) is currently an important component of the Banff criteria for diagnosis of antibody mediated rejection. There is a perception of significant variations in anti-HLA antibody (HLA-Ab) testing practices and interpretation algorithms among different laboratories. In this study, we aimed to survey large transplant centers worldwide to evaluate similarities and differences in HLA-Ab testing practices . METHOD An online survey was sent electronically to 120 histocompatibility laboratories and we received data from 72 laboratories (60% response rate). The survey focused on defining sensitization status, method of reporting HLA-Abs, and post-transplant DSA monitoring practices. Responses from laboratories that support more than 25 transplants per year were analyzed ( n = 66). RESULTS: More than 80% of surveyed laboratories use Luminex based single antigen bead assay to test for HLA-Abs. In non-sensitized patients, absence of HLA-Abs was defined by screening beads (11% of labs), phenotype beads (11%) by single antigen beads (30%) and 2 different assays (48%). For sensitized patients 54% of the laboratories used a combination of assays. Definition of highly sensitized patients varied widely among labs with thresholds ranging from 30% to 80% CPRA. Fifty-five percent of the labs reported MFI values associated with HLA-Abs while 30% reported relative strength (weak, moderate, strong) of the antibody. The definition of relative strength varied among labs with MFI ranges of 500–5000 and 3000–10,000 defining weak and strong antibodies, respectively. Only 39% of the laboratories routinely evaluated sera for the presence of interfering substances/prozone effect. Only 44% of the labs routinely screened for DSA post-transplant; 24% screened only at the time of for-cause and surveillance biopsies and 9% do not monitor for DSA at all. CONCLUSION There is great variability in testing, interpretation and reporting practices among laboratories. Similarly, the definition of a highly sensitize patient varies from center to center. Lack of consistency in practices and definitions hinder our ability to compare patient outcomes from one center to another and highlights the need for standardization and evaluation of clinical relevance of different practice patterns.

Background In flow cytometry crossmatches (FCXM), sera from AB blood group male donors with none or minimal anti-HLA IgG antibodies are used as the negative control serum (NCS). As NCS can give higher median channel fluorescence (MCF) on certain donor cells, it is advised to use cells treated with phosphate buffer saline (PBS) as an additional negative control for comparison. Aim We examined whether using PBS or NCS as the negative control and subsequent staining with FITC-goat-anti-human IgG polyclonal antibody (g α IgG pAb; Jackson Immunoresearch Laboratory; Cat# 109-096-098) or FITC-mouse-anti-human IgG monoclonal antibody (m α IgG mAb; BD biosciences; Cat# 555786; Clone: G18-145) impacts FCXM results. Methodology Peripheral blood ( N = 5) and serum ( N = 5) were collected from human subjects. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient separation. In 96 well V-bottom plate, 300,000 PBMC were incubated with 30 μl of serum for 20 min at 22 °C, washed and stained with 10 μl each of anti-CD3 PerCP and of anti-CD19 PE, and with either 10 μl (1:100 diluted) of FITC-gαIgG pAb or 60 μl of FITC-mαIgG mAb for 30 min at 4 °C. The cells were washed and flow cytometry was performed using BD FACS Canto II instrument and the data was analyzed using the DiVa software. A channel shift of >40 from the NCS for T cell and a shift of >50 for B cells is considered positive here. Results PBMCs treated with PBS gave positive B cell channel shift when stained with g α IgG pAb, but not when stained with m α IgG mAb ( Fig. 1 ). Unlike m α IgG mAb, the g α IgG pAb seems to bind non-specifically to B cells in the absence of human serum. Conclusion This observation cautions that in a clinical FCXM testing, the non use of NCS can lead to a false positive FCXM result. It is also anticipated that a technical error of not adding patient’s serum to the donor cells can lead to positive B cell crossmatch reporting. Using m α IgG mAb or any other α IgG antibody that does not give non-specific binding to donor cells is recommended for FCXM testing.

Aim The AbSorber flow cytometry crossmatch (FXM) XM-ONE 3:1 (XM3) test measures binding of IgG and IgM antibodies (ab) in recipient’s serum to donor-T, -B, and -Tie2+ EPC. The XM3 and our laboratory’s Clinical FXM (CFXM) were compared to examine the feasibility of the XM3’s use in clinical settings. Methods Peripheral blood (N=5) and serum (N=5) were collected from human subjects. Tie2+ EPC and lymphocytes were isolated using manufacturer’s instructions. Cells were incubated with either test serum, negative control serum (NCS) or positive control serum (PCS), washed, and stained with a combination of anti-CD3, -CD19, and ab directed at human -IgG, or -IgM. The median channel shift (MCS) from the NCS for T cell, B cell, and EPC FXM for IgG was considered positive if > 40, >100, and >50, respectively. The MCS cut-off for IgM on T cell, B cell, and EPC was considered positive if >80 MCS. The FXM were acquired in a BD FACSCanto II instrument and the data analysis was performed using the DiVa software. Results 1) The XM3 and CFXM for CD3+IgG+ T cells had similar Median Channel Fluorescence (MCF) (271 ± 19 vs. 241 ± 26) and MCS (9 ± 16 vs. 22 ± 19). All crossmatches were negative; 2) The XM3 and CFXM for CD19+IgG+ B cells also had similar MCF (301 ± 40 vs. 273 ± 19) and MCS (114 ± 77 vs. 105 ± 69). Two B cell FXM were positive and the remaining crossmatches were negative by both methods; 3) Three FXM for CD3+IgM+ T cell and CD19+IgM+ B cell by XM3 and CFXM were negative by both methods; and 4) Of five EPC crossmatches performed by XM3, two subjects were positive for IgG and all five subjects were negative for IgM. Conclusions In addition to facilitating the detection of conventional analysis of donor T and B cell specific IgG ab, the XM3 helps identify donor T and B cell specific IgM ab in recipient’s serum. In contrast to anti-HLA ab, donor-EPC specific IgG or IgM ab occur in fewer recipients, but their identification may impact allograft survival. The XM3 identifies EPC specific IgG or IgM ab in human serum.

Aim Luminex based solid phase testing has become the assay of choice for detecting and identifying IgG HLA antibodies (Abs) and has become a key component in virtual crossmatching and a front line test for many transplant centers. Based on the strength (MFI values) of the donor specific HLA Abs, a patient is ruled in or out as the renal graft recipient. We address one of the existing challenges in the clinical use of Luminex platform as the screening tool; specifically, when low or negative HLA DSA by Luminex solid phase assay result is associated with a positive CDC XM result. We investigated whether EDTA treatment of sera unmasks the presence of IgG HLA Abs and provides a more accurate view of anti-HLA repertoire. Methods Representative sera were selected for the evaluation of EDTA treatment: Sera previously characterized to be Negative for anti-HLA; Sera previously characterized to be Positive for anti-HLA; Sera previously characterized to be Negative for anti-HLA by Luminex and positive by CDC, Masked sera. Two aliquots of each serum, untreated & treated with EDTA (5 μl EDTA/95 μl serum), were tested for HLA Abs using One Lambda Class I/II Single Antigen Bead assay. The trimmed mean MFI values were compared between untreated & EDTA treated samples. Results The major findings from our study were: 1) EDTA treatment of known Negative sera did not convert negative to positive results 2) EDTA treatment of known Positive sera did not convert positive to negative results; however some of the positive sera gave higher MFIs after treatment 3) EDTA treatment of sera previously found to be Luminex negative and CDC positive resulted in a substantive increase in the MFIs in the sera with low MFIs. Conclusions Our encouraging results advance the idea that EDTA treatment of serum may help resolve the paradox of low MFI with the donor HLA by the Luminex platform and positive CDC and FCXM results.

Aim The “virtual crossmatch” is advanced as a predictor of HLA reactivity/compatibility between the recipient and the potential donor. HLA laboratories and transplant centers rely on this practice. Antibody detection however is limited by the makeup of the alleles in the test panel and the genetic makeup of the donor pool is endless. A laboratory with limited resources, specimen access or time may need to make an immediate “virtual” call about a donor/recipient pairing with low resolution molecular HLA typing paired with allelic level HLA Antibody information. Knowing that low resolution molecular HLA typing kits are geared towards identifying the most common alleles for each locus, we wanted to survey the actual frequency that these low resolution SSO kits identified the high resolution typing of these loci with a “single pass”. Methods We surveyed the last 7 ASHI high resolutions PT challenges. We compared our initial low resolution typing (and most likely implied high resolution typing) with the graded high resolution typing returned for each of these surveys. We then determined the percentage of matched results between the predicted high resolution typing with the actual high resolution typing at each of the following HLA loci: A, B, C, DRB1, DRB3,4,5 and DQB1. Results Percentage matched, Predicted High Resolution Vs. Actual High resolution typing A locus: 96% matched B locus: 96% matched Cw locus: 91% matched DRB1 locus: 81% matched DRB3, 4, 5 locus: 48% matched DQB1 locus: 89% matched. Conclusions Our survey has resolved that HLA typing based on low resolution SSO kits results in 90% matched HLA typing as ascertained by high resolution HLA-A, B and Cw typing; our study also demonstrates that HLA class II typing by low resolution, especially DRB3,4,5 typing, leaves much to be desired. The advantages and disadvantages unmasked by our study may be of value in informing clinical decisions and associated risks and benefits.