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Background: Currently ethnic minority patients comprise 60% of patients listed for kidney transplantation in the US; however, they receive only 55% of deceased donor renal transplants and 25% of living donor renal transplants. Ethnic disparities in access to kidney transplantation result in increased morbidity and mortality for minority patients with end-stage renal disease. Because these patients remain dialysis dependent for longer durations, they are more prone to the development of HLA antibodies that further delay the possibility of receiving a successful kidney transplant. Study Design: Two to 4 pretransplant and post-transplant plasma exchanges and IV immunoglobulin were used to lower donor-specific antibody levels to less than 1:16 dilution; cell lytic therapy was used additionally in some cases. Match pairing by virtual cross-matching was performed to identify the maximal exchange benefit. Sixty candidates for renal transplantation were placed into 4 paired kidney exchanges and/or underwent antibody reduction therapy. Results: Sixty living donor renal transplants were performed by paired exchange pools and/or antibody reduction therapy in recipients whose original intended donors had ABO or HLA incompatibilities or both (24 desensitization and 36 paired kidney exchanges). Successful transplants were performed in 38 ethnic minorities, of which 33 were African American. Twenty-two recipients were white. Graft and patient survival was 100% at 6 months; graft function (mean serum creatinine 1.4 g/dL) and acute rejection rates (20%) have been comparable to traditional live donor kidney transplantation. Conclusions: Paired kidney donor exchange pools with antibody reduction therapy can allow successful transplant in difficult to match recipients. This approach can address kidney transplant disparities. [ABSTRACT FROM AUTHOR]

Highlights from the article: Antibody testing is a cornerstone of all HLA laboratories. Solid phase antibody assays, such as single antigen (SAB) and phenotype (LSPRA) beads can characterize complex antibodies in a recipient's pre-transplant serum.

Aim RT-PCR is a rapid method to determine HLA antigens and many laboratories utilize this assay for deceased donor typing. However, HLA-DPB1 allele level results are typically reported to UNOS without confirmation by high-resolution typing methods. Our aim was to compare the accuracy of RT-PCR to sequence based testing (SBT) in order to asses the frequency of incorrect reporting. Methods HLA-DPB1 typing on 79 deceased donors previously typed using RT-PCR reagents (Linkage Bioscience) was confirmed using SBT methods. DPB1 typing results obtained by the different methods were compared. All discrepant results were verified by LabType SSO. Results In 99% (78/79) of the cases, RT-PCR produced the same allele pairing results obtained by SBT; in the remaining case, discrepant results were obtained between RT-PCR and SBT. LabType SSO determined that the SBT result was inaccurate. The manufacturer suggested that the erroneous SBT result was most likely due to the lack of exon 3 sequence information for the allele in question. In 43% (34/79) of the cases, RT-PCR detected an ambiguous combination that included multiple common and well-documented (CWD) alleles. In 21 of the ambiguous combinations, the alternative allele was in the same G group as the reported allele; in 13 cases, the alternative allele was in a different G group and three were reported incorrectly. In the remaining non-ambiguous cases, one incorrect tying was chosen based on frequencies of the alleles, however, the CWD allele was later ruled out by SBT (Fig. 1). Conclusions In general, RT-PCR methods are an efficient and accurate way to determine HLA-DPB1 alleles prior to organ allocation. However, in some situations, higher resolution methods such as SBT or SSO may be required to resolve common ambiguities and ensure that accurate typing results are reported. A procedure for revising reported alleles in the UNOS database may need to be adopted to help quantify the true prevalence of HLA-DPB1 alleles in the population. [ABSTRACT FROM AUTHOR]

Aim In order to facilitate rapid turnaround, exchange programs such as the NKR require preliminary flow cytometric crossmatching (FCXM) using frozen cells. However, we’ve found that questionable FCXM results occur more often with frozen cells compared to results obtained with fresh donor lymphocytes. In this study, we investigated the frequency of such discrepant results. We compared 17 donor/recipient pairs who were crossmatched using both frozen and fresh donor cells. Methods Fresh lymphocytes were isolated using EasySep™ (StemCell Technologies). Frozen cells were thawed following a validated protocol (ASHI Laboratory Manual). All cells were treated with pronase. FCXM were performed using a rapid protocol ( Liwski, et al., H.I. 72, 2011) . In all cases, a screening FCXM was performed with frozen cells and a final FCXM was performed with fresh donor cells. For final FCXM, one specimen tested in the initial screening FCXM was included. Donor specific antibodies (DSA) were analyzed using multiple solid phase platforms. Results Six out of the 17 FCXM comparisons were discrepant. Among the discrepant FCXM, 4/6 were T & B cell positive using frozen cells and negative using fresh cells; 1/6 had elevated, but within negative range, MCS using frozen cells and clearly negative MCS (<10) using fresh cells; 1/6 was negative using frozen cells but strong positive with fresh cells (Table 1). Conclusions The problem with unreliable frozen cell crossmatches is that false positive results can lead to unnecessary donor denial and false negative results can bring about last minute exchange failure. When a recipient clearly has no DSA, a positive FCXM using frozen cells is not typically problematic and most often a repeat FCXM using fresh cells is negative. On the other hand, for patients with DSA, the intent of an exploratory FCXM with frozen cells is to rapidly evaluate whether or not DSA strength is at an acceptable level. However, in order to be clinically informative, it is important that FCXM results using frozen cells correlate with fresh cells. [ABSTRACT FROM AUTHOR]

Aim The LABScreen single antigen bead (SAB) assay is routinely used for HLA antibody detection and allows for testing of multiple patient samples. Despite this, standard SAB assay is time intensive, taking 1.5–2 hr to perform, which is not optimal for urgent testing. Utilizing a multicenter study we compared the rapid optimized SAB (ROB) protocol (Liwski et al. ASHI 2014), with a 30 min turn-around time (TAT), to standard SAB LABScreen method. Methods Ten HLA labs (BCM, DCI, Edmonton, Halifax, Georgetown, Winnipeg, METC, PSBC, SWI, UPMC) participated in the study. Five ASHI 2014 AC-1 survey sera (AC 460-464) were tested in each lab with standard SAB LABScreen (SSL) vs the ROB protocol. Class I LS1A04 (lot 8) and class II LS2A01 (lot 10) HLA kits were used. Mean fluorescence intensity (MFI) values were collected. Protocols were compared with respect to the mean MFI, specificities reaching consensus (⩾90% labs; 2000 MFI cutoff) and coefficient of variation (%CV). Results HLA antibody reactivity patterns were similar between the protocols with an excellent MFI correlation (r2>0.99) and signal to noise differential. Average MFI was slightly reduced with the ROB protocol (by 5.0%). The number of specificities reaching consensus (⩾90% labs; 2000 MFI cutoff) was similar between protocols (97 SSL vs 94 ROB). When the ROB protocol cutoff was lowered to 1500 MFI, the number of consensus specificities improved to 99, including all 97 SSL specificities (Fig. 1). Importantly, the average %CV of raw bead MFI was significantly improved with the ROB protocol (ROB: 21.2+/−3.4 vs SSL: 34.4+/−5.6; p< 0.001; Fig. 2). A slight increase in background was noted with the ROB protocol (AC 462 serum) in some labs. However, this had no impact on antibody assignment and improved with one additional wash step. Conclusions ROB protocol reduces SAB LABScreen assay time by 70% and improves precision without compromising sensitivity or specificity. Implementation of the ROB protocol will expedite antibody testing in urgent cases and improve TAT for routine testing. [ABSTRACT FROM AUTHOR]

Aim: Reagent receipt and quality control is very important in each laboratory, our aim was to implement an easy and efficient system that would document the receipt of each reagent and automatically trigger QC testing prior to use. Methods: The following steps were implemented: 1) One key technologist was identified to oversee the process. 2) An electronic Excel-based reagent tracking spreadsheet was developed. The information recorded on the tracking spreadsheet includes: reagent name, lot number, order date, receipt date, date of QC, technologist performing QC, date of QC review, date the reagent was put in-use, and, last date the reagent was in-use. The spreadsheet is simple to complete and a filtering option was added for easy searching. 3) A procedure was written and competency was documented. 4) QC testing to be completed within one week of reagent receipt was assigned by the key technologist to a specific workbench. 5) A simplified reagent QC guideline was developed. 6) QC data were reviewed for acceptability by the key technologist. 7) All QC data were then approved by the supervisor. 8) An audit of the tracking system was performed monthly. Results: In January of 2013, after six months of implementing the system, we performed an audit of thirty random patient charts focusing on the completeness and documentation of reagent QC. The results of this audit revealed that all reagents used were documented and QC testing was complete. This represented a significant improvement over audit findings prior to the implementation of the tracking system. Conclusions: Streamlining reagent receiving and quality control testing is extremely important in every laboratory. Its successful implementation requires everyone’s participation. We have found that our restructured systematic process is not only easily monitored, but also 1)easily adhered to by all personnel, 2)saves time and facilitates inventory control, 3)avoids midnight reagent crises, and (best of all), 4)we are always ready for inspection. [Copyright &y& Elsevier]

Aim: Preliminary clinical data suggest that the C1q assay is a promising noninvasive technique for prediction of antibody mediated rejection (AMR). However, conflicting reports exist suggesting that assay performance and sensitivity vary between laboratories. Our aim was to evaluate a modified protocol for the commercially available C1qScreen™ assay to achieve increased sensitivity. We hypothesized that sensitivity of the assay could be increased by preincubation of serum with LABScreen beads prior to incubation with C1q. The rationale for this modification was that individual heads on a C1q molecule bind to the Fc region with weak affinity. Antibody aggregation on an antigenic surface facilitates binding of several heads significantly enhancing C1q affinity to low-MFI antibody. Methods: The standard and modified C1q assays were performed in parallel. The difference between protocols was that in the standard C1qScreen™ protocol, heat-inactivated serum was spiked with human C1q and incubated with LABScreen beads for 20 min at RT. In the modified protocol, heat-inactivated serum was first incubated with LABScreen beads for 20 minutes followed by a 20 minute incubation with C1q. The rest of the assay was performed as described in the standard protocol. Results: Of 30 samples tested, 17 samples that were positive for class I and 19 samples that were positive for class II C1q-binding antibody by both protocols. Some specificities that were weak or negative for C1q-binding using the standard protocol were strongly positive using the modified protocol. Also, of 13 ClassI and 11 ClassII samples that were negative for C1q-binding antibody by the standard protocol, 5 and 2 samples, respectively, were positive by the modified protocol. Conclusions: Preincubation of serum with LABScreen beads prior to addition of human C1q facilitates increased sensitivity for detection of C1q-binding antibody. Further investigation will reveal whether increase in sensitivity offers better prediction of AMR. [Copyright &y& Elsevier]