Optimized FC testing methods that are comparable in accuracy to standard CAT and tube methods are described. When used with filter plates, this methodology should allow rapid and cost-effective immunohematology testing of both patient and donor samples in an automated workstation format. The same workstation should support automation of other pretransfusion assays that can be analyzed by FC.

As RBC antibody screens are done every Monday, Wednesday, and Friday on this BMT service and as RBC antibody formation is low in these patients, screening for unexpected antibodies might be possible on a more infrequent basis. Also, the rate of HLA alloimmunization in this population receiving filtered blood components is low.

Grifols' Erytra analyzer showed reliable efficacy compared with equivalent FDA-licensed reagents and FDA-cleared instruments.

An improved method for FC immunohematology testing has been described. This assay was comparable in accuracy to standard CAT techniques, but had better sensitivity for detecting weak antibodies and was superior in detecting mixed-field reactions (p < 0.005). The FC method demonstrated excellent reproducibility. The compatibility of this assay with the PCA-96 capillary cytometer with plate-handling capabilities should simplify development of a completely automated platform.

Cell-associated HHV-8 appeared to be effectively removed by leukoreduction filtration. Cell-free HHV-8 was present in 42% of subjects as 1% to 20% of the total virus which was not removed by filtration.

No abstract

Background: TRALI is a potentially fatal complication of transfusion. Although the pathogenesis is not completely understood, and may involve a “multi-hit” mechanism, transfusion of donor plasma containing anti-HLA and/or anti-neutrophil antibodies appears to play an important role. In previous studies, up to 22% of all blood components were found to contain HLA antibodies (Bray et al, Human Immunology, 65, 240–4, 2004). Based on the possible involvement of HLA antibodies in TRALI, some blood services no longer provide plasma from female donors for transfusion, since these donors are more likely to have these antibodies due to pregnancy. An alternative, more specific approach to prevent transfusion of HLA antibody-containing plasma, and possibly reduce the risk of TRALI, is to screen plasma prior transfusion. However, no automated method is yet available with sufficient throughput to screen blood donors for HLA antibodies. Methods: The 3Ti Aegis is a novel open-architecture automated blood typing platform that uses fluorescence cytometry. The Aegis was readily configured for detecting anti-HLA Class I antibodies in donor plasma by modifying the commercially-available FlowPRA Class I Screening Test (One Lambda, Canoga Park, CA) to include a phycoerythrin (PE)-labeled secondary antibody. Positive and negative control samples, as well as patient specimens previously demonstrated to contain HLA antibodies, were evaluated to determine the accuracy of the automated Aegis method. Results: Positive and negative controls were readily distinguished from one another whether testing was performed manually (according to manufacturer’s instructions) and acquired on a BD FACScan cytometer, or performed using the fully-automated method on the 3Ti Aegis. For the Aegis, the mean fluorescence intensities for positive and negative controls were 156.2 and 14.4, respectively. For the manual method, the signal to noise separation was comparable (692.5 and 28.9, respectively). Six additional patient samples, which had previously identified anti-Class I antibodies, also tested positive with the Aegis system. Estimated staining and acquisition times on the Aegis for 96 samples are 2 hours and 1.5 hours, respectively, which is comparable to the times for manual testing Conclusions: The Aegis can perform accurate, high throughput, completely automated screening of plasma donations for HLA Class I antibodies prior to transfusion. Future work will focus on developing combined Class I and II screening beads, as well as beads for detecting common anti-neutrophil antibodies, to produce a comprehensive TRALI antibody detection package for the Aegis platform. The Aegis is the first device to make pretransfusion HLA antibody testing practical, and may reduce the incidence of TRALI and accompanying transfusion fatalities.

Background A critical barrier to progress in allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been a lack in understanding regarding why some transplant recipients of HLA-matched transplant grafts develop severe graft-versus-host disease (GvHD) while other recipients have relapse of their cancer without GvHD. Patients who develop a modest degree of acute and/or chronic GvHD have less relapse and optimal survival after allogeneic BMT. Thus, a mechanistic understanding of regulation of donor T-cell activation after allo-HSCT is needed. Using mouse models, Desmarets et al. have shown that pre-transplant leukoreduced RBC transfusions can cause recipient immunization against minor histocompatibility antigens (miHA) and activation and expansion of recipient T-cells that recognize donor miHA, contributing to rejection of subsequent allo-HSCT (Blood. 2009; 114:2315). Preliminary data from our lab suggest that leukoreduced RBC transfusions given concurrently with allo-HSCT can also increase post-transplant activation and expansion of donor T-cells, an effect which may lead to increased GvHD after transplant. Here, we have conducted a retrospective study of post-transplant RBC transfusions and acute GvHD (aGvHD) in allo-HSCT patients. We hypothesized that increased numbers of transfusions during the 30-day post-transplant period would be correlated with increased severity of aGvHD in these patients. Methods We conducted a retrospective analysis of RBC transfusion records and aGvHD data collected for 181 adult allo-HSCT patients who received their transplants at Emory University Hospital (EUH) between 2004 and 2009. Nine patients were excluded who died < 50 days post-transplant without developing aGvHD, since this was too early to determine aGvHD occurrence. Of the remaining 172 patients studied (median age 48 yrs at time of transplant, range 18-72), 88 (49%) were male and 84 (51%) were female. Patients had received either matched related HSCT (n=69, 40%) or matched unrelated HSCT (n=103, 60%) for treatment of SAA (n=7), BAL (n=2), ALL (n=18), AML (n=69), hemolytic anemia (n=2), CLL (n=6), CML (n=8), HD (n=5), MDS (n=23), myelofibrosis (n=6), MM (n=7) or NHL (n=19). For pts who developed aGvHD, the onset time ranged from 1 to 139 days post-transplant, with a median of 30 days. No aGvHD (grade 0) was diagnosed in 58 pts (34%), while 37 pts (21%) developed grade 1 aGvHD and 77 pts (45%) developed grade 2-4 aGvHD. The number of ABO matched, irradiated RBC units transfused 0 - 30 days post-transplant was tallied for each patient, ranging from 0 (no transfusions, n=13, 7.6% of pts) to 26 units, with an average of 5.6 and median of 4 units. All transfusions during this timeframe were administered at EUH. The median follow up time was 22 months post-transplant (range, 1.1 – 96.1 months). Results Pts were assigned to two groups, those who developed grade 0-1 aGvHD (n=95, 55%) or grade 2-4 aGvHD (n=77, 45%) within 140 days post-transplant. This study did not include analysis of late-onset aGvHD or chronic GvHD past this time point. Patients with grade 2-4 aGvHD had a higher average number of transfusions 0 - 30 days post-transplant compared with patients having grade 0-1 aGvHD (6.5 vs. 4.9 units, p = 0.02). Receiver-operator characteristics (ROC) analysis showed that a cutoff value of > 4 transfusions 0 - 30 days post-transplant had 56% sensitivity and 65% specificity to predict development of grade 2-4 aGvHD. When tested by logistic regression in a multivariate model, this cutoff value had a highly significant correlation with grade 2-4 aGvHD, with an odds ratio of 2.83 and p value = 0.0024. Other covariates including patient age, gender, and type of transplant (related vs. unrelated) were not significantly associated with aGvHD outcome. Conclusion Our retrospective analysis identified a significant positive correlation between the number of post-transplant RBC transfusions and severity of aGvHD after allo-HSCT. Additional studies are planned to determine whether RBC transfusions 0 - 30 days post-transplant stimulate allo-reactive T-cells via allo-antigen presentation or by otherwise promoting inflammation, and if one or both of these mechanisms contribute to increased GvHD. If so, it may be possible to develop strategies for optimization of RBC transfusion practices to reduce the risk of severe aGvHD after allo-HSCT. Disclosures: No relevant conflicts of interest to declare.