DiGuardo, Margaret A

AbstractBackgroundFlow cytometry protocols for counting fresh CD34+ cell samples are not ideal for cryopreserved products due to cryoprotectant cytotoxicity. For cryopreserved samples, often large volumes of hypotonic solutions, which can cause cell death, are used to remove the cryoprotectant with a post‐thaw wash. We recently developed a novel multistep dilution method with subsequent flow cytometry analysis to allow for accurate and reproducible results. The previous method involved washing steps which invalidate the ability to enumerate cell recovery, and success had to be gauged solely on viability. The new method allows for assessment of total cell recovery and viable cell recovery.Study Design and MethodsApheresis products were cryopreserved in 10% DMSO at a target WBC concentration of 300 × 106/mL. Cryovials from these products were thawed at 37°C, and samples were diluted 1:2 by three additions of 1/3 sample volume using 1%‐Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. A 1:10 dilution was performed to obtain the correct cell concentration for flow cytometric analysis resulting in a 1:20 dilution. End WBC concentrations were ~15 × 106/mL with DMSO diluted to 0.5%.ResultsFifty‐two samples were tested with this new method. Total and viable cell recoveries were calculated based on pre‐cryopreservation data. Median total cell recoveries for CD34 and CD3 were >85%, while median viable cell recoveries were >75%.DiscussionCryopreserved samples can be reliably prepared for flow cytometric testing using a step‐wise dilution to preserve cell integrity and robust recoveries.

AbstractBackgroundStandard flow cytometry protocols for CD34+ cell enumeration designed for fresh samples are not appropriate for cryopreserved products. Special protocols have been developed to remove the cryoprotectant by quickly washing a freshly thawed sample. Exposing cells to a large volume of hypotonic solution and subsequent washing process was hypothesized to cause lab‐induced cell death. Moreover, standard gating strategies must be altered to avoid reporting falsely high viabilities.Study Design and MethodsWe developed a novel method whereby thawed samples were diluted step‐wise to 1:2 by 3 additions of 1/3 sample volume using 1% Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. An additional 1:10 dilution was required to obtain a desired cell concentration for flow cytometry testing resulting in a 1:20 dilution.ResultsTwenty samples were tested simultaneously in a method comparison; the new method demonstrated significant increases in mean cell viabilities for white blood cells, hematopoietic progenitor cells, and T cells as well as reduced standard deviations for each parameter.DiscussionSlow, step‐wise dilutions of freshly thawed samples of cryopreserved apheresis products to 1:20 yielded higher and more precise viability measurements compared to quickly washing samples to remove DMSO.

Purpose: molecular testing is often indicated for recently transfused patients. However, there are no guidelines regarding the potential interference from donor DNA or whether it is necessary to wait for a period of time post-transfusion prior to genetic testing. While the majority of patients are transfused in the non-trauma setting using leukoreduced (LR) red blood cell products, the degree of leukoreduction varies among centers and is not universally practiced. Methods: whole blood units collected from anonymous donors were used in an in vitro transfusion model. One unit was split: half being leukoreduced simulating a leukopenic recipient and half left untreated. Donors were simulated by leukoreduced, partially leukoreduced (PLR), or non-leukoreduced units, transfused in 2, 5, or 16 unit equivalents. DNA from the combinations were subjected to short tandem repeat (STR) analysis for chimerism detection. Results: donor DNA was not detectable in any of the LR combinations, but detected in the PLR combinations, ranging from 0.1 to 1.5% donor DNA in the immunocompetent recipient and 6.3–27.8% in the leukopenic recipient. Non-LR donor DNA was also detected (13–95%). Conclusion: donor-derived DNA from leukoreduced blood products is unlikely to interfere with the interpretation of germline genetic testing in immunocompetent recipients but may interfere in immunocompromised recipients.

BackgroundProzone is a known phenomenon affecting immunoassays causing falsely low or negative results when excess target is present in the test system. For assays used to evaluate immune‐mediated platelet (PLT) transfusion refractoriness, prozone‐like phenomenon has been described in solid‐phase human leukocyte antigen (HLA) antibody testing and can be mitigated by diluting samples or pretreating samples with ethylenediaminetetraacetic acid (EDTA) or dithiothreitol. Prozone phenomenon has not yet been described in solid‐phase red blood cell (RBC) adherence PLT crossmatch assays.Case ReportA 40‐year‐old female with myeloid sarcoma and PLT transfusion refractoriness underwent repeated solid‐phase PLT crossmatches; however, crossmatch‐compatible PLTs units did not yield adequate PLT count responses. Class I HLA antibody testing with neat, diluted, and EDTA‐pretreated serum demonstrated significant prozone‐like effect and the presence of numerous high strength HLA antibodies. Based on this HLA antibody profile, HLA antigen–negative PLTs gave an adequate PLT count response. It was noted that the HLA types of her crossmatch‐compatible PLTs were incompatible with her HLA antibody profile (eg, HLA‐A2). With ABO‐identical, HLA‐A2–positive PLT units, a solid‐phase PLT crossmatch was repeated using undiluted and diluted EDTA plasma. Undiluted EDTA plasma demonstrated negative or weakly positive PLT crossmatches while the diluted EDTA plasma demonstrated strongly positive PLT crossmatches.ConclusionThe prozone phenomenon can cause false‐negative results in solid‐phase RBC adherence PLT crossmatch assays, which can be mitigated with sample dilution. In immune‐mediated PLT transfusion–refractory patients with high‐strength HLA antibodies, sample dilution should be considered to correctly identify compatible PLT inventory.

Background Daratumumab (DARA), a human IgG1 K monoclonal antibody targeting CD38, is used to treat refractory multiple myeloma patients. CD38 is expressed on many cell types (RBCs, granulocytes, lymphocytes, etc.), and thus, DARA can interfere with serological tests. Information regarding how DARA affects anti‐granulocyte antibody (AGA) testing and optimal neutralization of DARA will help laboratories perform accurate testing. Methods Screening of AGA was performed by the granulocyte agglutination test (GAT) and the flow cytometric granulocyte immunofluorescence test (Flow‐GIFT). Samples were tested from patients on DARA ( n = 7), non‐transfused blood donors (healthy controls, n = 7) and AGA reactive samples (positive controls, n = 5). Two neutralization experiments, CD38 removal with DTT and DARA epitope blockage with mouse anti‐CD38, were evaluated. Results Positive reactivity of human IgG binding was observed in 5/7 DARA cases when tested by Flow‐GIFT; however, all 7 cases had negative GAT agglutination results. Further studies by Flow‐GIFT revealed DARA concentrations >0·63 μg/ml bound to granulocytes. DARA binding was negated by DTT though a reduced Flow‐GIFT sensitivity was observed in positive control samples due to increased background detection of human IgG. Mouse anti‐CD38 neutralized the detection of human IgG observed in DARA‐treated patient serum without effecting controls. Conclusion We established that DARA can interfere with AGA testing, leading to false positive Flow‐GIFT results without causing GAT agglutination. DTT treatment increased background binding of secondary antibodies causing a decrease in Flow‐GIFT sensitivity. In comparison, blockage of the DARA binding epitope using mouse anti‐CD38 antibody was effective in neutralizing DARA interference while maintaining Flow‐GIFT sensitivity.