Kreuter, Justin D.

AbstractBackgroundSolid‐phase platelet crossmatch (PXM) testing is used to help manage patients with platelet transfusion‐refractoriness. Recently, we published the first report of false‐negative PXM results from prozone effect that was mitigated using sample dilution. This study aimed to describe the prevalence of PXM prozone effect and the levels of class I HLA antibodies (HLA‐Abs) associated with positive PXM results and with false‐negative PXM results from prozone effect.Study Design and MethodsA cross‐sectional study of patients undergoing PXM testing from July 2019 through December 2020 was performed. All PXM tests were run simultaneously using undiluted and 1:4 diluted patient plasma. Prozone effect was defined as a negative PXM result using undiluted patient plasma but a positive PXM result using 1:4 diluted patient plasma.ResultsAmong 59 patients, 830 individual ABO‐compatible PXM results yielded an overall positivity rate of 25.8% (214/830) and a false‐negative rate from prozone effect of 4.7% (10/214). Among the 28 patients with class I HLA‐Ab testing and no other anti‐platelet antibodies, maximum HLA‐Ab mean fluorescence intensity (MFI) was significantly associated with a positive PXM result (p < .0001; AUC approx. 0.9) and categorized into negative (<3700), indeterminate (3700–10300), and positive (>10300) maximum HLA‐Ab MFI zones. Maximum HLA‐Ab MFI, however, was not associated with prozone effect (p = .17; AUC approx. 0.6).DiscussionWhile there is a strong predictive association between class I HLA‐Ab levels and positive PXM results, PXM prozone effect is a common occurrence not associated with class I HLA‐Ab levels, so additional testing with diluted samples should be considered.

Abstract Background The two major types of cold autoimmune hemolytic anemias are cold agglutinin disease (CAD) and paroxysmal cold hemoglobinuria (PCH). In CAD, the cold agglutinin is usually IgM with anti-I specificity. In PCH, the Donath-Landsteiner (DL) antibody is a cold reacting IgG with anti-P specificity. In this study, we describe the clinical and serological characteristics of patients with autoimmune hemolytic anemia positive for both cold agglutinin and DL antibodies. Methods On review of our immunohematology reference laboratory database, we identified patients over a 16-year period (January 2000- March 2016) with the following: i) age ≥18 years, ii) hemoglobin (Hb) &lt;12g/dL, iii) positive direct antiglobulin test (DAT) with hemolysis (increased lactate dehydrogenase/low serum haptoglobin/ elevated indirect bilirubin), and iv) tested for both CAD and DL antibodies. We classified the patients into 3 cohorts. Cohort 1 included patients positive for both cold agglutinin (titer ≥1:64) and DL antibodies. Cohort 2 consisted of patients with DL antibody but no cold agglutinin (titer &lt;1:64), while Cohort 3 was comprised of patients with cold agglutinin but no DL antibody. We evaluated the clinical response based on the GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) criteria (Barcellini W, et al . Blood 2014) defined as i) complete (CR): hemoglobin ≥12 g/dL with normalization of at least one previously abnormal hemolytic marker; ii) partial (PR): hemoglobin 10-12 g/dL with hemolysis; or iii) none (NR): if any of the above criteria were not met. Results Seven patients had cold autoimmune hemolytic anemia with concomitant cold agglutinin and DL antibodies (Cohort 1). The clinical and serological characteristics are described in table 1. The median age at diagnosis was 68 years (range: 59-78) and the median hemoglobin at onset was 10.3 g/dL (range: 8.2-10.6). Six had red cell agglutination in the blood smear. Two patients had a recent history of infection (1 with Mycoplasma pneumoniae and 1 with upper respiratory tract infection). The median follow-up was 4.8 months (range: 1.0-166.4). Five patients received steroid/other immunosuppressants and two were managed conservatively. The clinical response to treatment was CR in 28.6% patients, PR in 42.9%, and NR in 14.3%. In comparison, Cohort 2 had a 20% CR, 20% PR and 40% NR, while Cohort 3 had a 50% CR, 25% PR and 25% NR, respectively. Conclusion Our study is the first series describing patients with cold autoimmune hemolytic anemia with concomitant cold agglutinin and DL antibodies. In terms of clinical response, patients with negative DL antibodies and cold agglutinin titers &gt;1:64 had better response (50% CR, Cohort 3) to immunosuppressants compared to patients with positive DL antibody (20-30% CR, Cohorts 1 and 2). Large scale studies may be warranted to determine the treatment strategies among patients with concomitant DL and high cold agglutinin titers. Disclosures Kay: Agios: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Tolero Corporation: Research Funding; Gilead: Research Funding. Winters: Fresenius Kabi USA: Consultancy; Eliaz Therapeutics Inc: Membership on an entity's Board of Directors or advisory committees; Sanofi Inc: Other: Moderated opinion leader's forum; Wiley Blackwell: Employment; Regional Health Inc: Consultancy; Mayo Clinic: Employment; Grifols International SA: Consultancy.

AbstractBackgroundPlatelet (PLT) transfusion refractoriness increases bleeding complications, hospital stays, and PLT inventory usage. Immune‐mediated refractoriness can be evaluated for using a physical PLT crossmatch with ABO‐compatible inventory and, if positive, managed with HLA‐compatible PLT inventory and donors. Manual completion of these complex tasks can be time‐consuming and potentially error‐prone. This study was conducted to determine if a Web‐based software application could improve process efficiency and accuracy.Study Design and MethodsWorkflow analysis was performed to identify process, data, and analytic requirements for a software application for three PLT transfusion‐refractoriness associated tasks: (a) physical PLT crossmatch inventory selection, (b) HLA‐compatible inventory selection, and (c) HLA‐compatible donor selection. After software application development, a comparison study was performed over 10 consecutive days, with each task performed manually and with the software application (Platelet Virtual Crossmatch [PLT VXM]) for a different unique immune‐mediated PLT transfusion–refractory recipient. Task completion time, number of incompatible units/donors presented, and number of documentation errors were compared.ResultsPLT VXM is a Web‐based software application developed using R and the Shiny Web application framework. PLT VXM significantly reduced median task completion times by 4.5 (49%), 11.2 (79%), and 59.1 minutes (94%), respectively. PLT VXM did not present any incompatible PLT units or donors for user consideration. PLT VXM also had a lower number of documentation errors than the manual process, and none of these documentation errors were software generated.ConclusionComputer‐aided evaluation and management of immune‐mediated PLT transfusion–refractory recipients can significantly improve workflow and reduce manual errors in this complex process.

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.

Abstract Background: Platelet transfusions are important for supporting patients with hematologic disorders. Refractoriness to platelet transfusions may lead to serious complications and pose a challenge to clinical management. A variety of laboratory strategies to optimize transfusion support in this population are being used; however, data on clinical correlation and success of these interventions is limited. We report the laboratory testing, transfusion yield, and clinical correlation in a cohort of patients receiving cross-matched platelet units. Methods: All consecutive adult patients refractory to platelet transfusions (failure to increment platelet count by 10 x 10(9) times two) with platelet crossmatch performed between June 2014 and April 2016 at our institution were included. Detailed clinical information, laboratory data of the crossmatch results, and Single Antigen Bead (SAB; Luminex) for each platelet unit cross-matched was retrospectively collected. Mean fluorescence intensity (MFI) of antibodies directed towards donor platelet HLA antigens identified through SAB were grouped into zones according to previously validated laboratory data: green zone (<4,000 MFI), yellow zone (4000-10000) and red zone (>10,000). Adequate transfusion yield was defined as an absolute increase of at least 10 x 10((9)/L platelets on a post-transfusion (i.e., within 1 hour) sample. Patients with major bleeding per International Society on Thrombosis and Haemostasis (ISTH) criteria, palpable splenomegaly, fever during transfusion, DIC, active sepsis, or antibodies directed against human platelet antigens were excluded from the final analysis. Importantly, the Hematologist and Pathologist were blinded to the other's data until collection was completed. Data analysis correlating adequate transfusion yield and laboratory parameters was performed. Results: 22 patients (72% female) met our study criteria. All patients had a hematologic malignancy with prolonged pancytopenia after chemotherapy, 4 (18%) had undergone allogeneic bone marrow transplantation. A total of 422 platelet cross-matches were performed, with 17 patients ultimately receiving 46 cross-matched units. The indication for transfusion was: prophylactic 35 (76%), bleeding 4 (9%) and peri-procedural 7 (15%). Ten (22%) transfusion events were excluded: splenomegaly 3, fever 4 and active severe bleeding 2. In one case a post transfusion platelet count was not performed. The overall success rate of transfusion was 64% (23/36). All transfusions leading to an adequate yield had a negative crossmatch. Of the 13 ineffective transfusions, 2 had a positive crossmatch. Based on single antigen MFI assignment, 26 (74%) donor units were classified as green zone, 8 (23%) yellow and 1 (3%) red. The success rates according to the single MFI zone assignment are presented in Table 1. These MFI-defined assignments were predictive of clinically successful transfusions. Conclusion: Selection of appropriate donor platelet units in refractory patients is a challenge that is managed in a variety of ways. While crossmatching is sometimes necessary, a strategy of antigen-avoidance can dramatically increase the identification of potentially effective platelet units for many patients. Understanding of how to navigate HLA antibody data is essential for identifying which HLA antigens should be avoided. Based on these data, in general, our practice is to avoid transfusing a platelet when there is a donor specific antibody of 8,000 MFI or greater. Furthermore, in our dataset there were two patients with both successful and unsuccessful transfusions after receiving crossmatch-negative units with green zone assignment. This finding highlights the limitations of laboratory testing and emphasizes the importance of post-transfusion platelet counts for continuously improving the platelet support of each immune-refractory patient. Disclosures No relevant conflicts of interest to declare.

Abstract BACKGROUND: Non-hereditary hemolytic anemia of unknown etiology after an exhaustive work-up is commonly known as direct antiglobulin test (DAT)-negative hemolytic anemia. Due to its rarity, limited data are available regarding this condition. We performed this retrospective study to determine the frequency, associated medical conditions, treatments used, and clinical outcome of patients with DAT- negative hemolytic anemia. METHODS: We included adult (age >18 years) patients with DAT-negative hemolytic anemia seen at Mayo Clinic from 1997-2015 who met all of the following criteria: a) hemoglobin (Hb)< 12 g/dL; b) presence of hemolysis (reticulocytosis, elevated lactate dehydrogenase, decreased haptoglobin, and/or elevated indirect bilirubin); c) DAT-negative; and d) unknown etiology of hemolytic anemia after extensive work-up appropriate for clinical context. Remission status after appropriate therapy for perceived hemolysis was defined as follows: a) complete remission (CR; Hb ≥12g/dL and normalization of at least one previously abnormal hemolytic marker); b) partial remission (PR; Hb: 10-11.9 g/dL with evidence of ongoing hemolysis); c) no remission (NR; neither CR nor PR). In order to calculate the relative frequency of DAT-negative hemolytic anemia, we also determined the number of DAT-positive hemolytic anemia cases during the same time period using the above criteria for hemolytic anemia. RESULTS: During the 19-year study period, 24 and 484 patients were diagnosed with DAT- negative and DAT-positive hemolytic anemia, respectively, a ratio of 1:20. Among those with DAT-negative hemolytic anemia, the majority were males (67%) and the median age at diagnosis was 70 years (range, 22-87). Six patients (25%) had pre-existing autoimmune conditions including immune thrombocytopenia (n=3), Crohn's disease (n=2), and Grave's disease (n=1). All patients had negative DAT by conventional method with one patient turning positive at 104 days after initial diagnosis. Enhanced DAT technique using 4°C low-ionic strength saline wash was performed in one case and was negative. In general, peripheral blood smear showed polychromasia, occasional spherocytes, and no schistocytes or bite cells. Additional work-ups performed based on clinical context and proportion of patients tested (all results negative or normal) were as follows: hemoglobin electrophoresis (54%), flow cytometry for paroxysmal nocturnal hemoglobinuria (92%), glucose 6-phosphate dehydrogenase level (67%), osmotic fragility test/eosin-5'-maleimide stain for hereditary spherocytosis (54%), other red blood cell enzymes (46%), computed tomographic scan of the body to look for occult malignancy (46%), and bone marrow biopsy (50%). The median hemoglobin at diagnosis was 8.5 g/dL (range, 4.5-11.8). Fourteen patients (58.3%) required blood transfusion during the course of their disease. Sixteen patients (67%) received prednisone as initial therapy with 19% CR and 38% PR response rates. Subsequent therapies included: rituximab (n=6; 40% PR), cyclophosphamide (n=2; 100% NR), mycophenolate mofetil (n=1; 100% NR), intravenous immunoglobulin (n=2; 100% NR), and splenectomy (n=6; 50% CR and 33.3% PR), respectively. The median follow-up for all cases was 32.9 months (range, 2-433). Six patients (25%) developed hematologic malignancies after a median follow-up of 8.7 months (range, 0.7-29.3), which included primary myelofibrosis (n=2), myelodysplastic syndrome (n=1), mantle cell lymphoma (n=1), non-Hodgkin lymphoma-not otherwise specified (n=1), and T-cell large granular lymphocyte leukemia (n=1). At last follow-up, 9 patients (38%) were in CR and 8 (33%) in PR. Relapses occurred in 67% and 0% of patients who received prednisone and splenectomy, respectively. CONCLUSION: At our institution, DAT-negative hemolytic anemia was approximately 20 times less common than DAT-positive hemolytic anemia. Most patients were treated with either immunosuppressive agents with or without splenectomy. Splenectomy was the most effective treatment. At least a quarter of the patients were eventually diagnosed with hematologic malignancies mostly within a year of DAT-negative hemolytic anemia diagnosis. Routine upfront evaluation for hematologic malignancies might be necessary for these patients. Disclosures No relevant conflicts of interest to declare.

BACKGROUNDRecipients of hematopoietic stem cell transplantation (HSCT) are among the highest consumers of allogeneic red blood cell (RBC) and platelet (PLT) components. The impact of patient blood management (PBM) efforts on HSCT recipients is poorly understood.STUDY DESIGN AND METHODSThis observational study assessed changes in blood product use and patient‐centered outcomes before and after implementing a multidisciplinary PBM program for patients undergoing HSCT at a large academic medical center. The pre‐PBM cohort was treated from January 1 through September 31, 2013; the post‐PBM cohort was treated from January 1 through September 31, 2015.RESULTSWe identified 708 patients; 284 of 352 (80.7%) in the pre‐PBM group and 225 of 356 (63.2%) in the post‐PBM group received allogeneic RBCs (p < 0.001). Median (interquartile range [IQR]) RBC volumes were higher before PBM than after PBM (3 [2‐4] units vs. 2 [1‐4] units; p = 0.004). A total of 259 of 284 pre‐PBM patients (91.2%) and 57 of 225 (25.3%) post‐PBM patients received RBC transfusions when hemoglobin levels were more than 7 g/dL (p < 0.001). The median (IQR) PLT transfusion quantities was 3 (2‐5) units for pre‐PBM patients and 2 (1‐4) units for post‐PBM patients (p < 0.001). For patients with PLT counts of more than 10 × 109/L, a total of 1219 PLT units (73.4%) were transfused before PBM and 691 units (48.8%) were transfused after PBM (p < 0.001). Estimated transfusion expenditures were reduced by $617,152 (18.3%). We noted no differences in clinical outcomes or transfusion‐related adverse events.CONCLUSIONPatient blood management implementation for HSCT recipients was associated with marked reductions in allogeneic RBC and PLT transfusions and decreased transfusion‐related costs with no detrimental impact on clinical outcomes.

Discovery of a novel HLA‐B*45:22 allele in a Middle Eastern heart and lung transplant candidate.