Jacob, Eapen K.

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.

Background: Molecular genetic testing is increasingly utilized in clinical care, typically with peripheral blood as the preferred specimen. Despite the prevalence of therapeutic blood transfusions, whether donor DNA is present in a sufficient quantity to interfere with recipient genetic testing has not been systematically studied. Microchimerism secondary to transfusion of blood products with donor leukocytes has been well-documented in trauma patients receiving whole blood; however, most medical centers currently transfuse leukoreduced (LR) red blood cell products for non-trauma patients. The degree of leukoreduction varies among centers, but to meet AABB standards must be <5x106 white blood cells (WBC) in 95% of units tested. We sought to determine what quantity, if any, of infused LR or non-LR (NLR) products leads to detectable microchimerism that may impact genetic test results. Methods: We performed an in vitro spiking study utilizing four whole blood units collected from different anonymous donors. Three were leukoreduced at varying levels in order to establish a LR (per our institutional guidelines and standards), a partially leukoreduced (PLR) and a NLR unit, which were then considered "donors". The 4th unit was divided to generate two separate "recipients". The first half was left untreated simulating an immunocompetent recipient while the other was leukoreduced to mimic a leukopenic recipient. Based on a 70 kg patient, we calculated the volume of blood from each "donor" to mix with aliquots of each "recipient" to represent a transfusion of 2, 5, or 16 units, with 2 units corresponding to a double apheresis red blood cell (RBC) transfusion and 16 units corresponding to a near total volume transfusion. DNA was extracted from each individual unit as well as all "transfused" combinations within 24 hours of mixing. Chimerism analysis was then performed by STR analysis using the GlobalFiler PCR amplification kit followed by ChimerMarker analysis software. Results: None of the LR units, despite volume transfused, revealed any level of microchimerism in either the immunocompetent or leukopenic "recipients". The PLR transfused combinations displayed levels ranging from 0.14% to 1.52% of donor chimerism for the immunocompetent "recipient", which is below the limit of detection for most clinical assays evaluating germline genetic variation, and 6.3% to 27.78% of donor chimerism for the leukopenic "recipient", which would be expected to impact a subset of clinical genetic assays. The NLR transfused combinations displayed levels ranging from 13.28% to 95.66% of donor chimerism. Discussion:In vitro "transfusion" of LR RBCs into simulated immunocompetent or leukopenic samples does not lead to detectable donor DNA. However, "transfusion" of PLR and NLR units in an in vitro model reveals significant levels of microchimerism dependent on volume transfused and immune status of the "recipient" which implies possible risk for impact on clinical genetic tests. The minimum time required for clearance of donor leukocytes in the recipient is unknown; we were unable to fully evaluate this and other variables in our in vitro system, but follow-up in vivo studies addressing this question are planned. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 4332 Background: Hemolytic anemia caused by the passive transfer of isoagglutinins, typically anti-A or anti-B antibodies, is a rare and likely under-recognized complication of intravenous immunoglobulin (IVIG) therapy. The Immunohematology Reference Laboratory at Mayo Clinic Rochester has detected 12 cases of passively acquired isoagglutinins following IVIG administration during the first seven months of this calendar year (January-July 2011). This frequency of detection is much higher than that seen over the three prior calendar years, when only two cases were detected. Given this “cluster” of isoagglutinin detection, we analyzed these 12 cases to determine the clinical significance of this finding as well as characteristics of resulting cases of hemolytic anemia. Materials/Methods: Charts of patients identified as having passively acquired isoagglutinins following IVIG therapy were analyzed for evidence of a significant drop in hemoglobin (defined as >1 g/dL) as well as evidence of hemolysis (elevated lactate dehydrogenase, reticulocytosis, spherocytosis, decreased haptoglobin, indirect hyperbilirubinemia, hemoglobinemia and hemoglobinuria). Cases with a >1 g/dL drop in hemoglobin and two or more of the above findings consistent with hemolysis were considered “confirmed” cases. Those with passively acquired isoagglutinins but without evidence of hemolysis or with an alternative explanation for anemia were considered “asymptomatic”, and cases without a full hemolytic evaluation but still clinically suspicious were categorized as “suspected” cases. Results: Passively acquired isoagglutinins were detected in 11 patients following 12 separate treatments with IVIG (one patient had antibodies noted after two separate treatment episodes >3 weeks apart). Four of these 12 cases were categorized as “confirmed” cases, five as “suspected” and three as “asymptomatic”. The indication for IVIG use was a neurologic disorder in three of the four confirmed cases, with immune thrombocytopenic purpura (ITP) being the indication in the other case. In suspected cases, ITP was the indication in four of the five cases, with the fifth patient carrying a diagnosis of Kawasaki’s disease. During the 7 month period reviewed, 250 unique patients were treated with IVIG, meaning 1.6% of patients (4/250) had confirmed IVIG-associated hemolytic anemia and another 1.6% (4 patients; 5 events) had suspected hemolytic anemia during this period. The mean decrease in hemoglobin was 4.1 g/dL in confirmed cases (range 1.3–7.1 g/dL) and 2.7 g/dL in suspected cases (range 1.7–4.2 g/dL). Eight of nine patients reviewed had blood type A positive; the other was B positive. The median time from initiation of therapy to hemoglobin nadir was eight days in confirmed and three days in suspected cases. The mean dose of IVIG administered in confirmed cases was 300 g (range 180–400 g) and was 162 g (range 50–300 g) in suspected cases. Red cell transfusion was required in three of the four confirmed cases. Privigen™ was the IVIG product utilized in all four confirmed and the first three suspected cases. After removal of Privigen™ from the Mayo Clinic formulary given concerns regarding these cases, two suspected cases of IVIG-associated hemolytic anemia occurred with Gamunex-C™, including one in a patient who previously suffered confirmed hemolysis after receipt of Privigen™. Conclusions: Hemolytic anemia is a rare complication of therapy with IVIG. The rate of hemolytic anemia following IVIG administration over the evaluated period is consistent with previous reports, but it should be noted that these cases were detected without the aid of an active surveillance policy, indicating the actual rate of hemolytic anemia associated with IVIG use was likely greater than that detected by this review. Our series suggests that those with blood type A may be at increased risk for this complication; further work is ongoing to better define risk factors for development of this potentially significant complication of IVIG therapy. Disclosures: Off Label Use: IVIG use in various off-label indications.

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.

Abstract Background: Little data exists on the incidence rate of transfusion reactions and red cell antibody formation following allogeneic (allo) hematopoietic progenitor cell transplant (HPCT) recipients. Study Design and Methods: Consecutive related and unrelated Allogeneic HPCTs from 2006 to 2013 were retrospectively reviewed regardless of graft source. Allo HPCT recipients were analyzed as a whole as well as divided into ABO compatibility groups of identical, minor mismatch, and major/bidirectional mismatch. Transfusions, transfusion reactions and antibodies were analyzed for the first year post-transplant while survival was recorded throughout the available follow-up period. Results: During the study period, 573 patients ranging in age from 0.6 to 72 years underwent first allo HPCT for various malignant and non-malignant etiologies: 278 acute leukemias, 89 lymphomas, 40 multiple myelomas, 134 other hematologic malignancies, 32 non-neoplastic etiologies. Of these, 52% underwent myeloablative conditioning and 48% reduced intensity conditioning. Transfusion reactions were reported in 93 of the 573 allo HPCT recipients. The incidence rate of transfusion reactions per 10,000 transfusion episodes was 45.54 in the ABO identical, 61.28 in the minor mismatch, and 29.95 in the major mismatch groups (p=0.04). The most common reaction type reported in all groups was allergic followed by febrile non-hemolytic, transfusion associated circulatory overload, and delayed hemolytic or serologic reactions. No acute hemolytic, transfusion related acute lung injury (TRALI), post transfusion purpura, transfusion associated graft-versus-host disease, or transfusion associated sepsis reactions were reported. A total of 43 unique patients(7.5%) developed new antibodies post-transplant: 16 allo HPCT recipients developed clinically significant antibodies, 17 developed clinically insignificant antibodies, and 17 developed auto antibodies post-transplant. Including those patients with pre-transplant antibodies the overall prevalence of red blood cell antibodies was 14.5%. See Table 1 for incidence rate for forming new rbc antibodies post-transplant. Table 1. New RBC Antibody Formation Incidence Rate Post-Transplant ABO Compatibility Group Clinically Significant Antibody Incidence /10,000 RBC Transfusions Clinically Insignificant Antibody Incidence/ 10,000 RBC Transfusions, Auto Antibody Incidence/10,000 RBC Transfusions Total Antibody Incidence/10,000 RBC Transfusions Identical 39.33 16.85 39.33 95.51 Minor Mismatch 35.87 57.39 43.04 136.30 Major Mismatch 8.34 16.67 8.34 33.35 A statistically significant difference (p=0.001) was seen in total antibody incidence when comparing the ABO compatibility groups. The most common, clinically significant allo antibodies formed were Anti-E, Anti-Jka, Anti-A1, Anti-K, Anti-D, and Anti-C. Survival of allo HPCT recipients as a whole was not statistically different for those who formed new rbc antibodies post-transplant following adjustment for ABO compatibility, age at transplant, diagnosis, graft source, and pre-transplant rbc infusions (p=0.068). Allo HPCT recipients that formed new rbc antibodies were not shown to have prolonged engraftment. Engraftment, neutrophils (ANC) and platelets (PLT), were not statistically different (p=0.34 ANC; p=0.20 PLT) when comparing all allo HPCT recipients to those who formed a new rbc antibody. Conclusion: Transfusion reaction and rbc antibody incidence rates were higher in the ABO minor mismatch when compared to the ABO identical and ABO major mismatch groups. The reaction etiology (predominantly antibody mediated) and the formation of new antibodies indicates a surprisingly active humoral immune system. The overall prevalence of antibody formation is likely affected by high exposure to transfusions. Development of a new rbc antibody did not affect engraftment or survival outcomes. 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.

BACKGROUNDABO‐incompatible platelet transfusions are common, and transfusions with ABO‐incompatible plasma are increasing with the use of group A plasma and group O whole blood (WB) in emergencies. Many centers screen blood products for anti‐A and/or anti‐B titers to help prevent hemolysis from ABO‐incompatible transfusions, yet titer methods and definition of high titers are not standardized.STUDY DESIGN AND METHODSThis international multicenter study collected data on anti‐A and anti‐B titer practices for plasma, apheresis platelet (AP), and WB units from January 2015 through December 2017 to determine the prevalence of high‐titer units using local definitions.RESULTSA total of 87,701 plasma, AP and WB units were screened for high‐titer anti‐A and/or anti‐B. High‐titer detection rates for group A plasma ranged 0%–13.6%; group A AP 2.7%–9.3%; group O AP 2.3%–65.7%; and group O WB 6.4%–20.7%. At the one center that collected group B AP, the high‐titer rate was 10.9%. High‐titer rates varied from month to month, as well as between years for a given month. There was no clear pattern of when high‐titer units were donated.CONCLUSIONThe prevalence of high‐titer plasma, AP, and WB units varies by titer method and local definition of high titer. Even at the lowest titer threshold of 50, a significant proportion of units had a high‐titer antibody, although the clinical relevance of this finding needs further investigation.

AbstractObjectiveHLA‐DPB1 matching may impact allogeneic hematopoietic stem cell transplantation (ASCT) outcomes; however, this locus is not in linkage disequilibrium with the remainder of the HLA genes. After classifying HLA‐DPB1 mismatches based on T‐cell epitope, avoiding non‐permissive mismatches may impact survival. We tested this hypothesis at a single academic institution.MethodsRetrospective HLA‐DPB1 genotyping was performed on 153 adult patients who underwent ASCT and unrelated donors matched for HLA‐A, B, C, DRB1, and DQB1 loci (10/10). Using the ImMunoGeneTics/HLA T‐cell epitope matching algorithm, mismatch status was classified as permissive or non‐permissive.ResultsOf 153 donor‐recipient pairs, 22 (14.4%) were HLA‐DPB1 matches, 64 (42.8%) permissive mismatches, and 67 (43.8%) non‐permissive mismatches. DPB1 mismatch increased risk of chronic graft‐versus‐host disease (cGVHD; RR 2.89 [1.19‐9.53], P=.016) compared with DPB1‐matched transplants, but there were no differences in overall mortality, risk of relapse, or acute GVHD (aGVHD). Combining matches and permissive mismatches and comparing to non‐permissive mismatches, there was no significant difference in overall survival or relapse; however, patients receiving non‐permissive mismatched transplants experienced greater risk of aGVHD overall and severe aGVHD (RR 1.66 [1.13‐2.44], P=.010 and RR 1.97 [1.10‐3.59], P=.024, respectively).ConclusionIn this single‐center study, HLA‐DPB1 matching influenced outcomes of patients undergoing ASCT for hematologic malignancy.

BackgroundThe use of hematopoietic progenitor cell (HPC) transplant has risen over the past two decades. A variety of adverse events (AEs) of varying severity have been noted during HPC infusions. These AEs have been associated with several factors such as the amount of dimethyl sulfoxide and white blood cells in the HPC product. We performed a single‐institution retrospective analysis to determine the effect of two different HPC infusion techniques, manual push with syringes versus infusion from bags with the aid of gravity, on the occurrence of infusion‐related AEs.Study Design and MethodsInfusions between December 2008 and November 2010 involving peripheral blood HPCs were reviewed. Pertinent clinical and HPC product–related information was recorded. Data were analyzed to determine the incidence of infusion‐related AEs and its association with patient and product‐related variables.ResultsWe found 461 AEs in 645 patients during the study period. A total of 325 (50%) experienced at least one AE. Flushing was the most common type of AE followed by nausea and hypertension. The use of syringe infusion was more commonly associated with AEs (odds ratio, 1.82 [95% confidence interval, 1.32‐2.50]; p = 0.002). Other independent risk factors were cryopreserved products and the amount of polymorphonuclear leukocytes in the product.ConclusionTo our knowledge, this is the first study examining the effect of two different infusion techniques on infusion‐related AEs. Our findings suggest that the use of bags for infusion protected the patients from AEs.