Chen, Dong

Introduction: The 2013 ASH Choosing Wisely campaign recommends against thrombophilia testing in patients with major transient risk factors for venous thromboembolism (VTE). Our Special Coagulation Laboratory (SCL) offers an algorithmic approach to thrombophilia testing which includes assays for lupus anticoagulant, dysfibrinogenemia, anticoagulant proteins (protein C, protein S, antithrombin), activated protein C resistance with reflex to factor v Leiden (if indicated), and prothrombin G20210A mutation. Samples are received through Mayo Clinic Laboratories (MCL), national and international reference laboratory (often with limited or no clinical information) and from internal Mayo Clinic practice. We hypothesized that thrombophilia testing would decline in cases where it was recommended against following the publication of testing guidelines. Methods: We audited the external thrombophilia testing samples between2013-2019 and internal samples between 2014-2019 (periods during which they were available). For the internal samples, complete test volumes were only available 2014-2019. Because external clients may either adopt internal testing or contract with a different reference laboratory, many clients may not have been retained over the entire observed period. To better understand the ordering practices of consistent clients, external clients which did not have thrombophilia testing sent to MCL each year of the observed period were excluded. We separated internal ordering practices by hematology and oncology or thrombophilia clinic staff and trainees contrasted with those of other specialties. Results: MCL received 18,529 external thrombophilia testing samples from 322 external healthcare systems during the observed period. From 37 clients, 5,878 (38.2%) samples met inclusion criteria. Annual volume of samples ranged from 890 in 2013 to 861 in 2019 (861-1046). Special coagulation lab processed 11,639 internal thrombophilia tests during the observed periods. There was a consistent small annual increase in testing with 1,398 performed in 2014 and 2,430 in 2019. Of 11651 tests ordered, only 18.6% (2167) were ordered by people most likely to be familiar with ASH choosing wisely campaign. Annual thrombophilia testing ordered by hematology and oncology or thrombophilia clinic staff increased from 307 in 2014 to 387 in 2019 (307-432). However, the ordering practices of these providers as a proportion of overall practices declined from 22.0% (307/1398) in 2014 to 15.9 % (387/2430) in 2019. Table Discussion: Our preliminary data showed no significant trend in thrombophilia ordering practices among included external clients since publication of the ASH Choosing Wisely guidelines on thrombophilia testing. Internally we found a consistent small rise in numbers of thrombophilia tests ordered since 2014 but this may reflect changes in patient volume. We observed that the majority of internal thrombophilia testing was ordered by non-hematology/oncology or thrombophilia providers who are perhaps less likely to be familiar with ASH Choosing Wisely guidelines. The proportion of testing ordered by hematology & oncology or thrombophilia providers declined during the observed period. Our findings are limited by lacking information on the indication and appropriateness for testing as well as possibility of change in patient population. However the overall trend in test volumes and specialty of ordering providers deserves attention and highlight the value in educating other medical societies on ASH Choosing Wisely recommendations for thrombophilia testing. Future work will focus on appropriateness for thrombophilia testing including the indication and time (remote from anticoagulation and acute thrombotic episode), location of testing (inpatient vs. outpatient), as well as investigating if testing has changed patient management, which may help in creating new Choosing Wisely recommendations for thrombophilia testing. Figure 1. Disclosures Pruthi: HEMA Biologics: Honoraria; Bayer Healthcare: Honoraria; Merck: Honoraria; Instrumentation Laboratory: Honoraria; Genentech Inc.: Honoraria; CSL Behring: Honoraria.

Introduction: In patients with SARS-CoV-2 COVID-19 infection elevated D-dimers are associated with a poor prognosis. Although suggestive of DIC, the majority of patients do not fulfil ISTH criteria for DIC. Studies have demonstrated that when compared to D-dimer, fibrin monomers are more sensitive and specific when differentiating overt DIC from non-overt DIC. Our special coagulation laboratory (SCL) routinely measures soluble fibrin monomer complex (SFMC) as part of our DIC profile in samples with elevated D-dimers. Here we aimed to investigate the percentage of patients with COVID-19 infection with elevated SFMC as performed as part of the DIC profile. Methods: Between April 1st 2020 and July 26, 2020 inpatients at the Mayo Clinic Rochester, MN campus with COVID-19 infection who underwent DIC testing in SCL were identified through SCL database. Patients were excluded if SARS-CoV2 PCR results were not available in the Mayo Clinic electronic medical record (EMR). DIC profile components include prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen and D-dimer. Reflex SFMC is performed if D-dimer is elevated. Data abstracted from EMR included results of laboratory tests, date of COVID-19 symptom onset, date of SARS-CoV2 PCR positivity, and death within 30 days of hospital admission. The ISTH DIC score was calculated and patients were labeled as overt DIC or non-overt DIC based off of ISTH DIC score ≥5. Results: 35 patients (19 female), median age 59 years (28-91) met our study criteria DIC profiles were obtained 6.5 (1-37) days after COVID-19 symptom onset (n=33) with 28 obtained within 14 days of symptom onset. Time to DIC profile from SARS-CoV2 PCR testing was 4 (0-37) days (n=35) with 31 having testing completed within 14 days of PCR positivity. 8 patients were intubated during hospitalization and of the remaining 27, 67% required supplemental oxygen. 21/35 (60%) patients had an elevated fibrinogen (526.5 mg/dL (201-800)) while 19/35 (54%) had a prolonged PT (12.8 seconds (10.1-99.6) and 5/35 (14%) had a prolonged APTT (31 seconds (24-68)). 17 patients with prolonged PT and 3 patients with prolonged APTT had factor activity assays performed (Figure 1A). 9/35 (25%) had thrombocytopenia (230 x 109/L (12-709) and 27/35 (77%) patients had an elevated D-dimer (895 ng/ml FEU (220- >100,000). D-dimer was higher in males than in females (p: 0.049). Of those with elevated D-dimer only 5/27 (18.5%) had an elevated SFMC (5 mcg/ml (5- >1100) (Figure 1B). 4/35 (11%) patients had ISTH DIC scores ≥ 5 and all had score of 5. All of these patients had D-dimer > 2000 ng/ml while only 2 had elevated SFMC however SFMC in both of these patients was < 15 mcg/ml. Of these, one patient with SFMC of 12 mcg/ml had known cirrhosis and the other with an SFMC of 11 mcg/ml had acute decompensation and associated shock liver. This patient also had venous thrombosis 3 days prior to DIC profile and died from complications related to COVID-19 infection. Of the two other patients with high ISTH score and normal SFMC, one had previous chronic thrombocytopenia of < 50 x109/L while the other had concomitant admission for acute and recurrent pancreatitis in the setting of alcoholism with acute intoxication. Six patients died within 30 days of admission and in 2/5 performed SFMC was elevated (Figure1C). Conclusion: Compared to 77% of patients with elevated D-dimers, of those tested only 18.5 % of patients had elevated SFMC. It has been hypothesized that the elevated D-dimer noted in COVID-19 pulmonary infection is a direct consequence of acute lung injury and not overt DIC. Although preliminary, the small percentage of patients with overt DIC by ISTH criteria and normal SFMC in the majority of the current cohort support this hypothesis; however studies in a larger more controlled cohort are needed to confirm these findings. Disclosures Sridharan: Alexion: Honoraria. Pruthi:Merck: Honoraria; Instrumentation Laboratory: Honoraria; HEMA Biologics: Honoraria; Bayer Healthcare: Honoraria; Genentech Inc.: Honoraria; CSL Behring: Honoraria.

Introduction Germline predisposition syndromes (GPS) are inherited disorders associated with germinal aberrations that increase the risk of malignancies. While aberrations in certain genes increase the risk for all types of malignancies (Tp53, ATM, CDKN2A, CHEK2), there is a growing list of genes associated specifically with hematological malignancies (GATA2, RUNX1, DDX41, ETV6, ANKRD26). At our institution, we have established a hematology GPS clinic to diagnose and manage GPS and with this report, detail our experience with 130 patients. Methods GPS were investigated in pediatric and adult patients with one or more first degree relatives with hematological/visceral malignancies or in those with antecedent thrombocytopenia (ANKRD26, RUNX1, ETV6), or with specific syndromic features (short telomere syndromes/STS, GATA2 haploinsufficiency, Fanconi anemia/FA, Shwachman-Diamond syndrome/SDS). Depending on the phenotype, specific functional assays such as flow-FISH for telomere length assessment and chromosomal breakage assays were ordered. After informed consent and genetic counselling, germline testing was carried out on peripheral blood mononuclear cell, skin fibroblast, or hair follicle-derived DNA. A custom-designed marrow failure NGS panel (200 genes) was used in most cases and interrogation of variants, in silico studies, and functional assays were carried out as previously described (Mangaonkar et al MC Proc 2019). Copy number variations were identified by aCGH. At the time of progression/worsening cytopenias, bone marrow/lymph node biopsies and NGS (next generation sequencing) were carried out where indicated. Results 130 patients with germline predisposition have been identified to date. The spectrum of disorders seen include STS 29 (22%), FA 17 (13%), GATA2 16 (12%), Diamond Blackfan anemia/DBA 13 (10%), RUNX1-FPD 12 (9%), ATM deletions/mutations 11 (8%), ANKRD26 6 (5%), SDS 5 (4%), DDX41 4 (3%), MPL 3 (2%), CHEK2, MECOM, Tp53 mutations 2 (2%) each, and CBL, CEPBA, ELANE, NF1, CDKN2A, CSF3R, ETV6, and GATA1 mutations, 1 (1%) each. Evidence for clonal evolution (CCUS) and hematological malignancies were seen in 51 (39%) patients, involving all the aforementioned genes/syndromes with the exception of DBA, CBL, ETV6, MPL, CSF3R, and GATA1. Seven (64%) of 11 patients with germline ATM deletions/mutations developed lymphoid malignancies; homozygous ATM (Follicular NHL-1, Burkitt lymphoma-1, T-ALL-1, T-LPD-1) and heterozygous ATM (T-PLL-1, DLBCL-1, CLL-1). Clonal evolution occurred in 11 (69%) of 16 GATA2 haploinsufficient patients (CCUS-2, MDS-3, CMML-1, AML-5) and in 7 (58%) of 12 RUNX1-FPD patients (CCUS-1, MDS-1, MDS/MPN-3, AML-2). Five of 29 (17%) STS patients had clonal progression (CCUS-2, MDS-2, AML-1), and 5 (29%) of 17 FA patients progressed to MDS-2 or AML-3. JMML was seen in one patient with a germline NF1 mutation, while 1 (20%) of 5 SDS patients progressed to AML. NGS data at progression was available in 24 (55%) of 44 myeloid/CCUS progressions, with somatic truncating ASXL1 mutations being most frequent (29%), followed by RAS pathway mutations (15%). AML/MDS progressions in STS, FA, and SDS were universally associated with complex/monosomal karyotypes, translating to refractory disease. Seventeen (39%) of 44 patients with myeloid predisposition underwent allogenic HCT (GATA2-7, FA-3, RUNX1-FPD-3, STS-2, NF1-1, Tp53-1), with 10 (59%) being alive at last follow up (Table 1). Conclusion We demonstrate the spectrum of germline aberrations associated with predisposition to hematological malignancies and outline the phenotypic heterogeneity of clonal transformation. The advent of NGS allows identification of clonal progression earlier than morphological changes, with mutations in ASXL1 and RAS pathway genes being commonly implicated. This study supports the universal development of dedicated germline predisposition clinics. Disclosures Pruthi: CSL Behring: Honoraria; Genentech Inc.: Honoraria; Bayer Healthcare: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria.

BackgroundPlatelet (PLT) transfusions are an important component of hemostatic resuscitation. The AABB has published several guidelines recommending that PLT units should not be infused through blood warming devices.Study Design and MethodsThirty‐one units of hospital blood bank apheresis PLTs were obtained. PLT‐rich plasma (PRP) aggregometry and thromboelastography (TEG) were performed on the unit samples before and after the units were infused through a Ranger blood/fluid warming device.ResultsThere were no differences in any of the aggregometry results before and after infusion of the PLTs through the blood warmer (all P > .32). There was a significant reduction in the TEG maximum amplitude (MA) of 69.8 ± 7.9 mm before and 66.0 ± 8.8 mm after (P < .001) infusion of the PLTs through the blood warmer and α angle 61.8 ± 9.4° before and 59.3 ± 8.2° after (P = .044) infusion of the PLTs through the blood warmer, although both mean values were within normal range for the TEG and not clinically significant. There were very good correlations of aggregometry and TEG results before and after infusion of the PLTs through the blood warmer device.ConclusionThis study did not demonstrate significant deleterious effect on PLT function from infusing apheresis PLT units through a blood warming device by PRP aggregometry. We did detect a statistically significant—but not clinically significant—reduction in TEG MA and α angle. The prohibition of transfusing PLT units though the Ranger blood warming device is not indicated.

Abstract Heparin-induced thrombocytopenia (HIT) is a life-threatening, prothrombotic, antibody-mediated disorder. To maximize the likelihood of recovery, early and accurate diagnosis is critical. Widely available HIT assays, such as the platelet factor 4 (PF4) heparin enzyme-linked immunosorbent assay (ELISA) lack specificity, and the gold-standard carbon 14–labeled serotonin release assay (SRA) is of limited value for early patient management because it is available only through reference laboratories. Recent studies have demonstrated that pathogenic HIT antibodies selectively activate PF4-treated platelets and that a technically simpler assay, the PF4-dependent P-selectin expression assay (PEA), may provide an option for rapid and conclusive results. Based upon predefined criteria that combined 4Ts scores and HIT ELISA results, 409 consecutive adults suspected of having HIT were classified as disease positive, negative, or indeterminate. Patients deemed HIT indeterminate were considered disease negative in the primary analysis and disease positive in a sensitivity analysis. The ability of PEA and SRA to identify patients judged to have HIT was compared using receiver operating characteristic curve statistics. Using these predefined criteria, the diagnostic accuracy of PEA was high (area under the curve [AUC], 0.94; 95% confidence interval [CI], 0.87-1.0) and similar to that of SRA (AUC, 0.91; 95% CI, 0.82-1.0). In sensitivity analysis, the AUCs of PEA and SRA were also similar at 0.88 (95% CI, 0.78-0.98) and 0.86 (95% CI, 0.77-0.96), respectively. The PEA, a technically simple nonradioactive assay that uses ∼20-fold fewer platelets compared with the SRA, had high accuracy for diagnosing HIT. Widespread use of the PEA may facilitate timely and more effective management of patients with suspected HIT.

AbstractTransplant‐associated thrombotic microangiopathy (TA‐TMA) has a wide range of presentations after hematopoietic stem‐cell transplantation (HSCT). We retrospectively studied the risk factors and outcomes of patients with early (≤day 100) and late (>day 100) TA‐TMA. Among the 1451 HSCT recipients, early TA‐TMA occurred in 45 (3.1%) patients at a median of 27 (3‐91) days, and late TA‐TMA in 39 (2.7%) patients at a median of 303 (122‐2595) days. Patients with early TA‐TMA were more likely to have high blood calcineurin‐inhibitor levels (P < .001) and acute graph‐vs‐host disease (GVHD, P < .001), while late TMA patients were more likely to have chronic GVHD (P < .001). The estimated median overall survival after onset of TMA for the entire cohort was 6 months. The estimated median overall survival was not reached in patients with an improvement of TMA vs 2 months in patients with no improvement (P < .001). In the early TMA group, older age (for every 10 years, HR 1.40; 95% CI 1.00‐1.94; P = .049) and bacterial infection (HR 2.42; 95% CI 0.98‐6.00; P = .056) were positively associated with mortality. Switching to MMF treatment (HR 0.40; 95% CI 0.16‐0.99; P = .047) and improvement of TMA (HR 0.08; 95% CI 0.03‐0.25; P < .001) were negatively associated with mortality in the multivariate analysis. In the late TMA group, the improvement of TMA was the only independent predictor associated with a lower risk of death (HR 0.05; 95% CI 0.02‐0.19; P < .001). Mortality rates in both early and late TMA remain unacceptably high. Future studies are needed for early diagnosis, trigger identifications, and use of targeted treatments.

Abstract Objectives Despite more than 40 years of experience performing the Bethesda assay (BA), poor intra- and interlaboratory precision remains the biggest laboratory challenge to date. Methods The BA procedure was modeled using stochastic simulation techniques to determine the precision of the BA up to dilutions of 1:4,096, to estimate the minimum significant relative change at various inhibitor titers, and to understand the laboratory procedural variables that could significantly affect the performance of the BA at high dilutions. Results Selecting the lowest dilution tube with a residual activity closest to 25% for calculating the reported Bethesda titer (BT), using a factor activity assay with a coefficient of variation less than or equal to 7.5% in the range of 15% to 50% factor activity level, performing the factor activity measurement in replicates, and minimizing pipette volumetric error resulted in the lowest imprecision in the reported BT. The factor neutralization kinetics of the inhibitor appear to have little impact on the precision of the assay if the incubation time is greater than 90 minutes. Conclusions This in silico model will assist future laboratory efforts in standardizing the quantification of specific coagulation factor inhibitors and improving the precision of the reported results.

Abstract Deletion 5q in Acute Myeloid Leukemia (AML): a Clinicopathological Description of 79 Cases From a Single Institution Rafael Santana-Davila, Shernan G. Holtan, Rhett P. Ketterling, Ryan A. Knudson, David P. Steensma, Dong Chen, James D. Hoyer, Curtis A. Hanson, Ayalew Tefferi. Background: Deletion 5q (del(5q)) is a lenalidomide-sensitive cytogenetic abnormality commonly seen in patients with myeloid disorders. In this study we analyze the clinicopathological characteristics of patients with AML and del(5q). Methods: Patients with del(5q) and AML were identified through a database query of our institutional cytogenetic database from May, 1992 through December, 2005. Both peripheral blood and bone marrow specimens from each patient were morphologically examined by a hematopathologist (DC, JDH and/or CAH). A morphologic diagnosis was given based on current WHO criteria. A critical review of the patient history was performed for each patient. Results: A total 79 patients were identified. Median age was 71 (range 39–87) years, 56% (n=43)were female. The most common subtype according to the FAB classification was M2 (n=23, 29%). In 35 (44%) patients AML developed a median of 9.4 (range 1–294) months since the diagnosis of a pre-existing myeloid malignancy, of which myelodysplastic syndrome accounted for 24 (9 cases of MDS NOS, 6 of RA, 6 of RAEB1, 2 of RAEB-2, and a single RARS), a myeloproliferative neoplasm in 10 (4 cases of a CMPD NOS, 4 cases of PV and a single case of ET and PMF each) and 1 case of a prior history of AML with a t(8;21). The del(5q) was documented in 7 of this 24 patients in the antecedent hematologic disorder. Neither a history of prior exposure to cytotoxic agents nor a prior myeloid disorder was found in 44% (n=35) of the patients. Del(5q) was commonly associated with a complex karyotype (n=67), however in 9 patients it was found to be the sole abnormality. The most frequent breakpoint was q13q33 (n=47) followed by q15q33 (n=12). Initial pathological description did not identify any morphological abnormalities in 6 cases, in contrast myelodysplasia involving at least two cell lines was appreciated in 58% (n=46) of the patients. The median survival was 2.8 months. The majority of the subjects (n=51, 65%) received palliative treatment, 7 patients were lost to follow up and no treatment information was available for review and the remaining 21 received induction chemotherapy with a curative intent, in this group median survival was 8.8 months with 52% (n=11) of patients achieving a complete response. Surprisingly the only factor that appeared to alter survival in the whole group was the presence of the breakpoint in chromosome 5 being distal to q15 (median 2.8 vs 4.1 months, p=0.025) Conclusions: This study constitutes the largest review of del(5q) in AML, its presence is currently associated with advanced age and a poor survival. Close to half of the patients in our series were classified as de novo AML. Although more commonly encountered as part of a complex karyotype it can also be present as a single abnormality. The different cytogenetic characteristics of the del(5q) may impact survival and this deserves further investigation. Figure. Survival of patients with AML and del(5q) in which the breakpoint was found to be distal to q15 (straight line) compared with patients in which the breakpoint was proximal to q13 (dotted line). Figure. Survival of patients with AML and del(5q) in which the breakpoint was found to be distal to q15 (straight line) compared with patients in which the breakpoint was proximal to q13 (dotted line).