Zehnder, James L.

Abstract Brains of 42 COVID-19 decedents and 107 non-COVID-19 controls were studied. RT-PCR screening of 16 regions from 20 COVID-19 autopsies found SARS-CoV-2 E gene viral sequences in 7 regions (2.5% of 320 samples), concentrated in 4/20 subjects (20%). Additional screening of olfactory bulb (OB), amygdala (AMY) and entorhinal area for E, N1, N2, RNA-dependent RNA polymerase, and S gene sequences detected one or more of these in OB in 8/21 subjects (38%). It is uncertain whether these RNA sequences represent viable virus. Significant histopathology was limited to 2/42 cases (4.8%), one with a large acute cerebral infarct and one with hemorrhagic encephalitis. Case-control RNAseq in OB and AMY found more than 5000 and 700 differentially expressed genes, respectively, unrelated to RT-PCR results; these involved immune response, neuronal constituents, and olfactory/taste receptor genes. Olfactory marker protein-1 reduction indicated COVID-19-related loss of OB olfactory mucosa afferents. Iba-1-immunoreactive microglia had reduced area fractions in cerebellar cortex and AMY, and cytokine arrays showed generalized downregulation in AMY and upregulation in blood serum in COVID-19 cases. Although OB is a major brain portal for SARS-CoV-2, COVID-19 brain changes are more likely due to blood-borne immune mediators and trans-synaptic gene expression changes arising from OB deafferentation.

Abstract Background: Coagulation factor V (FV) is an essential cofactor of the prothrombinase complex, catalyzing conversion of prothrombin to thrombin. Severe FV deficiency is a rare coagulation defect with a prevalence in the general population of approximately 1 in 1,000,000 and a variable bleeding risk (Asselta R et al. J Thromb Haemostas 2006). Genetic mutations underlying a FV-deficient hemorrhagic diathesis have been described in a limited number of cases, display considerable allelic heterogeneity, and have been due to point mutations in the FV gene located at chromosome 1q23. Here, we report a 15 year-old girl with psychomotor delay, CNS abnormalities, and a solitary left kidney - features consistent with a congenital 1q deletion including band 1q23 - who was found to have severely reduced plasma FV activity after a routine preoperative laboratory evaluation prior to an elective spinal stabilization surgery revealed a mildly prolonged prothrombin (PT, 13.2 seconds; normal 8.4 – 12 seconds) and activated partial thromboplastin (aPTT, and 38 seconds; normal 21 – 33 seconds) time. She had undergone minor surgical procedures in the past without reports of significant bleeding, and her family members were also clinically unaffected. Methods: FV procoagulant activity level was measured in the patient with an MDA-180 automated coagulation analyser (Trinity Biotech, Ireland). The patient’s peripheral blood leukocytes were separated by density centrifugation and fixed in methanol:glacial acetic acid (2:1). We then performed interphase fluorescent in-situ (FISH) analysis using probes covering the FV gene and the more telomeric HPC1 gene as a control. Genomic DNA was also isolated from the patient, and the coding region (25 exons) of the FV gene amplified by polymerase chain reaction (PCR) and sequenced. Results: FV activity assays revealed a severely reduced level of 9% (normal range: 60–130%). Factor V activity levels in the patient’s family members were not assessed. Peripheral blood karyotyping revealed an interstitial deletion of 1q: 46,XX,del(1)(q24.2q25.3 or q23.3q25.1). Interphase FISH analysis was performed. Red signals corresponded to the FV probe and green signals corresponded to HPC1, which is approximately 13 MB telomeric to FV. In contrast to healthy controls, the patient had a signal pattern of only 1 green HPC1 signal and 1 red FV signal, corresponding to heterozygous loss of the FV gene and adjacent chromosomal material. Sequencing of the retained allele revealed a novel hemizygous S234W mutation (encoded by exon 6). The lost serine is part of a conserved “SGP” peptide segment predicted to be a solvent-exposed region of the heavy chain of the FV protein. Conclusion: Most mutations causing FV deficiency are autosomal recessive and introduce frame shifts resulting in the generation of a premature termination codon. Karyotypic and FISH analysis of this patient revealed a novel mechanism for FV deficiency: loss of an entire FV allele as part of a chromosomal deletion, coupled with a point mutation in the other allele. Structural modeling of the consequences of the S234W point mutation, which is predicted to alter the hydrophobicity of the protein, is ongoing. Syndromes involving an interstitial deletion of 1q have been described in association with ATIII deficiency (Pallotta, Dalpra et al. 2001), but to our knowledge, this is the first description of a 1q deletion with FV deficiency.

Abstract Abstract 1415 Poster Board I-438 Introduction: Critical laboratory values (CVs) are those that indicate the patient is in danger of death unless corrective action is initiated immediately. CVs are an important feature of laboratory practice parameters, standards of regulatory agencies (including the Joint Commission's National Patient Safety Goals), and national and institutional quality improvement studies. However, there has been no research into CVs in coagulation laboratories. Our objectives were to determine: 1) The current policies on CVs in specialized coagulation laboratories 2) Current patterns of practice for reporting CVs for coagulation parameters; and 3) If there is any consensus, based on practice, on CVs for coagulation tests. Methods: A web-based survey on CVs was distributed to members of the North American Specialized Coagulation Laboratory Association in February 2009. Results: 38 laboratories participated. 97% had CVs for coagulation tests in place. 85% of laboratories used local opinion or consensus to set their CV policies, likely reflecting a lack of published data. 9% of laboratories were unsure where their CVs came from. CV reporting practices were fairly uniform. 97% of surveyed laboratories authorized technologists to report CVs, and only a minority (<40%) involved senior technologists or laboratory physicians in reporting. CV policies at almost all sites (97%) authorized the patient's nurse or MD, or the ordering MD to receive CV results. Medical students, clerical staff, or uninvolved staff nurses were rarely authorized to receive CVs (3%, 13%, and 37%, respectively). Policies in all participating laboratories required documentation of CV communication. Telephone was the primary method of communication, with 39% of sites used electronic broadcast or forced print as an adjunct. Inconsistency was noted in strategies to handle repeat CVs on the same patient, ranging from reporting all CVs, to reporting worsening CVs, to not reporting CVs on certain patient populations (e.g. critical FVIII:C levels in known hemophiliacs). After-hours communication of CVs on outpatients and referred clients was identified as challenging. All sites first attempted to telephone the patient's on-call MD, and if unsuccessful, sent a report by facsimile, contacted the Laboratory Chief of Service, or delayed reporting until the next day. When laboratories were asked about their CV values, over 80% had CVs for the INR (mean > 5, range > 2 - 6), aPTT (mean > 100 sec, range > 45 - 150 sec) and fibrinogen level (mean < 90 mg/dL, range < 50 - 100 mg/dL). Fewer laboratories (< 25%) had CVs for less commonly performed coagulation tests, including anti-Xa levels, factor and inhibitor assays, and thrombophilia testing. These CVs were more heterogeneous. Conclusion: This survey identified several potential areas for improvement in the way specialized coagulation laboratories handle CVs. Consensus data would assist laboratories in standardizing their policies, and establishing benchmarks for CV reporting and policy review. It would also ensure that a laboratory's CVs are in line with those of its peers, and are clinically relevant. The workload associated with CVs for coagulation assays is unknown, and it is possible that some of the current reporting strategies lead to inefficiencies and delays (e.g. reporting CVs for tests that are not truly life threatening; lack of established policies for after-hours handling of CVs when an on-call physician cannot be reached). Our study identifies an important need for further evaluation and standardization of CVs for coagulation tests. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1732 Background: In addition to chronic myelogenous leukemia (CML), the classic myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2 V617F mutation (V617F) can be detected through genomic analysis of peripheral blood in 95% of PV and 50–60% of ET and MF patients (pts). These MPNs are often associated with debilitating symptoms and decreased quality of life (QOL). Systemic mastocytosis (SM) is an uncommon MPN in which proliferation of mast cells can result in mediator symptoms, as well as organ damage in advanced disease. We sought to compare the symptom burden of individuals with CML and SM to pts with classic MPNs in a more quantitative manner using a web-based recruitment design. Methods: Individuals with MPN were recruited through a web-based outreach effort. Participants gave IRB-approved consent, and completed on-line surveys of demographic information and questions based on the validated MPN-symptom assessment form (MPN-SAF). In addition, pts submitted saliva samples which were genotyped with SNP arrays based on the Illumina OmniExpress BeadChip, including probes for V617F. Regression analysis was used to evaluate the correlation between symptom responses and genotype data for 495 pts (118 PV, 121 ET, 97 MF, 85 SM, and 74 CML). For each MPN diagnosis, mean scores were calculated based on six fatigue-focused questions (Fatigue-6) and for six other symptoms (Symptom-6): early satiety, abdominal pain/discomfort, inactivity, night sweats, pruritis, and bone pain (ranked from 0–10; 10 = worst). ET pts' scores were used as the reference in the regression analysis based on prior data showing that ET pts have the lowest symptom burden among the classic MPNs. Individual symptoms, Fatigue-6, Symptom-6, and QOL (ranked 0–10; 10=worst) were analyzed using regression analysis against age, gender, diagnosis and V617F status. Results: Median age was 56 years (7–87), with 70% women. V617F was detected in 63% of classic MPN pts (87% PV, 46% ET, 56% MF). Analysis of Fatigue-6 scores revealed that age in years was negatively correlated with reported fatigue (β=-0.007 per year, P=0.032), while female gender and SM diagnosis were positively correlated with fatigue (female: β=0.33, P=2.4×10−4; SM: β=0.46, P=7.6×10−4). This trend was also seen for the Symptom-6: age (β=-0.025, P=5.8×10−4), female gender (β=0.73, P=1.4×10−4), and SM (β=1.8, P=5.0×10−10). Both Fatigue-6 and Symptom-6 were positively correlated with QOL, but Fatigue-6 had a much stronger association with QOL than did Symptom-6 (β=1.3, P=1.5×10−28 vs. β=0.36, P=1.2×10−11). When analyzed individually, abdominal pain/discomfort, pruritis, and bone pain were significantly correlated with V617F (β=0.676, P=0.0371; β=0.768, P=0.0241; β=0.791, P=0.0341, respectively), though these associations were not significant when all six symptoms were evaluated together (Symptom-6: β=0.315, P=0.171). There was no statistically significant correlation between V617F and Symptom-6. In addition, there was no correlation between any symptom and a germline variant in telomerase reverse transcriptase (TERT) rs2853677, identified as a novel predisposition allele for MPNs using a genome-wide association study of this cohort. Conclusion: Using a novel, web-based recruitment design combining MPN symptom assessment and genotyping of saliva samples, we identified significant correlations between 3 specific symptoms (pruritis, abdominal pain/discomfort, and bone pain) with V617F. Fatigue was also more strongly associated than other symptoms with QOL. There was no association between symptoms and TERT. This design is a powerful tool for future studies seeking to correlate symptoms with genotyping analysis. Disclosures: Hinds: 23andMe: Employment, Equity Ownership, Patents & Royalties. Barnholt:23andMe, Inc.: Employment. Kiefer:23andMe, Inc.: Employment. Do:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Eriksson:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Mountain:23andMe, Inc.: Consultancy, Employment, Honoraria, Patents & Royalties, Research Funding. Francke:23andMe, Inc.: Employment, Honoraria, Research Funding; Locus Development: Consultancy, Membership on an entity's Board of Directors or advisory committees. Tung:23andMe, Inc.: Employment. Zehnder:23andMe, Inc.: Unpaid advisor and collaborator Other. Gotlib:23andMe, Inc.: Unpaid advisor and collaborator Other. Mesa:23andMe, Inc: Unpaid advisor and collaborator Other.

Abstract Abstract 1737 Background: The somatic JAK2 V617F (V617F) mutation is found in approximately 95% of polycythemia vera (PV) and 50–60% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. Detection of this mutation occurs primarily through genomic analysis of peripheral blood samples of myeloproliferative neoplasm (MPN) patients. This study demonstrates a novel broad screening mechanism for detection of V617F from myeloid cells in saliva samples from an online cohort of MPN patients and from a broader database of genotyped individuals. Methods: MPN patients were recruited primarily through social media and patient-mediated outreach efforts. Patients gave IRB-approved consent and completed surveys through an online 23andMe account. Participation in the MPN research initiative was free. In collaboration with an uncompensated panel of academic experts, online surveys were developed to collect patient-reported data on diagnosis and results of genetic testing for the V617F mutation. Participants were genotyped on SNP arrays based on the Illumina Omni Express, with additional custom content, including four probes for the V617F mutation. We estimated V617F mutation burden as the median across the four probes of B/(A+B) where A and B were normalized intensities for the reference and mutated alleles, respectively. We estimated sensitivity and specificity of our estimated V617F allele ratio, using the self-reported V617F-positive individuals to approximate the distribution of true positives, and 23andMe members from the broad database (outside of the MPN research cohort) and younger than 40 as likely true negatives. Results: Web-based recruitment efforts led to the accrual of over 900 MPN patients of different subtypes over 12 months. Of these participants, 745 patients have been genotyped and provided MPN-related health history and outcomes through online surveys. The cohort is disproportionately female (71%), with an average age of 53.1 ± 14.9 years. More than 57% of patients indicated a classic MPN diagnosis (PV, ET, PMF, or post PV/ET MF), 19.2% reported an atypical MPN diagnosis (systemic mastocytosis or hypereosinophilic syndrome), 14.5% indicated chronic myelogenous leukemia, and 9.3% of the cohort reported other diagnoses. From this MPN cohort, a total of 345 participants reported mutation test results. Consistent with expectations, the V617F variant was reported by patients in nearly all PV cases (105 of 110 patients with a self-reported diagnosis of PV), and at a lower rate in ET and MF cases (59 of 95 patients and 48 of 61 patients, respectively). In addition to self-reported data, we were specifically able to detect the V617F mutation using SNP array data. We determined a detection threshold for the V617F variant at which 86% of self-reported V617F carriers were scored as positive (i.e. test sensitivity), and 99.975% of young 23andMe customers were scored as negative (i.e. test specificity). Participants with a classic diagnosis in the MPN cohort who carry the V617F variant tended to be older (mean age=59.5 versus 54.6 years) and were less likely to be female (62.5% versus 72.0%) than non-carriers. At this threshold, we detected 133 individuals who likely carry the V617F mutation among a broader set of 23andMe participants (n= 99,700) who were not specifically recruited for the MPN study, corresponding to a prevalence of 0.14%. We estimated the positive predictive value of the test in this cohort, or the expected proportion of positive test results that are true, as 81%. Within the general 23andMe cohort, the frequency of the V617F variant increases with participant age, from a rate of 0.017% for age 20 to 30, to 1.1% for age 80 to 90. We also observed that patients with a high allele burden of V617F frequently had acquired uniparental disomy of chromosome 9p, as determined from calculated heterozygosity scores and no-call rates for this chromosomal arm. Conclusion: We can detect V617F with high specificity and sensitivity from myeloid cells in saliva. This non-invasive source for DNA extraction offers new possibilities for detecting genomic alterations and for finding novel genetic associations in MPN patients as well as the general population. This study demonstrates the feasibility of online technology to significantly accelerate recruitment and research progress in MPNs and other rare diseases. Disclosures: Barnholt: 23andMe, Inc.: Employment. Hinds:23andMe: Employment, Equity Ownership, Patents & Royalties. Kiefer:23andMe, Inc.: Employment. Do:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Eriksson:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Mountain:23andMe, Inc.: Consultancy, Employment, Honoraria, Patents & Royalties. Francke:23andMe, Inc.: Employment, Honoraria, Research Funding; Locus Development: Consultancy, Membership on an entity's Board of Directors or advisory committees. Tung:23andMe, Inc.: Employment. Mesa:23andMe, Inc.: Unpaid advisor and collaborator Other. Gotlib:23andMe, Inc.: Unpaid advisor and collaborator Other. Zehnder:23andMe, Inc.: Unpaid advisor and collaborator Other.

Abstract Abstract 707 Background: Myeloproliferative neoplasms (MPNs) are disorders that result in unregulated overproduction of one or more myeloid blood cell types by the bone marrow. Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) comprise the three classic MPNs. The somatic JAK2 V617F mutation is present in 95% of PV patients, and 50–60% of ET and PMF patients. Past work has identified a germline haplotype in JAK2 associated with risk of developing a V617F-positive MPN (Kilpivaara et al; Olcaydu et al; Jones et al, Nat. Genet., 2009). Methods: We recruited a web-based participatory cohort of individuals with MPNs to better understand the genetic basis of these conditions. Participation in this MPN research initiative included free genotyping, and participants provided informed consent and responses to surveys online, under a protocol approved by the external AAHRPP-accredited IRB, Ethical & Independent Review Services. We enrolled and collected saliva samples from more than 700 participants with the following self-reported diagnoses: systemic mastocytosis (n=142), ET (n=145), PV (n=137), PMF (n=50), chronic myelogenous leukemia (n=108), and 97 with overlapping diagnoses (e.g. ET or PV with PMF). Participants were genotyped using a derivative of the Illumina HumanOmniExpress BeadChip with additional custom content, including probes for the JAK2 V617F variant. We performed a genome-wide association study (GWAS) of classic MPN diagnoses (at least one of ET, PV, or MF), using 407 cases from this MPN research initiative and 94,037 controls drawn from the broader 23andMe research participant community. Association was assessed by logistic regression adjusted for age, gender, and 5 principal components to model ancestry. Results: We replicated the known association between germline variation in JAK2 and classic MPNs (rs12340895: odds ratio=2.5, P=7.4 × 10−35). We also identified a novel genome-wide-significant association in TERT, or telomerase reverse transcriptase (rs2853677: odds ratio=1.6 per G allele, P=6.3 × 10−11). The association is consistent with an additive model on the logistic scale (odds ratio=2.6 for 2 versus 0 G alleles). Another variant in TERT, rs2736100, has previously been associated with several non-hematological cancers and with red blood cell count, and our lead SNP is in linkage disequilibrium with TERT variant rs2736100 (r2=0.54). While no other loci reached the threshold of genome-wide significance (P<5.0 × 10−8), we saw a suggestive association with a germline missense variant (F858L) in ATM, or ataxia telangiectasia mutated gene (rs1800056: odds ratio=2.6 per C allele, P=1.4 × 10−5). This variant has also been reported to be associated with breast cancer and chronic lymphocytic leukemia. The rs2853677 TERT variant was associated with each of ET, PV, and PMF, with similar effect sizes. It was not associated with JAK2 V617F mutation status within the MPN cohort (odds ratio=1.0, P=1.0), while the germline JAK2 variant was preferentially associated with V617F-positive MPNs (odds ratio=2.1, P=3.0 × 10−6). The high-risk allele of rs2853677 was also predictive of V617F status among 23andMe participants who had not been recruited into the MPN community (odds ratio=1.6, P=0.0012). These results suggest that while variation in TERT facilitates either acquisition or proliferation of V617F-positive myeloid cells, it also predisposes to MPNs through a V617F-independent mechanism. Conclusion: We have identified a germline variant in TERT which is a new predisposition allele for classic MPNs. These results demonstrate the potential for web-based recruitment methods to contribute to genetic research for uncommon diseases. Disclosures: Hinds: 23andMe: Employment, Equity Ownership, Patents & Royalties. Barnholt:23andMe, Inc.: Employment. Zehnder:23andMe, Inc.: Unpaid advisor and collaborator Other. Kiefer:23andMe, Inc.: Employment. Do:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Eriksson:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Mountain:23andMe, Inc.: Consultancy, Employment, Honoraria, Patents & Royalties, Research Funding. Francke:23andMe, Inc.: Employment, Honoraria, Research Funding; Locus Development: Consultancy, Membership on an entity's Board of Directors or advisory committees. Tung:23andMe, Inc.: Employment. Mesa:23andMe, Inc: Unpaid advisor and collaborator Other. Gotlib:23andMe, Inc.: Unpaid advisor and collaborator Other.

AbstractIntroductionPlatelet function disorders (PFD) are an important group of bleeding disorders that require validated and practical laboratory strategies for diagnosis.MethodsThis review summarizes the authors’ experiences, current literature, and an international survey to evaluate the practices of diagnostic laboratories that offer tests for PFD.ResultsBlood counts, blood film review, and aggregation tests are the most commonly performed investigations for PFD and help determine whether there is thrombocytopenia and/or defective platelet function due to a variety of causes. The performance characteristics of tests for PFD, and the level of evidence that these tests detect bleeding problems, are important issues to determine where tests are useful for diagnostic or correlative purposes, or research only uses. Platelet aggregation assays, and quantitative analysis of platelet dense granule numbers, are tests with good performance characteristics that detect abnormalities associated with increased bleeding in a significant proportion of individuals referred for PFD investigations. Lumiaggregometry estimates of platelet adenosine triphosphate release show greater variability which limits the diagnostic usefulness. Diagnostic laboratories report that fiscal and other constraints, including a lack of high‐quality evidence, limit their ability to offer an expanded test menu for PFD.ConclusionPFD are clinically important bleeding disorders that remain challenging for diagnostic laboratories to investigate. While some PFD tests are well validated for diagnostic purposes, gaps in scientific evidence and resource limitations influence diagnostic laboratory decisions on which PFD tests to offer.

SummaryIntroductionWhile the presence of disseminated intravascular coagulation (DIC) has been implicated in worse clinical outcome in acute leukemia, the relationship between different subtypes of acute leukemia and the clinicopathologic features of DIC has not been systematically well studied.MethodsIn this study, we retrospectively reviewed 149 cases of newly diagnosed acute leukemia and assessed the utility of evaluating red blood cell morphologic features, and coagulation parameters in determining the presence of DIC as well as differentiating subtypes of acute leukemia.ResultsReview of our cohort demonstrates a novel finding, that elevated D‐dimer concentrations ≥19 000 ng/mL fibrinogen equivalent units (FEU) are a sensitive diagnostic indicator of acute promyelocytic leukemia (APL) with moderate specificity, sensitivity 96%, specificity 92% in acute leukemia subtyping. Similar to other studies, APL showed an increased incidence of DIC (P < 0.01) compared to other subtypes of acute leukemia. Surprisingly, the presence of schistocytes on the peripheral blood smear was not a statistically significant indicator of DIC, sensitivity of 36% and specificity of 89%. Finally, the presence of DIC was not a significant indicator of poorer prognosis amongst all patients with AML.ConclusionOverall we identify elevated D‐dimer concentrations ≥19 000 ng/mL FEU are a sensitive indicator of acute promyelocytic leukemia (APL), with a sensitivity of 96% and specificity of 92% in the subtyping of acute leukemias, and that the presence of schistocytes in peripheral blood smears is not a diagnostically sensitive screening test for DIC with a sensitivity of 36%.

Key Points Germ line variants in TERT, SH2B3, TET2, ATM, CHEK2, PINT, and GFI1B are associated with JAK2 V617F clonal hematopoiesis and MPNs. Age-related JAK2 V617F clonal hematopoiesis is found in ∼2 out of 1000 individuals in the general population.

Abstract Platelet function testing is important for the diagnostic evaluation of common and rare bleeding disorders. Our study goals were to promote best practices and reduce unnecessary testing variances by developing North American guidelines on platelet function testing. Guidelines were developed by consensus for expert recommendations (minimum level for approval, 70%) that included recommendations on the evaluation and interpretation of light transmission platelet aggregometry (LTA). To assess consensus, medical opinions on recommendations were gathered from diagnostic laboratories that perform LTA, in collaboration with the Quality Management Program–Laboratory Services (QMP-LS) in Ontario, Canada (10 laboratories), and the North American Specialized Coagulation Laboratory Association (NASCOLA; 47 laboratories, 5 overlapping the QMP-LS group). Adequate consensus was achieved for all and 89% of recommendations for the QMP-LS and NASCOLA groups, respectively. The recommendations adopted provide North American laboratories with additional guidance on platelet function testing, including how to interpret LTA abnormalities.