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TP53 mutations in patients with AML and MDS frequently portend a poor prognosis, related to both p53 allele status and blast count. In 2022, the ICC and WHO released updated guidelines for classifying p53-mutated AML/MDS. The characteristics of p53 mutations, their associated co-mutations, and their effects on overall survival (OS) are not known in the context of these new guidelines. A retrospective chart review was undertaken for all patients with AML or MDS and at least one TP53 mutation detected on next generation sequencing (NGS) at Yale New Haven Hospital from 2015 to 2023. All patients (N = 210) met criteria for one of the 5 diagnostic classes based on WHO and ICC guidelines. Kaplan-Meier curves with associated log-rank testing and Cox proportional hazards model quantified the effects of clinical and molecular data on survival. Multi-hit pathogenic mutations were related to poorer OS in MDS but not AML using either the WHO (p = .02) or the ICC (p = .01) diagnostic criteria. The most significant predictors of OS in the sample overall were platelet count < 50 K (HR: 2.01, 95% CI [1.47, 2.75], p < .001) and TP53 VAF ≤ 40% (HR: 0.68, 95% CI[0.50, 0.91], p = .01). Blast count ranges, complex karyotype, and p53 mutation type or location, showed no association with OS. In our cohort defined by the 2022 ICC and WHO criteria, VAF and thrombocytopenia, rather than blast count or p53 mutation features, significantly predicted OS. These results speak to each criteria's ability to identify cases of similarly aggressive disease biology and prognosis., Competing Interests: Declarations. Ethical approval: This study was conducted in accordance with the Yale University Institutional Review Board policies. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Inherited platelet disorders (IPDs) are a heterogeneous group of conditions that present significant challenges in diagnosis and management. Here, we report two cases of patients presenting with clinically significant bleeding but with unclear etiologies by conventional clinical laboratory testing. Further evaluation, utilizing a combination of high-dimensional multiplexed mass cytometry and genetic sequencing, revealed the underlying causes of bleeding in both cases, leading to definitive diagnoses. These cases underscore the potential utility of combined multimodal approaches in evaluating patients with bleeding disorders. Moreover, these high-parameter methods can offer substantial mechanistic insights and can enhance our understanding of the molecular pathogenesis of IPDs. Future studies involving larger patient cohorts are needed to further validate this strategy, directly comparing its diagnostic yield and accuracy with current clinical laboratory testing approaches, which can ultimately improve patient care., Competing Interests: Declaration of competing interest No conflicts of interest to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Myeloid neoplasms (MNs) frequently harbor pathogenic mutations not detected by karyotyping and fluorescence in situ hybridization; hence, next-generation sequencing (NGS) is necessary for diagnosis, risk stratification, and therapy. If, however, NGS is not clinically indicated but still performed, the results may promote futile avenues of investigation, heighten patient distress, and increase cost., Methods: We created criteria to approve NGS testing for MN (MN-NGS) with the goal of maximizing actionable results. These actionable results include making a new MN diagnosis, characterizing a MN with baseline mutational status, and altering treatment plans. Approval criteria included clinical suspicion of new, relapsed, or worsening disease and end-of-induction chemotherapy. Cancellation criteria included the suspicion of non-myeloid disease only, no suspicion of progression of a known MN, no evidence for recurrence post-transplant, a diagnosis of chronic myeloid leukemia, and cases using blood when a concurrent bone marrow NGS is being performed. We applied these criteria to NGS tests ordered at our institution between August and December 2018 and determined whether any tests meeting our cancelation criteria yielded actionable results., Results: Consecutive MN-NGS orders ( n  = 174) were retrospectively categorized as appropriate (Group A, n  = 115), inappropriate (Group B, n  = 29), and appropriately canceled (group C, n  = 30). Seventy-five of the 115 (65%) Group A tests and none of the 29 (0%) Group B tests yielded actionable results ( p  < 0.0001)., Conclusion: Approximately one third (59/174) of MN-NGS test orders can be safely canceled using these criteria, which would result in $150,370 of Centers for Medicare and Medicaid Services-reimbursed savings annually., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

The Heidenhain variant of Creutzfeld-Jakob disease (CJD) is a rare form that initially presents with visual disturbances. In early stages, the presentation can mimic neuromyelitis optica spectrum disorders (NMOSD) and lead to unnecessary treatment modalities. Herein, we describe a case of a 66-year-old man who presented with bilateral vision loss and retro-orbital discomfort. In addition to immunosuppressive therapy, he received 4 rounds of therapeutic plasma exchange after his preliminary diagnosis of NMOSD. We were surprised to note that his condition did not show improvement but deteriorated, with severe neurocognitive symptoms. Eventually, CJD was suspected, and real-time quaking-induced conversion (RT-QuIC) was performed. By the time the diagnosis of Heidenhain variant of CJD was confirmed, the patient was discharged to hospice care and died shortly after., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The thrombotic risk with haemoglobin C trait (HbAC) or haemoglobin C disease (HbCC) is unclear. However, individuals with HbCC have demonstrated chronic haemolysis, higher blood viscosity and altered rheology when compared to individuals with wild-type haemoglobin (HbAA). These physiological alterations may theoretically translate to increased risk of thrombosis; therefore, a systematic literature review was performed to investigate the possible association between HbAC and/or HbCC and thrombosis. Twenty-two studies met inclusion criteria representing 782 individuals with HbAC (n = 694) or HbCC (n = 88). Fifteen studies described the presence/absence of venous thromboembolism (VTE) in patients with HbAC (n = 685) or HbCC (n = 79), while seven studies described patients with HbAC (n = 9) or HbCC (n = 9) and arterial thrombosis. Most (n = 20) studies were case reports or case series; however, two studies suggested a potential increased VTE risk with HbAC compared to HbAA in (i) all patients (OR 2.2, 95% CI: 0.9-5.5) and in (ii) pregnant individuals (RR 3.7, 95% CI 0.9-16). This review is the largest assessment of patients with HbC trait or disease and thrombosis to date; despite its limitations, the findings suggest HbC may be a predisposing risk factor to thrombosis. Prospective cohort studies are warranted to definitively elucidate the risk of thrombosis in this population., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

Guillain-Barré syndrome (GBS) is an immune-mediated polyradiculoneuropathy and the most common cause of acute flaccid paralysis worldwide. GBS classically presents with acute, progressive, ascending weakness, reduced to absent reflexes, and albuminocytological dissociation on cerebrospinal fluid (CSF) analysis. Botulism is a neurotoxin-mediated acute descending flaccid paralysis with cranial nerve palsies and dysautonomia. Botulism in adults is caused by ingestion/inhalation of botulinum toxin or wound infection with Clostridium botulinum. Both GBS and botulism can rapidly precipitate respiratory failure; thus, prompt diagnosis and treatment are crucial to mitigate poor outcomes. Herein, we describe a case of botulism initially diagnosed as GBS given classic laboratory features, and describe the importance of careful consideration of the most appropriate therapeutic modalities in cases of acute flaccid paralysis, particularly regarding empiric administration of botulinum antitoxin and use of intravenous immune globulin in lieu of plasma exchange for potential GBS to prevent removal of antitoxin., (© 2023 Wiley Periodicals LLC.)

Next-generation sequencing (NGS) is used to monitor genetically measurable residual disease (gMRD) following allogeneic stem cell transplantation (aSCT). It is unknown whether an upper limit of chimerism exists such that gMRD NGS testing can be safely forgone. We reviewed 61 patients with acute myeloid leukemia and 24 patients with myelodysplastic syndrome who had at least 1 NGS panel before and after aSCT between 2016 and 2020. Donor chimerism was quantified. Logistic regression characterized which factors predicted gMRD. Receiver operating characteristic (ROC) curves were used to determine the optimal chimerism threshold for which gMRD would not be detected. Data from an additional 22 patients with follow-up NGS testing in 2022 were also analyzed to validate our proposed threshold. A: s expected, donor chimerism was a significant predictor of gMRD (odds ratio, .38; 95% confidence interval, .10 to .62; P = .02). Age, sex, conditioning regimen, presence of a related donor, and diagnosis were not associated with gMRD. A chimerism threshold of 92.5% optimized sensitivity (97.7%) and specificity (95.4%) such that values >92.5% strongly predicted the absence of gMRD (area under the ROC curve [AUC], .986). The validation cohort demonstrated similarly strong predictive capability (AUC, .974) with appropriate sensitivity (100%) and specificity (90.9%). NGS monitoring of gMRD is redundant at chimerism values greater than a more conservative threshold of 92.5% after aSCT., (Copyright © 2023 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Objectives: Clinical experts recommend against testing for lupus anticoagulant (LAC) during anticoagulation., Methods: We quantitated the risk of a single-positive dilute Russell viper venom time (dRVVT) result or partial thromboplastin time-based phospholipid neutralization (PN) result on anticoagulation., Results: Any anticoagulation led to a fourfold greater likelihood of single-positive results, primarily by rivaroxaban (odds ratio [OR] = 8.6) and warfarin (OR = 6.6), resulting in a positive dRVVT test with a normal PN test. Heparin and apixaban were twofold more likely to show single-positive results, but enoxaparin did not show significant single positivity., Conclusions: Our results quantitatively support experts' avoidance of LAC testing during anticoagulation., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Acquired hemophilia A, caused by autoantibodies that bind to and neutralize the activity of coagulation factor VIII (FVIII), almost universally presents as a severe bleeding diathesis. Lupus anticoagulants (LAs), autoantibodies directed against phospholipids or protein-phospholipid complexes, manifest clinically with an increased risk of thrombosis. While these autoantibodies are uncommon, the distinctive clinical presentation in conjunction with the typical laboratory findings often enable straightforward identification of the underlying autoantibody. However, the presence of a concomitant acquired FVIII inhibitor and LA is exceedingly rare with fewer than 20 documented cases. All prior patients presented with life-threatening hemorrhage, thrombosis, or both, prompting comprehensive hematologic evaluation and subsequent identification of the pathologic antibodies. We describe a novel case of a patient with no signs of hemorrhage or thrombosis who was incidentally found to have both a FVIII inhibitor and LA during evaluation of a prolonged partial thromboplastin time (PTT). This finding resulted in FVIII inhibitor-directed management, including immunosuppressive therapy. The unique presentation of an incidental FVIII inhibitor and LA in an asymptomatic patient without thrombotic or bleeding complications highlights the potential challenge in elucidating the etiology of a prolonged PTT, as LAs and FVIII inhibitors both prolong the PTT, and each entity can interfere with assays designed to detect the presence of the other autoantibody. This case underscores the importance of recognizing that patients with major underlying disturbances in their hematologic physiology, but in whom clinical symptoms have yet to manifest, may potentially be overlooked until such symptoms are evident., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: Acute promyelocytic leukemia (APL) with variant RARA translocation, eg, t(11;17), is not sensitive to all-trans retinoic acid and requires distinct chemotherapy. However, there are some leukemic entities that may mimic aspects of the clinical and/or laboratory picture of APL and cause confusion because of karyotype nomenclature. Therefore, recognition of such entities may be of therapeutic and prognostic significance., Methods: We present 2 cases of acute myeloid leukemia (AML) with t(11;17) that were clinically concerning for APL based primarily on clinical presentation but were ultimately diagnosed as AML with monocytic differentiation., Results: Both leukemias harbored KMT2A translocations, one located near but not involving RARA and the other with SEPT9., Conclusion: In leukemias that clinically and/or immunophenotypically mimic APL, identification of specific gene translocations can lead to the correct diagnosis and may carry therapeutic/prognostic implications., (© American Society for Clinical Pathology 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Thrombotic complications occur at high rates in hospitalized patients with COVID-19, yet the impact of intensive antithrombotic therapy on mortality is uncertain. We examined in-hospital mortality with intermediate- compared to prophylactic-dose anticoagulation, and separately with in-hospital aspirin compared to no antiplatelet therapy, in a large, retrospective study of 2785 hospitalized adult COVID-19 patients. In this analysis, we established two separate, nested cohorts of patients (a) who received intermediate- or prophylactic-dose anticoagulation ("anticoagulation cohort", N = 1624), or (b) who were not on home antiplatelet therapy and received either in-hospital aspirin or no antiplatelet therapy ("aspirin cohort", N = 1956). To minimize bias and adjust for confounding factors, we incorporated propensity score matching and multivariable regression utilizing various markers of illness severity and other patient-specific covariates, yielding treatment groups with well-balanced covariates in each cohort. The primary outcome was cumulative incidence of in-hospital death. Among propensity score-matched patients in the anticoagulation cohort (N = 382), in a multivariable regression model, intermediate- compared to prophylactic-dose anticoagulation was associated with a significantly lower cumulative incidence of in-hospital death (hazard ratio 0.518 [0.308-0.872]). Among propensity-score matched patients in the aspirin cohort (N = 638), in a multivariable regression model, in-hospital aspirin compared to no antiplatelet therapy was associated with a significantly lower cumulative incidence of in-hospital death (hazard ratio 0.522 [0.336-0.812]). In this propensity score-matched, observational study of COVID-19, intermediate-dose anticoagulation and aspirin were each associated with a lower cumulative incidence of in-hospital death., (© 2021 Wiley Periodicals LLC.)

Pathologic immune hyperactivation is emerging as a key feature of critical illness in COVID-19, but the mechanisms involved remain poorly understood. We carried out proteomic profiling of plasma from cross-sectional and longitudinal cohorts of hospitalized patients with COVID-19 and analyzed clinical data from our health system database of more than 3300 patients. Using a machine learning algorithm, we identified a prominent signature of neutrophil activation, including resistin, lipocalin-2, hepatocyte growth factor, interleukin-8, and granulocyte colony-stimulating factor, which were the strongest predictors of critical illness. Evidence of neutrophil activation was present on the first day of hospitalization in patients who would only later require transfer to the intensive care unit, thus preceding the onset of critical illness and predicting increased mortality. In the health system database, early elevations in developing and mature neutrophil counts also predicted higher mortality rates. Altogether, these data suggest a central role for neutrophil activation in the pathogenesis of severe COVID-19 and identify molecular markers that distinguish patients at risk of future clinical decompensation., (© 2021 by The American Society of Hematology.)

A hallmark of lymphoid malignancies is the presence of a monoclonal lymphocyte population. Monoclonality of B- and T-cell populations can be established through immunoglobulin (IG) or T-cell receptor (TCR) gene rearrangement analysis, respectively. The biological rationale of IG and TCR gene rearrangement analysis is that due to the extensive combinatorial repertoire made possible by V(D)J recombination in lymphocytes, it is unlikely that any substantive lymphocyte population would share the same IG or TCR gene rearrangement pattern unless there is an underlying neoplastic or reactive origin. Modern IG and TCR gene rearrangement analysis is typically performed by polymerase chain reaction (PCR) using commercially available primer sets followed by gel capillary electrophoresis. This process is highly sensitive in the detection of nearly all lymphoid malignancies. Several pitfalls and limitations, both biological and technical, apply to IG/TCR gene rearrangement analysis, but these can be minimised with high quality controls, performance of assays in duplicate, and adherence to strict criteria for interpreting and reporting results. Next generation sequencing (NGS) will likely replace PCR based methods of IG/TCR gene rearrangement analysis but is not yet widespread due to the absence of standardised protocols and multicentre validation., (Copyright © 2020 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Background: Allogeneic platelet (PLT) infusion is a strategy to raise Factor V (FV) levels in patients with congenital FV deficiency. However, since FV is labile in vitro, we hypothesized that FV activity could be low in PLT units., Study Design and Methods: FV activity was tested using a prothrombin time-based platform in the supernatant and platelet lysate (PL) of apheresis PLT units (16 units stored in PLT additive solution with acetate and phosphate [PAS-C] and 10 units stored in plasma only), on post-collection days 3-6. Statistical analysis was performed using Student's t test (P < .05)., Results: FV activity was severely diminished in PAS-C PLTs (N = 16) supernatant (3.70% ± 1.02%) and PL (3.26% ± 1.02%). FV activity in plasma-only PLTs (N = 10) was lower in both supernatant (44.55% ± 6.46%) and lysate (39.67% ± 6.33%) relative to normal plasma levels, but both were significantly higher (P < .0001) compared to PAS-C PLTs. In a separate set of experiments, FV activity in PAS-C PLTs examined serially over storage time (N = 3 for these experiments) showed that FV levels were reduced by day 3 and not significantly different by day 5 of storage (Day 3 supernatant 5.03% ± 1.41%; Day 5 supernatant: 3.10% ± 0.57%; P = .2; Day 3 lysate: 3.89% ± 1.03%; Day 5 lysate: 2.61% ± 0.41%; P = .4)., Conclusion: Plasma should be considered over PLTs as first-line therapy for non-complex FV deficiency-associated hemorrhage. If PLTs are considered for transfusion, plasma-only PLT units should be preferentially utilized, as PAS-C PLT have near-absent FV activity., (© 2020 AABB.)

Background: Thrombotic complications occur at high rates in hospitalized patients with COVID-19, yet the impact of intensive antithrombotic therapy on mortality is uncertain., Research Question: How does in-hospital mortality compare with intermediate- versus prophylactic-dose anticoagulation, and separately with in-hospital aspirin versus no antiplatelet therapy, in treatment of COVID-19?, Study Design and Methods: Using data from 2785 hospitalized adult COVID-19 patients, we established two separate, nested cohorts of patients (1) who received intermediate- or prophylactic-dose anticoagulation ("anticoagulation cohort", N = 1624), or (2) who were not on home antiplatelet therapy and received either in-hospital aspirin or no antiplatelet therapy ("aspirin cohort", N = 1956). Propensity score matching utilizing various markers of illness severity and other patient-specific covariates yielded treatment groups with well-balanced covariates in each cohort. The primary outcome was cumulative incidence of in-hospital death., Results: Among propensity score-matched patients in the anticoagulation cohort (N = 382), in a multivariable regression model, intermediate- compared to prophylactic-dose anticoagulation was associated with a significantly lower cumulative incidence of in-hospital death (hazard ratio 0.518 [0.308-0.872]). Among propensity-score matched patients in the aspirin cohort (N = 638), in a multivariable regression model, in-hospital aspirin compared to no antiplatelet therapy was associated with a significantly lower cumulative incidence of in-hospital death (hazard ratio 0.522 [0.336-0.812])., Interpretation: In this propensity score-matched, observational study of COVID-19, intermediate-dose anticoagulation and aspirin were each associated with a lower cumulative incidence of in-hospital death., Competing Interests: Summary conflict of interest statements: No conflict of interest exists for any author on this manuscript.

As the global community strives to discover effective therapies for COVID-19, immunomodulatory strategies have emerged as a leading contender to combat the cytokine storm and improve clinical outcomes in patients with severe disease. Systemic corticosteroids and selective cytokine inhibitory agents have been utilized both as empiric therapies and in clinical trials. While multiple randomized, placebo controlled trials have now demonstrated that corticosteroids improve survival in patients with COVID-19, 1, 2 IL-6 inhibition, which gained significant early interest based on observational studies, has not demonstrated reliable efficacy in randomized, placebo controlled trials. 3, 4 To better understand the mechanistic basis of immunomodulatory therapies being implemented for treatment of COVID-19, we assessed longitudinal biochemical changes in response to such approaches in hospitalized patients with COVID-19. We demonstrate broad suppression of multiple immunomodulatory factors associated with adverse clinical outcomes in COVID-19 in patients who received corticosteroids, but no such response was seen in patients who either received tocilizumab or no immunomodulatory therapy. Our findings provide early insights into molecular signatures that correlate with immunomodulatory therapies in COVID-19 which may be useful in understanding clinical outcomes in future studies of larger patient cohorts.

In patients with decompensated cirrhosis, procedure-related bleeding is a potentially lethal complication. Routine coagulation tests such as international normalized ratio and platelet count do not predict bleeding risk. We investigated whether thromboelastography (TEG) can identify patients with cirrhosis who are at risk of procedure-related bleeding. As a part of a prospective study on hemostasis in decompensated cirrhosis, patients had TEG performed on admission and were followed prospectively during hospitalization for the development of procedure-related bleeding. Eighty patients with cirrhosis were included. Among the 72 who had procedures performed, 7 had procedure-related bleeding, which was major in three cases (two following paracentesis and one following thoracentesis). Conventional coagulation tests were comparable between bleeding and nonbleeding patients, whereas TEG parameters of k-time (4.5 minutes vs. 2.2 minutes; P  = 0.02), α-angle (34° vs. 59°; P  = 0.003), and maximum amplitude (37 mm vs. 50 mm; P  = 0.004) were significantly different (all indicative of hypocoagulability). TEG maximum amplitude (MA), a marker of overall clot stability, accurately discriminated between patients who had major, life-threatening bleeding (all with MA < 30 mm) and those who had mild or no bleeding (all with MA > 30 mm), whereas a platelet count < 50 × 10 9 /L could not discriminate between bleeding (minor or major) and nonbleeding patients. Conclusion : In a prospective cohort of hospitalized patients with decompensated cirrhosis, TEG parameters associated with hypocoagulability appeared to predict procedure-related bleeding, particularly a TEG MA < 30 mm. If results are validated in a larger cohort, this could be a threshold to identify patients with decompensated cirrhosis at higher risk for procedure-related bleeding, in whom to consider preprocedural prophylaxis., (© 2020 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of the American Association for the Study of Liver Diseases.)

Background and Aims: Recent evidence suggests that acute kidney injury (AKI) is the main predictor of postparacentesis bleeding in patients with cirrhosis. To assess the factors responsible for bleeding tendency in AKI, we performed a prospective study comparing all three aspects of hemostasis (platelets, coagulation, and fibrinolysis) in patients with decompensated cirrhosis with and without AKI., Approach and Results: Primary hemostasis assessment included platelet aggregation and secretion (platelet function markers) and von Willebrand factor. Secondary hemostasis assessment included pro-coagulant (factor VIII and factor XIII) and anti-coagulant (protein C, protein S, and antithrombin) factors and thrombin generation. Tertiary hemostasis assessment included fibrinolytic factors and plasmin-antiplasmin complex. Eighty patients with decompensated cirrhosis were recruited (40 each with and without AKI). Severity of cirrhosis and platelet count were comparable between groups. Median serum creatinine was 1.8 mg/dL and 0.8 mg/dL in patients with and without AKI, respectively. At baseline, patients with cirrhosis and AKI had lower platelet aggregation and secretion, indicative of impaired platelet function (increased bleeding tendency), without differences in von Willebrand factor. Regarding coagulation factors, factor VIII was higher, whereas protein C, protein S, and antithrombin were all lower, which, together with increased thrombin generation, indicate hypercoagulability. In contrast, factor XIII was lower in AKI (increased bleeding tendency). Finally, while both hypofibrinolytic and hyperfibrinolytic changes were present in AKI, a higher plasmin-antiplasmin complex indicated a hyperfibrinolytic state. After AKI resolution (n = 23 of 40), platelet function and coagulation improved to levels observed in patients with cirrhosis patients without AKI; however, fibrinolysis remained hyperactivated., Conclusions: In patients with decompensated cirrhosis, AKI is associated with both hypocoagulable and hypercoagulable features that can potentially increase the risk of both bleeding and thrombosis., (© 2020 by the American Association for the Study of Liver Diseases.)

Pathologic immune hyperactivation is emerging as a key feature of critical illness in COVID-19, but the mechanisms involved remain poorly understood. We carried out proteomic profiling of plasma from cross-sectional and longitudinal cohorts of hospitalized patients with COVID-19 and analyzed clinical data from our health system database of over 3,300 patients. Using a machine learning algorithm, we identified a prominent signature of neutrophil activation, including resistin, lipocalin-2, HGF, IL-8, and G-CSF, as the strongest predictors of critical illness. Neutrophil activation was present on the first day of hospitalization in patients who would only later require transfer to the intensive care unit, thus preceding the onset of critical illness and predicting increased mortality. In the health system database, early elevations in developing and mature neutrophil counts also predicted higher mortality rates. Altogether, we define an essential role for neutrophil activation in the pathogenesis of severe COVID-19 and identify molecular neutrophil markers that distinguish patients at risk of future clinical decompensation.

Background: An important feature of severe acute respiratory syndrome coronavirus 2 pathogenesis is COVID-19-associated coagulopathy, characterised by increased thrombotic and microvascular complications. Previous studies have suggested a role for endothelial cell injury in COVID-19-associated coagulopathy. To determine whether endotheliopathy is involved in COVID-19-associated coagulopathy pathogenesis, we assessed markers of endothelial cell and platelet activation in critically and non-critically ill patients admitted to the hospital with COVID-19., Methods: In this single-centre cross-sectional study, hospitalised adult (≥18 years) patients with laboratory-confirmed COVID-19 were identified in the medical intensive care unit (ICU) or a specialised non-ICU COVID-19 floor in our hospital. Asymptomatic, non-hospitalised controls were recruited as a comparator group for biomarkers that did not have a reference range. We assessed markers of endothelial cell and platelet activation, including von Willebrand Factor (VWF) antigen, soluble thrombomodulin, soluble P-selectin, and soluble CD40 ligand, as well as coagulation factors, endogenous anticoagulants, and fibrinolytic enzymes. We compared the level of each marker in ICU patients, non-ICU patients, and controls, where applicable. We assessed correlations between these laboratory results with clinical outcomes, including hospital discharge and mortality. Kaplan-Meier analysis was used to further explore the association between biochemical markers and survival., Findings: 68 patients with COVID-19 were included in the study from April 13 to April 24, 2020, including 48 ICU and 20 non-ICU patients, as well as 13 non-hospitalised, asymptomatic controls. Markers of endothelial cell and platelet activation were significantly elevated in ICU patients compared with non-ICU patients, including VWF antigen (mean 565% [SD 199] in ICU patients vs 278% [133] in non-ICU patients; p<0·0001) and soluble P-selectin (15·9 ng/mL [4·8] vs 11·2 ng/mL [3·1]; p=0·0014). VWF antigen concentrations were also elevated above the normal range in 16 (80%) of 20 non-ICU patients. We found mortality to be significantly correlated with VWF antigen (r = 0·38; p=0·0022) and soluble thrombomodulin (r = 0·38; p=0·0078) among all patients. In all patients, soluble thrombomodulin concentrations greater than 3·26 ng/mL were associated with lower rates of hospital discharge (22 [88%] of 25 patients with low concentrations vs 13 [52%] of 25 patients with high concentrations; p=0·0050) and lower likelihood of survival on Kaplan-Meier analysis (hazard ratio 5·9, 95% CI 1·9-18·4; p=0·0087)., Interpretation: Our findings show that endotheliopathy is present in COVID-19 and is likely to be associated with critical illness and death. Early identification of endotheliopathy and strategies to mitigate its progression might improve outcomes in COVID-19., Funding: This work was supported by a gift donation from Jack Levin to the Benign Hematology programme at Yale, and the National Institutes of Health., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Context.—: Developing skills related to use of computer-based tools is critical for practicing genomic pathology. However, given the relative novelty of genomics education, residency programs may lack faculty members with adequate expertise and/or time to implement training. A virtual team-based learning (TBL) environment would make genomic pathology education available to more trainees., Objective.—: To translate an extensively implemented in-person TBL genomic pathology workshop into a virtual environment and to evaluate both knowledge and skill acquisition., Design.—: Using a novel interactive simulation approach, online modules were developed translating aspects of the TBL experience into the virtual environment with a goal of acquisition of necessary computer-related skills. The modules were evaluated at 10 postgraduate pathology training programs using a pre-post test design with participants deidentified. A postmodule anonymous survey obtained participant feedback on module quality and efficacy., Results.—: There were 147 trainees who received an email request to voluntarily participate in the study. Of these, 43 trainees completed the pretest and 15 (35%) subsequently completed the posttest. Mean overall scores were 45% on the pretest compared with 70% on the posttest ( P < .001; effect size = 1.4). Posttest improvement of results was similar for questions testing acquisition of knowledge versus skills. Regarding the 19 participants who took the survey, 18 (95%) would recommend the modules to others and believed they met the stated objectives., Conclusions.—: A simulation-based approach allows motivated pathology trainees to acquire computer-related skills for practicing genomic pathology. Future work can explore efficacy in a nonvoluntary setting and adaptation to different specialties, learners, and computer tools.

Most fibrinogen replacement strategies focus on quantitative deficiencies. A thrombin time (TT) mixing study helped to assess qualitative defects caused by dysfibrinogens. Plasma samples were collected from non-anticoagulated subjects (n=6) meeting laboratory criteria for suspected dysfibrinogenaemia (TT > 22 s; fibrinogen activity <180) and from a control group. TT mixing studies were performed on subject plasma with increasing volumes of pooled normal plasma at 1:2, 1:4 and 1:5 dilutions. No subjects with dysfibrinogenaemia demonstrated a complete TT correction at 1:2, but 50% corrected at 1:4 and 100% at 1:5 dilution. Based on these data, a correction factor (CF), defined as the reciprocal dilution yielding complete correction, was incorporated into our clinical practice formula for fibrinogen dosing in patients with dysfibrinogenaemias. Our study incorporates TT mixing studies for assessment of dysfibrinogens. The addition of a mix-derived CF to classical formulae may better approximate dosing in patients with dysfibrinogenaemia., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2018. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: To increase awareness of potential diagnostic test interference associated with alemtuzumab, which is a therapeutic immunoglobulin G1 κ monoclonal antibody used in hematologic malignancies, autoimmune diseases, and transplant-related disorders., Methods: Bone marrow and blood from patients with T-cell prolymphocytic leukemia treated with alemtuzumab were evaluated by flow cytometry. Healthy donor blood was analyzed with or without in vitro treatment with alemtuzumab for comparison., Results: Immunophenotypic analysis of bone marrow collected 4 weeks after alemtuzumab treatment demonstrated artifactual surface κ light chain restriction in CD19+ B cells and CD3+ T cells. Similar findings were observed in blood from another patient in a specimen collected 3 days after alemtuzumab treatment. These findings were recapitulated in healthy donor blood incubated with alemtuzumab., Conclusions: Alemtuzumab can produce direct interference during flow cytometry analysis, resulting in false-positive evidence of light chain clonality. Clinicians and laboratorians should be cognizant of this risk to avoid misdiagnosis of B-cell neoplasms.

Purpose: Postoperative cognitive dysfunction (POCD) occurs frequently after cardiac surgery. The pathophysiology of POCD remains elusive, but previous work showed that intravenous lidocaine may be protective against POCD, possibly by modulating cerebral inflammation. We hypothesized that intravenous lidocaine would attenuate the cerebral inflammatory response to cardiopulmonary bypass (CPB) by reducing the transcerebral activation gradients of platelets, leukocytes, and/or platelet-leukocyte conjugates., Methods: We studied 202 patients undergoing cardiac surgery with CPB in this prospective randomized double-blinded placebo-controlled trial. Subjects were randomized to receive either intravenous lidocaine (bolus + 48-hr infusion) or placebo (identical infusion volume and duration). Paired jugular venous and radial arterial blood samples were drawn at several time points and analyzed by fluorescence-activated cell sorting to identify activated platelets and platelet-leukocyte conjugates. Transcerebral activation gradients were calculated by subtracting arterial values from venous values and were compared between groups using repeated measures regression models with covariate adjustment for age, sex, surgery type, and CPB duration., Results: Beginning after aortic cross-clamp release and peaking ten minutes after the termination of CPB, the mean (SD) transcerebral activation gradient of platelet-monocyte conjugates decreased in lidocaine-treated vs placebo-treated patients [-1.84 (11.47) mean linear fluorescence intensity (MLFI) vs 1.46 (13.88) MLFI, respectively; mean difference, -4.08 MLFI; 95% confidence interval, -7.86 to -0.29; P = 0.03). No difference was seen at any time point for activated platelets or for platelet-neutrophil conjugates., Conclusion: While lidocaine did not affect the systemic or transcerebral activation of platelets or leukocytes, we did observe a reduction in the transcerebral activation of platelet-monocyte conjugates after aortic cross-clamp release. This may be a manifestation of reduced cerebral inflammation during cardiopulmonary bypass in response to treatment with lidocaine. This trial was registered at ClinicalTrials.gov (NCT00938964).

Objectives: The biannual Fellow In-Service Hematopathology Examination (FISHE) assesses knowledge in five content areas. We examined the relationship between taking the FISHE and performance on it with outcomes on the first attempted American Board of Pathology Hematology subspecialty certifying examination (ABP-HE)., Methods: The pass rate between the ABP-HE candidates who took the spring FISHE and those who did not were compared. The likelihood of fellows passing the ABP-HE based on their percentiles on the FISHE was also assessed., Results: ABP-HE candidates who took the spring FISHE had a higher pass rate (96.4%) than those who did not (76.1%, P < .001). Spring FISHE performance, including total percentile and percentiles in four of five FISHE content areas, was only a weak predictor of passing the ABP-HE., Conclusions: Candidates who take the spring FISHE do better on the ABP-HE than those who do not. Most fellows passed the first attempted ABP-HE regardless of FISHE performance. Whether this is due to fellows making use of the FISHE as a self-evaluation tool to help identify and then correct their knowledge deficiencies remains to be determined., (© American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Identification of hemoglobinopathies in pediatric patients can be challenging and has important implications for the patient, as well as family members. Laboratory identification of uncommon hemoglobin (Hb) variants can pose a significant problem. Although many Hb variants can be largely identified using conventional electrophoresis and HPLC, confirmatory Hb DNA analysis may be necessary. This report provides an example of a pediatric patient with a complex heterozygous Hb by electrophoresis and HPLC, which necessitated identification by DNA analysis. Clinical and laboratory scenarios warranting Hb DNA analysis are additionally discussed.

Objective: If we can identify critically ill children at high risk for central venous catheter-related thrombosis, then we could target them for pharmacologic thromboprophylaxis. We determined whether factor VIII activity or G value was associated with catheter-related thrombosis in critically ill children., Design: Prospective cohort study., Setting: Two tertiary academic centers., Patients: We enrolled children younger than 18 years who were admitted to the PICU within 24 hours after insertion of a central venous catheter. We excluded children with a recently diagnosed thrombotic event or those anticipated to receive anticoagulation. Children with thrombosis diagnosed with surveillance ultrasonography on the day of enrollment were classified as having prevalent thrombosis. Those who developed catheter-related thrombosis thereafter were classified as having incident thrombosis., Interventions: None., Measurements and Main Results: We enrolled 85 children in the study. Once enrolled, we measured factor VIII activity with one-stage clotting assay and determined G value with thromboelastography. Of those enrolled, 25 had incident and 12 had prevalent thromboses. The odds ratio for incident thrombosis per SD increase in factor VIII activity was 1.98 (95% CI, 1.10-3.55). The area under the receiver operating characteristic curve was 0.66 (95% CI, 0.52-0.79). At factor VIII activity more than 100 IU/dL, which was the optimal threshold identified using Youden index, sensitivity and specificity were 92.0% and 41.3%, respectively. The association between factor VIII activity and incident thrombosis remained significant after adjusting for important clinical predictors of thrombosis (odds ratio, 1.93; 95% CI, 1.10-3.39). G value was associated with prevalent but not with incident thrombosis., Conclusion: Factor VIII activity may be used to stratify critically ill children based on their risk for catheter-related thrombosis.

Objectives: To determine the current state of pathology resident training in genomic and molecular pathology., Methods: The Training Residents in Genomics (TRIG) Working Group developed survey and knowledge questions for the 2013 pathology Resident In-Service Examination (RISE). Sixteen demographic questions related to amount of training, current and predicted future use, and perceived ability in molecular pathology vs genomic medicine were included, along with five genomic pathology and 19 molecular pathology knowledge questions., Results: A total of 2,506 pathology residents took the 2013 RISE, with approximately 600 individuals per postgraduate year (PGY). For genomic medicine, 42% of PGY-4 respondents stated they had no training, compared with 7% for molecular pathology (P < .001). PGY-4 residents' perceived ability, comfort in discussing results, and predicted future use as a practicing pathologist were reported to be less in genomic medicine than in molecular pathology (P < .001). Based on PGY, knowledge question scores showed a greater increase in molecular pathology than in genomic pathology., Conclusions: The RISE is a powerful tool for assessing the state of resident training in genomic pathology and current results suggest a significant deficit. The results also provide a baseline to assess future initiatives to improve genomics education for pathology residents such as those developed by the TRIG Working Group., (Copyright© by the American Society for Clinical Pathology.)

Objective: To assess the sensitivity and specificity of neutrophil CD64 as a diagnostic marker for clinical sepsis (based on a hematologic score) and as an additional marker with hematologic parameters for culture-proven sepsis in neonates., Study Design: Prospective observational cohort over 18 months in a single-center neonatal intensive care unit., Results: Hematologic and CD64 data were available on 1,156 sepsis evaluations done in 684 infants, of which 411 (36%) instances of positive clinical sepsis were identified. The CD64 index for clinical sepsis had an overall area under the receiver operating characteristic curve of 0.71. An optimum CD64 cut point value of 2.19 for late-onset clinical sepsis was calculated with a sensitivity of 78%, a specificity of 59%, and a negative predictive value of 81%. The birth weight-specific CD64 cut point for early onset clinical sepsis was 3.13, 2.34, and 2.05 for very low, low, and normal birth weight, respectively. Neutrophil CD64, in combination with the absolute neutrophil count or the absolute band count, had the highest sensitivity (91%) and specificity (93%), respectively, to diagnose culture-proven sepsis., Conclusion: We conclude that neutrophil CD64 index can be incorporated with specific hematologic criteria as an additional marker for diagnosis of neonatal sepsis., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Chronic lymphocytic leukemia (CLL) is a monoclonal B-cell lymphoproliferative disorder generally characterized by an indolent clinical course. However, some patients with CLL will have more aggressive disease progression, and identifying that subgroup may be important for early, or perhaps more aggressive, intervention. In addition, monoclonal B-cell lymphocytosis is often found on routine laboratory evaluation, and it is important to distinguish this entity from overt CLL. Moreover, since many patients with CLL are discovered incidentally and before significant disease progression, prognostic laboratory evaluation may become increasingly efficacious as therapeutic options replace the older strategy of expectant observation. Prognostication may be especially critical if it correctly identifies patients with early stage CLL who are at high risk of clonal evolution and/ or resistance to chemoimmunotherapy. Laboratory studies include surface CD38 and intracellular ZAP-70 expression by flow cytometry, serum β2-microglobulin, and immunoglobulin heavy-chain variable gene mutational status. Cytogenetics for targeted chromosome alterations may similarly aid in predicting outcome and guiding early intervention. This article concisely reviews the utility of commonly performed prognostic markers and addresses the laboratory evaluation in patients with incidentally discovered early stage CLL.

We assessed neutrophil CD64 as a diagnostic marker for neonatal sepsis. For early-onset sepsis, the CD64 index with a cut-point value of 2.38 had sensitivity, specificity and negative predictive values of 100%, 68% and 100%, respectively. For late-onset sepsis, the respective values were 3.62, 75%, 77% and 96%. Neutrophil CD64 index can be incorporated as a valuable marker for excluding neonatal sepsis.

Genomic technologies are dramatically changing the practice of medicine. Next-generation sequencing has allowed prognostic stratification of cancer patients, personalized drug therapy and the identification of genetic risk factors for a multitude of diseases. As the physicians who oversee tissue- and laboratory-based diagnostic testing, pathologists must understand and utilize this new technology for the benefit of patients; however, only a minority of pathology residency programs currently provide training in genomics. In response to this urgent need, the Training Residents in Genomics (TRIG) Working Group has made significant progress towards creating, implementing, evaluating and disseminating a national curriculum in genomic pathology. Although presented in the context of pathology training, the approach described in this review can serve as model for education in genomic medicine of students, trainees or professionals in other areas of healthcare.

Background: Male sex chromosome aneuploidies are underdiagnosed despite concomitant physical and behavioral manifestations., Objective: To develop a non-invasive, rapid and high-throughput molecular diagnostic assay for detection of male sex chromosome aneuploidies, including 47,XXY (Klinefelter), 47,XYY, 48,XXYY and 48,XXXY syndromes., Methods: The assay utilizes three XYM and four XA markers to interrogate Y:X and X:autosome ratios, respectively. The seven markers were PCR amplified using genomic DNA isolated from a cohort of 323 males with aneuploid (n = 117) and 46,XY (n = 206) karyotypes. The resulting PCR products were subjected to Pyrosequencing, a quantitative DNA sequencing method., Results: Receiver operator characteristic (ROC) curves were used to establish thresholds for the discrimination of aneuploid from normal samples. The XYM markers permitted the identification of 47,XXY, 48,XXXY and 47,XYY syndromes with 100% sensitivity and specificity in both purified DNA and buccal swab samples. The 48,XXYY karyotype was delineated by XA marker data from 46,XY; an X allele threshold of 43% also permitted detection of 48,XXYY with 100% sensitivity and specificity. Analysis of X chromosome-specific biallelic SNPs demonstrated that 43 of 45 individuals (96%) with 48,XXYY karyotype had two distinct X chromosomes, while 2 (4%) had a duplicate X, providing evidence that 48,XXYY may result from nondisjunction during early mitotic divisions of a 46,XY embryo., Conclusions: Quantitative Pyrosequencing, with high-throughput potential, can detect male sex chromosome aneuploidies with 100% sensitivity.

The Resident In-Service Examination (RISE) addresses 1 area of the Accreditation Council for Graduate Medical Education Outcome Project; RISE results demonstrate progressive attainment of pathology knowledge during training. We compared RISE scores with primary pathology board certification success for residents graduating in 2008 and 2009. Overall RISE and nearly all sectional scores in anatomic and clinical pathology were significantly higher for residents passing all certifying examinations at the first attempt vs residents who failed any examination. The risk of failing increased with each lower quartile of overall RISE score, such that 34% (2009) and 54% (2008) of residents in the lowest quartile failed at least 1 certifying examination. Two thirds of graduating residents with lowest quartile scores had a similar quartile ranking in the previous RISE, identifying them as at risk. Residents passing the American Board of Pathology certifying examinations have a higher level of medical knowledge in general and specific pathology disciplines as assessed by senior RISE scores.

Objective: Turner syndrome (TS) occurs when an X-chromosome is completely or partially deleted or when X-chromosomal mosaicism is present. Girls with TS benefit from early diagnosis and treatment with GH; however, many girls with TS are not detected until after 10 yr of age, resulting in delayed evaluation and treatment., Methods: We developed a high-throughput test for TS, based on a quantitative method of genotyping to detect X-chromosome abnormalities. This test uses pyrosequencing to quantitate relative allele strength (RAS) from single-nucleotide polymorphisms using 18 informative single-nucleotide polymorphisms markers that span the X-chromosome and one marker for the detection of Y-chromosome material., Results: Cutoff ranges for heterozygous, homozygous, or out-of-range RAS values were established from a cohort of 496 males and females. Positive TS scoring criteria were defined as the presence of homozygosity for all 18 markers or the presence of at least one out-of-range RAS value. To determine the validity of this rapid test for TS detection, we undertook a large-scale study using DNA from 132 females without TS and 74 females with TS for whom karyotypes were available. TS was identified with 96.0% sensitivity and 97.0% specificity in this cohort. We also tested buccal swab DNA from a group of 19 females without TS and 69 females with TS. In this group, TS was identified with 97.1% sensitivity and 84.2% specificity., Conclusions: These results demonstrate the validity of a high-throughput, pyrosequencing based test for the accurate detection of TS, providing a potential alternative to karyotype testing.

As the 100th anniversary of the Flexner report nears, medical student education is being reviewed at many levels. One area of concern, expressed in recent reports from some national health care organizations, is the adequacy of training in the discipline of laboratory medicine (also termed clinical pathology). The Academy of Clinical Laboratory Physicians and Scientists appointed an ad hoc committee to review this topic and to develop a suggested curriculum, which was subsequently forwarded to the entire membership for review. The proposed medical student laboratory medicine curriculum defines goals and objectives for training, provides guidelines for instructional methods, and gives examples of how outcomes can be assessed. This curriculum is presented as a potentially helpful outline for use by medical school faculty and curriculum committees.

Background: Erythropoietin mitigates myocardial damage and improves ventricular performance after experimental ischemic injury. This study assessed safety and efficacy markers relevant to the biological activity of recombinant human erythropoietin (rHuEpo) in patients with acute myocardial infarction (MI)., Methods: We conducted a prospective, placebo-controlled, randomized, double-blind trial to determine the effects of intravenous rHuEpo (200 U/kg daily for 3 consecutive days) on measures of platelet and endothelial cell activation, soluble Fas ligand, and peripheral blood mononuclear cell (PBMC) expression of angiogenesis signaling proteins in 44 subjects with acute MI treated with aspirin and clopidogrel after successful percutaneous coronary intervention., Results: Recombinant human erythropoietin did not alter bleeding time, platelet function assay closure time, von Willebrand factor levels, soluble P-selectin, or soluble Fas ligand levels when compared with placebo. By contrast, rHuEpo significantly increased expression of erythropoietin receptor, vascular endothelial growth factor receptor Flt-1, and phosphorylated phosphatidylinositol 3-kinase in PBMCs when compared with placebo (all Ps < .05)., Conclusions: In acute MI patients treated with aspirin and clopidogrel, short-term administration of rHuEpo did not alter markers of platelet and endothelial cell activation associated with thrombosis, yet did increase expression of angiogenesis signaling proteins in PBMCs when compared with placebo. These data provide preliminary evidence of safety and biologic activity of rHuEpo at this dosing and support continued enrollment in ongoing efficacy trials.

This article briefly details the physiologic and interdependent mechanisms of vascular hemostasis, with an eye toward how the laboratory can assist in diagnosing and maintaining the balance of procoagulant and anticoagulant functions. These functions include determining characteristics of the blood vessel wall, platelet components and receptor-ligand interactions critical for hemostasis, the regulation of thrombin generation and its effects, and the complex fibrinolytic pathways that complete the coagulation cascade.