In patients with immune thrombotic thrombocytopenic purpura (iTTP), autoantibodies against the metalloprotease ADAMTS13 lead to catastrophic microvascular thrombosis. However, the potential benefits of recombinant human ADAMTS13 (rADAMTS13) in patients with iTTP remain unknown. Here, we report the clinical use of rADAMTS13, which resulted in the rapid suppression of disease activity and complete recovery in a critically ill patient whose condition had proved to be refractory to all available treatments. We also show that rADAMTS13 causes immune complex formation, which saturates the autoantibody and may promote its clearance. Our data support the role of rADAMTS13 as a novel adjunctive therapy in patients with iTTP.

There is widespread interest in identifying interventions that extend healthy lifespan. Chronic continuous hypoxia delays the onset of replicative senescence in cultured cells and extends lifespan in yeast, nematodes, and fruit flies. Here, we asked whether chronic continuous hypoxia is beneficial in mammalian aging. We utilized the Ercc1 Δ/- mouse model of accelerated aging given that these mice are born developmentally normal but exhibit anatomic, physiological, and biochemical features of aging across multiple organs. Importantly, they exhibit a shortened lifespan that is extended by dietary restriction, the most potent aging intervention across many organisms. We report that chronic continuous 11% oxygen commenced at 4 weeks of age extends lifespan by 50% and delays the onset of neurological debility in Ercc1 Δ/- mice. Chronic continuous hypoxia did not impact food intake and did not significantly affect markers of DNA damage or senescence, suggesting that hypoxia did not simply alleviate the proximal effects of the Ercc1 mutation, but rather acted downstream via unknown mechanisms. To the best of our knowledge, this is the first study to demonstrate that "oxygen restriction" can extend lifespan in a mammalian model of aging.

Examination of red blood cell (RBC) morphology in peripheral blood smears can help diagnose hematologic diseases, even in resource-limited settings, but this analysis remains subjective and semiquantitative with low throughput. Prior attempts to develop automated tools have been hampered by their poor reproducibility and limited clinical validation. Here, we present a novel, open-source machine-learning approach (denoted as RBC-diff) to quantify abnormal RBCs in peripheral smear images and generate an RBC morphology differential. RBC-diff cell counts showed high accuracy for single-cell classification (mean AUC, 0.93) and quantitation across smears (mean R2, 0.76 compared with experts, interexperts R2, 0.75). RBC-diff counts were concordant with the clinical morphology grading for 300 000+ images and recovered the expected pathophysiologic signals in diverse clinical cohorts. Criteria using RBC-diff counts distinguished thrombotic thrombocytopenic purpura and hemolytic uremic syndrome from other thrombotic microangiopathies, providing greater specificity than clinical morphology grading (72% vs 41%; P < .001) while maintaining high sensitivity (94% to 100%). Elevated RBC-diff schistocyte counts were associated with increased 6-month all-cause mortality in a cohort of 58 950 inpatients (9.5% mortality for schist. >1%, vs 4.7% for schist; <0.5%; P < .001) after controlling for comorbidities, demographics, clinical morphology grading, and blood count indices. RBC-diff also enabled the estimation of single-cell volume-morphology distributions, providing insight into the influence of morphology on routine blood count measures. Our codebase and expert-annotated images are included here to spur further advancement. These results illustrate that computer vision can enable rapid and accurate quantitation of RBC morphology, which may provide value in both clinical and research contexts.

Microscopic examination of red blood cell (RBC) morphology in a peripheral blood smear is a standard clinical test with broad utility even in resource-limited settings. Yet this analysis remains subjective, semi-quantitative, and low throughput, limiting its potential in clinical practice and translational research. Prior attempts to develop quantitative RBC morphology tools have been hampered by limited scope, reproducibility challenges, and sparse clinical validation. Here, we present a novel, open-source machine-learning approach leveraging durable geometric features to quantify clinically relevant RBC morphologies-elliptocytes, microcytes, macrocytes, schistocytes, sickle cells, spiculated cells, and teardrop cells-and generate an RBC differential ('RBC-diff'), analogous to the widely used white cell differential. Analysis of >300 thousand smear images across two medical centers shows algorithm concordance with expert human morphology estimates at both the individual cell and full-field smear level, and when compared to standard clinical grading of smears. This algorithm illustrates high intra-sample reproducibility while providing greatly improved speed and resolution comparative to current standards. To test the utility of this new tool we examined: (i) diagnostic signatures in the RBC differential, (ii) large-scale cohort prognostics, (iii) single-cell mechanistic studies, and (iv) an animal model of hematologic disease. Diagnostic RBC differential signatures were discovered by applying the RBC-diff to a cohort of patients with thrombotic microangiopathy (TMA). In particular, the RBC-diff facilitated rapid identification and differential diagnosis of patients with thrombotic thrombocytopenic purpura (TTP) or hemolytic uremic syndrome (HUS), providing greater specificity than manual review (72% vs. 41%, p < 0.001), while maintaining high sensitivity (94-100%). Because TTP is a life-threatening disorder requiring immediate therapy, this new rapid diagnostic tool may provide significant benefit, as existing gold-standard diagnostic tests for TTP are slow (hours to days). Large-scale cohort prognostic value was uncovered through an analysis of smears from a general cohort of 58,950 inpatients that found that elevated schistocyte counts are associated with increased all-cause mortality (9.5% six-month mortality for >1% schistocytes vs. 4.7% for schistocytes < 0.5%, p < 0.001), after adjusting for patient demographics, comorbidities, and manual smear review flags. This tool also allowed for analysis of morphologically annotated single-cell volume distributions, providing insights into how dysmorphology can affect mean corpuscular volume (MCV) and the red cell distribution width (RDW); note that current technology provides only whole-population measures for these variables. For example, in iron-deficient patients who underwent intravenous iron transfusions, this technique highlighted that successful hematologic resolution involved volume increases for all cell types, rather than an isolated reduction in microcyte levels. Finally, without any retraining the model was successfully applied to animal-derived blood smears, highlighting expected differences between a wild-type (WT) and sickle cell disease mouse model (SCD) (0% sickle cells for WT, 1.2% for SCD), demonstrating potential value for basic research investigations. Usage of the red cell differential has the potential to greatly benefit hematologic research and clinical practice. Here we present the RBC-diff, an extensively validated method for efficient calculation of this differential from commonly collected blood smear images. As a resource to the community, we provide open-source code for calculating the RBC-diff. We also provide a repository of 5000 individual RBC images, and 50 full-field smears, each manually labelled by a team of expert pathologists, to facilitate further developments of improved computational methods of RBC classification. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

B Introduction: b Severe COVID-19 has been associated with aberrant coagulation factor activities, particularly in patients with a thrombotic event (TE). This study evaluates a point-of-care (POC), functional, clot-time-based coagulation test to detect the anticoagulant effect of therapeutic unfractionated heparin (UFH) in hospitalized SARS-CoV-2-positive patients who developed a TE. [Extracted from the article] Copyright of Critical Care Medicine is the property of Lippincott Williams & Wilkins and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

The authors declare that there is no conflict of interest. Data sharing is not applicable to this article.

The antioxidant function of the phospholipid hydroperoxide glutathione peroxidase (GPx4) is vital for the homeostasis of many cell types, from neoplastic cells to normal erythroid precursors. However, some functional proteins in erythroid precursors are lost during the development of red blood cells (RBCs); whether GPx4 is maintained as an active enzyme in mature RBCs has remained unclear. Our meta-analyses of existing RBC proteomics and metabolomics studies revealed the abundance of GPx4 to be correlated with lipid-anchored proteins. In addition, GPx4 anti-correlated with lyso-phospholipids and complement system proteins, further supporting the presence of active GPx4 in mature RBCs. To test the potential biological relevance of GPx4 in mature RBCs, we correlated the rate of hemolysis of human RBCs during storage with the abundance of GPx4 and other heritable RBC proteins. Of the molecules that anti-correlated with the rate of hemolysis of RBCs, proteins that mediate the cellular response to hydroperoxides, including GPx4, have the greatest enrichment. Western blotting further confirmed the presence of GPx4 antigenic protein in RBCs. Using an assay optimized to measure the activity of GPx4 in RBCs, we found GPx4 to be an active enzyme in mature RBCs, suggesting that GPx4 protects RBCs from hemolysis during blood bank storage.

Autoimmune hemolytic anemia (AIHA) results in red blood cell destruction by auto-antibodies directed against surface antigens and is rarely fatal. Here we describe a case of AIHA, refractory to both standard and experimental therapies, complicated by multiorgan failure, and rapidly leading to death.A 65 year-old man who presented with progressive dyspnea and jaundice was found to have hemolytic anemia. Diagnostic work-up revealed a positive direct antiglobulin test and a strong pan-reactive antibody in the plasma reacting to a titer of 1:1024 with strongest reactivity at 37 °C Coombs' phase with reagent anti-IgG. The red cell eluate contained a pan-agglutinin. The patient received multiple lines of treatment including glucocorticoids, intravenous immunoglobulin, rituximab, eculizumab, splenectomy and etoposide. Despite these interventions, he continued to experience brisk hemolysis and remained transfusion dependent. Repeat testing on day 16 demonstrated persistent high titer IgG auto-antibodies, suggesting minimal suppressive effect of therapy. His course was complicated by acute renal and liver failure, venous thrombosis, and worsening coagulopathy, and he ultimately died from multiorgan failure on day 18.Severe cases of AIHA can result in multiorgan failure and a fatal outcome. The rapid development of liver failure in this setting has been described in only few case reports to date, and represents an important complication for clinicians to be aware of when treating patients with AIHA.

Introduction: Severe COVID-19 has been associated with aberrant coagulation factor activities, particularly in patients with a thrombotic event (TE). Management of anticoagulant is critical in the care of hospitalized patients with COVID-19. Hypothesis: Evaluation of a point-of-care (POC), functional, clot-time-based coagulation test to detect the anticoagulant effect of therapeutic unfractionated heparin (UFH) in hospitalized SARS-CoV-2-positive patients who developed a TE. Methods: An IRB-approved analysis of 36 citrated plasma specimens from 26 SARS-CoV-2-positive patients and 10 matched negative controls was performed. A Clotting Time Score (CTS), a measure of factor-specific inhibition (i.e. anticoagulant activity), was derived for each patient. CTS results were compared with traditional coagulation tests. Five UFH COVID-19 samples with low CTS scores (<10) were spiked with uniform dosing of UFH, low molecular weight heparin (LMWH), apixaban, or argatroban and retested to assess anticoagulation response. Results: The CTS detected sub-therapeutic UFH anticoagulation levels more frequently in COVID-19 cases compared with controls (76% vs. 17%). Prothrombin Times, activated Partial Thromboplastin Times, anti-Xa levels, and antithrombin activity did not correlate with each other or with the CTS in the COVID-19 samples. CTS correlated with both FV and Factor X activity (R 2 =0.49, Spearman R=-0.68), which form the prothrombinase complex. The CTS was 94% sensitive and 67% specific for the occurrence of TEs in patients on UFH. CTS demonstrated a consistent anticoagulant response only to argatroban (100%) compared with other anticoagulants (60%). Conclusions: The CTS, generated using a novel, low-volume, rapid POC coagulation test is a strong indicator of the therapeutic effect of UFH anticoagulation in COVID-19 patients and may provide a predictive measure of TEs potentially occurring from anticoagulation resistance.

International Journal of Laboratory HematologyVolume 42, Issue 6 p. e248-e251 LETTER TO THE EDITOR Peripheral blood morphologic findings in patients with COVID-19 Sam Sadigh, Sam Sadigh orcid.org/0000-0003-2557-7415 Pathology Department, Massachusetts General Hospital, Boston, Massachusetts, USASearch for more papers by this authorLucas R. Massoth, Lucas R. Massoth Pathology Department, Massachusetts General Hospital, Boston, Massachusetts, USASearch for more papers by this authorBianca B. Christensen, Bianca B. Christensen Pathology Department, Massachusetts General Hospital, Boston, Massachusetts, USASearch for more papers by this authorJonathan A. Stefely, Jonathan A. Stefely Pathology Department, Massachusetts General Hospital, Boston, Massachusetts, USASearch for more papers by this authorJoan Keefe, Joan Keefe Pathology Department, Massachusetts General Hospital, Boston, Massachusetts, USASearch for more papers by this authorAliyah R. Sohani, Corresponding Author Aliyah R. Sohani [email protected] orcid.org/0000-0002-6307-4854 Pathology Department, Massachusetts General Hospital, Boston, Massachusetts, USA Correspondence Aliyah R. Sohani, Department of Pathology, Massachusetts General Hospital, 55 Fruit Street, Warren 219, Boston, MA, 02114-2696. Email: [email protected]Search for more papers by this author Sam Sadigh, Sam Sadigh orcid.org/0000-0003-2557-7415 Pathology Department, Massachusetts General Hospital, Boston, Massachusetts, USASearch for more papers by this authorLucas R. Massoth, Lucas R. Massoth Pathology Department, Massachusetts General Hospital, Boston, Massachusetts, USASearch for more papers by this authorBianca B. Christensen, Bianca B. Christensen Pathology Department, Massachusetts General Hospital, Boston, Massachusetts, USASearch for more papers by this authorJonathan A. Stefely, Jonathan A. Stefely Pathology Department, Massachusetts General Hospital, Boston, Massachusetts, USASearch for more papers by this authorJoan Keefe, Joan Keefe Pathology Department, Massachusetts General Hospital, Boston, Massachusetts, USASearch for more papers by this authorAliyah R. Sohani, Corresponding Author Aliyah R. Sohani [email protected] orcid.org/0000-0002-6307-4854 Pathology Department, Massachusetts General Hospital, Boston, Massachusetts, USA Correspondence Aliyah R. Sohani, Department of Pathology, Massachusetts General Hospital, 55 Fruit Street, Warren 219, Boston, MA, 02114-2696. Email: [email protected]Search for more papers by this author First published: 30 July 2020 https://doi.org/10.1111/ijlh.13300Citations: 17Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat REFERENCES 1Zhu N, Zhang D, Wang W, et al A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 2020; 382(8): 727–733. 10.1056/NEJMoa2001017 CASPubMedWeb of Science®Google Scholar 2Fan BE, Chong VCL, Chan SSW, et al Hematologic parameters in patients with COVID-19 infection. 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Am J Hematol. 2020; 95(7): 861–862. 10.1002/ajh.25834 CASPubMedWeb of Science®Google Scholar 8Jiang S, Hillyer C, Du L. Neutralizing Antibodies against SARS-CoV-2 and Other Human Coronaviruses. Trends Immunol. 2020; 41: 355–359. 10.1016/j.it.2020.03.007 CASPubMedWeb of Science®Google Scholar 9Terpos E, Ntanasis-Stathopoulos I, Elalamy I, et al Hematological findings and complications of COVID-19. Am J Hematol. 2020; 95(7): 834–847. 10.1002/ajh.25829 CASPubMedWeb of Science®Google Scholar 10Arachchillage DR, Laffan M. Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia. J Thromb Haemost. 2020; 18(5): 1233–1234. 10.1111/jth.14820 CASPubMedWeb of Science®Google Scholar 11Zuo Y, Yalavarthi S, Shi H, et al Neutrophil extracellular traps in COVID-19. JCI Insight. 2020; 5(11):e138999. PubMedWeb of Science®Google Scholar Citing Literature Volume42, Issue6December 2020Pages e248-e251 ReferencesRelatedInformation

Haemophagocytic lymphohistiocytosis (HLH) can be a rapidly fatal disease. Current treatment in adults is extrapolated from the HLH-2004 protocol that specifies a regimen of etoposide, dexamethasone and cyclosporine. However, HLH presents as a spectrum of disease severity. A therapeutic challenge arises for milder cases where the harms of potent chemotherapy such as etoposide may outweigh its benefit. We present a case of an adult with HLH who developed significant pancytopenia but was otherwise not critically ill and who responded to treatment with a chemotherapy-sparing approach consisting of intravenous immunoglobulins and corticosteroids alone. The case illustrates that tailored therapy may allow effective treatment of the disorder while minimising therapy-related toxicities.

An asymptomatic 47-year-old woman with a history of recurrent melanoma presented to the hospital after routine quarterly surveillance imaging, performed after resection of right axillary melanoma, radiation therapy, and pembrolizumab therapy, had revealed new hilar and mediastinal lymphadenopathy and new pulmonary nodules. Diagnostic tests were performed.

BACKGROUND:The in vivo recovery of transfused platelets is variable and often unpredictable. Although many recipient-dependent factors are well described, donor-dependent variables remain poorly understood.STUDY DESIGN AND METHODS:To explore donor-dependent variables we conducted 2 retrospective studies of platelet transfusion outcomes in repeat donors. One study analyzed multiple autologous, radiolabeled platelet transfusions, and a second study analyzed multiple clinical platelet transfusions from a small cohort of repeat donors.RESULTS:In 36 subjects, multiple within-subject determinations of recovery and survival of radiolabeled autologous platelets revealed a relative consistency in platelet recoveries within donors compared to the range of recoveries among donors. Intraclass correlation coefficients for platelet recovery were 43% to 93%. In 524 ABO-compatible clinical platelet transfusions derived from seven donors, a linear mixed-effects model revealed significant donor-dependent differences in corrected count increments for units stored for 4 or 5 days.CONCLUSIONS:These two studies indicate reproducible donor-dependent differences in transfused platelet recovery, suggesting a possible heritable influence on the quality of transfused platelets.

Coagulopathy causes morbidity and mortality in patients with coronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Yet, the mechanisms are unclear and biomarkers are limited. Early in the pandemic, we observed markedly elevated factor V activity in a patient with COVID-19, which led us to measure factor V, VIII, and X activity in a cohort of 102 consecutive inpatients with COVID-19. Contemporaneous SARS-CoV-2-negative controls (n = 17) and historical pre-pandemic controls (n = 260-478) were also analyzed. This cohort represents severe COVID-19 with high rates of ventilator use (92%), line clots (47%), deep vein thrombosis or pulmonary embolism (DVT/PE) (23%), and mortality (22%). Factor V activity was significantly elevated in COVID-19 (median 150 IU/dL, range 34-248 IU/dL) compared to contemporaneous controls (median 105 IU/dL, range 22-161 IU/dL) (P < .001)-the strongest association with COVID-19 of any parameter studied, including factor VIII, fibrinogen, and D-dimer. Patients with COVID-19 and factor V activity >150 IU/dL exhibited significantly higher rates of DVT/PE (16/49, 33%) compared to those with factor V activity ≤150 IU/dL (7/53, 13%) (P = .03). Within this severe COVID-19 cohort, factor V activity associated with SARS-CoV-2 load in a sex-dependent manner. Subsequent decreases in factor V were linked to progression toward DIC and mortality. Together, these data reveal marked perturbations of factor V activity in severe COVID-19, provide links to SARS-CoV-2 disease biology and clinical outcomes, and nominate a candidate biomarker to investigate for guiding anticoagulation therapy in COVID-19.

Macroautophagy/autophagy is suppressed by MTOR (mechanistic target of rapamycin kinase) and is an anticancer target under active investigation. Yet, MTOR-regulated autophagy remains incompletely mapped. We used proteomic profiling to identify proteins in the MTOR-autophagy axis. Wild-type (WT) mouse cell lines and cell lines lacking individual autophagy genes (Atg5 or Ulk1/Ulk2) were treated with an MTOR inhibitor to induce autophagy and cultured in media with either glucose or galactose. Mass spectrometry proteome profiling revealed an elevation of known autophagy proteins and candidates for new autophagy components, including CALCOCO1 (calcium binding and coiled-coil domain protein 1). We show that CALCOCO1 physically interacts with MAP1LC3C, a key protein in the machinery of autophagy. Genetic deletion of CALCOCO1 disrupted autophagy of the endoplasmic reticulum (reticulophagy). Together, these results reveal a role for CALCOCO1 in MTOR-regulated selective autophagy. More generally, the resource generated by this work provides a foundation for establishing links between the MTOR-autophagy axis and proteins not previously linked to this pathway. Abbreviations: ATG: autophagy-related; CALCOCO1: calcium binding and coiled-coil domain protein 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain protein 2; CLIR: MAP1LC3C-interacting region; CQ: chloroquine; KO: knockout; LIR: MAP1LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLN: MLN0128 ATP-competitive MTOR kinase inhibitor; MTOR: mechanistic target of rapamycin kinase; reticulophagy: selective autophagy of the endoplasmic reticulum; TAX1BP1/CALCOCO3: TAX1 binding protein 1; ULK: unc 51-like autophagy activating kinase; WT: wild-type.

Background Dual degree program MD/PhD candidates typically train extensively in basic science research and in clinical medicine, but often receive little formal experience or mentorship in clinical and translational research. Methods To address this educational and curricular gap, the University of Wisconsin Medical Scientist Training Program partnered with the University of Wisconsin Institute for Clinical and Translational Research to create a new physician-scientist preceptorship in clinical and translational research. This six-week apprentice-style learning experience—guided by a physician-scientist faculty mentor—integrates both clinical work and a translational research project, providing early exposure and hands-on experience with clinically oriented research and the integrated career of a physician-scientist. Five years following implementation, we retrospectively surveyed students and faculty members to determine the outcomes of this preceptorship. Results Over five years, 38 students and 36 faculty members participated in the physician-scientist preceptorship. Based on student self-assessments ( n = 29, response rate 76%), the course enhanced competency in conducting translational research and understanding regulation of clinical research among other skills. Mentor assessments ( n = 17, response rate 47%) supported the value of the preceptorship in these same areas. Based on work during the preceptorship, half of the students produced a peer-reviewed publication or a meeting abstract. At least eleven peer-reviewed manuscripts were generated. The preceptorship also provided a structure for physician-scientist mentorship in the students’ clinical specialty of choice. Conclusion The physician-scientist preceptorship provides a new curricular model to address the gap of clinical research training and provides for mentorship of physician-scientists during medical school. Future work will assess the long-term impact of this course on physician-scientist career trajectories.

Cells adapt to nutrient and energy deprivation by inducing autophagy, which is regulated by the mammalian target of rapamycin (mTOR) and AMP-activated protein kinases (AMPKs). We found that cell metabolism significantly influences the ability to induce autophagy, with mitochondrial complex I function being an important factor in the initiation, amplitude, and duration of the response. We show that phenformin or genetic defects in complex I suppressed autophagy induced by mTOR inhibitors, whereas autophagy was enhanced by strategies that increased mitochondrial metabolism. We report that mTOR inhibitors significantly increased select phospholipids and mitochondrial-associated membranes (MAMs) in a complex I-dependent manner. We attribute the complex I autophagy defect to the inability to increase MAMs, limiting phosphatidylserine decarboxylase (PISD) activity and mitochondrial phosphatidylethanolamine (mtPE), which support autophagy. Our data reveal the dynamic and metabolic regulation of autophagy.