Microangiopathic thrombocytopenia caused by vitamin B12 deficiency responding to plasma exchange.

A young adult African American female presented with normocytic microangiopathic haemolytic anaemia, elevated lactate dehydrogenase and thrombocytopenia. The patient responded to therapeutic plasma exchanges (TPE) for presumed thrombotic microangiopathy caused by thrombotic thrombocytopenic purpura (TTP). After relapsing, the patient was found to have pancytopenia, megaloblastic bone marrow and low vitamin B12 consistent with pernicious anaemia, which improved with intramuscular B12 and discontinuation of TPE. B12-deficient macrocytosis was not seen at presentation due to concomitant alpha-thalassaemia. Initial clinical/laboratory improvement is attributed to B12 present in TPE plasma. B12 deficiency can mimic TTP. Vigilance is needed regarding atypical presentations of pernicious anaemia.

Smart and Fast Blood Counting of Trace Volumes of Body Fluids from Various Mammalian Species Using a Compact, Custom-Built Microscope Cytometer.

We report an accurate method to count red blood cells, platelets, and white blood cells, as well as to determine hemoglobin in the blood of humans, horses, dogs, cats, and cows. Red and white blood cell counts can also be performed on human body fluids such as cerebrospinal fluid, synovial fluid, and peritoneal fluid. The approach consists of using a compact, custom-built microscope to record large field-of-view, bright-field, and fluorescence images of samples that are stained with a single dye and using automatic algorithms to count blood cells and detect hemoglobin. The total process takes about 15 min, including 5 min for sample preparation, and 10 min for data collection and analysis. The minimum volume of blood needed for the test is 0.5 µL, which allows for minimally invasive sample collection such as using a finger prick rather than a venous draw. Blood counts were compared to gold-standard automated clinical instruments, with excellent agreement between the two methods as determined by a Bland-Altman analysis. Accuracy of counts on body fluids was consistent with hand counting by a trained clinical lab scientist, where our instrument demonstrated an approximately 100-fold lower limit of detection compared to current automated methods. The combination of a compact, custom-built instrument, simple sample collection and preparation, and automated analysis demonstrates that this approach could benefit global health through use in low-resource settings where central hematology laboratories are not accessible.

Diagnosing B-cell acute lymphoblastic leukemia in 2 pediatric patients with recent SARS-CoV-2 infection.

COVID-19 infection is still a mystery in terms of its long-term effect on health and its consequences on hematological disorders. Prior studies including ours have shown the abnormal changes in hematopoietic cells in COVID-19 patients. In this article, we are presenting 2 cases of pediatric B-lymphoblastic leukemia (B-ALL) with a previous history of COVID-19 infection. The first case describes a 22-month-old boy presenting with lymphadenopathy, neutropenia, and anemia with concurrent COVID-19 infection without any evidence of a hematolymphoid neoplasm as per bone marrow and lymph node biopsy. However, he presented after 2 months with bone marrow biopsy confirming B-ALL. The second case is that of a 4-year-old girl presenting with B-ALL who has had asymptomatic COVID-19 infection 5 months before this current presentation. Both the cases had complete resolution of COVID-19 infection during the time of presentation with acute leukemia. There were notably 2 rare findings along the course of the patients' illnesses. First, the unusual plasmacytosis in the marrow during active COVID-19 infection in the first patient and the second, is predilection of development of B-ALL following COVID-19. In both the cases the fluorescence in situ hybridization (FISH) studies showed pathologic alteration of the RUNX1 gene. Overall, there are no literature to support a causal association between acute B-ALL and COVID-19. The diagnosis of B-ALL in these patients after COVID-19 infection may be totally unrelated. However, if we consider Greaves proposed 2-hit model for childhood acute leukemia, that an infectious agent can precipitate development of B-ALL in a genetically susceptible individual. Alteration of the RUNX1 gene in both the patients, opens a door for further exploration of the "second-hit" hypothesis regarding an infectious agent precipitating development of B-ALL in a genetically susceptible individual.

Coagulation profile of liquid-state plasma.

BACKGROUND: Use of liquid plasma (LP) has been reported as early as the mid 1930s. Unlike fresh-frozen plasma (FFP), LP is maintained at 1 to 6°C for up to 40 days after collection and processing. Despite its approved use by the US Food and Drug Administration, the coagulation profile of LP is incompletely described. In this study we evaluate the coagulation profile of LP stored up to 30 days. STUDY DESIGN AND METHODS: LP was prepared by removing plasma from nonleukoreduced whole blood within 24 hours of collection. Three LP units from each ABO group were collected and stored at 1 to 6°C. Plasma aliquots were obtained at Postcollection Days 1 to 5, 10, 15, 20, 25, and 30 and then stored at -70°C. Each aliquot was tested for prothrombin time, activated partial thromboplastin time, and other coagulation and fibrinolytic factors. RESULTS: There was a significant decrease in Factor (F)V, FVII, FVIII, von Willebrand factor (VWF), protein S (PS) activity, and endogenous thrombin potential on Day 15 compared with Day 1. No significant difference was observed for PS antigen, D-dimer, or thrombin-antithrombin complex. At least 50% activity of all measured factors was noted on Day 15, compared to Day 1. Considerable heterogeneity was observed between the different blood groups for FVII, FVIII, and VWF. CONCLUSION: These data demonstrate that LP maintains at least 50% of factor activity and thrombin-generating capacity up to 15 days of refrigerated storage. It may be more appropriate to limit LP storage and supplement with FFP when used for management of massively bleeding patients.

Measuring dabigatran concentrations using a chromogenic ecarin clotting time assay.

BACKGROUND: Clinicians managing patients receiving the direct thrombin inhibitor dabigatran may benefit in being able to determine the amount of drug present in selected situations. This may include assessment of accumulation, concurrent drug interactions, or adequate removal from circulation. The ability to estimate the amount of dabigatran present using the chromogenic ecarin assay (ECA) requires further clarification. OBJECTIVE: To describe the reliability of dabigatran measurements using a chromogenic ECA. METHODS: This was an evaluation of the ECA method that incorporated assessment of imprecision, linearity, accuracy, carryover, and lower limits of detection or blank. Pooled normal plasma enriched with dabigatran at concentrations of 0, 25, 50, 75, 100, 125, 150, 200, 300, 400, and 500 ng/mL were sent blinded to 3 laboratories in the United States to compare our ECA results with those of laboratories reporting dilute thrombin time methods (HEMOCLOT thrombin inhibitor assay) for measuring dabigatran. Trough and peak levels from 35 patients were also compared with mass spectrophotometry for assessing ECA accuracy. RESULTS: The within-run or day-to-day imprecision was less than 10%, with high linearity (R (2) = 0.989) and high degree of accuracy (R (2) = 0.985; slope = 0.908) for levels ranging between 18 and 470 ng/mL and no carryover at 0 ng/mL noted. The ECA approach appeared to be more reliable at lower dabigatran concentrations. CONCLUSIONS: The chromogenic ECA appears to be an effective approach to determine the amount of dabigatran present. Further insights are necessary to determine how it can be used to reduce thromboembolic or bleeding complications in patients receiving dabigatran.

Extraocular muscle enlargement leading to the diagnoses of Burkitt lymphoma and acquired immune deficiency syndrome.

Orbital involvement in nonendemic Burkitt lymphoma is rare. The authors report a unique case of a patient who sought treatment for extraocular muscle enlargement without a concurrent orbital mass, which subsequently led to the diagnoses of Burkitt lymphoma and acquired immune deficiency syndrome in an adult patient. The case report adhered to the principles of the Declaration of Helsinki and Health Insurance Portability and Accountability Act compliance. This single case report was institutional review board exempt, given that it does not meet the definition of human subjects' research.

Evaluation of thrombotic thrombocytopenic purpura and other thrombotic microangiopathies: Lessons learned from a 14-year retrospective study.

INTRODUCTION: Thrombotic thrombocytopenic purpura (TTP) is a clinical thrombotic microangiopathy (TMA) syndrome defined by the pentad of symptoms. Therapeutic plasma exchange with plasma replacement is an ASFA Category I modality that can reduce morbidity and mortality if initiated early. We describe a 14-year review of patients referred for plasma exchange with a suspected diagnosis of TTP. METHODS: For 70 patients referred for urgent plasma exchange, clinical, therapeutic, and laboratory data were retrospectively analyzed, and the diagnosis was determined. RESULTS: Fifteen of the patients were diagnosed with TTP based upon ADAMTS-13 activity with the other 51 patients having other non-TTP TMA diagnoses. The mortality rate was significant for both TTP and non-TTP TMAs. PLASMIC scores were also calculated retrospectively and were noted to have limited value. TMA is a diagnostic challenge and encompasses different syndromes with similar presentations. CONCLUSION: Determining an accurate diagnosis, including prompt ADAMTS-13 testing, makes it possible to initiate appropriate therapy for the multiple different TMAs that can be seen in clinical practice.

Human thrombomodulin knock-in mice reveal differential effects of human thrombomodulin on thrombosis and atherosclerosis.

OBJECTIVE: We sought to develop a murine model to examine the antithrombotic and antiinflammatory functions of human thrombomodulin in vivo. METHODS AND RESULTS: Knock-in mice that express human thrombomodulin from the murine thrombomodulin gene locus were generated. Compared with wild-type mice, human thrombomodulin knock-in mice exhibited decreased protein C activation in the aorta (P<0.01) and lung (P<0.001). Activation of endogenous protein C following infusion of thrombin was decreased by 90% in knock-in mice compared with wild-type mice (P<0.05). Carotid artery thrombosis induced by photochemical injury occurred more rapidly in knock-in mice (12±3 minutes) than in wild-type mice (31±6 minutes; P<0.05). No differences in serum cytokine levels were detected between knock-in and wild-type mice after injection of endotoxin. When crossed with apolipoprotein E-deficient mice and fed a Western diet, knock-in mice had a further decrease in protein C activation but did not exhibit increased atherosclerosis. CONCLUSION: Expression of human thrombomodulin in place of murine thrombomodulin produces viable mice with a prothrombotic phenotype but unaltered responses to systemic inflammatory or atherogenic stimuli. This humanized animal model will be useful for investigating the function of human thrombomodulin under pathophysiological conditions in vivo.

Hidden treasures: Incidental findings in two cases of chronic subdural hematoma.

Background: Extramedullary hematopoiesis (EMH) and plasmacytomas occurring within the cranium are rare entities. Case Description: We review two cases in which patients presented with subdural hematoma and underwent evacuation. On routine histopathologic examination of their membranes, both patients were subsequently found to have focal EMH, as well as a clonal plasma cell proliferation in one case. Conclusion: EMH is rare and usually found in individuals with profound and chronic anemia. However, this entity may be more common in chronic subdural hematomas. Solitary extraosseous plasmacytoma is exceedingly rare in the cranium, and its presence in chronic subdural hematoma membranes is of uncertain significance. The cytokine milieu that promotes organization of chronic subdural hematomas may play a role in the establishment of both of entities in this location.

Leukoerythroblastosis in a Sickle Cell Patient With Pregnancy: An Interesting Peripheral Blood Smear Finding.

The global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shaken the entire world. The social, health and financial impacts of this pandemic are beyond words. We have learnt a lot about this new disease in a short period of time, but still a long road to go to fully determine its pathogenic effect. The primary target of this virus is angiotensin-converting enzyme 2 (ACE2) receptor, which is prevalent in endothelial cells throughout the body. Immunocompromised patients such as patients with sickle cell disease are more vulnerable to severe respiratory infections, including infection with SARS-CoV-2. In addition, sickle cell disease patients are prone to vaso-occlusive crisis, and theoretically SARS-CoV-2 can worsen the situation as it also can cause endothelial dysfunction and thrombosis. Herein, we are sharing an interesting peripheral blood smear finding of an asymptomatic 31-year-old multigravida pregnant female with a history of sickle cell disease and found to have a positive COVID-19 polymerase chain reaction (PCR) test during her third trimester of pregnancy at a routine clinic visit. Two weeks after the initial positive test, she developed nausea, vomiting, constipation and a pain crisis affecting her extremities while her COVID-19 PCR test was still positive. She was hemodynamically stable, and lab workup revealed chronic anemia, leukocytosis with neutrophilia and lymphopenia. Morphologic examination of the peripheral blood smear showed a marked leukoerythroblastosis: rare myeloblasts, sickle cells, markedly abundant nucleated red blood cells (RBCs), metamyelocytes, and many large and giant platelets were seen. In this context, her previous peripheral blood smears (prior to positive COVID-19 test) did not show leukoerythroblastosis. She was managed conservatively with hydration and pain control and delivered at 36 weeks via cesarean section due to pre-term labor and intrauterine growth retardation. The unusual finding of leukoerythroblastosis in a pregnant sickle cell disease patient with an asymptomatic COVID-19 infection indicates further studies to determine its effect on hematopoietic system and elucidate its clinical significance.

Successful use of maternal blood in the management of severe hemolytic disease of the fetus and newborn due to anti-Kp(b).

BACKGROUND: The incidence of hemolytic disease of the fetus and newborn (HDFN) has decreased since the introduction of Rh immunoglobulin prophylaxis in Rh(D)-negative pregnant women. Thus, the relative incidence of rare alloantibody-related HDFN has increased. The lack of available maternally matched red blood cells for transfusion in these cases may create management difficulties. CASE: We report a case of anti-Kp(b) HDFN. Severe fetal anemia required intrauterine transfusion. Difficulty in obtaining Kp(b)-negative blood necessitated using the mother's donated RBCs. CONCLUSION: Severe HDFN with rare antibodies can be managed successfully using maternal blood.

B-cell lymphoma line (Raji) viability and surface marker expression minimally affected by 20- and 25-gauge vitrectomy systems analyzed by flow cytometry.

PURPOSE: The purpose of this study was to evaluate the effect of 20-gauge (20-G) and 25-gauge (25-G) vitrectomy on cell viability and diagnostic yield (surface marker expression) using flow cytometry and human lymphoma cells in culture. METHODS: Cultured human Burkitt lymphoma cells (Raji B-cell lymphoma line) were allocated into five study groups in Roswell Park Memorial Institute media. By using manual aspiration, cells were then processed by aspiration alone, by 20-G vitrectomy at 600 cuts per minute (cpm) and 1,500 cpm, or by 25-G vitrectomy at both 600 and 1,500 cpm. To assess cell viability and cell surface marker expression, samples underwent standard flow cytometry analysis for suspected lymphoma using 7-amino-actinomycin D and antibodies against CD45, CD19, lambda, and kappa light chains. RESULTS: Twenty-five samples were processed after being divided into four vitrectomy groups and one nonvitrectomy group (control). The mean cell viability was 98.5 for both the nonvitrectomized and vitrectomized specimens. The percentage of cells positive for CD45 or kappa light chain was the same in the nonvitrectomized and vitrectomized groups. In addition, the level of expression of these molecules was not significantly different in all five groups. Similarly, no difference was seen for these markers between 20-G and 25-G vitrectomy at either a cut rate of 600 or 1,500 cpm. The percentage positive for CD19 was significantly lower for the 20-G vitrectomy at 1,500 cpm compared with the 25-G vitrectomy at both 600 and 1,500 cpm. Percentage of CD19 cells was greater for the 25-G vitrectomy at 600 cpm than the nonvitrectomy group. CONCLUSION: Compared with simple aspiration, both 20-G and 25-G vitrectomy seem to have no significant effect on cell viability or diagnostic yield for B-cell lymphoma cells (Raji cell line) in suspension based on flow cytometry. Further studies need to be conducted to study and compare 20-G versus 25-G vitrectomy on lymphoma cells in human vitreous or in an animal model.

Polyclonal CD5+/CD19+ B1a lymphocytes after allogeneic stem cell transplantation: a potential diagnostic pitfall.

In adults, B-lymphocytes comprise approximately 10% of circulating lymphocytes. The majority of peripheral B cells are B2 cells ("Mature" B-cells), which function as part of the humoral adaptive immune system. B1 cells ("Innate-like" B cells) are another sub-class of B lymphocytes, considered as innate immune cells with a characteristic phenotype (CD20+, CD27+, CD43+, CD70-, CD11b+, sIgM++, sIgD+) which can be divided into two subtypes; B1a (CD5+): spontaneously produce broadly reactive natural IgM, and B1b (CD5-): can generate T-cell independent, long-lasting IgM. There is very limited data available, indicating a correlation between allogeneic bone marrow transplantation and an increase in B1a cells. Here we present a case of a 17-year-old female with homozygous sickle cell disease (HbSS disease) who underwent hematopoietic stem cell transplant (HSCT). Approximately seven months post-transplant, she was found to have 16% immature mononuclear cells on complete blood count (CBC)-differential report. A follow-up peripheral blood flow cytometry showed that these cells were polyclonal CD5+/CD20+ B-cells, and comprised 66% of lymphocytes. Further workup and follow up failed to reveal any lymphoproliferative disorders. It is important not to misdiagnose these cells as an atypical CD5+ lymphoproliferative disorder. The presence of B1a cells has not been widely reported in non-neoplastic post-stem cell transplanted patients. This case also adds to and expands our knowledge regarding the presence of increased circulating B1a cells after stem cell transplant in a patient with no history of hematological malignancy.

Value of ADAMTS13 activity and inhibitor in the postmortem diagnosis of thrombotic thrombocytopenic purpura.

BACKGROUND AND OBJECTIVES: Thrombotic thrombocytopenic purpura (TTP) is a clinical diagnosis that can be difficult to establish in severely ill patients. We report a case of fulminant TTP in a woman who died before receiving plasma exchange. An autopsy plasma sample was analyzed for ADAMTS13 activity and inhibitor for correlation with the diagnosis of TTP. Recognizing that hemolysis in postmortem blood samples could interfere with ADAMTS13 activity, plasma samples from four additional decedents not suspected of having TTP were analyzed and correlated with their autopsy results. The purpose of this study was to assess whether testing postmortem samples for ADAMTS13 is useful in the postmortem diagnosis of TTP. MATERIAL AND METHODS: Plasma samples from the index case and four non-TTP decedents were analyzed for ADAMTS13 activity, ADAMTS13 inhibitor levels, and plasma free hemoglobin (PFH). Autopsy tissues were evaluated for evidence of platelet microthrombi in all five cases. RESULTS: The ADAMTS13 activity level in the index patient was <4%, and the inhibitor level was 1.0 inhibitor unit. Microthrombi were prominent in the heart, kidneys, pancreas, and adrenal glands, consistent with the clinical diagnosis of TTP. ADAMTS13 activity levels in the four non-TTP decedents ranged from 4 to 82% (3/4 < or = 26%), and inhibitor was present in two of the four samples. Postmortem PFH levels in the four non-TTP decedents ranged from 64 to 3,917 mg/dL. No microthrombi were observed. CONCLUSION: Low postmortem ADAMTS13 activity and evidence of inhibitor can occur in decedents without clinical or histologic evidence of TTP. Postmortem ADAMTS13 activity levels may not be valid in establishing a diagnosis of TTP, and high inhibitor levels in this setting may be related to elevated PFH. Caution must be used in the interpretation of ADAMTS13 testing in the presence of hemolysis.