Biomarkers of coagulation, endothelial, platelet function, and fibrinolysis in patients with COVID-19: a prospective study.

Prospective and sequential evaluation of homeostatic changes leading to thrombosis across COVID 19 disease severity spectrum are limited. In this prospective observational study, haemostasis was evaluated in patients with mild, moderate-severe, and critical COVID-19 infection. Markers of endothelial activation [Soluble thrombomodulin (sTM), von Willebrand Factor (VWF)], platelet activation [Soluble P-selectin, beta-thromboglobulin (BTG)] and global haemostasis [Rotational thromboelastometry (ROTEM)] were evaluated on days 1 and 5 after admission. The study cohort comprised of 100 adult patients (mild = 20, moderate-severe = 22, critical = 58). Sixty-five patients received anticoagulation for 10 (7-14) days. Thrombotic events were seen in 9 patients. In-hospital mortality was 21%. Endothelial activation markers were elevated at baseline in all subgroups, with levels in moderate-severe (sTM = 4.92 ng/ml, VWF = 295 U/dl) [reference-ranges: sTM = 2.26-4.55 ng/ml; Soluble P-selectin = 13.5-31.5 ng/ml; BTG = 0.034-1.99 ng/ml] and critical patients (sTM = 6.07 ng/ml, VWF = 294 U/dl) being significantly higher than in the mild group (sTM = 4.18 ng/ml, VWF = 206 U/dl). In contrast, platelet activation markers were elevated only in critically ill patients at baseline (Soluble P-selectin = 37.3 ng/ml, BTG = 2.51 ng/ml). The critical group had significantly lower fibrinolysis on days 1 and 5 when compared with the moderate-severe arm. COVID-19 infection was associated with graded endothelial activation and lower fibrinolysis that correlated with illness severity.

Cytogenetic profile of 1791 adult acute myeloid leukemia in India.

BACKGROUND: Cytogenetic analysis continues to have an important role in the management of acute myeloid leukemia (AML) because it is essential for prognostication. It is also necessary to diagnose specific categories of AML and to determine the most effective form of treatment. Reports from South Asia are few because the availability of cytogenetic services is relatively limited. METHODS: We performed a retrospective analysis of the cytogenetic findings in adults with AML seen consecutively in a single centre in India. The results were categorised according to the 2022 World Health Organisation (WHO), International Consensus Classification (ICC) and European LeukemiaNet (ELN) classifications. RESULTS: There were 1791 patients aged 18-85 years (median age 42, 1086 males). Normal karyotypes were seen in 646 (36%) patients. The 1145 (64%) abnormal karyotypes comprised 585 (32.7%) with recurrent genetic abnormalities (RGA), 403 (22.5%) with myelodysplasia-related cytogenetic abnormalities (MRC), and 157 (8.8%) with other abnormalities. There were 567 (31.7%) patients with solitary abnormalities and 299 (16.7%) with two abnormalities. Among the 279 (15.6%) patients with ≥ 3 abnormalities, 200 (11.2%) had complex karyotypes (CK) as per the WHO/ICC and 184 (10.3%), as per the ELN definition. There were 158 (8.8%) monosomal karyotypes (MK). Patients with normal karyotypes had a higher median age (45 years) than those with abnormal karyotypes (40 years, p < 0.001), and those with ≥ 3 abnormalities (43 years), than those with fewer abnormalities (39 years, p = 0.005). Patients with CK (WHO/ICC) and monosomal karyotypes had a median age of 48 years. Those with RGA had a lower median age (35 years, p < 0.001) than MRC (46 years) or other abnormalities (44 years). The t(15;17) was the most common abnormality (16.7%),followed by trisomy 8 (11.6%), monosomy 7/del 7q (9.3%), t(8;21) (7.2%), monosomy 5/del 5q (6.7%) and monosomy 17/del 17p (5.2%). CONCLUSION: Our findings confirm the lower age profile of AML in India and show similarities and differences with respect to the frequencies of individual abnormalities compared to the literature. The frequencies of the t(15;17), trisomy 8 and the high-risk abnormalities monosomy 7 and monosomy 5/del 5q were higher, and that of the inv(16), lower than in most reports.

Comparing two techniques of performing an epidural catheter-assisted epidural blood patch using a 20 ml syringe versus a 5 ml syringe and its effect on clotting time, the strength of clot retraction and haemolysis - A prospective in vitro study (EC-EBP study).

Background and Aims: Epidural blood patch (EBP) is performed by injecting autologous blood into the epidural space using a Tuohy needle. Certain clinical scenarios mandate an epidural catheter (EC)-assisted EBP. Collecting blood in a 20-ml versus 5-ml syringe appears to influence the quality of the clot. This in vitro study compared the techniques of performing the EC-assisted EBP using 20-ml versus 5-ml syringe on clotting time (CT), clot retraction (CR) and haemolysis. Methods: This in vitro study was performed in a haematology laboratory. Five consented adult healthy male volunteers donated blood. In the 5-ml syringe technique, blood was injected through an EC, and as it flowed out of the tip, it was collected at the beginning and the end of 1 min. With the 20-ml technique, blood was collected at the beginning and end of the first, second and third minute. The samples were tested for CT, CR and haemolysis by measuring the plasma-free haemoglobin (PFHb). Results: Five injections were made using a 5-ml syringe, and another five with a 20-ml syringe. Injection time was shorter in the 5-ml technique (80.80 ± 5.89 vs. 272 ± 28.4 s, P < 0.0001). With the 20-ml technique, CT progressively increased (>15 min), whereas, with the 5-ml syringe, the CT was normal. Both techniques caused mild, insignificant haemolysis (PFHb >0.005 g/dl), without affecting the quality of CR. Conclusion: EC-assisted EBP using a 5-ml syringe technique shortens the injection time and deposits fresh blood quickly without affecting CT and CR.

Utility of Cryohemolysis Test in the Diagnosis of Hereditary Spherocytosis.

Diagnosis of Hereditary spherocytosis (HS) often requires time-consuming and/or expensive tests. Cryohemolysis test (CHT) is a simple and easy to perform test with high predictive value for HS diagnosis. In this prospective study, we evaluated the diagnostic utility of CHT for the diagnosis of HS. We included 60 suspected HS patients, 18 patients with Autoimmune hemolytic anemia (AIHA) and 120 healthy controls. Among the 60 suspected cases, there were 36 HS cases and 24 with other hemolytic anemias. The mean CHT (%) ± SD for controls, AIHA, other hemolytic anemias, and HS was 6.63 ± 2.79, 6.79 ± 4.36, 6.61 ± 2.76 and 26.7 ± 8.9, respectively. The CHT % was significantly higher in HS group when compared to controls (p = < 0.0001), AIHA (p = < 0.0001) and other hemolytic anemia groups (p = < 0.0001). At a CHT cut off of > 18.3%, the sensitivity, specificity, positive predictive value and negative predictive value for diagnosis of HS in our study were 97.1%, 94.4%, 97.2% and 90.3%, respectively. CHT is a simple and sensitive test for the diagnosis of HS but remains underutilized. The addition of CHT in the diagnostic workup of HS will be very useful, especially in a resource limited setting.

Stability and utility of flow cytometric platelet activation tests: A modality to bridge the gap between diagnostic demand and supply.

Light transmission aggregometry (LTA) is the gold standard for the diagnosis of platelet function disorders (PFDs). The requirement of customized aggregometer, large blood volume, normal platelet count and processing within 4 hours of venipuncture for LTA makes platelet function testing inaccessible to wider population. Flow cytometric platelet activation test (PACT) may overcome these limitations. This study compares the performance of PACT with LTA, characterizes diagnostic patterns of PFDs on PACT and assesses the stability of PACT beyond 4 hours of venipuncture in controls (n = 5) at different temperature conditions. LTA and PACT were performed in 121 healthy controls and 66 patients with suspected PFD. PACT had excellent agreement (kappa = 0.93) with LTA and 94.1% sensitivity, 98.5% specificity. PACT had distinct patterns in Bernard Soulier Syndrome (n = 10), Glanzmann Thrombasthenia (n = 24), δ-granule disorder (n = 7), and other PFDs (n = 12). PACT could assess platelet function in patients (14%) with thrombocytopenia/lipemia wherein LTA was inconclusive. PACT was stable up to 24 hours in samples stored/transported at 2-8◦C. The results of utility and stability are only valid for the specific markers, agonist concentrations, and conditions investigated in this paper. PACT is a useful modality for the diagnosis of PFD, especially in children, thrombocytopenia cases or in the setup where an aggregometer is not readily available.

Utility of International Society on Thrombosis and Hemostasis Bleeding Assessment Tool (ISTH-BAT) in Patients with Inherited Bleeding Disorders: A Cross-Sectional Study from Southern India.

The International Society on Thrombosis and Hemostasis bleeding assessment tool (ISTH-BAT) was developed to record bleeding symptoms and aid in patient diagnosis. This study was done to investigate the utility of ISTH-BAT in patients suspected to have inherited bleeding disorders. This cross-sectional study was conducted in a tertiary care hospital in Southern India over 3 and 1/2 years. A trained investigator administered the ISTH-BAT questionnaire to 432 patients undergoing evaluation for inherited bleeding disorder prior to routine coagulation screening and confirmatory tests and to 131 healthy volunteers as controls. Among patients, 42(9.7%) had primary hemostatic defect, 150(34.7%) had secondary hemostatic defects and 229(53%) had normal screening coagulogram with mean bleeding scores (BS) being 5.9, 6.9, and 4.2 respectively and the proportion of patients with abnormal BS being 69%, 88.7% and 59.4% respectively; the latter qualifying as unknown hemostatic defect. 11(2.5%) with acquired hemostatic defect on workup were excluded. The mean BS was 1.52 among healthy volunteers. Common bleeding patterns were epistaxis (73.8%), cutaneous bleeding (52.4%), hematuria (54.8%), menorrhagia (50%) in primary hemostatic defect; cutaneous bleeding (72%), muscle hematoma (58.7%), hemarthrosis (46.7%), menorrhagia (58.7%) in secondary hemostatic defects and epistaxis (45.9%), cutaneous bleeding (62.4%), menorrhagia (30.7%) in normal screening coagulogram group. Grade of bleeding was mostly 2 and sometimes 4 in primary, 2-4 in secondary and 1-2 in normal screening coagulogram group. ISTH-BAT is a valuable tool to record lifelong bleeding history. The pattern and score give clues regarding the nature and severity of the bleeding disorder.

Laboratory characterization of obligate carriers of type 3 von Willebrand disease with a potential role for Platelet Function Analyzer (PFA-200).

INTRODUCTION: Type 3 von Willebrand disease (VWD) is a rare autosomal recessive disorder characterized by undetectable von Willebrand Antigen (VWF:Ag). Carriers of type 3 VWD carry one null allele and have von Willebrand factor (VWF) at about 50% of normal. The aim of this study was to characterize type 3 VWD carriers and to study the role of Platelet Function Analyzer (PFA-200) in this cohort. METHODS: This was a cross-sectional study where data were collected from carriers (parents/offspring) of type 3 VWD patients and evaluated with activated partial thromboplastin time, factor VIII, blood group, ristocetin cofactor assay (VWF:RCo), VWF:Ag, and closure time on PFA-200 with collagen/epinephrine (COL/EPI), and collagen/ADP (COL/ADP). RESULTS: One hundred carriers were included in the study of which 85 were included for PFA-200 analysis. The mean (SD) of VWF:Ag (IU/ml) and VWF:RCo (IU/ml) was 0.63 (0.24) and 0.61 (0.26), respectively. Among the 100 carriers, based on VWF levels (VWF:Ag and/or VWF:RCo) and bleeding history, there were 7 type 1 VWD, 10 type 2 VWD, 25 borderline VWF (0.30-0.50 IU/ml and no bleeding), and 58 normal VWF (>0.50 IU/ml). PFA-200 was prolonged in 71% of the carriers, all carriers with type 1 and type 2 VWD phenotype, 80% carriers with borderline VWF, and 59% with normal VWF. COL/EPI was more sensitive than COL/ADP and showed better correlation with VWF parameters than COL/ADP. CONCLUSION: Carriers of type 3 VWD can have a variable laboratory phenotype. PFA-200 showed good sensitivity among the carriers at VWF levels <0.50 IU/ml.

Sickle cells are not necessarily protective against falciparum- A case report.

The relation between sickle cell disease (SCD) and malaria is captivating where sickling of the infected red blood cells (RBCs) causes premature hemolysis and parasite death. Although patients with sickle cell trait are relatively protected, malaria can often lead to marked anemia in them due to hemolysis. We report an unusual case of a child with homozygous SCD presenting with falciparum malaria and had hyper parasitemia and severe anemia which completely resolved following treatment. Clinical suspicion in our case arose considering the endemic nature of malaria in our country. The two overlapping injuries to spleen reduced the clearance of parasites by the spleen as evidenced by high parasite load. Our case report reinforces malaria as a cause of clinical worsening of SCD and highlights the importance of a multifactorial approach in the management of worsening anemia in SCD.

Unusual Morphological and Automated Hematology Analyzer Features in 3 Cases of B-cell Malignancy-associated Type I Cryoglobulinemic Vasculitis.

Type I cryoglobulins are monoclonal immunoglobulins produced due to underlying hematological malignancy. Cryoglobulins spontaneously precipitate from serum and plasma at low temperatures and become soluble again on rewarming to 37 °C. Processing of blood at temperature lower than 37 °C in the laboratory may cause precipitation of cryoglobulins resulting in interferences in the automated cell counter analysis. We report three patients with cryoglobulinemic vasculitis wherein each case had different morphology of cryoglobulin precipitates on peripheral blood film, like needle shaped bluish-gray crystals, amorphous weakly basophilic extracellular deposits extraneously indenting red blood cells and basophilic neutrophilic inclusions respectively. The effect of cryoglobulins on two technologically different automated cell counters based on principles of impedance, Volume-Conductivity-Scatter (VCS) and fluorescence flow cytometry was assessed. This case series provides interesting insight into the varying morphological features of cryoglobulins on May-Grunwald-Giemsa stained blood films and interference caused by cryoglobulins in different automated cell counter analysis resulting in pseudo-leucocytosis, pseudo-thrombocytosis, abnormal histograms and scatterplots. Identification of these hematologic abnormalities and artifacts induced by cryoglobulins is necessary since it may be the first clue leading to the timely diagnosis of cryoglobulinemia and hence the underlying hematological malignancy, as in our cases.

Systematic review: role of elevated plasma von-Willebrand factor as predictor of mortality in patients with chronic liver disease.

In this systematic review, we aimed to assess role of plasma von-Willebrand factor (vWF), an endothelial activation marker, as prognostic marker in patients with chronc liver disease [cirrhosis and acute-on-chronic liver failure (ACLF)]. We searched published databases using predefined keywords to identify all studies up to June 2018, in which plasma vWF (antigen or activity assay) was used as prognostic marker predicting mortality in patients with chronic liver disease. Relevant extracted data from selected studies were narratively summarized. The individual study's area under ROC curve for plasma vWF as a predictor of mortality was pooled and meta-analyzed. Six studies (cirrhosis: 5; ACLF: 1) with an aggregate data of 765 patients (cirrhosis: 715 patients; ACLF: 50 patients) were included. Baseline plasma vWF-antigen was an independent predictor of medium-term mortality in patients with cirrhosis (summary area under the curve: 0.74; 95% confidence interval: 0.70-0.79) with an optimal cutoff of 318% (216-390%; median, range) over a period of 25.6 months (23.6-33 months). Plasma vWF also predicted short-term (over 7 days) mortality in patients with ACLF. Plasma vWF levels correlated with Child's score, model for end-stage liver disease (MELD) score and hepatic venous pressure gradient and performed as well as MELD score in predicting mortality in patients with cirrhosis and ACLF. Baseline plasma vWF level predicts mortality over a medium term (1-3 years) in cirrhosis and over a short term (1 week) in ACLF patients. The marked elevation of baseline plasma vWF levels in ACLF patients was associated with drastic truncation of survival when compared with cirrhosis patients.

D-dimer levels in patients with thromboangiitis obliterans.

Background: Thromboangiitis obliterans (TAO) or Buerger disease is a recurring progressive segmental vasculopathy that presents with inflammation and thrombosis of small and medium arteries and veins of the hands and feet. The exact cause remains unknown, with tobacco use (primarily smoking but also smokeless tobacco) being highly associated with the disease. The diagnosis is clinical and the lack of a diagnostic gold standard is a deterrent to diagnosing it in patients with atypical presentations. Obliterative endarteritis occurs perhaps due to a mixture of thrombosis and inflammation. The diagnostic sensitivity and specificity of D-dimer as a biomarker for thrombosis is well reported from its use in other areas such as deep vein thrombosis. Identification of a biomarker linked to the causation yields a diagnostic adjunct with a role in therapeutic decision-making, aiding diagnosis in atypical presentation, monitoring disease activity and gauging response to therapy. Methods: Between April 2014 and May 2015, we studied serum D-dimer (a marker of thrombosis) in 62 patients with TAO and compared this to 330 normal age- and sex-matched controls. We included all patients with peripheral arterial disease clinically diagnosed to have TAO according to the Shionoya criteria. There was no history of thrombosis or arterial disease in the control group. The control group was matched for baseline characteristics such as age and sex. All patients underwent a standard diagnostic protocol including blood tests (haemoglobin and creatinine), electrocardiogram, chest X-ray and ankle brachial pressure index. Blood was collected using an evacuated tube system into a citrate anticoagulant tube for testing D-dimer. Results: All the 62 patients diagnosed to have TAO were men with an average age of 40 years (range 18-65 years). They all had a history of tobacco use and did not have other atherogenic risk factors (part of the diagnostic criteria). Medium-vessel involvement was present in 53 patients (85%) and the rest presented with additional involvement of the popliteal and femoral vessels. Upper limb involvement or superficial thrombophlebitis was present in 95% of patients. Laboratory and imaging studies were consistent with TAO. The groups were well matched for age (p = 0.3). The median and interquartile range for D-dimer values were 61 ng/ml and 41-88 ng/ml in controls (n = 330) and 247 ng/ml and 126478 ng/ml in patients (n = 62), respectively (p<0.001). Conclusions: D-dimer levels are considerably elevated in patients with TAO. This indicates an underlying thrombotic process and suggests its potential role as a diagnostic adjunct. It also leads us to hypothesize a potential therapeutic benefit of anticoagulants in this disease.

Analytical performance of a point-of-care device in monitoring patients on oral anticoagulation with vitamin K antagonists.

BACKGROUND: [Please check the following sentence for clarity: "Point-of-care devices measuring international normalized ratio have clinical appeal, reports of 'off-label' in-hospital/primary care use report improved time to intervention/dose adjustment."]Point-of-care devices measuring international normalized ratio have clinical appeal, reports of 'off-label' in-hospital/primary care use report improved time to intervention/dose adjustment. We evaluated the accuracy and precision of a device for such multiple patient use compared to a reference laboratory. METHODS: The point-of-care international normalized ratio result of patients on oral anticoagulation at the Vascular Surgery clinic was compared to the reference to check for statistical and clinical correlation. This was a prospective case-control study design with sample size calculated for sensitivity of 87.5%, precision 5% and desired confidence level 95%. RESULTS: There were 168 patients tested; 55% were male, the mean age was 45.4. Sixty per cent were in the target international normalized ratio range. Tests were done for statistical and clinical correlation. The international normalized ratio range using the point-of-care device was 0.8-7.5 (reference lab 0.8-10), mean international normalized ratio was 2.22 ± 1.6 (point-of-care device) compared to 2.46 ± 1.3 (reference lab). The mean absolute difference was 0.79 ± 0.92 and the mean relative difference was 8.1% ± 1.03. Data was analysed using a Bland-Altman plot yielding a mean of 0.738 (standard deviation 0.92). Concordance between the tests was 75% with r2 = 0.52 on linear regression. Using an error grid plot, excellent clinical correlation was seen in 63.8%. In 5.4% major corrective action was needed but potentially missed if relying on the point-of-care device. CONCLUSION: The accuracy and precision of this point-of-care device is moderate. It may have potential utility only where access to a reference lab is difficult.

Evaluation of Factor VIII as a Risk Factor in Indian Patients with DVT.

Introduction. Elevated factor VIII population in the Indian population has not been studied as a possible risk factor for deep vein thrombosis (DVT). High factor VIII level is considered a predisposing factor for DVT and its recurrence. However it is known to vary between populations and its exact role in the etiopathogenesis of thrombophilia remains unknown. Material and Methods. Factor VIII levels of patients with DVT who had undergone a prothrombotic workup as a part of their workup was compared to normal age matched controls in a 1 : 3 ratio. Results. There were 75 patients with DVT who had undergone a prothrombotic workup in the course of their treatment for lower limb DVT. In these, 64% had levels of factor VIII more than 150 as compared to 63% of normal controls (p > 0.05, not significant). Conclusion. Elevated factor VIII in the Indians may not be associated with the same thrombotic risk as seen in the West. We find a variation in the levels of factor VIII with a different "normal" than what is reported in other populations. This needs further study to elucidate the role of factor VIII in the evaluation and treatment of thrombophilia.

Preliminary experience with use of recombinant activated factor VII to control postpartum hemorrhage in acute fatty liver of pregnancy and other pregnancy-related liver disorders.

Control of postpartum hemorrhage is difficult in patients with coagulopathy due to acute liver failure. Recombinant activated factor VII (rFVIIa) can help in control of bleed; however, it has short duration of action (2-4 h). The study aimed to report the use of rFVIIa in this setting. We retrospectively analyzed all patients with acute liver failure secondary to pregnancy-related liver disorders who received rFVIIa for control of postpartum hemorrhage (six patients, all six met diagnostic criteria for acute fatty liver of pregnancy). One dose of rFVIIa achieved adequate control of bleeding in five patients, while one patient needed a second dose. rFVIIa administration corrected coagulopathy and significantly reduced requirement of packed red cells and other blood products. No patient had thrombotic complications. In conclusion, rFVIIa was a useful adjunct to standard management in postpartum hemorrhage secondary to acute liver failure of pregnancy-related liver disorders.

Role of minimal residual disease monitoring in acute promyelocytic leukemia treated with arsenic trioxide in frontline therapy.

Data on minimal residual disease (MRD) monitoring in acute promyelocytic leukemia (APL) are available only in the context of conventional all-trans retinoic acid plus chemotherapy regimens. It is recognized that the kinetics of leukemia clearance is different with the use of arsenic trioxide (ATO) in the treatment of APL. We undertook a prospective peripheral blood RT-PCR-based MRD monitoring study on patients with APL treated with a single agent ATO regimen. A total of 151 patients were enrolled in this study. A positive RT-PCR reading at the end of induction therapy was significantly associated on a multivariate analysis with an increased risk of relapse (relative risk = 4.9; P = .034). None of the good risk patients who were RT-PCR negative at the end of induction relapsed. The majority of the relapses (91%) happened within 3 years of completion of treatment. After achievement of molecular remission, the current MRD monitoring strategy was able to predict relapse in 60% of cases with an overall sensitivity and specificity of 60% and 93.2%, respectively. High-risk group patients and those that remain RT-PCR positive at the end of induction are likely to benefit from serial MRD monitoring by RT-PCR for a period of 3 years from completion of therapy.