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.

Comparison of Point-of-Care PT-INR by Hand-Held Device with Conventional PT-INR Testing in Anti-phospholipid Antibody Syndrome Patients on Oral Anticoagulation.

Evidence on agreement of point-of-care (POC) INR testing with laboratory testing in APS patients on oral anticoagulation (OAC), is scarce. This study assessed agreement of paired PT INR testing by a POC device vs. conventional platform-based laboratory test, in APS patients on OAC using a pre-determined definition of agreement. Simultaneous paired PT INR estimation in 92 APS patients was carried out, during October 2020-September 2021. POC INR was performed on capillary blood (pin prick) using the qLabs® PT-INR hand-held device, while laboratory INR estimation was performed using citrated blood (venepuncture) on STA-R Max Analyzer® using STA-NeoPTimal thromboplastin reagent®. Concordance was defined no greater than ± 30% (as per international standards ISO 17593:2007 guidelines) for each paired INR estimation. Agreement between the two was defined as ≥ 90% of paired INR measurements being concordant. 211 paired estimations were performed, within which 190 (90%) were concordant. Good correlation was seen between the 2 methods of INR estimation on Bland Altman plot analysis with an Intra-class correlation coefficient (95% CI) of 0.91(0.882, 0.932). Lab INR range > 4 (P = 0.001) was a significant predictor of higher variability between both methods of INR estimation. Lupus anti-coagulant, other anti-phospholipid antibodies (APL) or triple APL positivity did not result in any statistically significant variation in paired measurements. This study demonstrated good correlation between POC INR measurement and Lab INR estimation and agreement was ascertained between the 2 methods in APS patients on OAC.

Rituximab therapy for neuro - inflammatory disorders - Real world experience from South India.

BACKGROUND AND OBJECTIVES: To determine the real-world data regarding the use of Rituximab (RTX) in neuroinflammatory disorders (NIDS) and assess the outcomes following RTX treatment. METHODS: A cohort of consecutive patients with NIDS started on RTX (2018-2020) was included. The outcomes assessed were the proportion with favourable clinical response (FCR) as defined by clinical remission/ improvement using disease specific disability scores, comparative efficacy based on timing of initiation and B cell kinetics. RESULTS: A total of 97 patients with NIDS were included. The mean age was 36.43 (±14.4) years and median duration of follow - up being 15 months (IQR 12-16). Forty patients (41.2%) were initiated on RTX "early" in disease course. Favourable clinical response at last follow-up was seen in 94.9% (n = 92). The mean change in disability score (mRS) was 1.89 (SD 1.30) (p < 0.001). RTX appeared more effective when initiated "early" with higher remission rates (75% vs. 42%, p 0.015). B cell kinetics varied across NIDS, with 73% having adequate depletion at 6 months. Minor adverse events including infusion related reactions were reported in 9%. CONCLUSIONS: RTX has a favourable efficacy and safety profile. Future prospective studies are needed to establish the optimal timing of initiation and need for disease-based dosage regimens.

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.

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.

Diagnostic utility of flow cytometry based coated-platelets assay as a biomarker to predict thrombotic or hemorrhagic phenotype in acute stroke.

BACKGROUND: Coated-platelets are sub-population of platelets "coated" with highly procoagulant proteins and phosphatidylserine that sustains thrombin generation. They are produced upon dual agonist stimulation by collagen and thrombin. This study was conducted to assess if there was any difference in the levels of coated-platelets in patients with primary intracranial hemorrhage (PICH) and ischemic stroke due to large artery atherosclerosis (LAA) as compared to healthy controls, and to see if coated-platelet levels had any influence on the hemorrhagic transformation (HT) of ischemic stroke. METHODS: Coated-platelet levels were determined by flow cytometry using fluorescently tagged Annexin V antibody to identify phosphatidylserine exposed on the surface of platelets activated by dual agonists (convulxin and thrombin) in cross-sectional cohort of 75 patients with stroke and 34 controls. RESULTS: Patients with PICH (n = 35) had significantly lower coated-platelets than the controls (adjusted mean ± SE, 21.0 ± 1.9% vs. 36.1 ± 1.7%, p < 0.001), while patients with LAA (n = 30) had significantly higher coated-platelets than controls (adjusted mean ± SE, 51.9 ± 1.5% vs. 36.1 ± 1.7%, p < 0.001). Patients with subsequent HT of ischemic stroke (n = 10) had significantly lower coated-platelet levels at admission compared to those without HT (adjusted mean ± SE, 18.1 ± 2.6% vs. 51.9 ± 1.5%, p < 0.001). CONCLUSIONS: Coated-platelet levels are significantly different in patients with hemorrhagic and ischemic stroke as compared with controls. Lower levels of coated-platelets measured by flow cytometry may be earliest predictor of subsequent HT in patients with ischemic stroke even before the radiological changes suggestive of HT are visualized.

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.

Pediatric systemic lupus erythematosus with lupus anticoagulant hypoprothrombinemia syndrome-A case series with review of literature.

INTRODUCTION: Lupus anticoagulant hypoprothrombinemia syndrome (LAHPS) is a rare phenomenon that leads to concomitant thrombosis and hemorrhage in children with SLE. LAHPS in pediatric SLE (pSLE) has a protracted course requiring long-term immunosuppressive therapy. Due to the rarity of this syndrome and paucity of reported cases, there is lack of standardized management. We herewith report 5 children with pSLE with LAHPS.Methodology: We retrospectively reviewed clinical features, laboratory features, treatment and outcome for 5 children with lupus anticoagulant hypoprothrombinemia syndrome with SLE and a review of literature of similar cases published. RESULTS: Mean age of presentation was 10.2 ± 2.38 years (mean ± SD) and female to male ratio was 4:1. All children presented with mild to severe bleeding manifestations like gum bleed, epistaxis, hematuria, menorrhagia and subarachnoid bleed. Coagulation profile revealed prolonged PT and aPTT, with low prothrombin levels and positive Lupus anticoagulant in all children. Mixing studies were characteristic in these children. On comparing laboratory parameters majority had low C3, C4 levels, ANA and anti-DsDNA antibody positivity and three children had anticardiolipin positivity. One child had lupus nephritis along with LAHPS at presentation. All responded well to steroids and supportive measures. CONCLUSION: High index of suspicion is needed when child with lupus presents with bleeding manifestations for early diagnosis and treatment.