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Objective: Hypercoagulation and high incidence of thrombosis during COVID-19 is well established. However, there is a lack of data, how it changes over time. The main purpose of our study was to access different parts of hemostasis in few months after acute disease. Material and methods. Patients discharged from our hospital were invited for follow up examination in 2,3-3,8 (group 1 - 55 pts) or 4,6-5,7 months (group 2 - 45 pts) after admission. Control group (37 healthy adults) had been collected before pandemic started. Standard coagulation tests, aggregometry, thrombodynamics and fibrinolysis results were compared between groups. Result(s): D-dimer was significantly higher, and was APPT was significantly lower in group 2 compared to group 1, while fibrinogen, prothrombin levels didn't differ. Platelet aggregation induced by ASA, ADP, TRAP, spontaneous aggregation didn't differ significantly between groups. Thrombodynamics revealed hypocoagulation in both group 1 and group 2 compared to control: V, mum/min 27,3 (Interquartile range (IQR) 26,3;29,4) and 28,3 (IQR 26,5;30,1) vs. 32,6 (IQR 30,4;35,9) respectively;all p < 0,001. Clot size and density in both group 1 and group 2 were significantly lower than in control group. Fibrinolysis appeared to be enhanced in x2 compared to control and group 1. Lysis progression, %/min was higher: 3,5 (2,5;4,8) vs. 2,4 (1,6;3,5) and 2,6 (2,2;3,4) respectively, all p < 0,05. Lysis onset time in both group 1 and group 2 was significantly shorter compared to control. Conclusion(s): We revealed normalization of parameters of clot formation process in 2-6 months after COVID-19, while fibrinolysis remained still enhanced. Further study is required to investigate the clinical significance of these changes.Copyright © Creative Cardiology 2021.
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Background: The impact of COVID-19 infection or COVID-19 vaccination on the immune system of people living with HIV (PLWH) is unclear. We therefore studied the effects of COVID-19 infection or vaccination on functional immune responses and systemic inflammation in PLWH. Method(s): Between 2019 and 2021, 1985 virally suppressed, asymptomatic PLWH were included in the Netherlands in the 2000HIV study (NCT039948350): 1514 participants enrolled after the start of the COVID-19 pandemic were separated into a discovery and validation cohort. PBMCs were incubated with different stimuli for 24 hours: cytokine levels were measured in supernatants. ~3000 targeted plasma proteins were measured with Olink Explore panel. Past COVID-19 infection was proven when a positive PCR was reported or when serology on samples from inclusion proved positive. Compared were unvaccinated PLWH with and without past COVID-19 infection, and PLWH with or without anti-COVID-19 vaccination excluding those with past COVID-19 infection. Result(s): 471 out of 1514 participants were vaccinated (median days since vaccination: 33, IQR 16-66) and 242 had a past COVID-19 infection (median days since +PCR: 137, IQR 56-206). Alcohol, smoking, drug use, BMI, age, latest CD4 count and proportion with viral blips were comparable between groups. Systemic inflammation as assessed by targeted proteomics showed 89 upregulated and 43 downregulated proteins in the vaccinated participants. In contrast, individuals with a past COVID-19 infection display lower levels of 138 plasma proteins compared to the uninfected group (see figure). 'Innate immune system' and 'cell death' were upregulated in pathway analysis in vaccinated PLWH, but downregulated in COVID-19 infected participants. The increased systemic inflammation of the COVID-19 vaccinated group was accompanied by lower TNF-alpha and IL-1beta production capacity upon restimulation with a range of microbial stimuli, while production of IL-1Ra was increased. In COVID-19 infected PLWH only a reduced production of TNF-alpha to S. pneumonia was significant. Vaccinated PLWH also showed upregulation of platelet aggregation pathways. Conclusion(s): COVID-19 vaccination in PLWH leads to an increased systemic inflammation, but less effective cytokine production capacity of its immune cells upon microbial stimulation. This pattern is different from that of COVID-19 infection that leads to a decreased inflammatory profile and only minimal effects on cytokine production capacity. (Figure Presented).
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Background: Advances in computational methodologies have enabled processing of large datasets originating from imaging studies. However, most imaging biomarkers suffer from a lack of direct links with underlying biology, as they are only observationally correlated with pathophysiology. Purpose(s): To develop and validate a novel AI-assisted image analysis platform, by applying quantitative radiotranscriptomics that quantifies cytokinedriven vascular inflammation from routine CT angiograms (CTA) performed as part of clinical care in COVID-19. Method(s): We used this platform to train the radiotranscriptomic signature C19-RS, derived from the perivascular space around the aorta and the internal mammary artery in routine chest CTAs, to best describe cytokinedriven vascular inflammation, defined using transcriptomic profiles from RNA sequencing data from human arterial biopsies (A). This signature was validated externally in 358 clinically indicated CT pulmonary angiograms from patients with or without COVID-19 from 3 different geographical regions. Result(s): First, 22 patients who had a CTA before the pandemic underwent repeat CTA <6 months post COVID-19 infection (B). Compared with 22 controls (matched for age, gender, and BMI) C19-RS was increased only in the COVID-19 group (C). Next, C19-RS was calculated in a cohort of 331 patients hospitalised during the pandemic, and was higher in COVID-19 positives (adjusted OR=2.97 [95% CI: 1.43-6.27], p=0.004, D). C19-RS had prognostic value for in-hospital mortality in COVID-19, with HR=3.31 ([95% CI: 1.49-7.33], p=0.003) and 2.58 ([95% CI: 1.10-6.05], p=0.028) in two testing cohorts respectively (E, F), adjusted for clinical factors and biochemical biomarkers of inflammation and myocardial injury. The corrected HR for in-hospital mortality was 8.24 [95% CI: 2.16-31.36], p=0.002 for those who received no treatment with dexamethasone, but only 2.27 [95% CI: 0.69-7.55], p=0.18 in those who received dexamethasone subsequently to the C19-RS based image analysis, suggesting that vascular inflammation may have been a therapeutic target of dexamethasone in COVID-19. Finally, C19-RS was strongly associated (r=0.61, p=0.0003) with a whole blood transcriptional module representing dysregulation of coagulation and platelet aggregation pathways. Conclusion(s): We present the first proof of concept study that combines transcriptomics with radiomics to provide a platform for the development of machine learning derived radiotranscriptomics analysis of routine clinical CT scans for the development of non-invasive imaging biomarkers. Application in COVID-19 produced C19-RS, a marker of cytokine-driven inflammation driving systemic activation of coagulation, that predicts inhospital mortality and identifies people who will have better response to anti-inflammatory treatments, allowing targeted therapy. This AI-assisted image analysis platform may have applications across a wide range of vascular diseases, from infections to autoimmune diseases.
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Introduction: Herbal medicines have been used for the treatment of human disorders and associated secondary complications since a very early age. Herbal medicines are composed with a variety of medicinal plants and their derived products. Coumarin class phytochemicals have an important role in medicine due to its anti-coagulant, anti-cancer, anti-HIV and anti-inflammatory activity. Herbal medicines have been gaining popularity in the modern system of medicine mainly due to its safety and efficacy. Columbianadin is an active phytochemical of Angelica pubescens and Heracleum candolleanum. Columbianadin have analgesic, anti-inflammatory, calcium-channel blocking properties and platelet aggregation inhibitory potential. Method(s): Present work described the medicinal importance and pharmacological activities of columbianadin in medicine supported by their traditional medicinal application and pharmacological activities. Scientific data of columbianadin has been collected from PubMed, Google Scholar, Google, Scopus, and Science Direct. However, scientific data of columbianadin published in Journals, books and scientific report have also been collected in the present paper. Analytical data of columbianadin have also been described to know the significance of analytical techniques for the isolation, and identification of columbianadin. Result(s): Scientific data of columbianadin signified the biological importance and therapeutic potential of columbianadin in medicine. Scientific data of columbianadin revealed their biological potential against inflammation, neuropathic pain, cancer, hepatic complications, and immune system disorders. However, biological effectiveness of columbianadin on blood-brain barrier permeability, body tissue, channels and platelet has also been discussed in the present work. Moreover, its therapeutic effectiveness against nematodes has been also summarized in this work. Analytical data for the isolation and identification of columbianadin in different samples has also been presented in this work. Discussion(s): Present work signified the biological importance and therapeutic potential of columbianadin in medicine, which could be used for the treatment of human disorders and associated secondary complications.Copyright © 2022 The Author(s)
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Infection by beta-coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coron-avirus-2) alters the homeostasis of the vascular endothelium, promoting an inflammatory state which causes damage and favors the prothrombotic state. The direct viral cytotoxicity induced by the SARS-CoV-2 leads to endothelial cell death;thus, altering the vessel functions. Moreover, SARS-CoV infection induces endothelial dysfunction (ED) and reduces the levels of nitric oxide (NO);thus, aggravating the vascular injuries, which promotes thrombotic events due to an altera-tion in the homeostasis. NO is a pleiotropic molecule that induces vasodilation, regulates the immune response, inhibits platelet aggregation, and decreases the cellular adhesion to vascular en-dothelium. Moreover, NO acts directly against invasive agents, exhibiting antibacterial, antiviral, and antifungal activity. High levels of NO result in an increase in the ED, causing an inflammatory amplification that aggravates the disease through undesirable positive feedback. The objective of this review was to present and discuss the involvement of NO on ED in SARS-CoV-2 infections. This review may also highlight new perspectives for therapeutic interventions through the supple-mentation of exogenous NO. The maintenance of homeostatic NO levels could represent a useful approach in the prevention of coronavirus-induced ED.Copyright © 2021 Bentham Science Publishers.
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Background: To assess the efficacy of various anticoagulants being prescribed in the COVID 19 induced hypercoagulability, so as to know optimally effective anticoagulant. Methods: This was a Indian observational study conducted in our covid centre at vijayawada,Andhra Pradesh between june 2020 to January 2021 . Results: A total of 100 COVID 19 subjects were included. The patients were found to be matched with respect to age, gender, diet and past history of various illnesses. Gender wise more males (60 patients)are affected when compared to females(40 patients). Age group more affected are less than or equal to 50yrs . Comorbidites like Diabetes(67patients),cardiac problems(62patients), dyslipidemia(62patients) were seen. Risk factors like smoking(52patients), alcoholism(50patients) noticed. Almost all subjects are RTPCR positive. IL- 6,CRP,LDH high in most subjects. Ferritin and PT/INR are normal in more subjects. Out of 100 patients oxygen is required in 48 subjects and BIPAP/CPAP required in 26 subjects. Death occurred in 24 patients (2 with CVA,22 with myocardial infraction). Mortality rate is more in vegetarians. More patients in our study belongs to CORADS score 4 and 5. D-dimer are increased in 67subjects. IL-6 are increased in 68patients . Frequency of subjects with raised D-dimer (p = 0.049) and CRP (p = 0.002) levels were found to be benefitted on receiving nattokinase. However, no other parameters such as IL-6 (p = 0.068) ferritin (p = 0.396), ESR (p = 0.278), PT/INR (p = 0.47) LDH (p = 0.34) or CORADS staging achieved such significant association. Also need of interventions such as Oxygen (p = 0.001), BIPAP/CPAP (p < 0.0001) were low in patients on nattokinase. No significant difference was noted in follow up investigations such as PT/INR (p = 0.31) and other markers (D-dimer, IL-6, LDH, CRP) (p = 0.55). No bleeding episodes were reported in subjects on nattokinase. Significant low rate of death was found in subjects who received nattokinase (p < 0.0001) and rivaroxaban (p < 0.0001). Also, significantly higher mortality rate was observed in subjects who required to be put on oxygen (p < 0.0001) as well as BIPAP/CPAP (p < 0.0001). Conclusions: Nattokinase simultaneously effects several key favourable benefits for thrombosis, hypertension, atherosclerosis, hyperlipidaemia, platelet aggregation, and neuroprotection in patients with COVID 19 infection. (Figure Presented).
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Purpose: Release and circulation of pro-inflammatory cytokines or “cytokine storm,” a pathophysiologic component of severe COVID-19, is associated with thrombosis and clot embolization. Compromised patients often require extracorporeal oxygenation and mechanical circulatory support (MCS), imparting blood flow disturbances and exogenous shear stress, further amplifying thrombotic potential. Central in these processes is the platelet. The dynamic interaction of MCS flow/shear and inflammatory cytokines and their propensity for altering platelet function remains unknown. We hypothesized that platelet function is modified in an MCS + pro-inflammatory cytokine environment. We examined platelet aggregation as a function of time, exposing platelets to COVID-19-associated cytokines under MCS flow in vitro. Methods: An Impella5.5® was affixed in a closed loop and positioned with outflow cannula in a 1-inch tube region, maintained at differential 60mmHg pressure. Alternatively, a CentriMag® was affixed in series with a similar closed loop. Porcine PRP, obtained via centrifugation of fresh, ACD-A anticoagulated whole blood was used as circulating fluid. A cytokine “COVID cocktail” of porcine IL-6 (4.5 ng/mL), IL-1β (0.5 ng/mL), IL-8 (2.7 ng/mL), and TNFα (1 ng/mL) was added to PRP and circulated at 5 L/ min. After 5, 60 and 240min of circulation, platelet samples were taken and measured for aggregation with ADP (20uM), and expression of activation markers (CD62P, AnnV) via flow cytometry. Samples were measured in duplicate from N ≥ 2 pigs per experiment. Results: The addition of COVID Cocktail cytokines led to an increase in overall aggregability of platelets over time. In contrast, the addition of shear via MCS devices led to a decrease in platelet aggregability despite Cytokine addition (Fig 1). Notably, platelet aggregability was more greatly reduced with CentriMag (85% reduction) than with Impella (65% reduction). There was no significant difference in platelet activation (AnnV binding, CD62P exposure) between CentriMag and Impella 5.5 in the cytokine environment. (Figure Presented).
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Background and aims: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare syndrome of unclear aetiology occurring after vaccinations against COVID-19. The aim of this study was to investigate the DNA vaccine-encoded Sars-cov-2 soluble spike protein (SP) as a potential trigger of platelet activation in VITT. Methods: We studied three VITT patients and seven healthy controls (HCs) within 3 weeks from the first dose of ChAdOx1 nCoV-19. Serum levels of SP, soluble angiotensin-converting enzyme 2 (sACE2), and platelet response to VITT serum stimulation were studied. A thrombus retrieved from middle cerebral artery during mechanical thrombectomy of one VITT patient, was analysed by immunohistochemistry for SP and ACE2. Neutrophil extracellular traps (NETs) markers and coagulation parameters were also measured. Results: We detected SP and sACE2 in all VITT patients, and in two and three out of 7 HCs, respectively. VITT sera markedly activated platelets and this activation was inhibited by both anti-SP and anti-FcγRIIA blocking antibodies. The retrieved thrombus showed positive immunohistochemical labelling of platelets using an anti-SP antibody with reduced ACE2 expression, compared to a thrombus from a pre-pandemic stroke patient. Markers of endothelial dysfunction, NETs and hypercoagulability state were present in VITT sera. Conclusions: The present data provide first evidence that DNA vaccineencoded Sars-cov-2 SP is detectable in VITT sera (up to several weeks post-vaccination) and in a platelet-rich thrombus, and suggest that SP may contribute to the initial platelet stimulation in VITT patients. Anti-PF4/ polyanion antibodies development could represent an epiphenomenon, which amplifies platelet aggregation, NETosis, and coagulation cascade.
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RATIONALE: The proteomic responses of hospitalized patients with SARS Co-V-2 infection may provide insight into risk, time course, and mechanisms associated with this infection. We used a high throughput proteomic platform to examine proteins that were differentially expressed relative to the length of hospital stay (LOS). METHOD:26 patients, hospitalized with SARS CoV-2 infection (mean age 48 yrs, 44% women) had blood samples obtained within 72 hours of admission. Initial plasma samples were analyzed from patients who were hospitalized for < 3 days (n=6), < 7days (n=12) and > 7 days (n=8) of LOS and compared to healthy controls (HC, n=8). Samples were analyzed with the modified aptamer-based array (SomaScan) that measures more than 7,000 human proteins representing different molecular pathways and gene families. Differentially regulated proteins with > 1.5 fold change and a false discovery rate of 5% were analyzed using the Ingenuity Pathway Analysis (IPA). Unique protein categories associated with LOS were assessed. RESULT: Compared to HC, differentially expressed proteins were detected among the 3 groups: 461 at < 3 days, 1,635 proteins at < 7 days and 1,738 proteins in >7 days. 407 proteins were common among all hospitalized COVID 19 individuals independent of LOS and 12, 250 and 361 proteins were uniquely present at < 3 days, < 7 days and > 7 days respectively compared to HC. The table below demonstrates the top highly enriched canonical pathway, molecular function and upstream regulator of differentially expressed proteins. The temporal sequence of these protein networks varied with LOS. Representative examples include early responses;platelet membrane glycoprotein GP6 signaling pathway that involves the FcR gamma-chain and the Src kinases linked to platelet aggregation, signaling involved in T cell receptor-mediated IL-2 production (TEC kinase). Less than 7 days include diacylglycerol associated with T cell activation, carnitine palmitoyltransferase associated with mitochondrial beta-oxidation of long chain fatty acids. CXCR4 a receptor for stromal -cell derived factor 1 and associated with COVID-19 prognosis. Late responses after 7 days include pathways involved in remodeling of epithelial adherens junctions. CONCLUSIONS : A high throughput proteomic approach provides insight into the dynamic regulation of protein pathways associated with the progression of SARS-Co-V2 infection. This may provide additional insight into risk and mechanisms associated with outcomes in COVID. (Table Presented).
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COVID-19 leads to disruption of the blood coagulation system, to thrombosis, hypercoagulability, as a result, to an increased risk of strokes and heart attacks. During COVID-19, endothelial dysfunction develops associated with NO deficiency with decrease in the level of SH compounds. Tiazotic acid (Thiotriazoline) has immunomodulatory, anti-inflammatory, antioxidant, anti-ischemic, cardio- and endothelioprotective, antiplatelet, hepatoprotective activity. Our studies conducted at the National Research Medical Center “University Clinic of ZSMU” with the participation of 57 patients (from 30 to 65 years old) with post-COVID syndrome, who received thiotriazol with basic therapy in either tablets (200 mg each) or suppositories Dalmaxin (0.2 g each) twice a day for 30 days. Inclusion criteria for the study were a positive PCR test for COVID-19;if the PCR test was negative, then the presence of IgM COVID-19 or IgG COVID-19 (with radiologically confirmed pneumonia). The following biochemical parameters were studied: C-reactive protein - by immunoturbodimetric method;D-dimer - by enzyme immunoassay;ferritin - by immunochemiluminescent method;endothelial NO-synthase (eNOS) - by ELISA method;alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), total bilirubin;international normalized ratio (INR) and determination of platelet aggregation. During treatment with thiotriazoline, significant increase in the eNOS content was recorded, which indicated the presence of endotheliopro-tective activity of the drug. Thiotriazoline significantly reduced the level of D-dimer in the blood of patients, and also led to the normalization of INR. The established effects testified to the presence of antiplatelet and fibrinolytic action of thiotriazoline and its ability to reduce the risks of heart attacks and strokes in post-COVID syndrome. Thiotriazoline led to an objective improvement in general clinical parameters in patients with post-COVID syndrome, complaints of palpitations disappeared, blood pressure stabilized.
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Basingstoke and North Hampshire hospital is part of the Southern Haemophilia Network (SHN) and is the Comprehensive Care Centre for the network. It spans a wide area and also receives regional referrals for further investigations of suspected platelet disorders. Platelet aggregation is a second-line test to investigate patients with possible bleeding disorders. It is usually performed after clotting factors are established to be normal and there is still concern about an underlying bleeding disorder. The aim of this study was to assess whether platelet aggregation studies led to a formal diagnosis of a platelet disorder. Further outcomes were to assess how many bleeding assessment tool (BAT) scores were documented and whether these corresponded with diagnostic rates. Patient records were retrospectively analysed over 13 months (from 20 June to 21 July). Seventy-two patients were identified who underwent platelet aggregation studies. These included regional and local referrals. Patients identified had their electronic records analysed for the appropriate information. Of note, platelet aggregation studies were stopped at the beginning of the COVID pandemic and only restarted in summer 2020. Therefore, these numbers may not be fully representative of a normal year due to a waiting list which developed during the suspension of this test. The 72 patients were made up of 78% females ( n = 56) and 22% males ( n = 16). Out of the 72 patients reviewed, 63% ( n = 45) were regional referrals and 37% were local referrals ( n = 27). The average age of this group was 31.89 years. Median age was 31 years (range 74). The youngest patient was 2 years old and the oldest was 76. BSH guidelines recommend using the BAT as a standardised tool for assessing bleeding history. However, it is not sensitive in determining which patients require further testing including platelet aggregometry. Only one patient had a documented BAT score out of the 72 referrals (1.39%). Platelet aggregometry did not go on and lead to a diagnosis in this patient. All the samples for platelet aggregometry were taken on site in keeping with national guidance. Three of the four cases of thrombocytopenia, (75%) had a blood film reviewed locally. Platelet aggregometry led to a diagnosis in 17% of patients ( n = 12) with a higher number of diagnoses in males than females. Forty-four per cent ( n = 7) of males seen were diagnosed with a platelet disorder compared to 9% ( n = 5) of females. Other diagnoses included connective tissue disease at 8% ( n = 6), drug induced/acquired defect 5% ( n = 4), thrombocytopenia 5% ( n = 4) and other bleeding disorders (such as VWF or Factor XI deficiency) at 4% ( n = 3). Out of the 12 diagnoses of bleeding disorders, 100% ( n = 12) were registered with the national database. In total 67% ( n = 48) of patients were discharged followed their platelet aggregations studies. In this study, platelet aggregometry had a low rate of diagnostic yield. There was a higher rate of diagnosis in the male population which likely reflects the that women are more heavily investigated due to menorrhagia . Despite the low diagnostic rates, platelet aggregation studies lead to a high number of discharges and reassurance to a number of patients that there is no underlying bleeding disorder. Therefore, they continue to have important role in the diagnosis and exclusion of underlying bleeding disorders.
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Background: Since the first case of COVID19 infection in 2019, this RNA virus has led an unprecedented pandemic that infected more than 232 million people. Although the disease is studied extensively, much remains poorly understood. Here, we performed the first correlation study on the peripheral blood morphology and immunophenotype of the white blood cells (WBCs) from COVID19 patients. Design: A total of 52 samples from COVID19 patients and 15 blood samples as control group were analyzed. COVID19 patients were divided into two groups based on clinical severity, severe (respiratory failure) or non-severe (hospitalized but stable). The controls were the patients with negative COVID19 results by PCR and antibody tests. The WBC morphology was examined either by blood smear review or via CellaVision DM analyzer captured images. Navios flow cytometer and Beckman Kaluza C software were used for immunophenotype analysis. Two-tailed T-test was performed on the COVID19 groups and the control group Results: Almost all COVID19 patients showed marked neutrophilia and lymphopenia on the CBC tests. Morphologically, the neutrophils showed irregularities like hypogranulation, toxic granules and pseudo Pelger-Huet anomaly (Fig 1A). In severe COIVD19 group, there was an increase in neutrophils with immatures phenotypes, showing CD33 positivity while CD10, CD13 and CD16 negative (Fig 1B). Conversely, the CD10(+) mature neutrophils aberrantly expressed CD56 (Fig 1B). The percentage of CD56(+) neutrophils was significantly higher in both COVID19 groups, suggesting a stronger cellular adhesion and interaction. The monocytes from the COVID19 patients had increased cytoplasm with cytoplasmic protrusion and vacuolization (Fig 2A). Phenotypically they were positive for CD13, CD33, CD38 and HLA-DR. The lymphocytes were also atypical, including increased cytoplasm with large granules and vacuoles. Phenotypically, they are activated, expressing CD38, HLA-DR, and mainly α/β subtype. Giant platelets with cytoplasmic vacuoles and projections were easily seen. Platelet aggregations were observed (Fig 2B). These platelets were CD45(-) and expressed CD61 at lower-than-normal intensity, while expressing increased CD42b intensity when compared to the control group on a log scale. Conclusions: Despite being a small study, we were able to correlate the morphologic and phenotypic alterations of the WBCs in COVID19 patients. As such, this helped to explain some of the clinical hematologic manifestation of the disease. (Figure Presented).
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Introduction.Vaccine induced immune thrombocytopenia and thrombosis (VITT) following ChAdOx1 nCOV-19 vaccine has been described, associated with unusual site thrombosis, thrombocytopenia, raised D-dimer and high titre immunoglobulin-G (IgG) class anti-Platelet Factor 4 (PF4) antibodies. Laboratory management of suspected cases begins with a sensitive anti PF4 antibodies binding assay (PF4-ELISA). If the PF4 binding assay is negative, this patient does not have Heparin induced Thrombocytopenia (HIT) or VITT. If the PF4 binding assay is positive, the positivity should be confirmed with one or multiple HIT functional assays as available, such as the serotonin release assay (SRA), heparin-induced platelet activation assay, platelet aggregation (PAT) test, flow cytometry test.Methods.We summarized clinical and laboratory findings of 7 patients in Piedmont who developed thrombosis and thrombocytopenia following AZD1222 vaccination. Plasma from all patients was used to test for anti PF4 antibodies by 2 different ELISA assays (Immucor and Stago) and by 2 different HIT functional assays, PAT and flow cytometry (HIT alert test) both performed in the presence of heparin, PF4 or both.Results.The 7 patients [6 males and 1 female, median age: 38 (range:31-76)] presented with thrombosis 2 to 17 days post vaccination: 5 males had deep vein thrombosis not in unusual sites, 1 male had stroke and the female had cerebral venous thrombosis (CVT). None had received heparin prior symptoms onset. Only 2 out of 7 patients tested positive for anti PF4 ELISA antibodies with both assays: the men with stroke showed low positivity (OD = 0,56 and 0,41) and the female with CVT strong positivity (OD = 3,2 and 3,87). Only the female patient with CVT tested positive with both HIT functional assays, PAT and HIT alert cytometry test in the presence of PF4 independently of heparin. Both assays were inhibited by high concentrations of heparin.Conclusions. In our limited experience VITT demonstrated to be an extremely rare event in the context of AZD1222 COVID-19 vaccination even in the subset of patients with thrombosis and thrombocytopenia.
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Introduction: Infection with SARS CoV2 leads to respiratory failureand can lead to support of extracorporeal oxygenation (ECMO)leading to exposure to heparin. Exposure to heparin and developmentof thrombocytopenia raises the suspicion of HIT. The strong positiveIgG PF4-heparin antigen test by immunochromatography is followedby platelet aggregation test in our center. We present a case of covid-19 in which HIT was strongly positive on gel agglutination butfunctional assay was negative. Review of literature shows that this could be due to circulating platelet immune complexes in critically illcovid patients which simulate HIT antibodies.Aims &Objectives: Materials &Methods: 7 months pregnantfemale, non-vaccinated, asthmatic, COVID 19 positive patient wasadmitted to our hospital. On admission her fetal ultrasound wasnormal and was started on non-invasive ventilation along with supportive care. However she deteriorated and shifted to veno-arterialextracorporeal membrane oxygenation support (ECMO). Her plateletcount dropped by>50% at day 6 of heparin exposure with anintermediate probability for HIT (4Tscore 5). HIT gel agglutinationtest was positive for IgG antibodies for antiheparin/PF4 antibodies butfunctional assay based on heparin-induced platelet aggregation(HIPA) revealed no increase in aggregation of patient serum with0.5U/ml and 1U/ml heparin dosage. Heparin induced thrombocytopenia was ruled out due to PAT and patient continued on ECMO.This could be due to increased platelet activating immune complexesor anti-PF4 antibodies mimicking antiheparin/PF4 antibodies. However, patient deteriorated and succumbed to the disease.Result: Heparin induced thrombocytopenia was ruled out due to PATand patient continued on ECMO. This could be due to increasedplatelet activating immune complexes or anti-PF4 antibodies mimicking antiheparin/PF4 antibodies. However, patient deteriorated andsuccumbed to the disease.Conclusions: Pathophysiology of Covid 19 disease in critical caseshas shown exacerbated immune reactions, increased endothelialinjury, which causes increased release of PF4 leading to plateletactivation.3 A recent work has also shown significantly increasedplatelet apoptosis, secondary to IgG-mediated FccRIIA signaling, incritically ill COVID-19 patients.4 It is also possible that the circulating immune complexes may be formed by corona virus-antibodycomplexes (as seen in H1N1 viral infection) 0.5 A high titre of antiPF4/heparin antibody test may not strongly predict the presence ofclinically relevant HIT antibodies, thus a confirmatory functional testshould be performed.
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Introduction: An unexpected accumulation of thrombotic events with thrombocytopenia emerged in association with the AZD1222 vaccine against COVID-19. We report our initial experience with emphasis on presenting characteristics, treatment, and short-term outcome. Methods: This is a retrospective, consecutive cohort of all patients admitted to Hannover Medical School between 8th March and 4th April 2021 with known or suspected thromboembolic events and thrombocytopenia within 2 weeks after vaccination with AZD1222. Results: Five patients were admitted during the observation period. These were women between 41 and 67 years of age, who had received AZD1222 5 to 11 days before. Clinical manifestations ranged from cerebral sinus vein thrombosis, splanchnic vein thrombosis, arterial ischemic stroke, and thrombotic microangiopathy (TMA) to mild symptoms without abnormal imaging results. Thrombocytopenia ranged between 12 and 105 /nl. All patients had markedly elevated D-Dimer. Heparin-induced thrombocytopenia (HIT) workup revealed anti-platelet factor 4 autoantibodies in sera from all patients. Platelet aggregation by these antibodies was observed in the presence of buffer or AZD1222 but suppressed by heparin. Treatment consisted of anticoagulation (heparin or argatroban), dexamethasone and, in severe cases, intravenous immunoglobulin (IVIG) or eculizumab. Two patients treated with anticoagulation, dexamethasone and IVIG had subsequent major thromboembolic events. One patient presenting with a picture of TMA responded to anticoagulation and eculizumab. Another patient responded to thrombolysis and eculizumab after failure of anticoagulation and IVIG. Long-term sequelae are expected in two patients with severe cerebrovascular events while remaining three patients have fully recovered. Anti-PF4 antibodies declined in recovering patients over a period of 8 weeks. Conclusions: The triad of thromboembolic events, thrombocytopenia and anti-PF4 autoantibodies is characteristic of VITT. The spectrum of clinical manifestations ranged from mild to unusually severe. Anticoagulation alone is not always sufficient to prevent recurrent or progressive thromboembolic events. IVIG and eculizumab are potential treatment options but their effects are currently uncertain.
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Background/Introduction: There are numerous reports regarding the direct endothelial damage by the SARS-CoV-2 that can lead to activation of both plasma hemostasis and platelet aggregation. However, the mechanism of interaction between endothelium and haemostasis in COVID-19 remains unclear. Purpose: The aim of our study was to assess the relationship between each link of clot formation process (endothelial function, plasma coagulation, platelet aggregation) with the severity of the disease. Methods: 58 COVID-19 patients were included in our study. Patients were divided into moderate (n=39) and severe (n=18) subgroups. All patients underwent a flow-mediated dilation (FMD) test, impedance aggregation, rotational thromboelastometry, thrombodynamics and von Willebrand factor antigen (vWF: Ag) quantification. All measurements were repeated on days 3 (point 2) and 9 (point 3) of hospitalization. Results: COVID-19 patients demonstrated the enhanced plasma coagulation (clotting time, s 613,0 [480;820], clot growth rate, μm/min 32,75 [29,3;38,7]). At point 1 no significant difference in parameters of plasma coagulation between patients' subgroups was noted. At point 2 a significant decrease in the size (CS, μm 1278.0 [1216.5;1356.5] vs 965.0 [659.8;1098.0], p<0,01) and clot growth rate (μm/min 32,4 [29,2;35,0] vs 17,7 [10,3;24,4], p<0,01) under the influence of anticoagulants in the moderate subgroup compared with point 1 was observed. We didn't observe such phenomenon in severe subgroup. There was no significant difference in platelet aggregation between subgroups at point 1. During the course of the disease the patients in the moderate and severe subgroups demonstrated a significant increase in platelet aggregation induced by arachidonic acid and ADP (severe: AUC ARA 48,0 [25,0;59,0] vs 77,5 [55,8;92,7], p=0,04;AUC ADP 44,0 [41,0;56,0] vs 58,0 [45,5;69,0], p=0,04;moderate: AUC ARA 31,5 [19,8;50,7] vs 56,0 [39,0;76,0], p=0,01;AUC ADP 43,0 [20,0;59,0] vs 56,6 [50,3;70,5], p=0,04;), in moderate subgroup the significant increase in TRAP-induced aggregation was also noted (AUC TRAP 58,0 [41,0;69,5] vs 76,0 [58,3;81,5], p=0,048). There were no significant differences in the FMD-test results between the patient subgroups. FMD-test results were predominantly within the reference ranges (7,1 [4,0;8,8]). Patients in the severe subgroup had significantly higher levels of vWF: Ag (228,0 [205,3;240,7] vs 232,0 [226,0;423,0], p=0,03). Conclusion: SARS-CoV-2 infection was characterized by increased levels of vWF:Ag, that could represent the local endothelial damage, meanwhile there was no generalized endothelial dysfunction assessed via FMD-test in moderate to severe patients. At the same time the enhanced plasma coagulation in COVID-19 patients was observed.
ABSTRACT
Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen responsible for the coronavirus disease 2019 (COVID-19) pandemic. Aside from the pulmonary manifestations, COVID-19 is associated with increased risk of venous and arterial thrombotic complications. The actual impact of SARS-CoV-2 infection on platelet reactivity and whether this is mediated by a hyperinflammatory status has not been fully elucidated to date. Objective: To evaluate platelet reactivity in COVID-19 patients compared to healthy subjects and to assess the association between platelet reactivity and levels of inflammatory biomarkers among COVID-19 patients. Methods: This prospective observational investigation included COVID-19 patients admitted into a tertiary care hospital and adult healthy volunteers, all of them not receiving any antiplatelet therapy. Subjects were classified in three groups: 1) Healthy subjects (HS group);2) COVID-19 patients in a pulmonary phase (viral pneumonia and bilateral infiltrates) but without meeting criteria for systemic hyperinflammation (C19-Pulm group);and 3) COVID-19 patients in a hyperinflammation phase (C19-Infl group) meeting at least 2 of the following criteria: CRP>100mg/l, D-dimer >1000mcg/l, LDH>400U/l, ferritin>1000ng/ml, IL-6>70ng/l. Blood samples for platelet function testing and quantification of inflammatory parkers were collected at a single visit. Platelet function was measured with multiple electrode aggregometry using ADP (MEA-ADP, primary endpoint), arachidonic acid (AA) and thrombin receptor activating peptide (TRAP) as stimuli. Unadjusted analyses are presented. Results: A total of 60 patients were included in the present investigation (20 in each group). A significantly greater platelet reactivity, measured with MEA-ADP, was observed in both groups of COVID-patients compared to healthy subjects (HS: 634,9±53,5, C19-Pulm: 919,9±53,5 and C19-Infl: 931,6±53,5 AU∗min;p for C19-Pulm vs. HS <0,001, p for C19-Infl vs. HS <0,001, p for C19-Pulm vs. C19-Infl 0,878;Figure 1). Parallel findings were found when using AA as stimulus for platelet aggregation showing greater platelet aggregation in COVID-19 patients compared to healthy subjects, but numerical differences were not statistically significant when using TRAP. Among COVID-19 patients, when stratified by IL-6 levels splitted into tertiles, greater platelet reactivity was observed in patients with higher IL-6 concentrations (mid and upper tertile together) compared to those with values in the lower tertile, as assessed with MEA-ADP (lower tertile: 829,0±75,8, mid and upper tertile: 1028,7±56,2;p=0,043);a similar trend was observed with AA and TRAP as stimuli. Conclusion: Patients with severe COVID-19 disease have greater platelet reactivity than healthy subjects. Increased IL-6 levels might be associated with the observed heightened platelet reactivity among COVID-19 patients.
ABSTRACT
Introduction: Coagulopathy plays a significant role in COVID-19 pathogenesis. Benefit from anticoagulation is well established in hospitalized patients. But since there is a lack of data on coagulopathy resolution: there is no consensus in guidelines if extended anticoagulation is required. Purpose: The purpose of our work was to analyze coagulation abnormalities at 2 to 5 months after moderate to severe COVID-19. Methods: COVID-19 reconvalescents (CR), discharged from our hospital, were called for follow-up at 2-3 (CR1 group, 21 patients) or 5-6 (CR2 group, 26 patients) months after discharge. All CR were not on the anticoagulation therapy by that time. In addition to clinical examination and standard lab tests, we performed an FMD-test to analyze endothelial function, impedance aggregometry to analyze platelet aggregation, and a thrombodynamics test to assess thrombogenesis and fibrinolysis. The control group was recruited before the pandemic started. Results: All CR were free from thrombotic complications after discharge from the hospital. Endothelial function was not significantly impaired in CR compared with control, and was still in the normal range (7,07, IQR (3,36;11,56) vs. 7,87 (5,42;13,45)). Platelet aggregation was significantly lower in CR1 than in the control group in ADP-induced mode (37, IQR (19;47) vs. 46, IQR (41;50), p=0,02) and didn't differ in other groups and other modes (Asa, TRAP-induced). Thrombodynamics tests revealed suppression of the clot formation process in both CR1 and CR2 compared with control. There were decreased clot growth rates (μm/min) (CR1/CR2: 27,1, IQR (26,1;29,2)/27,6, IQR (26,4;30,0) vs. 32,2, IQR (30,0;35,1), both p<0,001);decreased clot size (μm) (CR1/CR2: 1099, IQR (1069;1194)/1199, IQR (1058;1221) vs. 1304, IQR (1164;1380), both p<0,001), and decreased optical density (arb units) (CR1/CR2: 21'607, IQR (20'363;24'545)/22'741, IQR (21'344;25'961) vs. 26'556, IQR (24'672;29'387, p<0,001 and p=0,09 respectively. Fibrinolysis was enhanced in CR groups compared with control (lysis progression was significantly higher for CR2 only, CR1/CR2: 2,9, IQR (2,5;3,8)/3,8, IQR (2,6;5,4) vs. 2,5, IQR (1,1;3,4) %/min, p=0,087 and p=0,007 respectively;expected clot lysis time was shorter in both CR1 and CR2: 36,5, IQR (29,8;44,2)/31,7, IQR (24,3;42,7) vs. 65,9. IQR (36,3;95,5) min, p=0,019 and p=0,016 respectively). There was no statistical difference in clot formation and in fibrinolysis between CR1 and CR2. Conclusion: In the deferred period (2-5 months) of COVID-19 the fibrinolysis process remains still active whereas the process of clot formation is mostly suppressed. Endothelial function assessed via the FMD test is within the normal range in the post COVID period.