Hemorrhagic Risks of Percutaneous Dilated Tracheotomy in Thrombocytopenia

The objective: to assess the incidence and influence of platelets level on the hemorrhagic complications during percutaneous dilated tracheotomy (PDT) in patients with thrombocytopenia.Subjects and Methods. The study included 85 consecutive patients with varying degrees of thrombocytopenia at the stages of hematopoietic stem cell transplantation. The control group included 56 patients who underwent classical tracheotomy. The study group included 29 patients who underwent PDT (Griggs method). The operations were performed for prolonged artificial pulmonary ventilation. When the platelets level was below 20 × 109/L, platelet concentrate transfusion was performed before the operation.Results. The incidence of hemorrhagic complications in patients with thrombocytopenia during PDT was 13.8% (95% CI 9.13–18.45%). In open tracheotomy, the bleeding rate was 3.8% (95% CI 2.65–4.49%). These results are comparable to the incidence of hemorrhagic complications in patients with normal platelet counts. The influence of the platelet level on the presence of hemorrhagic complications in both groups was not established.Conclusion. Thrombocytopenia is not a contraindication to performing PDT. However, platelet concentrate transfusion should be performed in patients with platelet counts less than 20 × 109/L. An experienced team of anesthesiologists and endoscopists can reduce the incidence of other complications.

Peculiarities of complete blood count indicators in patients with community-acquired pneumonia during the COVID-19 pandemic

The article presents the literature review data on the community-acquired pneumonia and the survey of patients with community-acquired pneumonia during the pandemic of the new coronavirus infection COVID-19. There are no changes in the rate of leukocytosis detection in patients with mild course of the di­sease within 10 days of hospital stay. There is a tendency to increase the proportion of people with a normal number of leukocytes and lymphocytes from day 4–6 of hospital treatment. Significantly, leukopenia was detected in 9.3 % of patients 1–3 days after hospitalization and was not observed in the future. Lymphopenia was registered in the first 1–6 days of hospital stay and was not observed from day 7–9. Granulocytosis was detected in 27.6 % of patients on day 1–3 in the hospital and in the following days their percentage decreased significantly; at the same time, the number of people with normal range of granulocytes increased significantly. Normal platelet counts were observed in most patients (93.8 %) on day 1–3 of hospital stay, and thrombocytopenia was recorded only in 6.3 % of individuals on day 1–3 after hospitalization. In moderate-to-severe form, 24.7 % of patients had leukocytosis on day 1–3 of hospital stay, and on day 7–9 of treatment, it was detected already in 28.8 % (p > 0.05). The majority of patients (66.7 %) had normocytosis in the first three days, and leukopenia was observed in 8.7 % of people on day 1–3. Significantly, the share of detected lymphocytosis increased, and lymphopenia — decreased from 4–6 days of hospital stay. Granulocytosis was registered in 23.2 % of patients on day 1–3 of hospital stay, followed by a significant decrease in the percentage of granulocytes from days 4–6. Band neutrophils were elevated in the first three days among 51.6 % of patients, and their percen­tage significantly decreased after the fourth day. Most individuals (77.3 %) had a normal platelet count on day 1–3 in the hospital, and thrombocytopenia at this time was found in 16.7 %. The majority of patients aged 20–30 years had a normal number of leukocytes (65.5 %), and 25.5 % had leukocytosis 1–3 days after hospitalization. Lymphocytosis in the first 3 days was registered among 9 % of people and then increased significantly, lymphopenia was observed in 31 % of patients on day 1–3 and subsequently decreased, granulocytosis — in 25 % of cases, elevated levels of band neutrophils — in 43.8 %, thrombocytosis — in 7.3 %, thrombocytopenia — in 10.9 %. Among patients aged 31–40 years in the first 3 days after hospitalization, leukocytosis was registered in 28 % of cases, leukocyte count was normal in 72 %, leukopenia was not detected, 48 % of patients had lymphopenia, 15.8 % — granulocytosis, levels of band neutrophils were elevated in 65.2 %, thrombocytosis was observed in 8 % of cases, thrombocytopenia — in 8 %. Most patients (66.7 %) aged 41–50 years on day 1–3 after hospitalization had normocytosis, 19 % — leukocytosis, 14.3 % — leukopenia, 14.3 % — lymphocytosis, 38.1 % — lymphopenia, 3 (25 %) people had granulocytosis, levels of band neutrophils were elevated in 65 % of cases, thrombocytosis was detected in 4.8 %, thrombocytopenia — in 28.5 %. Among patients aged 51–60 years in the first 3 days after hospitalization, the following indicators of complete blood count were found: 16.1 % of individuals had leukocytosis, number of leukocytes was normal in 83.9 % of cases, leukopenia was not detected, lymphocytosis was found in 16.1 % of people, lymphopenia — in 22.6 %, granulocytosis — in 20 %, elevated levels of band neutrophils — in 39.3 %, thrombocytosis — in 3.2 %, thrombocytopenia — in 16.2 %. Patients older than 60 years on days 1–3 after hospitalization had the following changes: 38.8 % — leukocytosis, white blood cell count was normal in 49 % of cases, 12.2 % of people had leukopenia, 12.2 % — lymphocytosis, 51.1 % — lymphopenia, granulocytosis was not observed, 25 % had granulocytopenia, 60 % — elevated levels of band neutrophils, 8.2 % — thrombocytosis, and 18.3 % — thrombocytopenia.

MPL-Positive Essential Thrombocytosis Presenting as Budd-Chiari Syndrome in a Middle-Aged Woman with an Initially Normal Platelet Count

Budd-Chiari syndrome (BCS) results from an occlusion of the hepatic venous flow which in turn leads to portal hypertension causing ascites and other signs of liver dysfunction. Here, we present the case of a 43-year-old woman with recurrent ascites who was found to have BCS secondary to an inferior vena cava thrombosis extending into the hepatic veins. Although she had a normal platelet count on admission, additional laboratory investigations revealed an MPL mutation. She was discharged on anticoagulation with apixaban and later found to have thrombocytosis on repeat blood work, confirming the diagnosis of essential thrombocytosis, following which she was started on myelosuppressive therapy with hydroxyurea.

Morphofunctional assessment of platelet activity correction in hyperbaric blood oxygenation

100 patients with functional class II-III exertional angina were examined. The subjects underwent a 10-day course of hyperbaric oxygenation (HBO) in the 1.2 ATA mode for 40 minutes, against the background of standard therapy for coronary heart disease (IHD). Before and after HBO, the platelet link of hemostasis and the elastic properties of the platelet membrane were assessed. Assessment of the effect of the HBO course on the functional state of platelets, depending on their aggregation activity, showed that in patients with initially normal platelet aggregation, there is a tendency to a decrease in spontaneous aggregation, in contrast to patients with initial hypoaggregation, HBO promoted a significant increase in spontaneous aggregation. The use of a 10-day course of HBO was accompanied by a reaction of reducing the elasticity of the platelet biomembrane. Key words: aggregation; atomic force microscopy; platelet membrane; hyperbaric oxygenation.

RNA Binding Protein SRSF3 confers an essential role in megakaryocyte maturation and platelet production

RNA processing is increasingly recognised as a critical control point in the regulation of different haematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that Serine-arginine rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterised by megakaryocyte maturation arrest, dramatically reduced platelet counts and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.

Near fatal stent thrombosis in an aneurysmatic RCX as first manifestation of heparin induced thrombocytopenia (HIT) without thrombocytopenia

Background Thrombosis resulting from heparin-induced thrombocytopenia (HIT) occurs in about 2% of patients without a significant decrease in platelet counts. We report on such a near fatal thrombotic event caused by coronary intervention. Case presentation A supposedly “completely healthy” 53-year-old patient was admitted to hospital with covered rupture of an aneurysm of the Aorta descendens. He was successfully operated on and underwent coronary angiography due to NSTEMI six days later. Immediately after intervention of a 90% RCX stenosis he developed ventricular flutter, was defibrillated, and re-angiography showed partial occlusion of the RCX stent. Lots of white thrombi could be retrieved by aspiration catheter and gave reason for a HIT without thrombocytopenia. The detection of platelet factor 4/heparin complex antibodies by immunoassay supported and the subsequent Heparin Induced Platelet Activation Assay proved this diagnosis. Conclusions The clinical event of an acute stent thrombosis should alarm the interventional team to the diagnosis of HIT even with a normal platelet count.

Severe Congenital Thrombocytopenia Characterized by Decreased Platelet Sialylation and Moderate Complement Activation Caused by Novel Compound Heterozygous Variants in GNE

BackgroundHereditary thrombocytopenias constitute a genetically heterogeneous cause of increased bleeding. We report a case of a 17-year-old boy suffering from severe macrothrombocytopenia throughout his life. Whole genome sequencing revealed the presence of two compound heterozygous variants in GNE encoding the enzyme UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, crucial for sialic acid biosynthesis. Sialic acid is required for normal platelet life span, and biallelic variants in GNE have previously been associated with isolated macrothrombocytopenia. Furthermore, sialic acid constitutes a key ligand for complement factor H (FH), an important inhibitor of the complement system, protecting host cells from indiscriminate attack.MethodsSialic acid expression and FH binding to platelets and leukocytes was evaluated by flow cytometry. The binding of FH to erythrocytes was assessed indirectly by measuring the rate of complement mediated hemolysis. Complement activation was determined by measuring levels of C3bBbP (alternative pathway), C4d (classical/lectin pathway) and soluble terminal complement complex assays.ResultsThe proband exhibited markedly decreased expression of sialic acid on platelets and leukocytes. Consequently, the binding of FH was strongly reduced and moderate activation of the alternative and classical/lectin complement pathways was observed, together with an increased rate of erythrocyte lysis.ConclusionWe report two previously undescribed variants in GNE causing severe congenital macrothrombocytopenia in a compound heterozygous state, as a consequence of decreased platelet sialylation. The decreased sialylation of platelets, leukocytes and erythrocytes affects the binding of FH, leading to moderate complement activation and increased hemolysis.

The evolution of clot strength in critically-ill COVID-19 patients: a prospective observational thromboelastography study

Background Few studies detail the evolution of COVID-19 associated coagulopathy. We performed serial thromboelastography (TEG) and laboratory coagulation studies in 40 critically-ill, mechanically ventilated COVID-19 patients over a 14-day period and analysed differences between 30-day survivors and non-survivors. Methods Single-center prospective, observational study including 40 patients with severe COVID-19 pneumonia admitted to the intensive care unit (ICU) for mechanical ventilation. TEG analysis was performed on days 1, 7 and 14 of ICU admission and laboratory coagulation studies were performed on days 1 and 14. Coagulation variables were evaluated for change over the 14-day observation period. Differences between survivors and non-survivors at 30-days were analysed and compared. Results On admission, TEG maximum amplitude (MA) with heparinase correction was above the upper limit of the reference range in 32 (80%) patients while 33 (82.5%) presented with absent clot lysis at 30 min. The functional fibrinogen MA was also elevated above the upper limit of the reference range in 37 (92.5%) patients. All patients had elevated D-dimer and fibrinogen levels, mildly prolonged prothrombin times (PT), normal platelet counts and normal activated partial thromboplastin times (aPTT). The heparinase MA decreased significantly with time and normalised after 14 days (p = < 0.001) while the increased fibrin contribution to clot strength persisted with time (p = 0.113). No significant differences in TEG analysis were noted between 30-day survivors and non-survivors at all time points. No patients developed disseminated intravascular coagulopathy (DIC) after 14-days, however thrombosis and bleeding were each reported in 3 (7.5%) patients. Conclusion Critically-ill patients with COVID-19 present in a hypercoagulable state characterised by an increased clot strength. This state normalises after 14 days despite a persistently increased fibrin contribution to clot strength. We were unable to demonstrate any significant differences in TEG parameters between 30-day survivors and non-survivors at all time points.

Determining the Rate of Anti-PF4 Antibody Positive Results in Patients Presenting with Venous Thrombosis but a Normal Platelet Count Following ChAdOx1 Ncov-19 Astrazeneca Vaccination: An Australian Combined State Testing Centre Experience

Introduction The CHaDOx1 nCov-19 AstraZeneca (AZ) vaccination has been associated with an antibody-mediated prothrombotic syndrome, termed "Thrombosis with Thrombocytopenia Syndrome" (TTS)[1-3]. The current diagnostic criteria for TTS are thrombosis (venous or arterial) within 4-42 days of AZ vaccine, thrombocytopenia and presence of an antibody to platelet factor 4 (PF4)[4, 5]. TTS commonly presents with cerebral venous sinus thrombosis (CVST) or splanchnic vessel thrombosis (SVT), but outside of TTS, CVST and SVT are uncommon, with an overall incidence of less than 0.5 per 100,000 [5-7]. Deep vein thrombosis (DVT) and pulmonary embolism (PE) are also associated with TTS, however the background incidence of venous thromboembolism (VTE) is much higher, with 1-2 events per 1000 patients per year[7, 8]. Therefore, many patients will present with new VTE and a recent exposure to the AZ vaccine, requiring consideration of investigation for TTS. Recent data suggests that PF4 antibodies can be seen in up to 8% of patients without thrombosis but following AZ vaccination[9]. We hypothesised in patients with recent AZ vaccination, new VTE but with a normal platelet count, that the incidence of a PF4 antibody is similar to this background rate of PF4 positivity. If confirmed, then presence of a normal platelet count despite new VTE and recent vaccination may exclude TTS without the need for PF4 antibody testing. We present our preliminary data on the rates of PF4 antibody positivity amongst patients with VTE, recent AZ vaccination and a normal platelet count at presentation. Aim and Methods To assess the incidence of PF4 ELISA positive results in patients with confirmed VTE, recent vaccination (within 4-42 days) with the first dose of AZ vaccine, and platelet count greater than 150x10 9/L. A retrospective audit of cases referred with suspected TTS to Monash Pathology, Melbourne, Victoria, and New South Wales Health Pathology at Royal Prince Alfred Hospital and St George Hospital sites Sydney, New South Wales, Australia, for testing for anti PF4 antibodies from 1 st April to 31 st July 2021. Patient sera were tested for the Anti-PF4 antibody using the STAGO Asserachrom HPIA IgG ELISA (Asnières sur Seine, France). For patients with a positive PF4 antibody test additional testing was sought for either the presence of platelet activating antibodies with a flow cytometry-based assay or the presence of spontaneous serotonin release without heparin in the serotonin release assay. Results From April 1 st to July 31 st 350 tests were run on 332 patients. 91 patients met our criteria, of whom 51 were female and 40 male, with a median age of 73 years. Median platelet count at presentation was 226x10 9/L, and median D dimer values were 10 times the upper limit of normal. 86 patients had either DVT, PE or both, including 2 with upper limb DVT, and 5 patients had PE with concurrent arterial events (1 axillary artery thrombosis, 3 arterial strokes, 1 coronary artery thrombosis). Further details are presented in table 1. 82 patient samples tested negative for anti-PF4 antibodies by ELISA, 5 were positive, and were 4 weak positive/equivocal (see table 2 for further details). Of the positive results, 3 had functional testing available, of which 2 were negative, and 1 showed discordant results, with a positive SRA but negative flow cytometry. None of the weak positive/equivocal cases had functional testing results available. Of the negative ELISA results, 5 patients had functional testing results available, of which 4 were negative. One of these cases had positive testing by flow cytometry, but negative by SRA (case included in table 2). Conclusion In our Australian cohort of patients with their first dose of AZ vaccine and new VTE within 4-42days, but a normal platelet count (therefore not fulfilling the clinical criteria of TTS), the incidence of a positive PF4 antibody test was 9/91 (9.9%, 95% CI 3.7-15.9%) and only one had evidence of platelet activating antibodies. This observed rate is similar to that observed in healthy patients without thrombosis who received AZ vaccination as described by Thiele et. al., 2021. Further confirmation in a larger cohort of VTE patients is required, but if confirmed, then PF4 ELISA testing in patients with VTE and normal platelet count post AZ vaccine may not be required, and should give clinicians confidence to institute routine management. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Prevention of Fetal/Neonatal Alloimmune Thrombocytopenia in a Preclinical Model By Monoclonal Anti-HPA-1a Antibody Prophylaxis: Structural and Functional Properties of Antibody Variant Isoforms

Maternal alloantibodies to paternally inherited platelet antigens can cause fetal/neonatal alloimmune thrombocytopenia (FNAIT), rendering the fetus or newborn prone to bleeding and intracranial hemorrhage (ICH) with risk of lifelong disabilities or death. In Caucasians, the vast majority of cases are due to antibodies to the Human Platelet Antigen (HPA)-1a epitope on glycoprotein (GP)IIIa. To date, there are neither any means to prevent or reduce the risk of alloimmunization and subsequent FNAIT, nor safe and efficient treatment during affected pregnancies. Thus, a prophylactic regimen by administration of monoclonal antibodies to women at risk would be highly beneficial. Knowledge about the optimal functional design of prophylactic monoclonal antibodies for antenatal or post-natal use is however still limited. We have previously isolated and characterized a fully human anti-HPA-1a monoclonal antibody, mAb26.4. In the current study we have explored the prophylactic potential of this antibody by testing a panel of different IgG1 designs, including the wild-type mAb26.4, variants with modified Fc-region N-glycans, as well as an effector silent variant. We performed analyses of properties relevant for immunosuppression: in vitro Fc-receptor binding and capacity to induce phagocytosis, in vivo half-life measurements in humanized FcRn mice and platelet clearance in a recently developed transgenic mouse strain with a recreated HPA-1a epitope on murine GPIIIa. The prophylactic capacity by antibody-mediated immune suppression in vivo, were further tested in the FNAIT pre-clinical murine model, in which BALB/c females can be immunized by transfusion of HPA-1a-expressing platelets from the transgenic mice and where subsequent breeding of pre-immunized mice with transgenic males cause thrombocytopenia in off-springs mimicking FNAIT. By intravenous administrations of mAb26.4 variants prior to platelet transfusions, the mice generated no or low anti-platelet responses compared to control mice, and normal platelet counts in pups upon subsequent breeding. Our data thus successfully demonstrates efficient immunosuppression and prevention of FNAIT by anti-HPA-1a human monoclonal variants, providing further support for potential use in humans. Disclosures Skogen: Prophylix Pharma AS: Current holder of individual stocks in a privately-held company. Newman: Rallybio: Consultancy, Research Funding.