Gravity sedimentation reveals functionally and morphologically different platelets in human blood.

In contrast to red blood cells, platelets float rather than sediment when a column of blood is placed in the gravitational field. By the analogy of erythrocyte sedimentation (ESR), it can be expressed with the platelet antisedimentation rate (PAR), which quantitates the difference in platelet count between the upper and lower halves of the blood column after 1 h of 1 g sedimentation. Venous blood samples from 21 healthy subjects were analyzed for PAR. After a 1-h sedimentation, the upper and lower fractions of blood samples were analyzed for platelet count, mean platelet volume (MPV), immature platelet fraction (IPF), and high-fluorescence IPF (H-IPF). The mechanisms behind platelet flotation were explored by further partitioning of the blood column, time-dependent measurements of platelet count and comparison with ESR. The structure and function of the platelets were assessed by electron microscopy (EM) and atomic force microscopy (AFM), and platelet aggregometry, respectively. Platelet antisedimentation is driven by density differences and facilitated by a size-exclusion mechanism caused by progressive erythrocyte sedimentation. The area under the curve (AUC) of the whole blood adenosine diphosphate (ADP) aggregation curves showed significant differences between the upper and lower samples (p < .005). AUC in the upper samples of 38% of healthy subjects exceeded the top of the normal range (53-122) suggesting that ascending platelets show an intensified ADP-induced aggregability ex vivo. H-IPF was significantly higher in the upper samples (p < .05). EM and AFM revealed that platelets in the upper samples were larger in volume and contained 1.6 times more alpha granules compared to platelets in the lower samples. Our results indicate that antisedimentation is able to differentiate platelet populations based on their structural and functional properties. Therefore, PAR may be a suitable laboratory parameter in various thromboinflammatory disorders.

It is less known that platelets do not sediment in response to gravitational force but float on the top of the blood column. This phenomenon is called antisedimentation, the rate of which, however, can be different, yet this feature has not been widely studied and used in clinical practice or diagnosis. We tested the idea that antisedimentation of platelets from venous blood samples can be a potential biomarker. We have found that platelet antisedimentation is driven by density differences and facilitated by a size-exclusion mechanism caused by progressive erythrocyte sedimentation and after 1-h upper and lower fractions develop. Interestingly, the aggregation curves showed significant differences between the upper and lower samples, suggesting that the ascending platelets show ex vivo hyperaggregability. Electron and atomic force microscopy revealed that platelets in the upper samples were larger in volume and contained more alpha granules than platelets in the lower samples. Subsequently, antisedimentation can be used to differentiate platelet populations based on their structural and functional properties; thus, it may be a promising biomarker for various thromboinflammatory disorders.

The Management of COVID-19-Related Coagulopathy: A Focus on the Challenges of Metabolic and Vascular Diseases.

The course of COVID-19 is highly dependent on the associated cardiometabolic comorbidities of the patient, which worsen the prognosis of coronavirus infection, mainly due to systemic inflammation, endothelium dysfunction, and thrombosis. A search on the recent medical literature was performed in five languages, using the PubMed, Embase, Cochrane, and Google Scholar databases, for the review of data regarding the management of patients with a high risk for severe COVID-19, focusing on the associated coagulopathy. Special features of COVID-19 management are presented, based on the underlying conditions (obesity, diabetes mellitus, and cardiovascular diseases), emphasizing the necessity of a modern, holistic approach to thromboembolic states. The latest findings regarding the most efficient therapeutic approaches are included in the article, offering guidance for medical professionals in severe, complicated cases of SARS-CoV-2 infection. We can conclude that severe COVID-19 is closely related to vascular inflammation and intense cytokine release leading to hemostasis disorders. Overweight, hyperglycemia, cardiovascular diseases, and old age are important risk factors for severe outcomes of coronavirus infection, involving a hypercoagulable state. Early diagnosis and proper therapy in complicated SARS-CoV-2-infected cases could reduce mortality and the need for intensive care during hospitalization in patients with cardiometabolic comorbidities.

Use of blood products during the first months of COVID-19 pandemic period: A single center report.

The novel coronavirus (SARS-CoV-2) outbreak exerted a serious effect on healthcare. Between 1st of January and May 31, 2020 due to the special regulations in Hungary, the number of reported COVID-19 infections were relatively low (3876 cases). The inpatient and outpatient care and the blood supply were significantly affected by the implemented regulations. The aim of this study was to evaluate the use of blood products amid the first five months of the pandemic situation. This investigation has observed a significant reduction of hospitalizations (37.35%). Analyzing individually the included units, pre-transfusion hemoglobin concentrations of transfused patients presented slight modifications, which were not statistically significant. The special regulations resulted major changes in the frequency of diagnoses at admissions in case of the Department of Surgery, while in case of the other specialities (Division of Hematology and Department of Anesthesiology and Intensive Therapy), there were no major changes compared to pre-pandemic period. Considering each department separately, transfused red blood cell concentrates (RBC) per patient, and the proportion of transfused patients did not change significantly. However, the combination of these modifications resulted in the significant decrease in RBC transfusions (p < 0.0001) compared to the pre-pandemic baseline. With regard to platelet and fresh frozen plasma (FFP), their usage was significantly reduced (44.40% platelet concentrates and 34.27% FFP). Our results indicate that the pandemic had an important effect on the blood product usage at the included departments by introducing different patient care policies and the temporary deferral of the elective surgical interventions. Despite the challenging circumstances of blood collection and blood product supply, the hospitalized patients received adequate care.

Plasma Fibrinogen Independently Predicts Hypofibrinolysis in Severe COVID-19.

High rates of thrombosis are present in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Deeper insight into the prothrombotic state is essential to provide the best thromboprophylaxis care. Here, we aimed to explore associations among platelet indices, conventional hemostasis parameters, and viscoelastometry data. This pilot study included patients with severe COVID-19 (n = 21) and age-matched controls (n = 21). Each patient received 100 mg aspirin therapy at the time of blood sampling. Total platelet count, high immature platelet fraction (H-IPF), fibrinogen, D-dimer, Activated Partial Thromboplastin Time, von Willebrand factor antigen and von Willebrand factor ristocetin cofactor activity, plasminogen, and alpha2-antiplasmin were measured. To monitor the aspirin therapy, a platelet function test from hirudin anticoagulated whole blood was performed using the ASPI test by Multiplate analyser. High on-aspirin platelet reactivity (n = 8) was defined with an AUC > 40 cut-off value by ASPI tests. In addition, in vitro viscoelastometric tests were carried out using a ClotPro analyser in COVID-associated thromboembolic events (n = 8) (p = 0.071) nor the survival rate (p = 0.854) showed associations with high on-aspirin platelet reactivity status. The platelet count (p = 0.03), all subjects. COVID-19 patients presented with higher levels of inflammatory markers, compared with the controls, along with evidence of hypercoagulability by ClotPro. H-IPF (%) was significantly higher among non-survivors (n = 18) compared to survivors (p = 0.011), and a negative correlation (p = 0.002) was found between H-IPF and plasminogen level in the total population. The platelet count was significantly higher among patients with high on-aspirin platelet reactivity (p = 0.03). Neither the ECA-A10 (p = 0.008), and ECA-MCF (p = 0.016) were significantly higher, while the tPA-CFT (p < 0.001) was significantly lower among patients with high on-aspirin platelet reactivity. However, only fibrinogen proved to be an independent predictor of hypofibrinolysis in severe COVID-19 patients. In conclusion, a faster developing, more solid clot formation was observed in aspirin 'non-responder' COVID-19 patients. Therefore, an individually tailored thromboprophylaxis is needed to prevent thrombotic complications, particularly in the hypofibrinolytic cluster.

Effect of SARS-CoV-2 pandemic on blood product usage at the University of Pécs / A SARS-CoV-2-pandémia hatása a vérkészítmény-felhasználásra a Pécsi Tudományegyetemen.

Összefoglaló. Bevezetés: A COVID-19-világjárvány betegellátásra gyakorolt hatása hazánkban is jelentos. A vérellátást nehezítette a járványügyi intézkedések következményeként a véradási események elmaradása, a csökkent véradási hajlandóság, továbbá a nehezen megítélheto vérkészítményigény . A "Patient Blood Management" irányelveinek az orvosi gyakorlatban történo egyre szélesebb köru alkalmazása elosegíti az optimális vérkészítmény-felhasználást a transzfúziók lehetoség szerinti elkerülésével. Célkituzés és módszer: Vizsgálatunk célja a Pécsi Tudományegyetem Klinikai Központjának Janus Pannonius Klinikai Tömbjében a vérkészítmény-felhasználás változásainak felmérése volt a 2020. év elso öt hónapjában. Eredmények: A járványügyi intézkedéseket követo idoszakban szignifikánsan csökkent a hospitalizált betegeknek (34,08%), a transzfúziót igénylo betegeknek (39,69%) és a felhasznált vörösvérsejt-készítményeknek (46,41%) a száma, valamint az egy betegre jutó felhasznált vörösvérsejt-koncentrátum átlaga (2,61-rol 1,97-re) is. Közel 30%-os arányban csökkent a felhasznált friss fagyasztott plazma egységeinek és a thrombocytakoncentrátumoknak a száma is. Következtetés: A szigorú korlátozások életbe léptetését követoen a nehézségek ellenére sikerült elegendo mennyiségu vérkészítményt biztosítani a betegeknek. Az Országos Vérellátó Szolgálat Pécsi Regionális Vérellátó Központja munkatársainak és a klinikusok erofeszítéseinek köszönhetoen a vérkészítményigény és -kínálat között új egyensúly alakult ki, mely megfelelo ellátást biztosított a feltétlenül szükséges transzfúziók kivitelezéséhez. Orv Hetil. 2021; 162(43): 1717-1723. INTRODUCTION: The impact of COVID-19 pandemic on patient care is pronounced also in Hungary. Blood supply was hindered by the reduction of public blood donation events, the reduced willingness to donate, and the difficult predictability of blood product demand as a result of the epidemiological regulations. The wider application of Patient Blood Management guidelines in the medical practice will promote optimal blood product utilization by avoiding transfusions where possible. OBJECTIVE AND METHOD: The aim of our study was to assess the changes in the usage of blood products in the first five months of 2020 at the Clinical Center of the University of Pécs, Janus Pannonius Clinical Building. RESULTS: In the period following the epidemiological measures, we found reduction in the number of hospitalized patients (34.08%), in the number of patients requiring transfusion (39.69%) and in the number of red blood cell products used (46.41%). The number of transfused red blood cell concentrates per patient was also significantly reduced (from 2.61 to 1.97) in this period. The number of transfused fresh frozen plasma units and platelet concentrates also decreased by approximately 30%. CONCLUSION: After the implementation of the strict restrictions, despite the difficulties, it was possible to provide patients with sufficient blood products. Due to the efforts of both the Regional Blood Transfusion Center of Pécs of the Hungarian National Blood Transfusion Service and of the clinicians, a new balance was established between the demand and the supply of blood products, which provided adequate care for the necessary transfusions. Orv Hetil. 2021; 162(43): 1717-1723.

O-GlcNAcylation in early stages of chronic lymphocytic leukemia: Protocol development for flow cytometry.

BACKGROUND: Recent studies proved that metabolic changes in malignant disorders have an impact on protein glycosylation, however, only a few attempts have been made so far to use O-GlcNAc analysis as a prognostic tool. Glucose metabolism is reported to be altered in hematological malignancies thus, we hypothesized that monitoring intracellular O-GlcNAc levels in Rai stage 0-I (Binet A) CLL patients could give deeper insights regarding subtle metabolic changes of progression which are not completely detected by the routine follow-up procedures. OBJECTIVE: In this proof of concept study we established a flow cytometric detection method for the assessment of O-GlcNAcylation as a possible prognostic marker in CLL malignancy which was supported by fluorescence microscopy. METHODS: Healthy volunteers and CLL patients were recruited for this study. Lymphocytes were isolated, fixed and permeabilised by various methods to find the optimal experimental condition for O-GlcNAc detection by flow cytometry. O-GlcNAc levels were measured and compared to lymphocyte count and various blood parameters including plasma glucose level. RESULTS: The protocol we developed includes red blood cell lysis, formalin fixation, 0.1% Tween 20 permeabilisation and employs standardized cell number per sample and unstained controls. We have found significant correlation between O-GlcNAc levels and WBC (R2= 0.8535, p< 0.0029) and lymphocyte count (R2= 0.9225, p< 0.0006) in CLL patients. Interestingly, there was no such correlation in healthy individuals (R2= 0.05664 for O-GlcNAc vs WBC and R2= 0.04379 for O-GlcNAc vs lymphocytes). CONCLUSION: Analyzing O-GlcNAc changes in malignant disorders, specifically in malignant hematologic diseases such as CLL, could be a useful tool to monitor the progression of the disease.

Much More Than a Pleasant Scent: A Review on Essential Oils Supporting the Immune System.

The augmenting acceptance and application of herbal medicine in prevention and treatment of diseases also involve the use of plant essential oils (EOs) through different routes of administration (aromatherapy). Scientific data supporting the efficacy of certain herbal products are continuously growing; however, the cumulative evidence is not always sufficient. The anti-inflammatory properties of EOs have been investigated more extensively and also reviewed in different settings, but so far, our review is the first to summarize the immune-supporting properties of EOs. Our aim here is to synthesize the currently available data on the immune function enhancing effects of EOs. An online search was conducted in the PubMed database, which was terminated at the end of July 2019. Other articles were found in the reference lists of the preselected papers. Studies that applied whole EOs with known components, or single EO constituents under in vitro or in vivo laboratory conditions, or in human studies, and de facto measured parameters related to immune function as outcome measures were included. Two specific fields, EO dietary supplementation for livestock and fish, and forest bathing are also explored. Some EOs, particularly eucalyptus and ginger, seem to have immune function enhancing properties in multiple studies.

Detection of high-risk thrombophilia with an automated, global test: the Coagulation Inhibitor Potential assay.

: The diagnosis of thrombophilia is a cost-consuming and time-consuming process, as each defect should be separately investigated. The Coagulation Inhibitor Potential (CIP) assay is a promising new global test, sensitive for most of the hereditary thrombophilias, developed for manual methodology. We adapt the original method to an optical coagulation analyser. By this automation, the test will be easier, faster and more precise, and it also allows carrying out 18 measurements simultaneously. The CIP assay was performed in 126 healthy subjects and 193 patients with different types of hereditary thrombophilia conditions. Detected with conventional laboratory tests high-risk thrombophilia was present in 70 patients: deficiencies of antithrombin (AT) (n = 12), protein C (PC) (n = 14), protein S (PS) (n = 6), homozygous factor V Leiden (FVL) mutation (n = 9) and combined types (n = 29). Low-risk thrombophilia was present in 123 patients: heterozygous FVL (n = 115) and FII G2010A mutation (n = 8). Significantly lower median CIP values were found for AT-,PC-, PS deficiencies, homozygous and heterozygous FVL mutations and combined thrombophilias (P < 0.01) as compared with healthy controls. There was no significant difference between the heterozygous FIIG20210A (P = 0.669) thrombophilia group and the healthy controls. The best performance of the test was achieved at the cut-off value of 90.0 U (area: 0.981) with 96% sensitivity and 92% specificity in the high-risk thrombophilia group estimated by receiver operating characteristic analysis. The new method seems to be appropriate and reliable for the detection of AT-, PC- and PS deficiencies, homozygous FVL mutation and also for combined deficiencies. The automated CIP test is insensitive to FII G2010A mutation.

Challenges in the evaluation of D-dimer and fibrinogen levels in pregnant women.

INTRODUCTION: Normal pregnancy is associated with hypercoagulable state. Elevated markers of coagulation and fibrinolytic system activation indicate increased thrombin activity and increased fibrinolysis following fibrin formation throughout pregnancy. These changes exceed the biological variability in most cases. Haemostatic reference intervals are generally based on samples from non-pregnant women. Thus, they may not be relevant to pregnant women, a problem that may hinder accurate diagnosis and treatment of haemostatic disorders during pregnancy. The aim of the study was to follow the changes of haemostatic parameters and to establish gestational age-specific reference intervals during normal pregnancy. MATERIALS AND METHODS: Blood samples of 83 pregnant women were collected at gestational weeks 16, 26 and 36. Fibrinogen, D-dimer, and C-Reactive Protein (CRP) were examined. Reference intervals were calculated for fibrinogen, D-dimer tests with two different methods (mean±2 SD or median and 2.5th and 97.5th percentiles with 90% confidence intervals). RESULTS: fibrinogen and D-dimer increased progressively throughout pregnancy. Mean fibrinogen levels were higher than the maximum of the conventional reference interval, already in the 16th week of pregnancy. D-dimer levels were at or above the conventional cutoff point (250ng/mL) throughout the pregnancy in 42% of pregnant women, while in the 36th week 98% of them displayed elevated D-dimer levels. CRP did not increase in normal pregnancy. CONCLUSIONS: There seems to be an emerging need to reconsider fibrinogen and D-dimer values from a different aspect in pregnancy compared to non-pregnant reference intervals. New reference ranges are suggested to be established in pregnancy.