Platelet Function: Meloxicam Intravenous in Whole Blood Samples From Healthy Volunteers

Abstract Background Rupture of an atherosclerotic plaque and subsequent exposure of the subendothelial prothrombotic matrix to blood cause arterial thrombosis. Circulating platelets play an indispensable role in the growth of arterial thrombi partially owing to their unique ability to adhere to the subendothelial matrix and to aggregate to each other under flow conditions. Recently, the Total Thrombus-formation Analysis System (T-TAS) was developed for ex vivo analysis of the thrombogenic potential of whole blood samples under flow conditions. Despite the potential clinical utility of the T-TAS in assessing the risk for thrombosis and bleeding, reference intervals for T-TAS analysis in healthy individuals have not been determined. Methods In total, 122 whole blood samples were collected from healthy volunteers ranging in age from 25 to 45 years. T-TAS analysis and hematological, physiological, and lifestyle assessments were conducted in these subjects. Whole blood samples anticoagulated with hirudin were perfused into a collagen-coated microchip (PL chip). The time to 10 kPa and the area under the flow pressure curve up to 10 min (AUC10) were analyzed as representative variables for thrombogenic potential. Reference intervals, which were defined as 2.5–97.5 percentiles, were determined. Additionally, univariate and multivariate analyses were performed to identify factors associated with the AUC10 in the T-TAS. Results The time to 10 kPa and the AUC10 widely varied, even in healthy volunteers. The reference intervals were 1.50–4.02 min and 223.4–456.8, respectively, at a shear rate of 1500 s− 1. Univariate and multivariate analyses showed that platelet counts were most significantly associated with the AUC10 of the T-TAS. The presence of one or more cardiovascular risk factors of a high body mass index, a high pulse pressure, high fasting serum glucose levels, high low-density lipoprotein-cholesterol levels, a history of smoking, and no habitual exercise, had the second largest effect on the AUC10 of the T-TAS. Conclusions Healthy volunteers who had any cardiovascular risk factors showed augmented thrombogenicity, even in artificial uniform capillaries, compared with those without any risk factors in the T-TAS.

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A Strong Correlation of the HIF1a mRNA Expression Level with the Total Expression of JAK2, As Well As a Decrease in Their Level Is Observed in Leukocytes of Patients with MPN and Lymphoproliferative Cancer

Blood ◽

10.1182/blood-2019-122086 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5032-5032

Author(s):

Igor A. Olkhovskiy ◽

Aleksey S. Gorbenko ◽

Marina A. Stolyar ◽

Anna V. Komina ◽

Evgeniy V. Vasiliev ◽

...

Keyword(s):

Gene Expression ◽

Healthy Volunteers ◽

Mrna Expression ◽

Whole Blood ◽

Strong Correlation ◽

Blood Cells ◽

Expression Level ◽

Blood Samples ◽

Whole Blood Samples ◽

Lymphoproliferative Cancer

Background. The hypoxia inducible factor 1a (HIF1a) is an important transcription factor regulating gene expression for an adaptive response to low oxygen level in human cells. Earlier, we observed a decrease of HIF1a and CALR mRNA, but not MPL expression in whole blood samples of patients with Ph-negative myeloproliferative neoplasm (MPN) compared with healthy volunteers (Gorbenko A., et al., Haematologica, 2017; Gorbenko A., et al., Blood, 2016). Recent transcriptome studies in granulocytes and CD34+ blood cells from MPN patients confirmed a decrease of mRNA CALR, but also revealed an increase of the relative mRNA level of HIF1a and JAK2 (Cokic VP, et al., PLoS ONE, 2015). These changes in the regulation of number genes expression may depend on the mutation JAK2V617F as was also shown (Berkofsky-Fessler W, et al., Clin Cancer Res. 2010). But some studies demonstrated transcriptomes data of the isolated blood cells from patients with lymphoproliferative disease did not find significant changes in the expression of these genes (Liao W, et al., BMC Cancer, 2015). Aims. Investigate HIF1a, CALR and JAK2 mRNA expression in patients with lymphoproliferative and myeloproliferative cancer. Methods. 10 healthy volunteers (average age 42 years, range 20-63 years, 80% of men) and 80 (average age 54 years, range 30-83 years, 46% of man), patients with MPN, also 36 (average age 62 years, range 28-79 years, 78% of man) patients with lymphoproliferative cancer after signing an informed consent were included in this study. In our study, we investigated the expression level of mRNA genes in whole blood samples obtained in test tubes with an RNA stabilizer (LTD "Formula of gene", Russia) in order to exclude the factors of preanalytical cell hypoxia. Quantitative real-time PCR was performed to determine the levels of HIF1a, CALR and JAK2 mRNA transcripts using TaqMan probes on the CFX96 (Bio-Rad). The results were calculated by ΔCt method in the software package of "R". The threshold cycles (Ct) genes and housekeeping genes (TBP, GUS, ABL) was determined using Cy0 method. The results were normalized by these reference genes. Mann-Whitney U-test was used to evaluate significance of differences between the groups, the degree of correlation (r) was assessed using Spearman test. Results. We observed that HIF1a and JAK2 mRNA expression was significantly lower in whole blood samples of all patients with MPN and lymphoproliferative cancer compared with a group of healthy volunteers (p<0,001) (Figure). We discovered a strong correlation between JAK2 and HIF1a expression in all myeloproliferative and lymphoproliferative neoplasms (r=0.83 and r=0.93 respectively, p<0,001) (Figure). It should be noted that the correlation in the blood samples of patients with MPN was observed only when the total expression of wild and mutated transcripts JAK2 (JAK2+JAK2 V617F) was assessed. No correlation was found between the level of mRNA expression and the cellular number of granulocytes or lymphocytes. The expression level of CALR mRNA also decreases in the blood cells of MPN and lymphoproliferative cancer patients (p<0.05), but we did not observe its correlation with HIF1a or JAK2 mRNA. Conclusion. We assume that the studied gene expression changes reflect the regulated metabolic processes in the cancer stem cells. Probably, the activation of the associated signaling pathways HIF1a and JAK2/STAT in the white blood cells of patients with chronic blood cancer leads to the adoptive enhancement of autophagy, causing a chronic course of the disease. The assume that the opposite shifts of HIF1a and JAK2 in the microarray research (Cokic VP, et al., PLoS ONE, 2015) can be associated with the procedure of blood cells isolation and the absence of a group of healthy people as a control. Reduced expression of CALR mRNA in patient blood cells requires further investigation. Disclosures No relevant conflicts of interest to declare.

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Characterization of a Microchip Flow-Chamber System for the Analysis of Platelet Thrombus Formation Using Whole Blood Samples - Studies On Healthy Subjects

Blood ◽

10.1182/blood.v120.21.1144.1144 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1144-1144

Author(s):

Yusuke Yamaguchi ◽

Takanori Moriki ◽

Atsuko Igari ◽

Yumiko Matsubara ◽

Tomoko Ohnishi ◽

...

Keyword(s):

Platelet Function ◽

Whole Blood ◽

Healthy Subjects ◽

Thrombus Formation ◽

Flow Chamber ◽

Advisory Committees ◽

Blood Samples ◽

Platelet Aggregometry ◽

Platelet Thrombus ◽

Whole Blood Samples

Abstract Abstract 1144 Introduction: A flow-chamber system was developed to evaluate the growth of platelet thrombus formation (PTF) quantitatively using whole blood under various shear stress conditions. This device, T-TAS (Total Thrombus-formation Analysis System, Fujimori Kogyo Co., Yokohama, Kanagawa), analyzes the process of PTF by monitoring the continuous pressure increase in the capillary of microchip where whole blood flows, using two kinds of thrombogenic surfaces (PL chip: coated with collagen, AR chip: coated with collagen plus tissue factor). In the current study, we characterized this system using whole blood samples from healthy subjects by comparing the measurements with those of other standard platelet function tests. Materials and Methods: Whole blood samples were collected from 32 healthy volunteers with hirudin (PL chip) or 3.2% sodium citrate (AR chip) as anticoagulants. For AR chip, CaCl2 with corn trypsin inhibitor was mixed immediately before the testing. The samples were individually applied on the system to measure the PTF starting time (T10: time to reach 10 kPa), occlusion time (OT: T60, time to reach 60 kPa for PL chip; T80, 80 kPa for AR chip), and AUC (area under the flow pressure curve: AUC10, until 10 min for PL chip; AUC30, until 30 min for AR chip) under various shear rates (1000, 1500, 2000 s−1 for PL chip; 300 s−1 for AR chip). Platelet function of the blood sample was also tested using platelet aggregometry (collagen, ADP, ristocetin, and epinephrine as agonists), PFA-100 (C/EPI-, C/ADP-CT: closure time) and VerifyNow P2Y12 assay (PRU). Results: In PL chip, T10 was correlated with C/EPI- and C/ADP-CT, and AUC10 was correlated with C/EPI-CT under all of the shear conditions. The correlation was enhanced in accordance with the increase of the shear rates. In addition, T60 and AUC10 were correlated with AUC of collagen-induced aggregation curve of platelet aggregometry. In AR chip, T10–80, reflecting the rate of thrombus growth, was likely correlated with C/ADP-CT. Measured values from VerifyNow P2Y12 assay was not significantly associated with those from this system. Interestingly, platelet numbers were significantly correlated with all of the measurements with AR chip, and partially with those with PL chip. Conclusion: In healthy subjects, PTF starting time and AUC with PL chip, and the growth rate of PTF with AR chip, seemed associated with PFA-100 measurements, indicating its characteristics related to shear induced PTF. However, the values from this system showed a rare correlation with those from platelet aggregometry and VerifyNow P2Y12 assay. This system may allow us to identify the parameters of individuals' thrombogenicity independent of those related to other platelet function tests, under whole blood flow conditions. Disclosures: Matsubara: Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees. Ohnishi:Fujimori Kogyo Co.: Employment. Hosokawa:Fujimori Kogyo Co.: Employment. Murata:Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees.

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Shear stress induced activation of von Willebrand factor may predispose to gastrointestinal bleeding in syndrome of Heyde

European Heart Journal ◽

10.1093/ehjci/ehaa946.3820 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

Y.N Avtaeva ◽

I.S Melnikov ◽

A.E Komlev ◽

T.E Imaev ◽

S.D Okhota ◽

...

Keyword(s):

Shear Stress ◽

Gastrointestinal Bleeding ◽

Healthy Volunteers ◽

Whole Blood ◽

Platelet Adhesion ◽

Blood Samples ◽

Shear Rates ◽

Whole Blood Samples ◽

Heyde’S Syndrome ◽

Von Willebrand

Abstract Introduction Von Willebrand factor (vWF) changes conformation from globular to fibrillar in the range of shear rates above 5000 s-1. High shear rates, observed in severe aortic stenosis, create conditions for activation of vWF, which opens up access for platelets and coagulation factors to the previously hidden domains of the molecule. At the same time, vWF undergoes increased degradation by metalloproteinase ADAMTS13. Proteolytic cleavage of vWF leads to deficiency of hemostatically active high molecular weight multimers (HMWM) of vWF, while its mass concentration remains unaltered. This results in the development of acquired von Willebrand disease type 2A (vWD) and concomitant gastrointestinal bleeding. The combination of acquired vWD 2A and gastrointestinal bleeding, developed due to severe aortic stenosis, is called Heyde's syndrome. The correlation of shear stress activation of vWF and gastrointestinal bleeding in patients with Heyde's syndrome remains poorly studied. The aim of the study was to measure vWF-mediated platelet adhesion to fibrinogen-coated surfaces under shear rates higher than 5000 s-1 in whole blood samples of healthy volunteers and patients with Heyde's syndrome. Methods A microfluidic system simulating blood flow in vessels was used to assess platelet adhesion. Platelet adhesion was measured by an increase in the intensity of laser radiation scattered from a fibrinogen-coated surface during a 15 minutes circulation of whole blood samples through a flow chamber under shear rates higher than 5000 s-1. Platelets in whole blood samples were activated with 5 μM ADP prior to measurement. The study included 5 patients with Heyde's syndrome 55–80 years old. The control group included 6 healthy volunteers 25–55 years old. vWF-mediated platelet adhesion was detected by blocking platelet-vWF binding with anti-GPIb monoclonal antibody (mAb). Fibrinogen-mediated platelet adhesion was detected by blocking platelet GPIIb/IIIa receptors with mAb. Result The inhibition of GPIb vWF-receptor reduced platelet adhesion by 7.6±3.5% (p<0.05) in patients with Heyde's syndrome, and by 16.5±3.3% (p<0.05) in healthy volunteers. The inhibition of GPIIb/IIIa fibrinogen receptor reduced platelet adhesion by 96±7% (p<0.05) in patients with Heyde's syndrome, and by 80.2±6.6% (p<0.05) in healthy volunteers. Conclusion Significantly reduced contribution of vWF to platelet adhesion under shear rates higher than 5000 s-1 may indicate a decrease in hemostatically active HMWM of vWF. Shear stress activation of vWF in the range of high shear rates and its subsequent inactivation by ADAMTS13 may lead to functional vWF deficiency and the development of gastrointestinal bleeding in Heyde's syndrome. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): The Russian Science Foundation

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Steadiness/stability of antiepileptic drugs in serum, plasma and whole-blood samples under various storage methods

Neuropediatrics ◽

10.1055/s-0030-1265593 ◽

2010 ◽

Vol 41 (02) ◽

Author(s):

N Shazi ◽

A Böss ◽

HJ Merkel ◽

F Scharbert ◽

D Hannak ◽

...

Keyword(s):

Antiepileptic Drugs ◽

Whole Blood ◽

Blood Samples ◽

Storage Methods ◽

Whole Blood Samples

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A New Method to Assess Platelet Activation and Leukocyte-platelet Aggregates in Whole Blood Samples by Flow Cytometry for Clinical Studies

10.1055/s-0039-1680171 ◽

2019 ◽

Author(s):

O. Hidiatov ◽

R. Jouni ◽

I. Marini ◽

A. Straub ◽

T. Bakchoul

Keyword(s):

Flow Cytometry ◽

Platelet Activation ◽

Whole Blood ◽

Clinical Studies ◽

New Method ◽

Blood Samples ◽

Platelet Aggregates ◽

Whole Blood Samples

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Collection of Human Liver Biopsy and Whole Blood Samples From T1DM, TP or PP Patients for Potential Use for Insulin Producing Cells in Future Clinical Studies

Case Medical Research ◽

10.31525/ct1-nct04038931 ◽

2019 ◽

Author(s):

Keyword(s):

Liver Biopsy ◽

Whole Blood ◽

Clinical Studies ◽

Human Liver ◽

Blood Samples ◽

Insulin Producing Cells ◽

Whole Blood Samples ◽

Potential Use

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Simultaneous detection of Epstein - Barr virus and Cytomegalovirus DNA in Mini-pooling of Whole blood Samples from Iraqi blood donors by Multiplex Real-Time PCR Assay

10.36295/asro.2020.231441 ◽

2020 ◽

Vol 23 (14) ◽

Author(s):

Ghinwa S. Majid ◽

Ahmed S. Abdulamir ◽

Abbas M. Ahmed ◽

Iman M. Aufi ◽

Orooba I. Abdullah

Keyword(s):

Real Time Pcr ◽

Whole Blood ◽

Epstein Barr Virus ◽

Blood Donors ◽

Simultaneous Detection ◽

Pcr Assay ◽

Blood Samples ◽

Barr Virus ◽

Whole Blood Samples ◽

Epstein Barr

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Determination of 19 Psychoactive Substances in Premortem and Postmortem Whole Blood Samples Using Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry

Separations ◽

10.3390/separations8060078 ◽

2021 ◽

Vol 8 (6) ◽

pp. 78

Author(s):

Sevasti Karampela ◽

Jessica Smith ◽

Irene Panderi

Keyword(s):

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

Formic Acid ◽

Whole Blood ◽

High Performance ◽

Tandem Mass ◽

Psychoactive Substances ◽

Blood Samples ◽

Whole Blood Samples

An ever-increasing need exists within the forensic laboratories to develop analytical processes for the qualitative and quantitative determination of a broad spectrum of new psychoactive substances. Phenylethylamine derivatives are among the major classes of psychoactive substances available on the global market and include both amphetamine analogues and synthetic cathinones. In this work, an ultra-high-performance liquid chromatography-positive ion electrospray ionization tandem mass spectrometric method (UHPLC-ESI-MS/MS) has been developed and fully validated for the determination of 19 psychoactive substances, including nine amphetamine-type stimulants and 10 synthetic cathinone derivatives, in premortem and postmortem whole blood. The assay was based on the use of 1 mL premortem or postmortem whole blood, following solid phase extraction prior to the analysis. The separation was achieved on a Poroshell 120 EC-C18 analytical column with a gradient mobile phase of 0.1% formic acid in acetonitrile and 0.1% formic acid in water in 9 min. The dynamic multiple reaction monitoring used in this work allowed for limit of detection (LOD) and lower limit of quantitation (LOQ) values of 0.5 and 2 ng mL−1, respectively, for all analytes both in premortem and postmortem whole blood samples. A quadratic calibration model was used for the 12 quantitative analytes over the concentration range of 20–2000 ng mL−1, and the method was shown to be precise and accurate both in premortem and postmortem whole blood. The method was applied to the analysis of real cases and proved to be a valuable tool in forensic and clinical toxicology.

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