Effect of Pre-Analytical Conditions On the Results of Thrombodynamics Assay

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4393-4393
Author(s):  
Natalia M. Dashkevich ◽  
Tatiana V. Vuimo ◽  
Anna N. Balandina ◽  
Ruzanna A. Ovsepyan ◽  
Natalia P. Soshitova ◽  
...  
Keyword(s):  
Thin Layer ◽  
Healthy Volunteers ◽  
Sodium Citrate ◽  
Initial Rate ◽  
Volume Ratio ◽  
Blood Collection ◽  
Contact Activation ◽  
Stationary Rate ◽  
Significant Difference ◽  
Frozen Plasma

Abstract Abstract 4393 Introduction Thrombodynamics is a novel global coagulation assay based on spatial separation of clotting activation and propagation. In this assay fibrin clot grows from the TF-coated surface in a thin layer of non-stirred plasma. Clot formation is monitored via light scattering (Fig.1). The main parameters measured in the assay are: lag-time (Tlag), time between bringing plasma in contact with TF and actual start of clot growth: initial rate of clot growth (Vin), rate of clot growth during first 5 minutes; stationary rate of clot growth (Vst), rate of clot formation in 20 minutes after the beginning. (Fig.2) In this study we analyzed effect of the pre-analytic conditions (blood collection and plasma preparation) on the results of Thrombodynamics assay. Materials and Methods Blood was collected into sodium citrate tubes with 9:1 volume ratio. It was usually processed by two centrifugations to obtain PPP (1 600g, 15 min) and then PFP (10 000g, 5 min). 20 μg/ml of Corn Tripsin Inhibitor (CTI) was used to prevent contact activation. 20 mM of CaCl2 was added to plasma prior to the experiment. Coagulation was activated by tissue factor immobilized to plastic surface (100 pmol/m2). Results Sodium citrate venous blood collection tubes of three major manufacturers (BD Vacutainer, Greiner Vacuette and Sarstedt Monovette) and citrate concentrations 3.2% and 3.8% for Greiner Vacuette tubes were compared. Blood from 12 healthy volunteers was used for that purpose. All experiments were performed in duplicates. BD Vacutainer tubes showed significant hypercoagulation compared to the other tubes tested. Stationary rate was significantly higher (Mann-Whitney test p=0.05) in these tubes while three other types of tubes showed similar results (Table 1). There was no significant difference between 3.2% and 3.8% of sodium citrate for Vacuette tubes in all parameters measured. Sarstedt Monovette and BD Vacutainer tubes revealed an increased initial rate of clot growth compared to both Vacuette tubes (p=0.05). Lag times were similar for all the tubes tested. To estimate the possible error introduced by blood collection procedure, 3 independent collections from different veins were performed for 3 healthy volunteers. Standard deviation for the Thrombodynamics parameters were 12% for Tlag, 7% for Vin, 8% for Vst. Two centrifugation protocols were studied: PPP was centrifuged for 5 min at 10 000 g or for 20 min at 1600 g. No significant difference was observed for these methods (n=7, p=0.05). PPP can potentially be used in the Thrombodynamics assay as well as PFP with adjusted range of normal values. Clot growth rates increase in PPP compared to PFP (n=50, Vin =48.8±7.4μm/min and 45.6±12.8 μm/min for PPP and PFP respectively; Vin= 28.6±4.2 μm/min and 24.1±3.0 μm/min for PPP and PFP respectively). Inhibition of contact activation is important as thin layer of plasma is used and surface to volume ratio in experimental chamber is high. Addition of CTI to PPP with subsequent centrifugation induced significant decrease in initial rate (44.8±6.7 μm/min) compared to 51.1±7.6 μm/min when CTI was added to PFP directly, n=12, p=0.05. Stationary rate did not change significantly (25.8±3.6 μm/min and 28.2±4 μm/min respectively, p=0.05). Finally we studied the effects of sample storage. For healthy donors no significant change in Thrombodynamics parameters was observed during 3 hours (n=8) and 24 hours (n=3) of storage of PFP at RT. 50 samples of fresh and frozen plasma from healthy volunteers were compared. Clot growth rates increased for frozen plasma (Vst = 28.4±4.0μm/min) compared to non-frozen samples (24.1±3.0μm/min). Therefore frozen plasma can be used in the assay but normal ranges for frozen and non-frozen samples should be defined separately. These data represent a first step for standardization of Thrombodynamics assay and decrease of variability due to pre-analytic conditions. Disclosures: Dashkevich: HemaCore LLC: Employment. Vuimo:HemaCore LLC: Employment. Balandina:HemaCore LLC: Employment. Ovsepyan:HemaCore LLC: Employment. Soshitova:HemaCore LLC: Employment. Seregina:HemaCore LLC: Employment. Surov:HemaCore LLC: Employment. Lipets:HemaCore LLC: Employment. Panteleev:HemaCore LLC: Employment. Ataullakhanov:HemaCore LLC: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

1-Desamino-8-D-Arginine Vasopressin (DDAVP) Infusion in Type IIB von Willebrand’s Disease: Shortening of Bleeding Time and Induction of a Variable Pseudothrombocytopenia

1990 ◽  
Vol 64 (01) ◽  
pp. 117-120 ◽  
Author(s):  
Alessandra Casonato ◽  
M Teresa Sartori ◽  
Luigi de Marco ◽  
Antonio Girolami
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Count ◽  
Arginine Vasopressin ◽  
Calcium Concentration ◽  
Sodium Citrate ◽  
Bleeding Time ◽  
Blood Collection ◽  
Von Willebrand's Disease ◽  
Blood Samples ◽  
Von Willebrand’S Disease

SummaryWe have investigated the effects of 1-desamino-8-D-arginine vasopressin (DDAVP) infusion on platelet count and bleeding time in 4 patients with type IIB von Willebrand’s disease (vWd). Three of four patients showed a normalization of the bleeding time within 1 h after the infusion, while bleeding time was not modified in the fourth. In accordance with the literature, thrombocytopenia was observed after DDAVP infusion, but this thrombocytopenia was due to the anticoagulants used for blood collection. In two patients (F. I., G. F.) no thrombocytopenia was observed when platelets were counted by fingerstick method but there was a 20% platelet decrease in blood samples collected in sodium citrate and a 50% decrease in samples collected in EDTA. Dramatic falls in platelet counts (70–95%) were observed in the additional two patients (C. A., D.Z.) after DDAVP infusion, when both sodium citrate or EDTA were used as anticoagulants. In the latter two patients there was also a 50% decrease in platelet count when the fingerstick method was used. The decrease in the patient’s platelet count in EDTA samples after DDAVP infusion could be prevented, in part, by the previous additions of an anti GPIb monoclonal antibody and an anti GPIIb-IIIa monoclonal antibody.Thus, the thrombocytopenia observed in the four IIB vWd patients studied after DDAVP infusion seems to be, at least partially, a pseudothrombocytopenia depending on the calcium concentration in the blood samples and the availability of GPIb and GPIIb-IIIa receptors. These findings and the normalization of the bleeding time observed in three of the four patients has led us to reconsider the possible use of DDAVP in the treatment of our IIB vWd patients.

A Specific Immunologic Assay for Functional Plasminogen Activator Inhibitor 1 in Plasma – Standardized Measurements of the Inhibitor and Related Parameters in Patients with Venous Thromboembolic Disease

1992 ◽  
Vol 68 (05) ◽  
pp. 486-494 ◽  
Author(s):  
Malou Philips ◽  
Anne-Grethe Juul ◽  
Johan Selmer ◽  
Bent Lind ◽  
Sixtus Thorsen
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Normal Subjects ◽  
Tissue Type ◽  
Blood Collection ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Significant Difference ◽  
Activator Inhibitor ◽  
Pai 1

SummaryA new assay for functional plasminogen activator inhibitor 1 (PAI-1) in plasma was developed. The assay is based on the quantitative conversion of PAI-1 to urokinase-type plasminogen activator (u-PA)-PAI-l complex the concentration of which is then determined by an ELISA employing monoclonal anti-PAI-1 as catching antibody and monoclonal anti-u-PA as detecting antibody. The assay exhibits high sensitivity, specificity, accuracy, and precision. The level of functional PAI-1, tissue-type plasminogen activator (t-PA) activity and t-PA-PAI-1 complex was measured in normal subjects and in patients with venous thromboembolism in a silent phase. Blood collection procedures and calibration of the respective assays were rigorously standardized. It was found that the patients had a decreased fibrinolytic capacity. This could be ascribed to high plasma levels of PAI-1. The release of t-PA during venous occlusion of an arm for 10 min expressed as the increase in t-PA + t-PA-PAI-1 complex exhibited great variation and no significant difference could be demonstrated between the patients with a thrombotic tendency and the normal subjects.

scholarly journals Impact of Face Masks on 6-Minute Walk Test in Healthy Volunteers

2021 ◽  
Vol 11 (1) ◽  
pp. 204589402098843
Author(s):  
Kevin M. Swiatek ◽  
Charnetta Lester ◽  
Nicole Ng ◽  
Saahil Golia ◽  
Janet Pinson ◽  
...  
Keyword(s):  
Healthy Volunteers ◽  
Face Mask ◽  
Walk Test ◽  
Six Minute Walk Test ◽  
Significant Difference ◽  
6 Minute Walk Test ◽  
The Impact ◽  
Minute Walk

Our objective was to establish the impact of wearing a face mask on the outcome of six-minute walk test in healthy volunteers. In a study of 20 healthy volunteers who each completed two 6MWTs, one with a mask and one without, there was no difference in distance walked. However, there was a significant difference in perception of dyspnea between the two groups.

scholarly journals The Alterations in Methylene Blue/Light-Treated Frozen Plasma Proteins Revealed by Proteomics

2021 ◽  
pp. 1-8
Author(s):  
Tiange Wu ◽  
Xiaoning Wang ◽  
Kai Ren ◽  
Xiaochen Huang ◽  
Jiankai Liu
Keyword(s):  
Mass Spectrometry ◽  
Methylene Blue ◽  
Western Blot ◽  
Blue Light ◽  
Differentially Expressed ◽  
Enzyme Linked Immunosorbent Assay ◽  
Differentially Expressed Protein ◽  
Significant Difference ◽  
Modified Proteins ◽  
Frozen Plasma

Introduction: The aim of this study was to investigate the modified proteins in methylene blue/light-treated frozen plasma (MB-FP) compared with fresh frozen plasma (FFP) in order to gain a better application of MB/light-treated plasma in clinic transfusion. Methods: MB-FP and FFP were collected from Changchun central blood station, and a trichloroacetic acid/acetone precipitation method was used to remove albumin for the enrichment of lower abundance proteins. The plasma protein in MB-FP and FFP were separated using two-dimensional gel electrophoresis (2-DE) and the differentially expressed protein spots were analyzed using mass spectrometry. Finally, the differentially expressed proteins were tested using Western blot and enzyme-linked immunosorbent assay (ELISA). Results: Approximately 14 differentially expressed protein spots were detected in the MB-FP, and FFP was chosen as the control. After 2-DE comparison analysis and mass spectrometry, 8 significantly differentially expressed protein spots were identified, corresponding to 6 different proteins, including complement C1r subcomponent (C1R), inter-alpha-trypsin inhibitor heavy chain H4 (ITI-H4), keratin, type II cytoskeletal 1 (KRT1), hemopexin (HPX), fibrinogen gamma chain (FGG), and transthyretin (TTR). Western blot showed no significant difference in the expression level of KRT1 between MB-FP and FFP (p > 0.05). Both Western blot and ELISA indicated that the level of HPX was significantly higher in FFP than in MB-FP (p < 0.05). Conclusion: This comparative proteomics study revealed that some significantly modified proteins occur in MB-FP, such as C1R, ITI-H4, KRT1, HPX, FGG, and TTR. Our findings provide more theoretical data for using MB-FP in transfusion medicine. However, the relevance of the data for the transfusion of methylene blue/light-treated plasma remains unclear. The exact modification of these proteins and the effects of these modified proteins on their functions and their effects in clinical plasma infusion need to be further studied.

scholarly journals The effect of clamped and unclamped umbilical cord samples on blood gas analysis

2021 ◽  
Author(s):  
Elisabetta Colciago ◽  
Simona Fumagalli ◽  
Elena Ciarmoli ◽  
Laura Antolini ◽  
Antonella Nespoli ◽  
...  
Keyword(s):  
Blood Gas Analysis ◽  
Cross Sectional Study ◽  
Gas Analysis ◽  
Blood Collection ◽  
Large Sample Size ◽  
Blood Gas ◽  
Delayed Cord Clamping ◽  
Cross Sectional ◽  
High Risk Women ◽  
Significant Difference

Abstract Purpose Delayed cord clamping for at least 60 s is recommended to improve neonatal outcomes. The aim of this study is to evaluate whether there are differences in cord BGA between samples collected after double clamping the cord or without clamping the cord, when blood collection occurs within 60 s from birth in both groups. Methods A cross-sectional study was carried out, collecting data from 6884 high-risk women who were divided into two groups based on the method of cord sampling (clamped vs unclamped). Results There were significant decrease in pH and BE values into unclamped group compared with the clamped group. This difference remained significant when considering pathological blood gas analysis parameters, with a higher percentage of pathological pH or BE values in the unclamped group. Conclusion Samples from the unclamped cord alter the acid–base parameters compared to collection from the clamped cord; however, this difference does not appear to be of clinical relevance. Findings could be due to the large sample size, which allowed to achieve a high power and to investigate very small numerical changes between groups, leading to a statistically significant difference in pH and BE between samples even when we could not appreciate any clinical relevant difference of pH or BE between groups. When blood gas analysis is indicated, the priority should be given to the timing of blood collection to allow reliable results, to assess newborns status at birth and intervene when needed.

Safety evaluation of an antimalarial herbal product from Andrographis paniculata (AS201-01) in healthy volunteers

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Aty Widyawaruyanti ◽  
Arijanto Jonosewojo ◽  
Hilkatul Ilmi ◽  
Lidya Tumewu ◽  
Ario Imandiri ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Healthy Volunteers ◽  
Day Treatment ◽  
Phase 1 ◽  
Andrographis Paniculata ◽  
Control Group ◽  
Healthy Human ◽  
Double Blind ◽  
Random Blood Glucose ◽  
Significant Difference

Abstract Objectives Andrographis paniculata tablets (AS201-01) have previously been shown to have potent bioactivity as an antimalarial and to produce no unwanted side effects in animal models. Here, we present the phase 1 clinical trial conducted to evaluate the safety of AS201-01 tablets in healthy volunteers. Methods The study was a randomized, double-blind controlled cross-over, a placebo-controlled design consisting of a 4-day treatment of AS201-01 tablets. A total of 30 healthy human volunteers (16 males and 14 females) were divided into two groups, and each group was given 4 tablets, twice daily for 4 days. Group 1 received AS201-01, while group 2 received placebo tablets. Volunteers were given a physical examination before the treatment. The effects of AS201-01 on random blood glucose, biochemical, and hematological as well as urine profiles were investigated. Results There were no changes in observed parameters as a result of AS201-01 being administered. Statistical analysis showed no significant difference (p>0.05) between the test and control group regarding hematology profile, biochemical profile, and random blood glucose. Increased appetite and better sleep, which categorized as grade 1 adverse event was reported after treatment with AS201-01 tablet Conclusions The outcome supports our previous observation that the AS201-01 tablet, given twice a day for 4 days, is safe and nontoxic.

scholarly journals Serotonin and Tryptophan Serum Concentrations in Shelter Dogs Showing Different Behavioural Responses to a Potentially Stressful Procedure

2020 ◽  
Vol 8 (1) ◽  
pp. 1
Author(s):  
Giacomo Riggio ◽  
Chiara Mariti ◽  
Valeria Sergi ◽  
Silvana Diverio ◽  
Angelo Gazzano
Keyword(s):  
Behavioural Response ◽  
Blood Collection ◽  
Stressful Situation ◽  
Serum Concentrations ◽  
Chi Square ◽  
Significant Difference ◽  
Relaxation Stress ◽  
Shelter Dogs ◽  
Stress Signals ◽  
The Relationship

In mammals, serotonin (5-HT) levels depend on the availability of tryptophan (TRP). Low 5-HT concentrations have been linked to behavioural disorders in dogs. This study aimed at investigating possible differences in dogs’ serum TRP and 5-HT concentrations according to their behavioural response to a potentially stressful procedure. Thirty-nine physically healthy shelter dogs, 15 females and 24 males, mean age = 5.6 years, were categorized by a certified veterinary behaviourist according to their behavioural response to medical examination and blood collection, in: relaxation, stress signals, tension without growling, tension with growling, escape attempts, and aggression attempts. Extraction and quantification of 5-HT and TRP were performed using a HLPC method. Data were statistically analysed, applying Chi-square and Spearman tests. Results showed no significant difference in TRP (χ2 = 2.084, p = 0.555) nor 5-HT (χ2 = 0.972, p = 0.808) serum concentrations among different categories of dogs; however, some categories were underrepresented (relaxation = 20.5%, stress signals = 30.8%, tension without growling = 43.6%, tension with growling = 5.1%, escape attempts = 0%, aggression attempts = 0%). No correlation between serum TRP and 5-HT concentrations was found (ρ = 0.086, p = 0.602). Serum 5-HT levels do not seem to be associated with dogs’ behavioural response to a stressful situation nor with serum TRP concentrations. The relationship between serum TRP and 5-HT concentrations and behaviour needs further research.

Molar Absorptivity Values for Aflatoxins and Justification for Their Use as Criteria of Purity of Analytical Standards

1970 ◽  
Vol 53 (1) ◽  
pp. 96-101 ◽  
Author(s):  
J V Rodricks ◽  
L Stoloff ◽  
W A Pons ◽  
J A Robertson ◽  
L A Goldblatt
Keyword(s):  
Thin Layer ◽  
Statistical Study ◽  
Molar Absorptivity ◽  
Confidence Limits ◽  
Components Of Variance ◽  
Significant Difference ◽  
Single Determination ◽  
Comparison Measurements ◽  
Analytical Standards

Abstract Measurements of molar absorptivities in methanol were carried out by two laboratories on samples of anatoxins Bi and Gi prepared and purified independently in four laboratories and on samples of aflatoxins B2 and G2 prepared and purified independently in three laboratories. Molar absorptivities of pure aflatoxins Bi, B2, G1, and G2 in benzene-acetonitrile (98 + 2) were determined at two laboratories. With the exception of aflatoxin G2, no significant difference between aflatoxin samples could be demonstrated. Molar absorptivity values and absorbance ratios for each aflatoxin, based on these data, are given. Statistical 95% confidence limits were established for a single determination of molar absorptivity. A statistical study was made of the components of variance which contribute to the accuracy of the determination of molar absorptivity. A comparison of the fluorescence intensities of spots of various aflatoxin preparations, developed on silica gel-coated thin layer plates, with the amount of aflatoxin in each spotting solution as determined by absorbance measurements, demonstrated the validity of using absorbance and fluorescence comparison measurements for aflatoxin quantitation.

Abstract 51: Peripheral Regulatory T cells and Th17 Cells Is Associated With Pathogenesis of Moyamoya Disease

Stroke ◽  
2016 ◽  
Vol 47 (suppl_1) ◽  
Author(s):  
Leihua Weng ◽  
Xiang Chen ◽  
Yun Xu
Keyword(s):  
Healthy Volunteers ◽  
Moyamoya Disease ◽  
Peripheral Blood ◽  
Th17 Cells ◽  
Treg Cells ◽  
Peripheral Blood Lymphocytes ◽  
Pathological Process ◽  
Medium Term ◽  
Significant Difference ◽  
And Gender

Background: Despite unclear pathogenesis, previous studies have suggested immune responses may play a pivotal role in the process of Moyamoya disease (MMD), a rare cerebrovascular occlusive disorder. The objective of this study is aimed to explore the change of peripheral Treg/Th17 in MMDpatients and whether the change is associated with pathogenesis of MMD. Methods: In the present study, we collected 26 MMD patients diagnosed by angiography according to the diagnostic criteria of definitive MMD and recruited 32 healthy volunteers. To explore the balance of peripheral Treg/Th17 in MMD patients, lymphocytes in peripheral blood were harvested and flow cytometry was used to measure the percentage of Treg and Th17in CD4+ Tcells, respectively. Meanwhile, relevant cytokines in serum were measured to evaluate the function of Treg and Th17 cells. Results: According to Suzuki’s angiographic staging of moyamoya disease, patients were divided into subgroups of the preliminary-term, medium-term and late-term. Cerebral hemorrhage is thefirstsymptom of onset occuringin half of patients, followed bycerebral ischemia.Our data revealed that both the percentage of Treg and Th17 cells in peripheral blood lymphocytes was increased in MMD patients compared with volunteer group. Meanwhile, the levels of IL-6, IL-10,IL-12, IL-17, TNF-α, VEGF and TGF-β in serum were significantly increased in MMD patients. In this study, the level of HMGB-1, a middle-late period inflammation biomarker, in serum of MMD patients is obviously elevated compared with volunteers. However, the ratio of Treg/Th17 had no significant difference in MMD patients compared to healthy volunteers. Intriguingly, our data revealed that ratio of Treg/Th17 was significantly increased in late-term MMD patients compared with medium-term patients as evidenced by elevated percentage of Treg cells.. In addition, TGF-β level in later-term MMD patients was significantly higher than this in medium-term MMD patients. No difference was observed in the way of onset and gender between two groups. Conclusion: Enhanced peripheral Treg and Th17 in MMD patients suggested that there may be an immunological component in the pathogenesis of MMD. Peripheral Treg may be associated with pathological process of MMD.

scholarly journals Evaluation of ubiquitin C-terminal hydrolase-L1 enzyme levels in patients with epilepsy

2020 ◽  
Vol 78 (7) ◽  
pp. 424-429
Author(s):  
Ibrahim Halil YASAK ◽  
Mustafa YILMAZ ◽  
Murat GÖNEN ◽  
Metin ATESCELIK ◽  
Mehtap GURGER ◽  
...  
Keyword(s):  
Healthy Volunteers ◽  
Epileptic Seizure ◽  
Epileptic Seizures ◽  
Healthy Individuals ◽  
Roc Curve Analysis ◽  
Remission Period ◽  
Significant Difference ◽  
Epileptic Attack ◽  
Patients With Epilepsy

ABSTRACT Objective: Ubiquitin C-terminal Hydrolase-L1 (UCH-L1) enzyme levels were investigated in patients with epilepsy, epileptic seizure, remission period, and healthy individuals. Methods: Three main groups were evaluated, including epileptic seizure, patients with epilepsy in the non-seizure period, and healthy volunteers. The patients having a seizure in the Emergency department or brought by a postictal confusion were included in the epileptic attack group. The patients having a seizure attack or presenting to the Neurology outpatient department for follow up were included in the non-seizure (remission period) group. Results: The UCH-L1 enzyme levels of 160 patients with epilepsy (80 patients with epileptic attack and 80 patients with epilepsy in the non-seizure period) and 100 healthy volunteers were compared. Whereas the UCH-L1 enzyme levels were 8.30 (IQR=6.57‒11.40) ng/mL in all patients with epilepsy, they were detected as 3.90 (IQR=3.31‒7.22) ng/mL in healthy volunteers, and significantly increased in numbers for those with epilepsy (p<0.001). However, whereas the UCH-L1 levels were 8.50 (IQR=6.93‒11.16) ng/mL in the patients with epileptic seizures, they were 8.10 (IQR=6.22‒11.93) ng/mL in the non-seizure period, and no significant difference was detected (p=0.6123). When the UCH-L1 cut-off value was taken as 4.34 mg/mL in Receiver Operating Characteristic (ROC) Curve analysis, the sensitivity and specificity detected were 93.75 and 66.00%, respectively (AUG=0.801; p<0.0001; 95%CI 0.747‒0.848) for patients with epilepsy. Conclusion: Even though UCH-L1 levels significantly increased more in patients with epilepsy than in healthy individuals, there was no difference between epileptic seizure and non-seizure periods.

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