A simple clot based assay for detection of procoagulant cell-derived microparticles

2016 ◽  
Vol 54 (5) ◽  
Author(s):  
Rucha Patil ◽  
Kanjaksha Ghosh ◽  
Shrimati Shetty
Keyword(s):  
Flow Cytometry ◽  
Calcium Chloride ◽  
Gold Standard ◽  
Screening Method ◽  
Detection Methods ◽  
Clinical Settings ◽  
Clotting Time ◽  
Recalcification Time ◽  
Free Plasma

AbstractBackground:Cell-derived microparticles (MPs) are important biomarkers in many facets of medicine. However, the MP detection methods used till date are costly and time consuming. The main aim of this study was to standardize an in-house clot based screening method for MP detection which would not only be specific and sensitive, but also inexpensive.Methods:Four different methods of MP assessment were performed and the results correlated. Using the flow cytometry technique as the gold standard, 25 samples with normal phosphatidylserine (PS) expressing MP levels and 25 samples with elevated levels were selected, which was cross checked by the commercial STA Procoag PPL clotting time (CT) assay. A simple recalcification time and an in-house clot assay were the remaining two tests. The in-house test measures the CT after the addition of calcium chloride to MP rich plasma, following incubation with Russell viper venom and phospholipid free plasma.Results:The CT obtained by the in-house assay significantly correlated with the results obtained by flow cytometry (RConclusions:Though preliminary, the in-house assay seems to be efficient, inexpensive and promising. It could definitely be utilized routinely for procoagulant MP assessment in various clinical settings.

scholarly journals Detection of Movement Events of Long-Track Speed Skating Using Wearable Inertial Sensors

Sensors ◽  
2021 ◽  
Vol 21 (11) ◽  
pp. 3649
Author(s):  
Yosuke Tomita ◽  
Tomoki Iizuka ◽  
Koichi Irisawa ◽  
Shigeyuki Imura
Keyword(s):  
Gold Standard ◽  
Detection Method ◽  
Standard Measure ◽  
Detection Methods ◽  
Phase Identification ◽  
Foot Pressure ◽  
Pressure System ◽  
Speed Skating ◽  
Stance Time ◽  
Integrated Detection

Inertial measurement units (IMUs) have been used increasingly to characterize long-track speed skating. We aimed to estimate the accuracy of IMUs for use in phase identification of long-track speed skating. Twelve healthy competitive athletes on a university long-track speed skating team participated in this study. Foot pressure, acceleration and knee joint angle were recorded during a 1000-m speed skating trial using the foot pressure system and IMUs. The foot contact and foot-off timing were identified using three methods (kinetic, acceleration and integrated detection) and the stance time was also calculated. Kinetic detection was used as the gold standard measure. Repeated analysis of variance, intra-class coefficients (ICCs) and Bland-Altman plots were used to estimate the extent of agreement between the detection methods. The stance time computed using the acceleration and integrated detection methods did not differ by more than 3.6% from the gold standard measure. The ICCs ranged between 0.657 and 0.927 for the acceleration detection method and 0.700 and 0.948 for the integrated detection method. The limits of agreement were between 90.1% and 96.1% for the average stance time. Phase identification using acceleration and integrated detection methods is valid for evaluating the kinematic characteristics during long-track speed skating.

scholarly journals O46 Flow cytometry- the new ‘gold standard’ for Coeliac Disease diagnosis?

2021 ◽  
Author(s):  
Jeremy Woodward ◽  
Hannah Creasey ◽  
Jennifer Stevens ◽  
Nina Bruggeman ◽  
David Bloxham
Keyword(s):  
Flow Cytometry ◽  
Coeliac Disease ◽  
Gold Standard ◽  
Disease Diagnosis

scholarly journals An Evaluation of Three Kinematic Methods for Gait Event Detection Compared to the Kinetic-Based ‘Gold Standard’

Sensors ◽  
2020 ◽  
Vol 20 (18) ◽  
pp. 5272
Author(s):  
Nicole Zahradka ◽  
Khushboo Verma ◽  
Ahad Behboodi ◽  
Barry Bodt ◽  
Henry Wright ◽  
...  
Keyword(s):  
Event Detection ◽  
Gold Standard ◽  
Kinetic Data ◽  
Force Plate ◽  
Detection Methods ◽  
Initial Contact ◽  
Typically Developing ◽  
Typically Developing Children ◽  
Sensor Signals ◽  
Video Sensor

Video- and sensor-based gait analysis systems are rapidly emerging for use in ‘real world’ scenarios outside of typical instrumented motion analysis laboratories. Unlike laboratory systems, such systems do not use kinetic data from force plates, rather, gait events such as initial contact (IC) and terminal contact (TC) are estimated from video and sensor signals. There are, however, detection errors inherent in kinematic gait event detection methods (GEDM) and comparative study between classic laboratory and video/sensor-based systems is warranted. For this study, three kinematic methods: coordinate based treadmill algorithm (CBTA), shank angular velocity (SK), and foot velocity algorithm (FVA) were compared to ‘gold standard’ force plate methods (GS) for determining IC and TC in adults (n = 6), typically developing children (n = 5) and children with cerebral palsy (n = 6). The root mean square error (RMSE) values for CBTA, SK, and FVA were 27.22, 47.33, and 78.41 ms, respectively. On average, GED was detected earlier in CBTA and SK (CBTA: −9.54 ± 0.66 ms, SK: −33.41 ± 0.86 ms) and delayed in FVA (21.00 ± 1.96 ms). The statistical model demonstrated insensitivity to variations in group, side, and individuals. Out of three kinematic GEDMs, SK GEDM can best be used for sensor-based gait event detection.

Research activities for the monitoring of genetically modified organisms in Japan

2010 ◽  
Vol 82 (1) ◽  
pp. 139-147
Author(s):  
Kazumi Kitta
Keyword(s):  
Quality Control ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Screening Method ◽  
Simultaneous Detection ◽  
Cauliflower Mosaic Virus ◽  
Detection Methods ◽  
Internal Quality ◽  
Research Activities ◽  
Labeling System

The Japanese government introduced a labeling system for genetically modified (GM) foods. To ensure the authenticity of the labeling system, we have developed and validated detection methods for newly approved GM events. One was the development of quantitative analytical methods utilizing plasmid DNAs as calibrators, which enabled us to obtain an unlimited supply of calibrators of consistent quality and also to obtain a stable standard curve to quantify GM organisms (GMOs) in samples. The significance of quality control has been recognized among relevant stakeholders, and in response we launched a project to distribute certified reference materials (CRMs) to the users of our methods for the purpose of internal quality control. In addition to these activities, we have developed time- and cost-effective detection methods, such as a new screening method to simultaneously detect the sequence of Cauliflower mosaic virus 35S promoter (p35S) and the construct-specific sequence of GA21 event utilizing multiplex real-time polymerase chain reaction (PCR). We also developed a qualitative nonaplex PCR detection method, which allows the simultaneous detection of eight events of GM maize lines. Because the influx of any unapproved and unknown GMOs into the Japanese market is not permitted, we continue to explore this issue.

Role of erythrocytes and platelets in the hypercoagulable status in polycythemia vera through phosphatidylserine exposure and microparticle generation

2013 ◽  
Vol 109 (06) ◽  
pp. 1025-1032 ◽  
Author(s):  
Chunyan Gao ◽  
Xue Yang ◽  
Jianan Li ◽  
Wei Wang ◽  
Jinxiao Hou ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Cells ◽  
Significant Inhibition ◽  
Polycythaemia Vera ◽  
Factor V ◽  
Healthy Controls ◽  
Clotting Time ◽  
Thrombotic Risk ◽  
Few Data

SummaryThe development of thrombosis in polycythaemia vera (PV) involves multifactorial processes including pathological activation of blood cells. Release of microparticles (MPs) by activated cells in diseases is associated with thrombotic risk, but relatively few data are available in PV. The aim of the present study was to investigate the increase in MP release and exposure of phosphatidylserine (PS) on the outer membrane of MP-origin cells in patients with PV, and to analyse their procoagulant activity (PCA). PS-positive MPs and cells were detected by flow cytometry, while PCA was assessed with clotting time and purified coagulation complex assays. We found that PV patients had elevated circulating lactadherin+ MPs, which mostly originating from erythrocytes, platelets, granulocytes, and endothelial cells, as well as increased PS exposing erythrocytes/platelets as compared to secondary polycythaemia patients or healthy controls. These PS-bearing MPs and cells were highly procoagulant. Moreover, lactadherin competed factor V and VIII to PS and inhibited about 90% of the detected PCA in a dose-response manner while anti-TF antibody did no significant inhibition. Treatment with hydroxyurea is associated with a decrease in PS exposure and lactadherin+ MP release of erythrocytes/platelets. Our data demonstrate that PV patients are characterised by increased circulating procoagulant MPs and PS exposing erythrocytes/platelets, which could contribute to the hypercoagulable state in these patients.

scholarly journals Clinical Evaluation of thei-STATKaolin Activated Clotting Time (ACT) Test in Different Clinical Settings in a Large Academic Urban Medical Center

2011 ◽  
Vol 135 (5) ◽  
pp. 741-748 ◽  
Author(s):  
Elizabeth Lee Lewandrowski ◽  
Elizabeth M. Van Cott ◽  
Kimberly Gregory ◽  
Ik-Kyung Jang ◽  
Kent B. Lewandrowski
Keyword(s):  
Clinical Evaluation ◽  
Medical Center ◽  
Clinical Settings ◽  
Clotting Time ◽  
Activated Clotting Time ◽  
Act Test

scholarly journals A Smartphone-Based Approach to Screening for Sudden Sensorineural Hearing Loss: Cross-Sectional Validity Study

10.2196/23047 ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. e23047
Author(s):  
Heng-Yu Haley Lin ◽  
Yuan-Chia Chu ◽  
Ying-Hui Lai ◽  
Hsiu-Lien Cheng ◽  
Feipei Lai ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Sensorineural Hearing Loss ◽  
Gold Standard ◽  
Pure Tone ◽  
Pure Tone Audiometry ◽  
Sudden Sensorineural Hearing Loss ◽  
Sensorineural Hearing ◽  
Clinical Settings ◽  
Cross Sectional ◽  
Scale Difference

Background Sudden sensorineural hearing loss (SSNHL) is an otologic emergency that warrants urgent management. Pure-tone audiometry remains the gold standard for definitively diagnosing SSNHL. However, in clinical settings such as primary care practices and urgent care facilities, conventional pure-tone audiometry is often unavailable. Objective This study aimed to determine the correlation between hearing outcomes measured by conventional pure-tone audiometry and those measured by the proposed smartphone-based Ear Scale app and determine the diagnostic validity of the hearing scale differences between the two ears as obtained by the Ear Scale app for SSNHL. Methods This cross-sectional study included a cohort of 88 participants with possible SSNHL who were referred to an otolaryngology clinic or emergency department at a tertiary medical center in Taipei, Taiwan, between January 2018 and June 2019. All participants underwent hearing assessments with conventional pure-tone audiometry and the proposed smartphone-based Ear Scale app consecutively. The gold standard for diagnosing SSNHL was defined as the pure-tone average (PTA) difference between the two ears being ≥30 dB HL. The hearing results measured by the Ear Scale app were presented as 20 stratified hearing scales. The hearing scale difference between the two ears was estimated to detect SSNHL. Results The study sample comprised 88 adults with a mean age of 46 years, and 50% (44/88) were females. PTA measured by conventional pure-tone audiometry was strongly correlated with the hearing scale assessed by the Ear Scale app, with a Pearson correlation coefficient of .88 (95% CI .82-.92). The sensitivity of the 5–hearing scale difference (25 dB HL difference) between the impaired ear and the contralateral ear in diagnosing SSNHL was 95.5% (95% CI 87.5%-99.1%), with a specificity of 66.7% (95% CI 43.0%-85.4%). Conclusions Our findings suggest that the proposed smartphone-based Ear Scale app can be useful in the evaluation of SSNHL in clinical settings where conventional pure-tone audiometry is not available.

scholarly journals Characterization and Use of an Antibody Detecting the CBFβ-SMMHC Fusion Protein in inv(16)/t(16;16)-Associated Acute Myeloid Leukemias

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1882-1890
Author(s):  
David S. Viswanatha ◽  
I.-Ming Chen ◽  
Pu Paul Liu ◽  
Marilyn L. Slovak ◽  
Cathy Rankin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Type A ◽  
Detection Methods ◽  
Rt Pcr ◽  
Cytogenetic Abnormalities ◽  
Permeabilized Cells ◽  
Flow Cytometric ◽  
Acute Myeloid

The inv(16)(p13q22) and t(16;16)(p13;q22) cytogenetic abnormalities occur commonly in acute myeloid leukemia (AML), typically associated with French-American-British (FAB) AML-M4Eo subtype. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been recently developed to detect the presence of several variants of the resultant CBFB-MYH11 fusion gene that encodes a CBFβ-smooth muscle myosin heavy chain (SMMHC) fusion protein. We have now determined the clinical use of a polyclonal antibody [anti-inv(16) Ab] directed against a junctional epitope of the most common type of CBFβ-SMMHC fusion protein (type A), which is present in 90% of inv(16)/t(16;16) AML cases. Using flow cytometry, reproducible methods were developed for detection of CBFβ-SMMHC proteins in permeabilized cells; flow cytometric results were then correlated with cytogenetics and RT-PCR detection methods. In an analysis of 42 leukemia cases with various cytogenetic abnormalities and several normal controls, the anti-inv(16) Ab specifically detected all 23 cases that were cytogenetically positive for inv(16) or t(16;16), including a single AML case that was RT-PCR–negative. In addition to detecting all type A fusions, the anti-inv(16) Ab also unexpectedly identified the type C and type D CBFβ-SMMHC fusion proteins. Molecular characterization of one RT-PCR–positive and Ab-positive t(16;16) case with a non-type A product showed a novel previously unreported CBFB-MYH11 fusion (CBFB nt 455-MYH11 nt 1893). Flow cytometric results were analyzed using the Kolmogorov-Smirnov statistic D-value and the median value for positive samples was 0.65 (range, 0.35 to 0.77) versus 0.07 (range, −0.21 to 0.18) in the negative group (P < .0001). The overall concordance between cytogenetics and RT-PCR was 97%, whereas the concordance between flow cytometry and cytogenetics was 100%. Thus, using the anti-inv(16) Ab, all cytogenetically positive and RT-PCR–positive AML cases with inv(16) or t(16;16) could be rapidly identified. This study demonstrates the use of this antibody as an investigational tool in inv(16)/t(16;16) AML and suggests that the development of such reagents may have potential clinical diagnostic use.

Role of Trisodium Citrate in the Unclottability by Thrombin of Fibrinogen Metz

1975 ◽  
Author(s):  
C. Soria ◽  
J. Soria ◽  
M. Samama ◽  
E. Poirot ◽  
G. Kling
Keyword(s):  
Calcium Chloride ◽  
Whole Blood ◽  
Blood Clotting ◽  
Negative Charge ◽  
Trisodium Citrate ◽  
Thrombin Time ◽  
Clotting Time ◽  
Fibrin Formation ◽  
Blood Clotting Time

In a case of homozygous dysfibrinogenemia, the whole blood clotting time was moderately prolonged, while the thrombin clotting time was infinite, whatever dose or nature of thrombin used. Besides, the bleeding syndrome in this case was very weak.We observed also that only after trisodium citrate addition to purified fibrinogen, the abnormal fibrinogen became unclottable by thrombin even after addition of calcium chloride, since without trisodium citrate thrombin time was only prolonged.By immunoelectrophoresis and by isofocusing in the presence or in the absence of trisodium citrate, we therefore undertook to show that trisodium citrate reacts more strongly with the abnormal fibrinogen than with normal one. Thus, trisodium citrate conferring a negative charge to the pathological molecule, the abnormal fibrinogen became resistant to clotting with thrombin. Protamine sulfate, by positiving the charges of fibrinogen, partially corrects the defect in fibrin formation.

scholarly journals Factor XII-Deficient Chicken Plasma as a Useful Target for Screening of Pro- and Anticoagulant Animal Venom Toxins

Toxins ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 79
Author(s):  
Benedito C. Prezoto ◽  
Nancy Oguiura
Keyword(s):  
High Sensitivity ◽  
Factor Xii ◽  
Dependent Manner ◽  
Clotting Time ◽  
Natural Factor ◽  
Rotational Thromboelastometry ◽  
Factor Xii Deficiency ◽  
Dose Dependent ◽  
Recalcification Time ◽  
Coagulation Pathway

The sensitivity of vertebrate citrated plasma to pro- and anticoagulant venom or toxins occurs on a microscale level (micrograms). Although it improves responses to agonists, recalcification triggers a relatively fast thrombin formation process in mammalian plasma. As it has a natural factor XII deficiency, the recalcification time (RT) of chicken plasma (CP) is comparatively long [≥ 1800 seconds (s)]. Our objective was to compare the ability of bee venom phospholipase A2 (bvPLA2) to neutralize clot formation induced by an activator of coagulation (the aPTT clot) in recalcified human and chicken plasmas, through rotational thromboelastometry. The strategy used in this study was to find doses of bvPLA2 that were sufficient enough to prolong the clotting time (CT) of these activated plasmas to values within their normal RT range. The CT of CP was prolonged in a dose-dependent manner by bvPLA2, with 17 ± 2.8 ng (n = 6) being sufficient to displace the CT values of the activated samples to ≥ 1800 s. Only amounts up to 380 ± 41 ng (n = 6) of bvPLA2 induced the same effect in activated human plasma samples. In conclusion, the high sensitivity of CP to agonists and rotational thromboelastometry could be useful. For example, during screening procedures for assaying the effects of toxins in several stages of the coagulation pathway, such as clot initiation, formation, stability, strength, or dissolution.

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