Citrated blood collected from a donor was incubated in the absence or presence of 1 µM AYA1809002 at room temperature. After 2 h incubation, the indicated concentration of the reverse complement strand was added to the citrated blood sample. After an additional incubation time of 2 h, plasma was obtained through centrifugation. Factor II activity was measured using human plasma immunodepleted of Factor II for the quantitative determination of Factor II activity in citrated plasma based on the prothrombin time (PT) assay, for which an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory (Bedford, MA, USA) was used. The PT time was measured using RecombiPlasTin 2G for quantitative determination in human citrated plasma of PT and Fibrinogen on an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory (Bedford, MA, USA). As a control, 1 µM of dabigatran was incubated with the citrated blood.
Acltop coagulation analyzer
Manufactured by Werfen
Sourced in United States
The ACLTOP coagulation analyzer is a laboratory instrument designed to perform automated analysis of coagulation tests. It is capable of measuring various coagulation parameters, such as prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen. The ACLTOP provides accurate and reliable results to support clinical decision-making in the field of hemostasis and thrombosis.
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9 protocols using acltop coagulation analyzer
1
Factor II Activity Measurement in Plasma
2
Measurement of Coagulation Factors
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The von Willebrand factor antigen (VWF: Ag) level was measured by an in-house sandwich ELISA assay using rabbit polyclonal anti-human VWF antibodies (Dako, Glostrup, Denmark). Commercial reference plasma (Diagnostica Stago, Taverny, France) was used as calibration in each experiment. In addition, assay control samples (both pooled normal and commercially available plasma) were used in each experiment.
Human tissue plasminogen activator inhibitor type I (PAI-I) concentrations were measured in citrated plasma samples with a commercially available ELISA kit according to manufacturer's instructions (Hyphen BioMed, Neuville– Sur-Oise, France).
Plasma fibrinogen was measured on an ACL TOP coagulation analyzer (Instrumentation Laboratory, Milano, Italy) using the prothrombin-derived method, according to the manufacturer's instructions.
Human tissue plasminogen activator inhibitor type I (PAI-I) concentrations were measured in citrated plasma samples with a commercially available ELISA kit according to manufacturer's instructions (Hyphen BioMed, Neuville– Sur-Oise, France).
Plasma fibrinogen was measured on an ACL TOP coagulation analyzer (Instrumentation Laboratory, Milano, Italy) using the prothrombin-derived method, according to the manufacturer's instructions.
3
Comprehensive Hepatitis B Biomarker Profiling
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Patient data were obtained from the electronic medical record database of the hospital. The prothrombin time and international normalized ratio were measured with an ACL TOP coagulation analyzer (Instrumentation Laboratory, Beckman Coulter, Sydney, NSW, Australia). Platelet count was monitored with an LH 750 Automated Hematology Analyzer (Instrumentation Laboratory, Beckman Coulter). Alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, albumin, total bilirubin, and creatinine levels were measured using an AU5800 Clinical Chemistry Analyzer (Instrumentation Laboratory, Beckman Coulter). The serum HBV-DNA level was quantified by polymerase chain reaction using a Roche LightCycler (Roche Diagnostics, Basel, Switzerland). HBsAg and hepatitis B e-antigen were determined using an AutoLumo A2000Plus Chemiluminescence Detector (Autobio, Zhengzhou, China).
4
Thrombin Aptamer Anticoagulant Activity
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Human citrated plasma (0.5 mL) was incubated in the absence or presence of 2 µM thrombin aptamers or dabigatran for 2 h. PT time was measured using RecombiPlasTin 2G for quantitative determination in human citrated plasma of PT and Fibrinogen on an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory (Bedford, MA, USA). To assess the activity of the aptamers in a dose-dependent manner in whole blood, human blood samples were collected in citrate-treated tubes. The blood samples were then incubated at room temperature for 2 h in the absence or presence of varying concentrations of the thrombin aptamers. To measure the Prothrombin Time (PT) on the ACLTOP coagulation analyzer, plasma was obtained by centrifuging the samples at 1500× g for 10 min at 4 °C. For the dose dependence study, concentrations of 1 µM, 2 µM, 3 µM, and 4 µM of AYA1809002 and AYA1809004 were utilized.
5
Thrombin Aptamer Evaluation in Plasma and Blood
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For screening in vitro activity of the aptamers, human citrated plasma (0.5 mL) was incubated in the absence or presence of 2 µM thrombin aptamers or dabigatran for 2 h. Factor II activity was measured using human plasma immunodepleted of Factor II for the quantitative determination of Factor II activity in citrated plasma based on the prothrombin time (PT) assay, for which an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory (Bedford, MA, USA) was used. To evaluate the dose-dependent activity of the aptamers in whole blood, human blood samples were collected in citrate-treated tubes. Subsequently, the blood samples were incubated at room temperature for 2 h in both the absence and presence of various concentrations of thrombin aptamers. To measure the Factor II activity using the ACLTOP coagulation analyzer, plasma was separated from the cells by centrifugation at 1500× g for 10 min at 4 °C. For the dose-dependent study, concentrations of 1 µM, 2 µM, 3 µM, and 4 µM of AYA1809002 and AYA1809004 were utilized.
6
Plasma vWf Ag Measurement Protocol
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All participants had a day off from training before each experimental session. Blood samples were collected in the morning after an overnight fast. Blood samples were drawn, through venipuncture using a 21-gauge needle, and immediately transferred into plastic tubes containing trisodium citrate 3.2%. All samples were transferred to the laboratory and centrifuged for 10 minutes at 1,800× g within 2 hours. All the platelet-poor plasma samples were stored at −70°C until analysis. On the day of the analysis, the samples were thawed at 37°C for 20 minutes and centrifuged again.
Plasma vWf Ag was measured using a latex-particle-enhanced immunoturbidimetric assay (Von Willebrand Antigen Kit; Instrumentation Laboratory, Bedford, MA, USA) on an ACL Top coagulation analyzer (Instrumentation Laboratory).
Plasma vWf Ag was measured using a latex-particle-enhanced immunoturbidimetric assay (Von Willebrand Antigen Kit; Instrumentation Laboratory, Bedford, MA, USA) on an ACL Top coagulation analyzer (Instrumentation Laboratory).
7
Thrombin Aptamer Effect on APTT
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Human citrated plasma (0.5 mL) was incubated in the absence or presence of 2 µM thrombin aptamers or dabigatran for 2 h. APTT time was measured using the synthetic phospholipid reagent SynthASIil for quantitative determination in human citrated plasma on an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory (Bedford, MA, USA).
8
Coagulation Profile in Dialysis
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Demographic and clinical data were collected, and coagulation parameters [i.e. activated partial thromboplastin time (APTT) and aXa activity] were assessed before and after the dialysis. Pre-dialysis blood samples were drawn from the access needle immediately following needle insertion. Post-dialysis blood samples were drawn from the arterial blood line exactly 30 s after setting the blood pump at 50 mL/min to mitigate any access recirculation. To measure the APTT values and aXa activity, tests were conducted using an ACL TOP Coagulation Analyzer by HemosIL SynthASil (Instrumentation Laboratory, Bedford, MA, USA). The established reference range for APTT in the laboratory was 27–38 s, whereas that for aXa activity was 0–0.01 U/mL.
9
Routine Blood Cell and Coagulation Analysis
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Number of red blood cells and thrombocytes, hemoglobin (mmol/l), and the volume percentage of red blood cells (hematocrit %) were assessed using an ADVIA 2120i analyzer (Siemens), as part of routine clinical practice. Citrated plasma was analyzed for fibrinogen (g/l) using the ACL-top coagulation analyzer (Instrumentation Laboratory) based on the Clauss method (CV = 5.5%).
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