Common genetic risk factors were evaluated, and the activity deficiency subjects were considered to have inherited risk factors. Since factor V Leiden and G20210A are very rare in the Chinese population, only the loss of function mutations involving natural anticoagulant factors was evaluated. Blood samples of VTE patients were collected into Na-citrate-containing vacutainer tubes. Plasma was obtained by centrifugation at 4 °C for 15 min at 2000 g and stored at − 80 °C until use. The protein C (PROC) and antithrombin (SERPINC1) activity levels were determined by the chromogenic method assay on an IL coagulation system (Werfen, Bedford, USA) according to the manufacturers’ instructions. Their activity was quantified with a synthetic chromogenic substrate. Then, the absorbance was detected at 405 nm on a Werfen ACLTOP 700 system (Bedford, USA). The protein S activity was correlated with the prolongation of the clotting time. The normal range of PROS1 was evaluated using the protein S activity kit on representative members with the ACL TOP family systems. The normal range of PROC and antithrombin activity levels were based on hundreds of clinical subjects.
Acl top 700 system
Manufactured by Werfen
Sourced in United States
The ACL TOP 700 System is a fully automated coagulation analyzer designed for clinical laboratory use. It is capable of performing a wide range of coagulation tests, including prothrombin time (PT), activated partial thromboplastin time (aPTT), and other specialized coagulation assays. The system offers efficient workflow, high-throughput testing, and reliable results.
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Lab products found in correlation
Cobas 8000 analyser, by Roche (2 mentions)
Xn 9000 system, by Sysmex (2 mentions)
Synthasil reagent, by Werfen (1 mentions)
Recombiplastin 2g, by Werfen (1 mentions)
Bilirubin total dpd gen 2 kit, by Roche (1 mentions)
Recombiplastin 2g kit, by Werfen (1 mentions)
Creatinine plusver 2, by Roche (1 mentions)
Cobas 6000, by Roche (1 mentions)
Dxr smart raman spectrometer, by Thermo Fisher Scientific (1 mentions)
8 protocols using acl top 700 system
1
Inherited Anticoagulant Deficiency Assessment
2
Antithrombin Activity Determination Protocol
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Blood samples were collected into trisodium citrate by venopuncture. Plasma samples were obtained by centrifugation at 2,000g for 15 min at 4°C and stored at -80°C until analysis. The antithrombin anticoagulant activity was determined by automatic chromogenic assay on the IL Coagulation systems according to the manufacturer’s instructions (Cat.0020300400, Werfen, Bedford, MA). Briefly, the plasma was first incubated with Factor Xa reagent in the presence of an excess of heparin, followed by incubation with a synthetic chromogenic substrate. Quantification of the residual Factor Xa was performed by detecting the absorbance at 405 nm by ACLTOP 700 system (Werfen). The absorbance is inversely proportional to the antithrombin level in the test sample. The activation levels were calculated by the standard curve of each detection. The established normal range of plasma antithrombin levels was 83–128%, which was based on our hospital pathology reference from thousands of previously diagnosed clinical subjects.
3
Clinical Characteristics of COVID-19 Patients
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Information included demographic data, comorbidities, symptoms and signs, laboratory findings and chest computed tomographic (CT) scans were reviewed. All data were collected with a customized form from the electronic hospital information system. The medical records of the patients were reviewed by three investigators (JC, XD and YJ) independently to verify data accuracy.
Peripheral venous blood samples were assessed at the central laboratory of Zhongnan hospital following standard operative procedures. The routine blood tests (including white blood cell count [WBC], leukocyte subtypes, hemoglobin count and platelet count) were measured with XN-9000 multi-function automatic blood analyzer of fluid (Sysmex Corporation, Kobe, Japan). The ARCHITCT ci16200 automatic biochemistry analyzer (Abbott Laboratories, Illinois, United States) was used to measure the biochemical parameters. Blood coagulation tests including plasma D-dimer, prothrombin time (PT), international normalized ratio (INR), activated partial prothrombin time (APTT), thrombin time (TT) were measured by ACL TOP 700 system (Instrumentation Laboratory, Milan, Italy).
Identifying of SARS-CoV-2: the throat swab samples were tested for SARS-CoV-2 with the Chinese Center for Disease Control and Prevention (CDC) recommended Kit (BioGerm, Shanghai, China) following WHO guidelines for qRT-PCR.11
,12
Peripheral venous blood samples were assessed at the central laboratory of Zhongnan hospital following standard operative procedures. The routine blood tests (including white blood cell count [WBC], leukocyte subtypes, hemoglobin count and platelet count) were measured with XN-9000 multi-function automatic blood analyzer of fluid (Sysmex Corporation, Kobe, Japan). The ARCHITCT ci16200 automatic biochemistry analyzer (Abbott Laboratories, Illinois, United States) was used to measure the biochemical parameters. Blood coagulation tests including plasma D-dimer, prothrombin time (PT), international normalized ratio (INR), activated partial prothrombin time (APTT), thrombin time (TT) were measured by ACL TOP 700 system (Instrumentation Laboratory, Milan, Italy).
Identifying of SARS-CoV-2: the throat swab samples were tested for SARS-CoV-2 with the Chinese Center for Disease Control and Prevention (CDC) recommended Kit (BioGerm, Shanghai, China) following WHO guidelines for qRT-PCR.11
,12
4
Automated Coagulation Measurements
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The coagulation measurements were performed automatically on an ACL TOP 700 system from Instrumentation Laboratory (Kirchheim, Germany). For the prothrombin time, we used RecombiPlasTin 2G and for partial thromboplastin time SynthASil reagent, both from Instrumentation Laboratory (Kirchheim, Germany). All laboratory tests were performed automatically at the control temperature of 37°C and the clotting formation was measured by a turbidimetric method for dabigatran determination and the color development was measured at 405 nm for rivaroxaban determination, respectively. For calibration and control commercially available calibrators for dabigatran and rivaroxaban, as well as quality-control samples for both drugs from Technoclone GmbH (Vienna, Austria) were used.
5
Calculating MELD Score from Laboratory Data
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Clinical chemistry routine parameters were available for each patient. Creatinine and bilirubin serum concentrations were determined by the application of enzymatic assay creatinine Plus Ver. 2 and Bilirubin Total DPD Gen.2 kit (both kits purchased from Roche, Mannheim, Germany), respectively according to manufacturer’s instructions. Both assays ran on the Cobas 6000 and 8000 analyzers (Roche, Mannheim, Germany). The RecombiPlasTin 2G kit (Instrumentation Laboratory, Lexington, USA) was used to determine the INR from citrated plasma using an ACL TOP 700 System (Instrumentation Laboratory, Lexington, USA).
MELD score was calculated according to the guidelines of the UNOS [12 ] using the following formula:
According to the guidelines, determined creatinine concentrations below 1.0 mg/dl or above 4.0 mg/dl were set on the defined minimum of 1.0 mg/dl or on the defined maximum of 4.0 mg/dl, respectively. The MELD score range is defined from 6 to 40.
MELD score was calculated according to the guidelines of the UNOS [12 ] using the following formula:
According to the guidelines, determined creatinine concentrations below 1.0 mg/dl or above 4.0 mg/dl were set on the defined minimum of 1.0 mg/dl or on the defined maximum of 4.0 mg/dl, respectively. The MELD score range is defined from 6 to 40.
6
Comprehensive Serum Biomarker Analysis
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All analyzed parameters are summarized in Supplemental Table 1 . ALT and AST, gamma glutamyl transferase (GGT), glutamate dehydrogenase (GLDH), creatinine and bilirubin serum concentrations were analyzed using Cobas 6000 and 8000 analyzers (Roche, Mannheim, Germany). The prothrombin assay was performed in citrate plasma to determine the INR using an ACL TOP 700 System (Instrumentation Laboratory, Lexington, USA).
7
Comprehensive Blood Parameter Analysis
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Serum parameters were measured on cobas 8000 analysers (Roche, Mannheim, Germany). The international normalized ratio (INR) was determined in citrate plasma using the ACL TOP 700 System (Instrumentation Laboratory, Lexington, USA). Blood cells were measured in EDTA whole blood using the XN 9000 system (Sysmex, Kobe, Japan).
8
Raman Spectroscopy for Serum Analysis
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Raman spectroscopy We used the Thermo Fisher Scientific DXR SmartRaman Spectrometer (Waltham, Massachusetts, USA) with an excitation wavelength of 780 nm for data collection. Outgoing Raman radiation was detected with a cooled CCD sensor (at -50 • C) in the range from 50 to 3500 cm -1 . After a period of thorough testing and comparing various settings, we finally chose the settings listed in Table S1 in the supplementary material.
A crucial parameter is the duration and the repetition of the measurements taken by the device. Obviously, a higher number of repetitions would be better than a lower number for statistics and, most importantly, for noise cancellation. However, substantial photo-bleaching was observed, especially in range between 1500 and 3000 cm -1 . The device's laser can be focused on different heights. We chose a height such that the background created by the carrier was minimal. The focus was slightly above the level of the coverslips.
Throughout the entire analysis, the dominant background was of obvious concern. Laboratory analysis Serum parameters were measured on cobas 8000 analysers (Roche, Mannheim, Germany). The international normalized ratio (INR) was determined in citrate plasma using the ACL TOP 700 System (Instrumentation Laboratory, Lexington, USA). Blood cells were measured in EDTA whole blood using the XN 9000 system (Sysmex, Kobe, Japan).
A crucial parameter is the duration and the repetition of the measurements taken by the device. Obviously, a higher number of repetitions would be better than a lower number for statistics and, most importantly, for noise cancellation. However, substantial photo-bleaching was observed, especially in range between 1500 and 3000 cm -1 . The device's laser can be focused on different heights. We chose a height such that the background created by the carrier was minimal. The focus was slightly above the level of the coverslips.
Throughout the entire analysis, the dominant background was of obvious concern. Laboratory analysis Serum parameters were measured on cobas 8000 analysers (Roche, Mannheim, Germany). The international normalized ratio (INR) was determined in citrate plasma using the ACL TOP 700 System (Instrumentation Laboratory, Lexington, USA). Blood cells were measured in EDTA whole blood using the XN 9000 system (Sysmex, Kobe, Japan).
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