Validation of Pre-analytical Stability of Specimens Requested for Various Routine Coagulation Tests

Abstract Introduction: Orgaran® (Org 10172) is a low molecular weight heparinoid which consists of natural sulphated glycosaminoglycans (heparan, dermatan, chondroitin sulphate). It has a mean molecular weight of approximately 6 kDa (4–10 kDa), an excellent bioavailability following subcutaneous administration and an anti-Xa/anti-IIa activity ratio superior to 22. It is the anticoagulant of choice in patients developping Heparin-Induced Thrombocytopenia (HIT), whereas its’ use is also proposed for surgical thromboprophylaxis. Orgaran® has no effect on routine coagulation tests (aPTT, PT, TT). Thrombin generation test(TG) is a global clotting assay proven to be sensitive to the anticoagulant effect of LMWHs and specific FXa inhibitors (i.e. fondaparinux and BAY-597939). In this in vitro study, we determined the tissue factor (TF)-induced TG inhibition potency of Orgaran® using the Thrombogram-Thrombinoscope® assay. Materials and Methods: TG was assessed after TF pathway activation in Platelet Rich Plasma (PRP) (1.5x105 platelets/μl) using diluted thromboplastin (Dade Innovin®, 1:1000 final dilution). The clotting process is provoked by a physiologically relevant TF concentration. Orgaran® was added to control plasma from 8 healthy volunteers at five different final concentrations (0.2, 0.4, 0.6, 0.8 and 1IU anti-Xa/ml). TG was initiated by adding the triggering solution containing CaCl2 and the fluorogenic substrate. The analyzed TG parameters are the lag time, the maximal concentration of thrombin (Cmax), the time to reach Cmax (Tmax), the TG velocity and the endogenous thrombin potential (ETP). Results: Orgaran® prolonged significantly the lag time and the Tmax at a concentration over 0.40 IU anti-Xa/ml (p<0.05). At the lowest studied concentration (0.20 IU anti-Xa/ml), lag time and Tmax were only prolonged by 12%, whereas their maximal prolongation (around 50%) was observed at 1IU anti-Xa/ml. Furthermore, Orgaran® inhibited ETP, Cmax and TG velocity in an almost linear dose dependent manner. A significant inhibition of ETP, Cmax and TG velocity was obtained at concentrations superior to 0.20 IU anti-Xa/ml. (p<0.05). At the highest studied concentration (1IU anti-Xa/ml) Orgaran® suppressed all TG parameters by about 80% (Table 1). Conclusion: Orgaran® exhibited a significant inhibitory activity of in vitro TG. At concentrations achieved in clinical practice (prophylactic or therapeutic dose), Orgaran® modified in vitro TG profile while it has no effect on routine coagulation tests. Thus, TG assay is a sensitive method for monitoring Orgaran® and this test requires a clinical prospective evaluation. Table 1. Determination of IC20 and IC50 anti-Xa inhibitory concentrations of Orgaran® on TG parameters Lag Time Tmax ETP Cmax Velocity IC: Inhibitory Concentration * or Concentration increasing 20% and 50% the lag time and the Tmax respectively IC 20 (IU/ml) 0.30 0.30 0.18 0.18 0.15 IC 50 (IU/ml) 0.83 >1 0.30 0.50 0.35 1IU anti-Xa/ml 53% 47% 68% 76% 84%

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Resolving Interference of Direct Oral Anticoagulants (DOACs) on Routine Coagulation, Thrombophilia, and Lupus Anticoagulant Testing Using Activated Carbon

Blood ◽

10.1182/blood-2021-152867 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 830-830

Author(s):

Pierre Toulon ◽

Mathilde Vannini ◽

Anny Appert-Flory ◽

Florence Fischer ◽

Didier Jambou ◽

...

Keyword(s):

Activated Carbon ◽

Room Temperature ◽

Oral Anticoagulants ◽

Test Results ◽

Clotting Time ◽

Median Concentration ◽

Coagulation Tests ◽

Before And After ◽

D Dimer ◽

Routine Coagulation

Abstract Background: Direct (anti-IIa and anti-Xa) oral anticoagulants (DOACs) have a significant impact on various coagulation test results. As the correct interpretation of these tests is mandatory to prevent misclassification and subsequent clinical consequences, withholding treatment could be necessary, with the associated risk of thrombosis. Aims: To evaluate the performance of the activated carbon DOAC-Remove (5-Diagnostics, Basel, Switzerland) in extracting DOACs from plasma samples and its effect on various routine and esoteric coagulation test results. Patients, Materials and Methods: Left-over plasmas from patients treated or not with DOAC obtained in the routine laboratory workload were evaluated. Briefly, 0.8 mL of plasma sample was incubated with 1 tablet of the activated carbon for 10 min at room temperature using a rotating shaker. After a 2 min-centrifugation at 2000x g and room temperature, the supernatant was pipetted before being analyzed. Tests were performed before and after such incubation. DOACs were measured using either a specific direct thrombin inhibitor or an anti-Xa assay with specific calibrations. Routine coagulation tests (PT, aPTT, and factor(F)V, FVIII:C, FIX, fibrinogen, and D-dimer) were performed, as well as thrombophilia panel [antithrombin, protein C (PC, chromogenic and clotting assays), PS (free PS antigen and clotting assays)], and lupus anticoagulant (LA) panel [silica clotting time (SCT), and dilute Russell venom clotting time (dRVVT)]. All assays were performed using reagents from Werfen (Bedford, MA, USA) on the ACL TOP 700 analyzer. As the distribution of data was not normal, test results were compared using non-parametric tests. Results: We evaluated a total of 756 plasma samples, obtained from patients treated with dabigatran (n=139, median concentration 129 ng/mL [range:18-905]), rivaroxaban (n=157, median concentration 159 ng/mL [range:19-815]), or apixaban (n=155, median concentration=154 ng/mL (range:11-510), and from 305 patients with various disease states and not on DOAC including 35 patients on coumadin and 32 patients on heparin (UFH: n=18 ; LMWH: n=14). In untreated patients, the DOAC-Remove had no significant impact on test results (n>30 for each parameter), except for D-dimer, free PS and FVIII:C. However, the mean biases, evaluated according to Bland-Altman, were below the accepted limits, and changes would not have had any clinical relevance. In the plasma from patients treated on DOAC, the DOAC-Remove eliminated all the three DOACs, with levels far below the detection limit of the techniques after a 10 min-incubation, leading to a dramatic correction of the DOAC-induced prolongations of PT and APTT, as well as of their lowering effect on FV, FVIII and FIX activities. The same applied to the DOAC-induced elevation of PC and PS anticoagulant activities (clotting assays), and antithrombin activity in the plasma from patients on rivaroxaban and apixaban. DRVVT screen/confirm ratios, which were above the normal ranges in 45% of the patients on dabigatran (n=31), and 78% of the patients on rivaroxaban (n=32), were normalized in most samples after a 10 min-incubation with the carbon and remained positive in only 10% of the patients on dabigatran and 12% of the patients on rivaroxaban. The impact of the DOACs on the SCT screen/confirm ratio was less noticeable with baseline positive test results in only one of the samples from patients on dabigatran (n=31), and in 2 patients on rivaroxaban (n=32). Treatment with the carbon leaded to a correction in the positive sample from patient on dabigatran and in one of the two positive samples from patients on rivaroxaban. Both dRVVT and SCT test results were within the normal range in the plasma from 32 patients on apixaban before and after treatment with the activated carbon. As expected, fibrinogen, PC activity evaluated using a chromogenic assay, free PS antigen concentration, VWF:RCo and VWF:Ag were not affected by any of the 3 DOACs, and test results were not significantly different before and after incubation with the activated carbon. Conclusion: The DOAC-Remove had no effect on test results obtained in the plasma from untreated patients and from patients on traditional anticoagulants. It effectively removed all 3 tested DOACs from the plasma of treated patients, allowing an accurate measurement of routine and esoteric coagulation tests in DOAC treated patients without withholding the treatment. Disclosures No relevant conflicts of interest to declare.

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Characteristics of routine coagulationtesting and thromboelastometry in polytrauma patients at admission

Ministry of Science and Technology, Vietnam ◽

10.31276/vjst.63(9).01-05 ◽

2021 ◽

Vol 63 (9) ◽

pp. 1-5

Author(s):

Thi Hang Tran ◽

◽

Thi Thu Hien Trinh ◽

Van Chinh Nguyen ◽

An Son Doan ◽

...

Keyword(s):

University Hospital ◽

Clot Lysis ◽

Whole Blood Assay ◽

Coagulation Parameters ◽

Blood Assay ◽

Coagulation Testing ◽

Coagulation Tests ◽

Polytrauma Patients ◽

Old Men ◽

Routine Coagulation

Background: polytrauma is one of the emergency surgeries with a high mortality rate. One of the leading causes of death is coagulopathy that is not detected early and treated promptly. Thromboelastometry (ROTEM) is a whole blood assay that evaluates the viscoelastic properties during clot formation and clot lysis. This method can detect coagulopathy rapidly and accurately, thereby improving the management of bleeding after trauma. Objectives: describing and evaluating the correlation between ROTEM parameters and routine coagulation tests in polytrauma patients at admission. Method: 110 patients admitted to the Emergency Department, Viet Duc University Hospital from May 2021 to July 2021 were diagnosed with polytrauma. All patients underwent routine coagulation testing and ROTEM parameters at admission. Result: the average age of patients is 41.4±14.7 years old, men accounts for 77.3%, average ISS score is 24.5±6.3. The proportion of the polytrauma patients with coagulopathy by routine coagulation testing was 50.9%. A significant correlation was found between routine coagulation parameters and ROTEM: between APTT with CFT-INTEM (r=0.65; p<0.01); between PT and CFT-EXTEM (r=0.64; p<0.01); between platelet count and MCF-INTEM (r=0.56; p=0.00) and MCF-EXTEM (r=0.57; p=0.00); between fibrinogen level and MCF-INTEM (r=0.71; p=0.00), MCF-EXTEM (r=0.71; p=0.00), and MCF-FIBTEM (r=0.91; p=0.00). Conclusion: proportion of the polytrauma patients with coagulopathy by routine coagulation testing was 50.9%. A significant correlation was found between routine coagulation parameters and ROTEM

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The effects of fructose diphosphate on routine coagulation tests in vitro

Scientific Reports ◽

10.1038/s41598-021-04263-y ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Tongqing Chen ◽

Duan Chen ◽

Lu Chen ◽

Zhengxu Chen ◽

Baolong Wang ◽

...

Keyword(s):

Platelet Aggregation ◽

Blood Coagulation ◽

Detection System ◽

Coagulation System ◽

Thrombin Time ◽

Aggregation Rate ◽

Coagulation Testing ◽

Coagulation Tests ◽

Routine Coagulation

AbstractTo evaluate the effects of fructose diphosphate (FDP) on routine coagulation tests in vitro, we added FDP into the mixed normal plasma to obtain the final concentration of 0, 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, 30 and 35 mg/mL of drug. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen (FBG) and thrombin time (TT) of samples were analyzed with blood coagulation analyzers from four different manufacturers(Sysmex, Stago, SEKISUI and Werfen) and their corresponding reagents, respectively. Before the experiment, we also observed whether there were significant differences in coagulation test results of different lots of reagents produced by each manufacturer. At the same time as the four routine clotting tests, the Sysmex blood coagulation analyzer and its proprietary analysis software were used to detect the change of maximum platelet aggregation rate in platelet-rich plasma after adding FDP (0, 1, 2, 3, 4, 5 and 6 mg/mL). The results of PT, aPTT and TT showed a FDP (0–35 mg/mL) concentration-dependent increase and a FBG concentration-dependent decrease. The degree of change (increase or decrease) varied depending on the assay system, with PT and aPTT being more affected by the Sysmex blood coagulation testing instrument reagent system and less affected by CEKISUI, TT less affected by CEKISUI and more affected by Stago, and FBG less affected by Stago and more affected by Sysmex. The results of PT, aPTT and TT were statistically positively correlated with their FDP concentrations, while FBG was negatively correlated. The correlation coefficients between FDP and the coagulation testing systems of Sysmex, Stago, Werfen and SEKISUI were 0.975, 0.988, 0.967, 0.986 for PT, and 0.993, 0.989, 0.990 and 0.962 for aPTT, 0.994, 0.960, 0.977 and 0.982 for TT, − 0.990, − 0.983, − 0.989 and − 0.954 for FBG, respectively. Different concentrations of FDP (0, 1, 2, 3, 4, 5 and 6 mg/mL) had different effects on the maximum aggregation rate of platelet induced by the agonists of adenosine diphosphate (ADP, 5 µmol/L), arachidonic acid (Ara, 1 mmol/L), collagen (Col, 2.5 µg/mL) and epinephrine (Epi,10 µmol/L), but the overall downward trend was consistent, that is, with the increase of FDP concentration, the platelet aggregation rate decreased significantly. Our experimental study demonstrated a possible effect of FDP on the assays of coagulation and Platelet aggregation, which may arise because the drug interferes with the coagulation and platelet aggregation detection system, or it may affect our in vivo coagulation system and Platelet aggregation function, the real mechanism of which remains to be further verified and studied.

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The Comparison of Manual and Automatic Interference Determination on Coagulometers ACL Top 750 CTS and Roche Cobas t511 for Routine Coagulation Tests

10.1055/s-0039-1680262 ◽

2019 ◽

Author(s):

L. Slavik ◽

K. Chasakova ◽

J. Ulehlova ◽

A. Pavlikova

Keyword(s):

Coagulation Tests ◽

Roche Cobas ◽

Routine Coagulation

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Acquired factor XIII deficiency in two patients with bleeding events during veno-venous extracorporeal membrane oxygenation treatment

Journal of Artificial Organs ◽

10.1007/s10047-019-01148-w ◽

2019 ◽

Vol 23 (3) ◽

pp. 283-287 ◽

Cited By ~ 2

Author(s):

Asami Ito ◽

Yoshiaki Iwashita ◽

Ryo Esumi ◽

Ken Sasaki ◽

Masahiro Yukimitsu ◽

...

Keyword(s):

Extracorporeal Membrane Oxygenation ◽

Factor Xiii ◽

Bloody Stool ◽

Ecmo Patient ◽

Bleeding Events ◽

Factor Xiii Deficiency ◽

Blood Tests ◽

Membrane Oxygenation ◽

Coagulation Tests ◽

Routine Coagulation

AbstractWe report two cases of acquired factor XIII deficiency with bleeding events during veno-venous extracorporeal membrane oxygenation (ECMO). Case 1: A 76-year-old man diagnosed with aspiration pneumonia after near-drowning was started on ECMO. Later, the patient presented with hemoptysis and anemia. Blood tests showed a decreased factor XIII activity of 29%. Although the patient recovered after receiving 1200 International Units of factor XIII concentrate, the patient had another episode of decreased factor XIII activity and bloody stool and was treated again with factor XIII concentrate. Case 2: A 48-year-old female diagnosed with pneumonia was started on ECMO. Soon after, she presented with hemoptysis and anemia. Blood tests showed a decreased factor XIII activity of 39%. The patient was treated with 720 IU of factor XIII concentrate with good recovery. Acquired factor XIII deficiency cannot be detected by routine coagulation tests, therefore it may be under-diagnosed in the ICU. Detection of acquired factor XIII deficiency is essential when treating a bleeding ECMO patient.

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