In vitro evaluation of a new viscoelastometry-based point-of-care analyzer.

INTRODUCTION: The VCM is a point-of-care analyzer using a new viscoelastometry technique for rapid assessment of hemostasis on fresh whole blood. Its characteristics would make it suitable for use in austere environments. The purpose of this study was to evaluate the VCM in terms of repeatability, reproducibility and interanalyzer correlation, reference values in our population, correlation with standard coagulation assays and platelet count, correlation with the TEG5000 analyzer and resistance to stress conditions mimicking an austere environment. METHODS: Repeatability, reproducibility, and interanalyzer correlation were performed on quality control samples (n = 10). Reference values were determined from blood donor samples (n = 60). Correlations with standard biological assays were assessed from ICU patients (n = 30) and blood donors (n = 60) samples. Correlation with the TEG5000 was assessed from blood donor samples. Evaluation of vibration resistance was performed on blood donor (n = 5) and quality control (n = 5) samples. RESULTS: The CVs for repeatability and reproducibility ranged from 0% to 11%. Interanalyzer correlation found correlation coefficients (r2) ranging from 0.927 to 0.997. Our reference values were consistent with those provided by the manufacturer. No robust correlation was found with conventional coagulation tests. The correlation with the TEG5000 was excellent with r2 ranging from 0.75 to 0.92. Resistance to stress conditions was excellent. CONCLUSION: The VCM analyzer is a reliable, easy-to-use instrument that correlates well with the TEG5000. Despite some logistical constraints, the results suggest that it can be used in austere environments. Further studies are required before its implementation.

Taipan snake venom time has high sensitivity for lupus anticoagulants in non-anticoagulated, triple positive antiphospholipid syndrome patients.

INTRODUCTION: Dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) are the mainstay assays in lupus anticoagulant (LA) detection yet they have limitations, particularly in relation to interferences and specificity. The recently validated Taipan snake venom time (TSVT) screening with ecarin time (ET) confirmatory assays overcome many of those limitations due to the innate specificity engendered from direct prothrombin activation, and insensitivity to the effects of vitamin K antagonists (VKA). The present study aimed to further evidence diagnostic utility of TSVT/ET by performing them in samples from 116 nonanticoagulated patients with established triple-positive antiphospholipid syndrome (APS). METHODS: Samples were identified in three expert centres who performed dRVVT, APTT and solid phase antiphospholipid antibody assays with reagents from a variety of manufacturers. All samples additionally received TSVT/ET analysis using standardised reagents. RESULTS: Ninety seven of 116 (83.6%) were dRVVT- and APTT-positive, 85/97 (87.6%) of which were TSVT/ET-positive, 9/116 (7.8%) were dRVVT-positive only, 6 of which were TSVT/ET-positive, and 10/116 (8.6%) were APTT-positive only, 5 of which were TSVT/ET-positive. 96/116 TSVT/ET-positivity returned a high sensitivity for LA of 82.8%. Low coefficients of determination revealed weak relationships between LA potency and anticardiolipin and anti-ß2-glycoprotein I antibody titres for all three LA assays. CONCLUSIONS: TSVT/ET has high sensitivity for the clinically significant LA found in triple positive APS patients. TSVT/ET can establish multiple LA assay positivity in nonanticoagulated patients negative for one of dRVVT or APTT, and is the only assay pairing insensitive to VKAs, the recommended anticoagulation for APS.

The influence of corn trypsin inhibitor on the contribution of coagulation determinants to the Technoclone Thrombin Generation Assay (TGA) and the Calibrated Automated Thrombogram (CAT).

In thrombin generation (TG) assays, regarded as global coagulation tests, contact activation is considered a major problem which can be eliminated by adding Corn Trypsin Inhibitor (CTI). In previous studies, however, venous thrombosis risk prediction using TG assays did not improve after CTI addition. However, it is unknown whether CTI addition could help to detect subtle but relevant nuances in determinants of TG, making the assay more suitable to detect disturbances in the coagulation system. This study's objective was to assess whether the addition of CTI is associated with a broader contribution of individual coagulation factors to the total amount of thrombin formed in Calibrated Automated Thrombogram (CAT) and Technoclone Thrombin Generation Assay (TGA). Thrombin generation was measured in 326 healthy individuals from THE VTE study at very low tissue factor concentrations, with and without addition of CTI prior to blood sampling. The influence of several coagulation factors on total amount of thrombin formed, i.e. area under the curve (AUC) or endogenous thrombin potential (ETP), was analysed using multiple linear regression with standardisation of all values resulting in Z-scores with 95% confidence intervals (95%CI). Association between coagulation factors and TG changed minimally after addition of CTI. Largest changes after CTI addition were found for following factors: for CAT: free protein S (from 0.00 (95%CI -0.12 to 0.12) to -0.29 (95%CI -0.43 to -0.15)) and protein S (from -0.05 (95%CI -0.18 to 0.08) to -0.21 (95%CI -0.37 to -0.05)); for TGA: antithrombin (from -0.11 (-0.23 to 0.02) to -0.19 (-0.30 to -0.07)), factor VIII (from 0.15 (0.03 to 0.27) to 0.24 (0.13 to 0.36)) and fibrinogen (from 0.12 (-0.01 to 0.26) to 0.19 (0.06 to 0.32)). In conclusion, there is no clear trend towards a broader contribution of coagulation factors in samples handled with CTI compared with those handled without CTI.

Using a low-molecular weight heparin-calibrated anti-factor Xa assay to assess the concentration of apixaban and rivaroxaban.

INTRODUCTION: Direct oral anticoagulant (DOAC)-inhibiting factor Xa (FXa-DOAC) are being increasingly used as prophylaxis of venous thromboembolism and for prevention of stroke in patients with atrial fibrillation. In contrast to vitamin K antagonists, DOACs do not require monitoring in general. However, it is sometimes of value in the acute setting, for instance when considering a reversal agent in uncontrolled bleeding in patients on DOAC. METHODS: We evaluated if a low-molecular weight heparin (LMWH)-calibrated anti-factor Xa assay could be used to estimate FXa-DOAC concentration in the concentration range <100 ng/mL by spiking known concentrations of FXa-DOAC and from those result calculate the FXa-DOAC concentration from the response of the LMWH assay. This procedure was then evaluated by comparing the result with a drug-calibrated chromogenic assay and liquid chromatography tandem mass spectrometry (LC-MS/MS) on clinical plasma samples from patients treated with apixaban or rivaroxaban. RESULTS: Although the measuring range was narrower for the LMWH-calibrated assay, concentrations recalculated from the LMWH assay was comparable with those measured by the drug-calibrated method when compared with LC-MS/MS. CONCLUSION: We suggest that an LMWH-calibrated anti-factor Xa assay can be used after characterization of the response of FXa-DOACs to give guidance on the concentration of apixaban and rivaroxaban. Shorter turnaround time than LC-MS/MS and the greater availability than drug-calibrated chromogenic assays could make this a valuable option in the acute setting.

Influence of direct oral anticoagulants on thrombin generation on Ceveron TGA.

INTRODUCTION: Monitoring of direct oral anticoagulants (DOACs) with calibrated anti-Xa assay is limited by the high intra- and interindividual variations of the test results. Thrombin generation (TG) is a global hemostatic assay that reflects the patient´s individual coagulation status. The aim of this study was to investigate the influence of DOACs on TG measured with a fully automated assay system. METHODS: All consecutive patients under apixaban and rivaroxaban coming to the outpatient coagulation center MVZ Limbach, Magdeburg, Germany between October 2017 and April 2020 were included. DOAC plasma levels were correlated with TG assessed using the fully automated Ceveron TG analyzer. RESULTS: A total of 703 rivaroxaban and 252 apixaban containing plasma samples were included. There was a significant correlation between DOAC plasma levels and all TG parameters except for lag time regarding apixaban. Time to peak and peak thrombin followed an exponential regression curve, while this was linear for the endogenous thrombin potential (ETP). Apixaban showed a lower correlation coefficient for all TG parameters compared with rivaroxaban, and thrombin generation was less influenced by apixaban than rivaroxaban at plasma levels >100 ng/ml. The sensitivity and negative predictive value of normal TG parameters for the prediction of DOAC plasma levels <30 ng/ml was >85%. CONCLUSION: The present data show a moderate predominantly nonlinear correlation between TG parameters and plasma levels of apixaban and rivaroxaban. Rivaroxaban has a stronger effect on TG than apixaban.

Impact of centrifugation time and pneumatic tube transport on plasma concentrations of direct oral anticoagulants.

INTRODUCTION: Rapid results are needed when plasma concentrations of direct oral anticoagulants (DOACs) are required in acute clinical settings. We evaluated the impact of centrifugation time and pneumatic tube transport on DOAC plasma concentrations with the overall aim of reducing turnaround time. METHODS: Blood samples were spiked with rivaroxaban, apixaban or dabigatran in a low and a high concentration prior to centrifugation for 25 minutes (3163 g) or 5 minutes (3000 g) (n = 20 for each DOAC). Both samples spiked with DOACs (n = 20 for each DOAC) and patient samples (n = 25 in total) were transported manually or by pneumatic tube system samples. RESULTS: For samples spiked with DOAC, statistically significant differences in DOAC plasma concentrations were found between centrifugation times for rivaroxaban in low (P < .05) and high (P < .05) concentrations. Relative bias was below 9% for all DOACs. Statistically significant differences were found between modes of transportation for rivaroxaban (P < .01) and dabigatran (P < .01) in high concentrations. Relative bias was 4%-23% for all DOACs. For patient samples, no statistically significant differences were found between modes of transportation, and relative bias was below 12% for all DOACs. CONCLUSION: Minor, clinically insignificant, differences regarding centrifugation times were found in DOAC plasma concentrations. Importantly, no significant differences were found according to transportation modes for samples collected from patients. Although statistically significant differences were found depending on mode of transportation of spiked samples, relative bias was clinically acceptable. Thus, reduced centrifugation time and pneumatic tube transport should be considered to reduce turnaround time for rapid measurement of DOAC plasma concentrations.

Reflex factor coagulation testing in patients with an unexplained prolonged aPTT: An institutional retrospective review.

BACKGROUND: We aim to determine the clinical utility of reflex coagulation investigations (RCI) for prolonged lupus insensitive activated partial thromboplastin time (aPTT) at our institution. METHODS: We retrospectively reviewed all potential RCI (lupus insensitive aPTT of ≥32s) from April 2014 to June 2019. Our diagnostic algorithm requires completion of RCI only if samples had no interfering medications to explain a prolonged aPTT and were either from a preoperative sample or from a patient presenting with unexplained bleeding. Appropriate RCI samples undergo further investigations with one-stage factor activity testing for factors 8(FVIII), 9(FIX), and 11(FXI) reflexively. Data were obtained through electronic medical records to capture clinical characteristics, laboratory findings, prophylactic hemostatic replacement, and bleeding outcomes. RESULTS: Three thousand and three hundreds seventeen samples from 2940 distinct patients were considered as potential RCI during the study period. 263/3317 (8%) samples had RCI completed. Of those, 55/263 (21%) had abnormal factor testing, with the majority from preoperative setting (43/55; 78%). 5/43 (12%) patients were referred to hematology for preoperative evaluation. 5/43 patients received preoperative hemostatic support. A total of 5 patients (5/43) developed postop bleeding. Six patients (6/55) had RCI for unexplained bleeding, and five patients (83%) had a newly identified clinically significant bleeding disorder. CONCLUSION: Reflex coagulation investigations benefited patients presenting with unexplained bleeding as this expedited the diagnosis and management of clinically significant bleeding disorders. RCI for preoperative evaluation infrequently led to additional hemostatic support/referral to hematology. The lack of additional workup for an abnormal factor activity level suggests laboratory alert fatigue as a potential contributory factor.

Study of in vitro thrombin generation after neutralization of heparin.

INTRODUCTION: Thrombin generation (TG) documents hypercoagulability. TG in platelet-poor plasma is exquisitely sensitive to heparins, which thus must be neutralized before testing. Heparinase and hexadimethrine bromide (polybrene) have been used for that purpose, but their effects per se on TG have been poorly studied so far. METHODS: (i) TG was studied in commercial normal pooled plasma (NPP; CryoCheck® , Cryopep) in absence or presence of neutralizing agents. (ii) NPP was spiked with increasing concentrations of unfractionated heparin (UFH; up to 1.0 IU/mL) or low-molecular-weight heparin (LMWH; enoxaparin up to 1.2 IU/mL) and TG studied after incubation of heparinase (Hepzyme® ; 15 minutes) or polybrene (0.025 mg/mL; 10 minutes). RESULTS: (i) With ThromboScreen reagent to initiate TG, addition of heparinase was associated with increased peak, whereas polybrene caused lengthening of lag time and time to peak, compared with nonsupplemented NPP. (ii) With polybrene, TG was completely restored over the whole range of UFH and LMWH studied. By contrast, heparinase failed to fully restore TG in presence of UFH concentrations ≥0.8 IU/mL or LMWH concentrations ≥1.0 IU/mL. Those effects were matched with detectable tiny residual amounts of non-neutralized heparin (as assessed with an anti-Xa assay) and were less pronounced with a higher picomolar concentration of tissue factor (DrugScreen reagent). CONCLUSION: Polybrene fully restored TG of heparinized plasma at the expense of an alteration of TG, pointing to the need to use adapted reference ranges. Heparinase failed to do so in presence of high concentrations of both heparins.

Quantification and optimization of clot retraction in washed human platelets by Sonoclot coagulation analysis.

INTRODUCTION: Clot retraction is a pivotal process for haemostasis, where platelets develop a contractile force in fibrin meshwork and lead to the increased rigidity of clot. The pathophysiological alteration in contractile forces generated by the platelet-fibrin meshwork can lead to haemostatic disorders. Regardless of its utter significance, clot retraction remains a limited understood process owing to lack of quantification methodology. Sonoclot analysis is a point-of-care technique used in clinical laboratories for whole blood analysis that provides in vitro qualitative as well as quantitative assessment of coagulation process from initial fibrin formation to clot retraction. METHODS: Human washed platelets were isolated by differential centrifugation method and analysed via optical imaging, microscopy and Sonoclot analysis using 1-2 × 108 /mL of washed platelets, 1 U/mL of thrombin, 1 mg/mL of fibrinogen and 1 mM of calcium chloride. RESULTS: In this study, we demonstrate the novelty of this instrument in the quantitative evaluation of clot retraction in washed platelets and attempted to optimize the reference range of Sonoclot parameters including ACT - 87.3 ± 20.997, CR - 16.23 ± 3.538 and PF - 3.57 ± 0.629, (n = 10). DISCUSSION: Sonoclot analysis provides a simple and quantitative method to better understand in vitro clot retraction and its modulation by retraction components including platelet count, fibrinogen and platelet-fibrin interaction compared with existing conventional methods. Sonoclot may prove to be a valuable tool in thrombus biology research to understand fundamental basis of blood clot retraction.

A single-institution retrospective study of causes of prolonged prothrombin time and activated partial thromboplastin time in the outpatient setting.

INTRODUCTION: An algorithmic approach, termed the prolonged clot time profile (PROCT), consisting of initial screening with prothrombin time (PT) and activated partial thromboplastin time (aPTT), reflexive mixing studies if indicated, and follow-up assays depending on initial testing results, offers an efficient approach to delineate the etiology of a prolonged PT/aPTT. Herein, we present the outcomes of the PROCT in the outpatient setting. METHODS: In this retrospective study, we reviewed medical records of consecutive outpatients who had prolonged PT and/or aPTT noted in the routine coagulation laboratory and who had PROCT ordered in our institutional Special Coagulation Laboratory between 2010 and 2017. RESULTS: One hundred and six patients, median age 55 years (IQR 30-67), met our study criteria. Twenty-nine patients had normal PT/aPTT, while 77 had persistent abnormalities and underwent reflexive testing. A prolonged PT, aPTT, or PT and aPTT was noted in 27 (35%), 27 (35%), and 23 (30%) respectively. Forty-nine (64%) had an acquired condition, 17 (22%) had a congenital condition, 7 (9%) had unclear etiology, and 4 (5%) were the result of laboratory artifact. The most common known cause of an isolated prolonged PT in our study was vitamin K deficiency in 8 (10%), the most common cause of an isolated prolonged aPTT was lupus anticoagulant in 4 (5%), and the most common cause of prolonged PT and aPTT was liver disease in 11 (14%). CONCLUSION: Prolonged PT/aPTT have a wide range of causes, including artifactual prolongation or abnormalities in secondary hemostasis due to both inherited and acquired conditions.

International Council for Standardization in Haematology (ICSH) recommendations for processing of blood samples for coagulation testing.

This guidance document has been prepared on behalf of the International Council for Standardization in Haematology (ICSH). The aim of the document is to provide guidance and recommendations for the processing of citrated blood samples for coagulation tests in clinical laboratories in all regions of the world. The following areas are included in this document: Sample transport including use of pneumatic tubes systems; clots in citrated samples; centrifugation; primary tube storage and stability; interfering substances including haemolysis, icterus and lipaemia; secondary aliquots-transport, storage and processing; preanalytical variables for platelet function testing. The following areas are excluded from this document, but are included in an associated ICSH document addressing collection of samples for coagulation tests in clinical laboratories; ordering tests; sample collection tube and anticoagulant; preparation of the patient; sample collection device; venous stasis before sample collection; order of draw when different sample types are collected; sample labelling; blood-to-anticoagulant ratio (tube filling); influence of haematocrit. The recommendations are based on published data in peer-reviewed literature and expert opinion.

A performance evaluation of chemiluminescence enzyme immunoassays on the Sysmex CN-6500 haemostasis analyser.

BACKGROUND: The Sysmex CN-6500 is a new haemostasis analyser with an integrated immunoassay module that performs chemiluminescence enzyme assay (CLEIA) in addition to coagulation, turbidimetric, chromogenic and platelet aggregation tests. AIMS: To evaluate the analytical performance of the CN-6500 against the predicate device (Sysmex HISCL-800) for soluble thrombomodulin (TM), thrombin-antithrombin (TAT), tissue plasminogen activator/plasminogen activator inhibitor 1 complex (tPAI-C) and plasmin α2 plasmin inhibitor complex (PIC) assays. METHODS: Imprecision was assessed by testing two levels of quality control plasmas 10 times on 5 separate days. Comparability was studied in 230 plasmas from normal donors (n = 30), patients with suspected disseminated intravascular coagulation (DIC, n = 100), sepsis (n = 20) or liver disease (n = 20), lipaemic (n = 20), haemolysed (n = 20) and icteric samples (n = 20). Limit of detection, limit of quantitation and linearity were determined by testing serial dilutions of normal plasma. Sample carryover was assessed by testing samples with high and low normal levels of the analytes concerned. RESULTS: The CN-6500 performed 21 CLEIA tests per hour, while simultaneously performing coagulation tests. Acceptable between-run imprecision was obtained using commercial controls with normal and high activity for each analyte (%CV <4%), for all four assays. Excellent linearity was observed (slope 0.89-1.03; r2 >0.99) across the measurement range. The lower limits of detection and quantitation were as follows: TM <0.3/0.6 TU/ml, TAT >0.1/<0.2 ng/ml, PIC <0.004/<0.008 µg/ml and tPAI-C < 0.01/<0.1 ng/ml, respectively. All four assays showed excellent correlation between analysers and were unaffected by haemolysis, icterus or lipaemia. No carryover was observed. CONCLUSIONS: Our data demonstrate that the performance of the CLEIA assays on the CN-6500 is comparable to that of a stand-alone immunoassay analyser.

Impact of centrifugation on thrombin generation in healthy subjects and in patients treated with direct oral anticoagulants.

INTRODUCTION: Double centrifugation before freezing is recommended before thrombin generation assays (TGA). However, this procedure is not mandatory for routine hemostasis tests, precluding the use of these samples for TGA. The aim of this study is to assess the impact of single and double centrifugation on TGA performed on frozen samples from healthy volunteers (HVs) and patients receiving direct oral anticoagulants (DOACs). METHODS: Forty HVs and 57 patients receiving a DOAC (dabigatran, rivaroxaban, apixaban, or edoxaban) were included in this prospective double-center observational study. Blood was collected into 109 mmol/L citrated tubes and frozen at -70°C before TGA using ST Genesia with STG-DrugScreen reagent. Four pre-analytical conditions were studied: (A) single centrifugation (2000 g, 15 minutes) before freezing; (B) one centrifugation before freezing and another after thawing (2000 g, 15 minutes for both); (C) one centrifugation before freezing(2000 g, 15 minutes) and another after thawing (2000 g, 10 minutes); (D) double centrifugation (2000 g, 15 minutes) before freezing (reference). Centrifugation conditions (A), (B), and (C) were compared with the reference condition (D). Acceptable relative differences were defined at 6%, 8%, and 10% for normalized lag time, endogenous thrombin potential, and peak height, respectively. RESULTS: Centrifugation conditions had a small but acceptable impact on HVs samples, but single centrifugation always resulted in unacceptable reductions in normalized lag times for DOAC samples. A second centrifugation after thawing permitted the recovery of acceptable differences for the three TGA parameters for edoxaban but not for apixaban, rivaroxaban, nor dabigatran. CONCLUSION: Double centrifugation before freezing should remain the recommended pre-analytical condition before TGA.

A retrospective analysis of discordances between international normalized ratio (INR) self-testing and INR laboratory testing in a pediatric patient population.

INTRODUCTION: The lack of quality control procedures for home point-of-care (POC) international normalized ratio (INR) devices is a concern. Concomitant laboratory and POC INR testing may be proposed to overcome the lack of quality control. However, a difference between the POC INR and the laboratory INR is not necessarily due to failure of the POC device. This study aimed to identify variables associated with a significant deviation between the POC INR and the laboratory INR. METHODS: Children included in this retrospective cohort study performed at least one concomitant laboratory and POC INRs. Clinical and laboratory variables were assessed for an association with significant deviation within pairs of INR. RESULTS: A significant deviation was noted for 30 (15.3%) of the 196 pairs of INR measurements from 124 children. Relative to patients without deviations, patients with deviations were younger (odds ratio =0.91; P = .020), less experienced in the use of POC INR devices (odds ratio =0.89; P = .098), and more likely to have received an INR result from a laboratory using animal thromboplastin (odds ratio =2.81 vs. 0.37 for laboratories using human thromboplastin; P = .016). In a multivariate analysis, younger age and the laboratory's use of animal thromboplastin were associated with significant deviations. CONCLUSIONS: Although most children had coherent pairs of INR values, the occurrence of deviations raises the question of the origin of the thromboplastin used in the laboratory and emphasizes the need to provide specific quality control procedures for POC INR devices.

Viscoelastic Coagulation Monitor (VCMVet) Reference Intervals and Sex Differences in Mature Adult Mice.

INTRODUCTION: Viscoelastic coagulation tests are useful to assess coagulation status in the clinical setting and to aid in understanding underlying pathophysiological mechanisms that affect coagulation status. Such tests also are useful for coagulation research. Because mouse models are widely used to study molecular mechanisms in fine detail, a simple viscoelastic coagulation test requiring small blood volumes would be convenient for such studies in mice. METHODS: We tested viscoelastic coagulation properties of normal healthy adult mice using a novel veterinary clinical point-of-care device, Viscoelastic Coagulation Monitor (VCM Vet™; Entegrion Corp.). Fresh whole blood was collected from 63 healthy mature adult C57 black 6N mice, with ultimately 54 mice, equal numbers of male and females, used to determine reference intervals (RIs) for VCM test parameters. RESULTS: RIs were determined for equal numbers of male and female mice: clot time: 43.0-353.0 s; clot formation time: 49.4-137.6 s; alpha angle: 54.4-62.2°; A10: 25.0-49.6 VCM units; A20: 31.0-56.5 VCM units; maximum clot firmness: 37.6-62.8 VCM units; Lysis Index 30 (Li30): 99.8-100.0%; and Li45: 99.7-100.0%. Significant differences were found between male and female subgroups, where females had higher mean A10 and A20 and median MCF values, indicating greater clot firmness in female versus male mice. CONCLUSION: VCM Vet is a feasible viscoelastic coagulation test device for studies with mature adult mice, including studying inherent sex differences in coagulation parameters. Inherent differences in coagulability of male and female mice warrant further investigation to determine if such differences underlie greater coagulopathic, hemorrhagic, or thromboembolic risk during trauma or other pathophysiologic conditions.

Thrombin generation in children using ThromboScreen reagent kit with ST Genesia-A pilot study.

INTRODUCTION: Thrombin generation assays assess overall coagulation system and are widely used in research; however, they still need standardization and clinical validation. The new ST Genesia is a benchtop, automated analyzer that normalizes each thrombin generation parameter using a reference plasma. The ThromboScreen reagent kit has two triggers, one of which contains thrombomodulin to assess the effect of the protein C pathway. This study aimed to make a pilot approach to the ThromboScreen reference range in children and evaluate the impact of sex, age, and pro- and anticoagulant plasma proteins on thrombin generation parameters. METHODS: This study included 55 healthy children from the following age groups: 1-6 years (n = 14), 7-11 years (n = 15), and 12-17 years (n = 26). Children younger than 1 year were excluded from the study. We measured thrombin generation using ThromboScreen, coagulation routine and test, pro- and anticoagulant proteins. RESULTS: Age did not influence ThromboScreen results. Males showed significantly lower endogenous thrombin potential and peak height values than females. The strongest determinants of endogenous thrombin potential were von Willebrand factor parameters, whereas for endogenous thrombin potential inhibition, the strongest determinants were protein C and protein S. No statistically significant differences were found between groups on temporal parameters. CONCLUSIONS: For the ThromboScreen reagent kit, it may not be necessary to subdivide reference ranges according to age for children (>1 year).

International Council for Standardisation in Haematology (ICSH) recommendations for collection of blood samples for coagulation testing.

This guidance document has been prepared on behalf of the International Council for Standardisation in Haematology (ICSH). The aim of the document is to provide guidance and recommendations for collection of blood samples for coagulation tests in clinical laboratories throughout the world. The following processes will be covered: ordering tests, sample collection tube and anticoagulant, patient preparation, sample collection device, venous stasis before sample collection, order of draw when different sample types need to be collected, sample labelling, blood-to-anticoagulant ratio (tube filling) and influence of haematocrit. The following areas are excluded from this document, but are included in an associated ICSH document addressing processing of samples for coagulation tests in clinical laboratories: sample transport and primary tube sample stability; centrifugation; interfering substances including haemolysis, icterus and lipaemia; secondary aliquots-transport and storage; and preanalytical variables for platelet function testing. The recommendations are based on published data in peer-reviewed literature and expert opinion.

Estimation of Measurement Uncertainty of Factor Assays Using the Monte Carlo Simulation.

OBJECTIVES: We aimed to quantify the measurement uncertainty (MU) for factor activities using the Monte Carlo simulation (MCS), which is a computational algorithm that simulates statistical sampling to obtain numerical results through complex mathematical calculations. METHODS: The uncertainties of factor V (FV), factor VIII (FVIII), and factor X (FX) were simulated with two coagulation testing systems: ACL TOP 750 CTS (Instrumentation Laboratory) and STA Compact (Diagnostica Stago). RESULTS: When the factor activities were 74% (FV), 68% (FVIII), and 89% (FX), the MUs were 3.5% (FV), 9.3% (VIII), and 2.8% (FX) for ACL TOP and 8.5% (FV), 18.2% (FVII), and 6.5% (FX) for STA Compact. MCS MUs were compared with MUs obtained from running actual samples (not simulated) using a method called the top-down approach. The MCS MU results were interchangeable with MUs from the top-down approach, except for FVIII from STA Compact. CONCLUSIONS: The MCS procedure is well suited for the quantification of MUs for factor assays over the entire measurement range.

Differential impacts of hemolysis on coagulation parameters of blood samples: A STROBE-compliant article.

ABSTRACT: This study aimed at investigating the impact of hemolysis on different coagulation parameters.A total of 216 venous blood samples without visible hemolysis were collected from adult patients at a tertiary referral center over six months. The plasma obtained was quantified for six coagulation parameters including prothrombin time, activated partial thromboplastin time, fibrinogen, D-dimer, antithrombin III, and protein C. The rest of the plasma from each blood sample was aliquoted into three tubes, each containing 1 mL of plasma with three different volumes of cell-free hemoglobin (i.e., 2, 4, 8 µL) from lysed RBCs to create simulated hemolyzed blood samples with hemoglobin concentration of approximately 0.1, 0.2, and 0.4 g/dL to mimic mild (1+), moderate (2+), and severe (3+) hemolysis, respectively, before repeating the coagulation tests to determine possible correlation between the simulated degree of hemolysis and the changes in test results of the coagulation parameters.Spearman correlation analysis showed significant decreases in the values of activated partial thromboplastin time, fibrinogen, D-dimer, and protein C values with an increasing degree of simulated hemolysis (all P < .01). Comparison of the percentage bias of biological variance showed significant positive associations of cell-free hemoglobin concentrations with the percentage bias of D-dimer and protein C. However, only the former was still within the range of biological variance under condition of simulated hemolysis. Besides, the presence of cell-free hemoglobin regardless of concentration had a notable impact on the percentage bias of activated partial thromboplastin time, whereas the influence was non-significant for prothrombin time, fibrinogen, and antithrombin III.The results showed different impacts of simulated hemolysis on six coagulation parameters, highlighting the dependence of clinical reliability on the coagulation parameter to be investigated in hemolytic blood samples.