Polymethyl Methacrylate-Based Smart Microfluidic Point-of-Care Testing of Prothrombin Time and International Normalized Ratio through Optical Detection.

The mechanical heart valve is a crucial solution for many patients. However, it cannot function on the state of blood as human tissue valves. Thus, people with mechanical valves are put under anticoagulant therapy. A good measurement of the state of blood and how long it takes blood to form clots is the prothrombin time (PT); moreover, it is an indicator of how well the anticoagulant therapy is, and of whether the response of the patient to the drug is as needed. For a more specific standardized measurement of coagulation time, an international normalized ratio (INR) is established. Clinical testing of INR and PT is relatively easy. However, it requires the patient to visit the clinic for evaluation purposes. Many techniques are therefore being developed to provide PT and INR self-testing devices. Unfortunately, those solutions are either inaccurate, complex, or expensive. The present work approaches the design of an anticoagulation self-monitoring device that is easy to use, accurate, and relatively inexpensive. Hence, a two-channel polymethyl methacrylate-based microfluidic point-of-care (POC) smart device has been developed. The Arduino based lab-on-a-chip device applies optical properties to a small amount of blood. The achieved accuracy is 96.7%.

Micro-mechanical blood clot testing using smartphones.

Frequent prothrombin time (PT) and international normalized ratio (INR) testing is critical for millions of people on lifelong anticoagulation with warfarin. Currently, testing is performed in hospital laboratories or with expensive point-of-care devices limiting the ability to test frequently and affordably. We report a proof-of-concept PT/INR testing system that uses the vibration motor and camera on smartphones to track micro-mechanical movements of a copper particle. The smartphone system computed the PT/INR with inter-class correlation coefficients of 0.963 and 0.966, compared to a clinical-grade coagulation analyzer for 140 plasma samples and demonstrated similar results for 80 whole blood samples using a single drop of blood (10 µl). When tested with 79 blood samples with coagulopathic conditions, the smartphone system demonstrated a correlation of 0.974 for both PT/INR. Given the ubiquity of smartphones in the global setting, this proof-of-concept technology may provide affordable and effective PT and INR testing in low-resource environments.

Evaluation of a new thromboplastin reagent STA-NeoPTimal on a STA R Max analyzer for the measurement of prothrombin time, international normalized ratio and extrinsic factor levels.

INTRODUCTION: We aimed at evaluating the performance of a new prothrombin time (PT) reagent (STA-NeoPTimal) with two other PT reagents (STA-Neoplastine R and STA-Neoplastine CI Plus) and the reference PT reagent used in our laboratory (ReadiPlasTin). METHODS: Evaluation consisted in intra- and interassay precision assessment, determination of sensitivity to unfractionated heparin (UFH) or enoxaparin in spiked samples and to direct oral anticoagulants (DOACs) in patients (n = 43). Method comparison of the 4 PT reagents, factor II, V, VII and X assays was tested on normal (n = 20) and abnormal samples: VKA (n = 47), preoperative (n = 23), liver failure (n = 12) and burned patients (n = 37). RESULTS: Analytical performance met manufacturers' criteria for all reagents. All PT reagents gave correlation coefficients >0.8 and even >0.9 in many situations. In some VKA samples, differences ≥ 0.5 INR units were found in samples within and above therapeutic ranges. For burned patients, PT correlations were good but with some minimal bias (<5.0%) while factor assays gave very consistent results (R > .8 and mainly >0.9). As expected, poor responsiveness of the PT to DOAC concentrations was observed with all four assays. CONCLUSION: The STA-NeoPTimal showed comparable performance to ReadiPlasTin, making it suitable for VKA control, detection of factors II, V, VII, X deficiency and assessment of liver disease coagulopathy. However, for patients receiving VKA, some significant differences were observed. We confirmed the inability of the PT assay to detect residual DOAC concentrations. Finally, burned patients results showed that recombinant thromboplastins were less sensitive to factor deficiencies in comparison to extraction thromboplastins.

Routine coagulation testing in Vacutainer® Citrate Plus tubes filled at minimum or optimal volume.

Background Filling of citrate tubes with appropriate amount of blood is essential for obtaining reliable results of coagulation testing. This study aimed to verify whether results of routine coagulation tests are comparable when the new Becton Dickinson Vacutainer® Citrate Plus tubes are filled at minimum or optimal volume. Methods The study population consisted of 133 patients (40 on oral anticoagulant therapy), who had blood collected for routine coagulation testing. Two sequential Vacutainer® Citrate Plus tubes of the same type and lot were drawn. The first tube was collected after a butterfly needle was inserted into the vein, so that the air in the tubing was aspirated into the tube before blood (minimum fill volume), whilst the second was drawn at optimal fill volume. Experiments were repeated using 2.7-mL (n = 86) and 1.8-mL (n = 47) tubes. Results Prothrombin time (PT) and fibrinogen values were slightly but significantly decreased in tubes with minimum than in those with optimal fill volume. The activated partial thromboplastin time (APTT) was slightly prolonged in tubes with minimum than in those with optimal fill volume, but the difference was not statistically significant. An identical trend was noted in separate analyses for the 2.7-mL and 1.8-mL tubes. Spearman's correlations between the two fill volumes were always >0.94 and bias was always within the quality specifications. Conclusions Blood drawing into Vacutainer® Citrate Plus tubes at minimum fill volume does not clinically bias routine coagulation testing.

Effects of hemolysis, bilirubin, and lipemia interference on coagulation tests detected by two analytical systems.

INTRODUCTION: Interference on biological assays due to hemolysis, icterus, or lipemia (HIL) could represent a significant source of analytical errors leading to inaccurate interpretation of results. The aim of this study was to assess the HIL interference on prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen, using mechanical and optical detection methods. METHODS: Control plasmas and plasmas from patients treated with vitamin K antagonists or unfractionated heparin, with or without HIL, were performed on two analytical detection systems in order to identify potential analytical biases. Whether HIL lead to significant biological interferences was also evaluated, and a cutoff point for HIL-induced analytical bias was determined. RESULTS: Hemolysis influenced PT and aPTT when hemoglobin was at 5 and 1.5 g/L in plasma, respectively. At 1.8 g/L, a positive relationship was found between the bias and the hemoglobin supernatant level only for fibrinogen measurement, using optical detection. For icteric interference, no significant bias was observed until a bilirubin concentration of 30 mg/dL. Lipamia (>500 mg/dL) led to analytical interference when using the optical analyzer. CONCLUSION: The present study detected analytical interferences such as lipemia (>500 mg/dL) on coagulation tests on the optical analyzer. We also found a biological impact on the results in case of hemolyzed sample: Fibrinogen was decreased when the hemoglobin level was superior to 1.8 g/L, PT was prolonged beyond 5 g/L, and aPTT was shortened beyond 1.5 g/L hemoglobin concentration, especially in patients treated with heparin. Above these thresholds, it is important not to give results that could influence the clinical decision.

Laboratory Testing for Activated Protein C Resistance (APCR).

Activated protein C resistance (APCR) describes a hemostatic disorder characterized by a poor anticoagulant response to activated protein C (APC). This results in an increased risk of venous thrombosis, including deep vein thrombosis and pulmonary embolism. Protein C is a natural anticoagulant that is synthesized in the liver and is activated to APC via proteolysis. APC then degrades Factors Va and VIIIa. APCR describes the reduced inability of APC to cleave Factors Va and VIIIa, which therefore promotes increased thrombin generation and potentially leads to a prothrombotic state. APCR may be hereditary or acquired. The most common hereditary defect is due to mutations in Factor V, predominantly the Factor V Leiden [FVL] mutation-a G1691A missense mutation at Arginine 506 that results in its replacement by a glutamine [R506Q] and the abolition of an APC inactivation cleavage site in Factor Va. Laboratory testing for APCR may be undertaken by a variety of methods, but this chapter describes an automated procedure using a commercial Russell Viper Venom-based clotting assay, and using CS-5100 and STA-R analyzers.

Clinical evaluation of whole blood prothrombin time (PT) and international normalized ratio (INR) using a Laser Speckle Rheology sensor.

Prothrombin time (PT) and the associated international normalized ratio (INR) are routinely tested to assess the risk of bleeding or thrombosis and to monitor response to anticoagulant therapy in patients. To measure PT/INR, conventional coagulation testing (CCT) is performed, which is time-consuming and requires the separation of cellular components from whole blood. Here, we report on a portable and battery-operated optical sensor that can rapidly quantify PT/INR within seconds by measuring alterations in the viscoelastic properties of a drop of whole blood following activation of coagulation with thromboplastin. In this study, PT/INR values were measured in 60 patients using the optical sensor and compared with the corresponding CCT values. Our results report a close correlation and high concordance between PT/INR measured using the two approaches. These findings confirm the accuracy of our optical sensing approach for rapid PT/INR testing in whole blood and highlight the potential for use at the point-of-care or for patient self-testing.

A performance evaluation of a novel human recombinant tissue factor prothrombin time reagent (Revohem™ PT).

INTRODUCTION: A new prothrombin time reagent (Revohem™ PT) based on recombinant human tissue factor produced by the silkworm-baculovirus expression system was tested. The aim of this study was to compare the performance of the new PT reagent with two widely used routine PT reagents. METHODS: All testing was performed on a Sysmex CS-5100 coagulometer. Revohem™ PT was tested for imprecision and stability using normal and abnormal lyophilized commercial control plasmas. Comparability was assessed with two widely used reagents: one containing recombinant human tissue factor (Reagent A) and the other a human placental thromboplastin (Reagent B) using a wide range of normal and abnormal plasmas and analyser-specific ISI values. RESULTS: Excellent between-day imprecision was obtained for Revohem™ PT (CV <1.0%) and acceptable open-vial on-board stability over 7 days. There was good agreement between methods in samples from patients with liver disease and patients receiving warfarin and no significant differences between methods with increasing INR values. Both recombinant reagents suffered less interference from lupus anticoagulant than the placental thromboplastin. Revohem™ PT had similar sensitivity to reagents A and B for FII, V, VII and X deficiency and demonstrated dose responsiveness to dabigatran, apixaban and rivaroxaban with steeper response curves than the comparison reagents. CONCLUSION: Revohem™ PT showed comparable or improved performance relative to two widely used reagents and is suitable for use in warfarin control, detection of inherited factor II, V, VII and X deficiency and assessment of liver disease coagulopathy.

Point-of-care testing for emergency assessment of coagulation in patients treated with direct oral anticoagulants.

BACKGROUND: Point-of-care testing (POCT) of coagulation has been proven to be of great value in accelerating emergency treatment. Specific POCT for direct oral anticoagulants (DOAC) is not available, but the effects of DOAC on established POCT have been described. We aimed to determine the diagnostic accuracy of Hemochron® Signature coagulation POCT to qualitatively rule out relevant concentrations of apixaban, rivaroxaban, and dabigatran in real-life patients. METHODS: We enrolled 68 patients receiving apixaban, rivaroxaban, or dabigatran and obtained blood samples at six pre-specified time points. Coagulation testing was performed using prothrombin time/international normalized ratio (PT/INR), activated partial thromboplastin time (aPTT), and activated clotting time (ACT+ and ACT-low range) POCT cards. For comparison, laboratory-based assays of diluted thrombin time (Hemoclot) and anti-Xa activity were conducted. DOAC concentrations were determined by liquid chromatography-tandem mass spectrometry. RESULTS: Four hundred and three samples were collected. POCT results of PT/INR and ACT+ correlated with both rivaroxaban and dabigatran concentrations. Insufficient correlation was found for apixaban. Rivaroxaban concentrations at <30 and <100 ng/mL were detected with >95% specificity at PT/INR POCT ≤1.0 and ≤1.1 and ACT+ POCT ≤120 and ≤130 s. Dabigatran concentrations at <30 and <50 ng/mL were detected with >95% specificity at PT/INR POCT ≤1.1 and ≤1.2 and ACT+ POCT ≤100 s. CONCLUSIONS: Hemochron® Signature POCT can be a fast and reliable alternative for guiding emergency treatment during rivaroxaban and dabigatran therapy. It allows the rapid identification of a relevant fraction of patients that can be treated immediately without the need to await the results of much slower laboratory-based coagulation tests. TRIAL REGISTRATION: Unique identifier, NCT02371070 . Retrospectively registered on 18 February 2015.

The microINR portable coagulometer: analytical quality and user-friendliness of a PT (INR) point-of-care instrument.

Regular measurement of prothrombin time as an international normalized ratio PT (INR) is mandatory for optimal and safe use of warfarin. Scandinavian evaluation of laboratory equipment for primary health care (SKUP) evaluated the microINR portable coagulometer (microINR®) (iLine Microsystems S.L., Spain) for measurement of PT (INR). Analytical quality and user-friendliness were evaluated under optimal conditions at an accredited hospital laboratory and at two primary health care centres (PHCCs). Patients were recruited at the outpatient clinic of the Laboratory of Medical Biochemistry, St Olav's University Hospital, Trondheim, Norway (n = 98) and from two PHCCs (n = 88). Venous blood samples were analyzed under optimal conditions on the STA-R®Evolution with STA-SPA + reagent (Stago, France) (Owren method), and the results were compared to capillary measurements on the microINR®. The imprecision of the microINR® was 6% (90% CI: 5.3-7.0%) and 6.3% (90% CI: 5.1-8.3) in the outpatient clinic and PHCC2, respectively for INR ≥2.5. The microINR® did not meet the SKUP quality requirement for imprecision ≤5.0%. For INR <2.5 at PHCC2 and at both levels in PHCC1, CV% was ≤5.0. The accuracy fulfilled the SKUP quality goal in both outpatient clinic and PHCCs. User-friendliness of the operation manual was rated as intermediate, defined by SKUP as neutral ratings assessed as neither good nor bad. Operation facilities was rated unsatisfactory, and time factors satisfactory. In conclusion, quality requirements for imprecision were not met. The SKUP criteria for accuracy was fulfilled both at the hospital and at the PHCCs. The user-friendliness was rated intermediate.

[Evaluation of Portable Coagulation Analyzer in Comparison with Conventional Hospital Laboratory Instrument with Stratification of International Normalized Ratios by Therapeutic Upper Limit].

Point of care devices have been widely applied to outpatients receiving anticoagulation therapy with warfarin for monitoring prothrombin time-international normalized ratio (PT-INR) regularly. However, accuracy in measurement with the device remains undetermined when PT-INR exceeds therapeutic range. We evaluated the performance of a portable CoaguChek XS coagulation analyzer in comparison with a conventional laboratory method according to therapeutic and supra-therapeutic PT-INR values in cardiac outpatients on oral vitamin K antagonists. All participants were classified into 2 groups on the basis of PT-INR 3.0 by the laboratory method; therapeutic group less than or equal to 3.0 (n=48) and supra-therapeutic group above 3.0 (n=8). The correlation coefficients in therapeutic and in supra-therapeutic groups were r=0.82 and r=0.78, respectively (p<0.05). The difference in PT-INR between the laboratory method and the CoaguChek XS was significantly larger in supra-therapeutic group than therapeutic group (1.03±0.73 versus 0.34±0.26, p=0.042). Our study indicates that CoaguChek XS can be useful handheld coagulation analyzer to determine PT-INR rapidly; however, the device may underestimate PT-INR in supra-therapeutic range.

A Point-of-Care Prothrombin Time Test on a Microfluidic Disk Analyzer Using Alternate Spinning.

In this study, we conducted a fully integrated point-of-care prothrombin time test on a microfluidic disk analyzer. The microfluidic functions integrated on the disk were capable of separating whole blood, decanting plasma, and mixing it with reagents in sequence under alternate spinning. The assay protocol was completed by alternate spinning without using microvalves or surface modification. Clinical sample tests on prothrombin time measurement were conducted by both the microfluidic disk analyzer and the reference instrument used in medical centers. The test results showed a good correlation and agreement between the two instruments.

Performance Evaluation of the Sysmex CS-5100 Automated Coagulation Analyzer.

BACKGROUND: Coagulation testing is widely applied clinically, and laboratories increasingly demand automated coagulation analyzers with short turn-around times and high-throughput. The purpose of this study was to evaluate the performance of the Sysmex CS-5100 automated coagulation analyzer for routine use in a clinical laboratory. METHODS: The prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (APTT), fibrinogen (Fbg), and D-dimer were compared between the Sysmex CS-5100 and Sysmex CA-7000 analyzers, and the imprecision, comparison, throughput, STAT function, and performance for abnormal samples were measured in each. RESULTS: The within-run and between-run coefficients of variation (CV) for the PT, APTT, INR, and D-dimer analyses showed excellent results both in the normal and pathologic ranges. The correlation coefficients between the Sysmex CS-5100 and Sysmex CA-7000 were highly correlated. The throughput of the Sysmex CS-5100 was faster than that of the Sysmex CA-7000. There was no interference at all by total bilirubin concentrations and triglyceride concentrations in the Sysmex CS-5100 analyzer. CONCLUSIONS: We demonstrated that the Sysmex CS-5100 performs with satisfactory imprecision and is well suited for coagulation analysis in laboratories processing large sample numbers and icteric and lipemic samples.

Correlation Between Home INR and Core Laboratory INR in Patients Supported with Continuous-Flow Left Ventricular Assist Devices.

It has been well established that patient self-testing (PST) of international normalized ratio (INR) using home monitoring devices increases the average therapeutic time and patient satisfaction. Long-term anticoagulation therapy with warfarin is used in patients with continuous-flow left ventricular assist device (CF-LVAD) to minimize the occurrence of thromboembolic events; however, PST devices have never been tested in patients with CF-LVADs. The purpose of this study was to determine the reliability of the PST device Alere INRatio 2 in patients supported with CF-LVADs. A correlation study was performed in 50 patients with CF-LVAD who were on stable warfarin therapy for a minimum of 3 weeks. Simultaneous INR values were determined from capillary whole blood samples using the Alere PST device and venous blood samples processed in the core laboratory at Columbia University Medical Center. There was a moderate correlation between the venous and the capillary INR values with a correlation coefficient of 0.83. The median difference between the methods was 0.39, with 44 of 50 patients recording higher INRs with Alere. Results remained unchanged after adjusting for use of amiodarone, abnormal hematocrit and liver enzymes, creatinine, and thyroid-stimulating hormone. Point of care testing with Alere correlates moderately well but consistently overestimates INR when compared with conventional laboratory testing in patients with CF-LVAD.

Influence of dabigatran and rivaroxaban on routine coagulation assays. A nationwide Belgian survey.

The Belgian national External Quality Assessment Scheme performed a nationwide survey using lyophilised plasma samples spiked with dabigatran or rivaroxaban to demonstrate to the Belgian clinical laboratories how these drugs affect their routine coagulation assays prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and antithrombin. Virtually all Belgian laboratories performing routine coagulation testing (189/192) participated in the survey. Both, dabigatran and rivaroxaban significantly prolonged the PT and aPTT in a concentration- and reagent-dependent manner. PT reagents were more influenced by rivaroxaban than by dabigatran and aPTT reagents more influenced by dabigatran than by rivaroxaban. Among PT reagents, Neoplastin R® was the most sensitive to rivaroxaban and Innovin® and Thromborel S® the least sensitive. Converting PT results to INR only increased the variability between reagents. Among aPTT reagents, Actin FSL® was the least sensitive to dabigatran while the other aPTT reagents showed slightly higher sensitivities. The presence of dabigatran led to falsely reduced fibrinogen concentrations when measured with a low thrombin concentration reagent. The presence of dabigatran caused an overestimation of the antithrombin level when measured with a thrombin-based activity assay and the presence of rivaroxaban an overestimation of the antithrombin level when measured with a FXa-based assay. Instrument-related differences were found for all tested parameters. In conclusion, this paper provides detailed information on the effect of dabigatran and rivaroxaban on routine coagulation assays as performed with a large number of reagent/instrument combinations.

A cartridge based sensor array platform for multiple coagulation measurements from plasma.

This paper proposes a MEMS-based sensor array enabling multiple clot-time tests for plasma in one disposable microfluidic cartridge. The versatile LoC (Lab-on-Chip) platform technology is demonstrated here for real-time coagulation tests (activated Partial Thromboplastin Time (aPTT) and Prothrombin Time (PT)). The system has a reader unit and a disposable cartridge. The reader has no electrical connections to the cartridge. This enables simple and low-cost cartridge designs and avoids reliability problems associated with electrical connections. The cartridge consists of microfluidic channels and MEMS microcantilevers placed in each channel. The microcantilevers are made of electroplated nickel. They are actuated remotely using an external electro-coil and the read-out is also conducted remotely using a laser. The phase difference between the cantilever oscillation and the coil drive is monitored in real time. During coagulation, the viscosity of the blood plasma increases resulting in a change in the phase read-out. The proposed assay was tested on human and control plasma samples for PT and aPTT measurements. PT and aPTT measurements from control plasma samples are comparable with the manufacturer's datasheet and the commercial reference device. The measurement system has an overall 7.28% and 6.33% CV for PT and aPTT, respectively. For further implementation, the microfluidic channels of the cartridge were functionalized for PT and aPTT tests by drying specific reagents in each channel. Since simultaneous PT and aPTT measurements are needed in order to properly evaluate the coagulation system, one of the most prominent features of the proposed assay is enabling parallel measurement of different coagulation parameters. Additionally, the design of the cartridge and the read-out system as well as the obtained reproducible results with 10 µl of the plasma samples suggest an opportunity for a possible point-of-care application.

A global quality control system to check PT-INR portable monitor for Antivitamin K antagonists.

INTRODUCTION: Although most guidelines for quality assessment of INR PMs recommend specific procedures, no clear regulation or methodology is required for outpatients in our country. We have developed a specific INR portable monitor (PM) quality control system within our telemedicine organization to check over time quality performances and plan corrective actions. METHODS: Based on current guidelines for laboratory QC, the following aspects were assessed: suitability of PM, defined in terms of imprecision and accuracy; intra-assay imprecision, defined according to monthly revision of Levey-Jennings cards with data from each peripheral healthcare unit (PHU), using an internal QC provided by the manufacturer (CV ± 20% considered as acceptable); quarterly accuracy study, for assessing agreement between analytical instruments, based on duplicate analysis of three samples with INR values reflecting different therapeutic ranges (differences ± 0.5 considered as acceptable); external quality assessment (NEQAS). RESULTS: In the nine PHU, 18 portable monitors were used to perform 22 929 test during year 2010. Analytical imprecision was low, showing CVs always <5%. Accuracy check showed two of 216 results out of range (0.92%), thus providing timely indication for instrument replacement. The external QC NEQAS showed optimal performance. CONCLUSION: The current protocol for INR PMs quality assessment was effective to establish and maintain a reliable control of devices, ensuring the quality of analytical data over time. National authorities should be prompted to guarantee and apply correct protocols for INR-PM use.