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RESEARCH ARTICLE |
https://doi.org/10.5005/jp-journals-10054-0174
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Analytical Performance Assessment of the Prothrombin Time and Activated Partial Thromboplastin Time Tests by Roche Cobas t511 Coagulation Analyzer
Corresponding Author: Murat CAN, Zonguldak, Turkey, Phone: +903722612839, e-mail: drcanmurat@yahoo.com
How to cite this article CAN Murat, Guven B, Tekin A. Analytical Performance Assessment of the Prothrombin Time and Activated Partial Thromboplastin Time Tests by Roche Cobas t511 Coagulation Analyzer. Indian J Med Biochem 2021;25(2):71–75.
Source of support: Nil
Conflict of interest: None
ABSTRACT
Aim and objective: The study aims to evaluate the performance of a newly installed fully automatic coagulation analyzer Roche t511.
Materials and methods: The prothrombin time (PT), activated partial thromboplastin time (aPTT), and international normalized ratio (INR) values obtained from 150 patients by a Roche t511 were compared with those obtained by Siemens Sysmex CS 2000i. Coagulation assays were performed under routine conditions using standard reagents and an analyzer. Reference intervals were established using 2.5th and 97.5th percentiles for hematologic analytes and minimum and maximum values for coagulation tests.
Results: In our study, within- and between-run precision CVs for PT and aPTT in Cobas t511 analyzer were excellent according to the criteria for acceptance. Reference intervals are reported.
Conclusion: The data reported here show that the PT and aPTT assay on the t511 fully automated analyzer is highly sensitive, accurate, and specific for the measurement of hemostasis.
Keywords: Activated partial thromboplastin time, Analytic performance, Coagulation analyzer, Prothrombin time, Roche t511.
INTRODUCTION
Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are often measured in clinical practice. They are commonly used in the assessment of coagulopathies, the activity of anticoagulant therapy, and preoperative screening. 1,2 The clinical interpretation of coagulation test results depends on established reference intervals derived from clinical studies in healthy populations.
A fully automated coagulation analyzer allows fast, precise, and reliable measurement of coagulation parameters in routine laboratories, thus being cost and labor-saving. In hemostasis testing, pre‐analytical issues are still challenging and functions of the coagulation analyzer include reagent preparation, primary tube sampling, cap piercing, dilution, automatic rerun, and reflex testing helps to solve most problems.
The study aims to evaluate the performance of a newly installed fully automatic coagulation analyzer t511 (Roche) and compare the consistency of its testing results with the confirmed clinical automatic coagulation analyzer Sysmex CS 2000i (Siemens) at our laboratory.
MATERIALS AND METHODS
The study was performed between April and June 2019 in the clinical laboratory of Zonguldak Bulent Ecevit University of Turkey. The study was approved by our institutional Ethics Committee and all participants gave written informed consent.
Blood Samples
One hundred fifty whole blood samples (each 1.8 mL) were collected into Vacuette® blood tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) each containing 3.2% (w/v) sodium citrate (0.109 M). Plasma was obtained via centrifugation for 15 minutes at 1,500 × g. All samples were assayed using the two systems within 1 hour of blood collection.
Instrument
The Cobas t511 system (Roche, Germany) is a fully automated coagulation analyzer with 20 incubation and 13 measurement channels, intended for clotting, chromogenic, and immunoturbidimetric assays (408, 588, 625, and 800 nm). According to the manufacturer, the Cobas t511 analyzer can hold 75 samples with 5 samples per rack and perform 195 tests PT/aPTT per hour. The analyzer has a cap-piercing property, which processes capped and uncapped sample tubes. For assessment of sample quality, the analyzer performs automatic hemolysis, icterus, and lipemia check with a preanalytical scan of patient samples performed at two wavelengths (575 and 660 nm) and a primary tube sample volume check identifies insufficient sample volume. Furthermore, the analyzer reconstitutes all reagents automatically and it has continuous reagent loading/unloading.
Reagents
Roche PT Rec reagent contains recombinant human thromboplastin with a heparin-neutralizing substance and calcium that initiates the activation of the extrinsic coagulation cascade when added to citrated human plasma. The time between the addition of reagent to the plasma and the formation of a fibrin clot is measured and reported in seconds. The reagent lot-specific ISI is used to convert the patient’s PT result in seconds into the international normalized ratio (INR) using the following formula: INR = (patient’s PT/mean normal PT)ISI. The ISI value for a specific thromboplastin reagent is determined by a method comparison of the thromboplastin reagent to be standardized to an international reference thromboplastin.
Roche aPTT Screen reagent contains a mixture of purified phospholipids and silicon dioxide particles as an activator that stimulates factor XIIa generation. Calcium chloride is then added which prompts the initiation of the intrinsic coagulation cascade. The time from the addition of calcium chloride until clot formation is measured. The aPTT Screen assay is designed to have the highest sensitivity toward unfractionated heparin (UFH) therapy.
Siemens PT was measured with Thromborel S reagent that contains lyophilized human placental thromboplastin and calcium. The coagulation process is triggered incubation of Thromborel S reagent and plasma. The time of formation of a fibrin clot is then measured.
Siemens aPTT was measured with actin FS reagent that contains purified soy phospholipids and ellagic acid as an activator. Intrinsic factors are activated by incubated actin FS reagent and plasma. The addition of calcium triggers the coagulation process, and the clotting time is then measured.
Reagents Reconstitution
We analyzed the accuracy and imprecision of the automatically resuspended lyophilized PT cassettes. The cassettes were weighed on the same precision balance (SBC32; Scaltec; linearity range: 0.01–120 g; imprecision: 0.001 g). The empty cassette was weighted and then resuspended with 33 mL distilled water automatically. The weight difference was finally calculated for each cassette.
Analytical Performance
The within-run and between-run precision of each assay was performed according to Clinical Laboratory Standards Institute (CLSI) EP05-A3 guidelines. 3 Each assay was independently evaluated on the Cobas t511 analyzer two-level quality control tests using materials supplied by either manufacturer (Roche CON1, catalog no. 07530331190; Roche CON2, catalog no. 07532997190). Within-run precision for each assay was evaluated in one run using two controls (PT Rec, aPTT screen) samples (n = 20 replicates per sample). Between-run was evaluated over 20 days using two controls (each measured two times daily).
Method Comparison
The study was designed to evaluate the Roche t511 in terms of quality and routine use. Therefore, patient samples were measured with both the t511 and the confirmed clinical automatic coagulation analyzer Sysmex CS 2000i at our laboratory according to CLSI EP09-A3 guidelines. 4
Reference Range Study
Reference ranges for each assay were determined using 115 samples from apparently healthy adult persons. Inclusion criteria were apparently healthy adults (aged 18–50 years) and able to provide written informed consent; exclusion criteria were self-declared pregnancy or breastfeeding, and use of anticoagulation medication including acetylsalicylic acid, phenprocoumon, and warfarin.
Statistical Analysis
The coefficient of variation (CV) was calculated for within- and between-run precision of each assay and evaluated against prespecified acceptance ranges (within-run precision: ≤3.0%; total reproducibility ≤25.0%) that in accordance with published guidelines. 5,6
Method comparison was performed by MedCalC using the Passing-Bablok regression analysis and Bland–Altman analysis [mean values and 95% confidence interval (95% CI)]. 7,8
Reference ranges (seconds) were presented as mean ± standard deviation and on the 2.5th and 97.5th percentile values of the series of measurements for each assay.
RESULTS
The precision of the automatic resuspension was 32.68 ± 0.03 g and CV% 0.09%. The difference between theoretical and measured filling volume for both PT was −0.32 ± 0.02.
The CVs for within- and between-run precision of PT, INR, and aPTT in Cobas t511 analyzer are presented in Table 1. Within- and between-run precision CVs were within the criteria for acceptance in evaluated assays.
We randomly collected 150 samples from the routine laboratory, which included 115 normal and 35 abnormal (with anticoagulant) samples. With Passing and Bablok regression analysis, for PT (Fig. 1A) yielded the equation of y = 1.015 × (95% CI, 0.870 to 1.144)–2.70 (95% CI, −4.168 to −0.904); INR (Fig. 1B) yielded the equation of y = 1.05 × (95% CI, 0.910 to 1.137)–0.0198 (95% CI, −0.111 to 0.121); aPTT (Fig. 1C) yielded the equation of y = 1.571 × (95% CI, 1.424 to 1.787) −8.029 (95% CI, −13.24 to −4.55). Calculation of Spearman’s correlation coefficient between the two methods showed positive correlation for PT, INR, and aPTT, respectively (r = 0.732, p %3C; 0.001; r = 0.736, p < 0.001; r = 0.787, p < 0.001).
Comparisons made via the Bland–Altman test between the two analyzers are shown in Figure 2. Difference of PT, INR, and aPTT levels between the averages was −1.6 (−20.8%), 0.09 (2.4%), and 7.0 (21.4%).
| Test | Sample |
Within run
|
Between run
|
Total CV | ||||
|---|---|---|---|---|---|---|---|---|
| Mean ± SD | CV (%) | Manufacturer CV (%) | Mean ± SD | CV (%) | Manufacturer CV (%) | |||
| PT (seconds) | CON1 (n = 10) | 8.70 ± 0.04 | 0.4 | 0.4 | 8.75 ± 0.07 | 0.8 | 0.7 | 0.9 |
| CON2 (n = 10) | 27.38 ± 0.11 | 0.5 | 0.8 | 27.52 ± 0.33 | 1.1 | 1.4 | 1.2 | |
| INR | CON1 (n = 10) | 0.98 ± 0.004 | 0.4 | 0.4 | 0.98 ± 0.009 | 0.9 | 0.7 | 1.0 |
| CON2 (n = 10) | 2.72 ± 0.01 | 0.4 | 0.7 | 2.79 ± 0.05 | 1.7 | 1.2 | 1.7 | |
| aPTT (seconds) | CON1 (n = 10) | 30.14 ± 0.16 | 0.5 | 0.4 | 30.3 ± 0.52 | 1.7 | 0.5 | 1.8 |
| CON2 (n = 10) | 52.57 ± 0.35 | 0.7 | 0.4 | 52.3 ± 0.65 | 1.2 | 0.6 | 1.4 | |
Figs 1A to C: Passing-Bablok regression analysis for PT (A), INR (B) and aPTT (C) results
References intervals of PT, INR, and aPTT were obtained from 115 healthy persons. The distribution of samples and detailed information about them is provided in Table 2.
DISCUSSION
Cobas t511 is a medium throughput (195 tests/hour) coagulation analyzer that provides performing a wide range of different coagulation assays on a single or multiple samples at the same time. Additionally, the analyzer stops working in the presence of expired material. The software was simple to use, but the expression of calibration and control lot changes in software should be improved.
One of the most important advantages of the t511 analyzer is automatical reagent reconstitution. Therefore, it provides eliminates the inherent risk of manual lyophilization of reagent and facilitates the monitoring of kit stabilization. Lippi et al. found the automatic resuspension of PT on the Cobas t711 platform with a variation of 0.04%. 9 In the present study, variation of automatic resuspension was 0.09% and the agreement between the two systems was excellent.
In our study, within- and between-run precision CVs for PT and aPTT in Cobas t511 analyzer were excellent according to the criteria for acceptance. Our results are similar to a previous study that within-run imprecision in Cobas t511 was comprised between 0.1 and 0.4% for PT, 0.2 and 0.8% for aPTT, total imprecision was comprised between 2.2 and 3.7% for PT, 0.8 and 6.3% for aPTT. 10 Despite these results, especially in the aPTT test, we noticed higher CV values than reported those of the manufacturer. Therefore, we suggest that producers check analytic performance criteria for the aPTT test in Cobas Roche t511.
In this study, we compared the blood coagulation analyzer Roche Cobas t511 system to the mid-volume blood coagulation analyzer Sysmex CS-2000i System for analytical performance. Totally, 150 samples including 115 normal and 35 high with anticoagulant were analyzed on two different analyzers to check the reliability of PT and aPTT. There was a positive correlation in PT, INR, and aPTT levels between Roche Cobas t511 and Sysmex CS-2000i System, even though the correlation of tests obtained on the two analyzers was unsatisfactory.
According to regression analysis and bias study, PT values measured by Cobas t511 showed a slight discrepancy with Sysmex CS 2000i measures. Compared with PT results of the Sysmex CS 2000i, the Cobas t511 had a negative bias (mean bias: −2.7) in normal PT levels (<12 sn) and a positive bias (mean bias: 1.7) in higher PT levels (%3E;30 sn). Lippi et al. found a study that revealed a proportional negative bias of approximately 20% of PT in Cobas t711 compared with ACL-TOP and STA-R MAX. 9 Cobas t711 is a high-throughput coagulation analyzer using the same reagent as Cobas t511. Our study provides also evaluating PT results in Cobas t511 via comparison of INRs, because PT data may vary depending on the thromboplastin used. International normalized ratio results standardizing the results of PT tests are demonstrated a good correlation with INR values close to 1, but a small proportional bias within the therapeutic range (>2.5 INR). On the other hand, the proportional difference between the two methods for aPTT was evident, also positive bias included the entire aPTT measuring range. Similar results for aPTT were reported in the study of Lippi et al. comparing IL ACL TOP 700 and Cobas t711. 9 The variation between the two analyzers is attributable to different aPTT reagents based on different contact activators. However, we suggest concordance evaluation in aPTT results of Cobas t511 and further assessment to verify the effect of this bias on clinical decision-making.
Figs 2A to C: Bland and Altman plot for PT (A), INR (B) and aPTT (C) results
| Test | 2.5th percentile | 97.5th percentile | Min–Max | Mean ± SD |
|---|---|---|---|---|
| PT (seconds) | 7.96 | 10.48 | 7.82–11.10 | 8.91 ± 0.61 |
| INR | 0.91 | 1.16 | 0.89–1.20 | 1.00 ± 0.06 |
| aPTT (seconds) | 22.6 | 35.4 | 21.5–36.7 | 28.5 ± 3.2 |
The reference range in healthy volunteers measured with the aPTT assay was slightly lower at 2.5th percentile (22.6 seconds) and higher at 97.5 percentile (35.4 seconds) with those reported by the manufacturer (23.9–33.2 seconds). The reference range for the PT Rec assay was slightly lower (8.0–10.5 seconds) than that previously reported by the manufacturer (8.4–10.6 seconds). This difference may be due to ethnic differences.
To conclude, the data reported here show that the PT and aPTT assay on the t511 fully automated analyzer is highly sensitive, accurate, and specific for the measurement of hemostasis.
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