1 Department of Translational Medicine & Centre for Thrombosis and Haemostasis, Malmö, Lund University, Sweden.
2 Department of Clinical Chemistry, Karolinska University Hospital, Stockholm, Sweden.
3 Coagulation Unit, Haematology Centre, Karolinska University Hospital, Stockholm, Sweden.
4 Hemostasis Department and Hemophilia Centre, Blood Transfusion Institute of Serbia.
5 Clinical Centre of Serbia & Faculty of Medicine, University of Belgrade, Belgrade, Serbia.
6 Coagulation Research, Institute for Molecular Medicine and Surgery, Karolinska Institute & Department of Clinical Chemistry, Karolinska University Hospital, Stockholm, Sweden.
Received: August 2, 2017
Accepted: October 9, 2017
Mediterr J Hematol Infect Dis 2017, 9(1): e2017064 DOI 10.4084/MJHID.2017.064
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thrombin-generation tests are available, but few have been directly
compared. Our primary aim was to investigate the correlation of two
thrombin generation tests, thrombin generation assay-calibrated
automated thrombogram (TGA-CAT) and INNOVANCE ETP, to factor VIII
levels (FVIII:C) in a group of patients with hemophilia A.
There are several thrombin generation tests on the market, and most of them have not been compared to each other. The first aim of this study was to investigate which of two tests: the thrombin generation assay-calibrated automated thrombogram (TGA-CAT) or the INNOVANCE ETP, correlated best with the factor VIII level in a group of persons with hemophilia (PWH). TGA-CAT uses a fluorogenic substrate while the INNOVANCE ETP uses a chromophore for detection of thrombin generation. For the latter test, the plasma samples are defibrinated by its ETP reagent containing a fibrin aggregation inhibitor. With the TGA-CAT method, this is not required.
Despite the advantages of TGTs, the TGA-CAT is used primarily in research laboratories due to the lack of standardization of the method and the large inter-center variability. However, in recent studies, a number of problems have been addressed, and promising improvements have been made in increasing the level of standardization for pre-analytical and analytical techniques.[14-16] Several investigations have also evaluated different standard reference plasmas’ ability to reduce TGA-CAT inter-center variability.[14,15,17] The second aim of this study was to investigate the inter-laboratory variability of the TGA-CAT method between two centers.
Material and Methods
Written informed consent was obtained from all subjects prior to the study. The study was approved by the Ethics Committee, Stockholm (Dnr 01-0003; 2006/778-32; 2013/263-32).
Blood samples and plasma preparation. Peripheral venous blood was collected into BD Vacutainer® plastic tubes (Becton Dickinson, Franklin Lakes, NJ, USA) with anticoagulant trisodium citrate (0.129 M, pH 7.4) (one part trisodium citrate and nine parts blood). Plasma was prepared within 60 minutes from venipuncture by centrifugation at 2000xG for 20 minutes at room temperature (RT), then divided into aliquots and stored at -70° C. Frozen samples on dry ice were transported from Belgrade to Stockholm and then from Stockholm to Malmö, and were still frozen and in good condition upon arrival.
Methods. TGA-CAT was measured according to the method described by Hemker et al.[19,20] Briefly, twenty microliters of PPP-reagent LOW (1 pM Tissue Factor (TF) and 4 µM phospholipids (Pl)(TS31.00) and twenty microliters of Thrombin Calibrator (TS20.00) were manually pipetted into the wells of a round-bottom 96 well-microtiter plate (Immulon 2HB, Thermo Scientific, Rochester, NY, USA). All three reagents were manufactured by Thrombinoscope BV, Maastricht, The Netherlands. Eighty microliters of plasma were added to each PPP-reagent well and its corresponding Thrombin Calibrator well.
The plate was then placed in a Fluoroscan Ascent reader (Thermo Labsystems, Shanghai, China) for a 10 minute, 37°C incubation. Following the incubation twenty microliters of the starting reagent, FluCa-kit (TS50.00), was automatically dispensed into each well by the fluorometer. The wavelengths of 390 nm (excitation) and 460 nm (emission) were used to detect the fluorescence intensity. Thrombin generation curves were calculated by a dedicated software program, Thrombinoscope (Thrombinoscope BV, Maastricht, The Netherlands) version: V220.127.116.112. The TGA-CAT setup, instrumentation, software version, method and reagents, were identical for both laboratories. The intra-assay and inter-assay coefficients of variation (CV) for endogenous thrombin potential (ETP) with the TGA-CAT, Malmö, are 2.3% (n=12) and 9.5% (n=5), respectively.
The second thrombin generation test, INNOVANCE ETP, a chromogenic ETP assay (Siemens Healthcare Diagnostics, Marburg, Germany), was performed in Stockholm on a BCS XP system using C-settings according to the manufacturer´s instructions and as previously described. Dilutions of Innovin, 1:555 and actin, 1:20 are mixed in the proportion 1:2 and this mixture is used as an activator. The intra-assay and inter-assay CV for the area under the curve (AUC) with the INNOVANCE ETP method (C-setting) are 4,7% (n=5) and 5,7% (n=4) respectively.
The FVIII:C was determined with a FVIII assay, Coamatic (Chromogenix, Instrumentation Laboratory SpA, Milano, Italy) on a BCS XP Instrument (Siemens Healthcare Diagnostics Inc., Deerfield, IL, USA) in Stockholm. In the test of agreement, the TGA-CAT parameters were normalized by dividing the value obtained when analyzing each patient’s plasma with that obtained when analyzing CryoCheck pooled normal plasma (Precision BioLogic, Dartmouth, Canada).
Statistical analysis. The associations between parameters were evaluated using Spearman´s (non-parametric) rank correlation test. For the statistical presentation and evaluation of the TGA-CAT inter-laboratory variability, Bland-Altman plots, 45-degree lines, and frequency plots were used.
Both methods (ETP and AUC) showed a similar correlation to FVIII:C (r=0.734 and r=0.701). The FVIII:C values were divided according to disease severity (severe, moderate and mild) and the results for FVIII:C to ETP and AUC at the group level are shown in Table 1. Results grouped by severity showed lower associations compared to the total sample.
|Table 1. Correlation coefficient (r) for ETP and AUC to FVIII:C in all patients and grouped by disease severity.|
To determine the precision in terms of severity, FVIII levels were grouped as severe, moderate and mild (laboratory severity) and plotted against ETP values (Figure 1). Substantial overlap in the groups was observed with both assays, indicating that neither of the methods was considerably better in discriminating among categories of disease severity.
|Figure 1. Patients grouped by severity of
hemophilia A and plotted against ETP for both methods, TGA-CAT Malmö
and ETP INNOVANCE (AUC).
Test of Agreement: TGA-CAT Malmö vs. Stockholm. When performing inter-laboratory tests of agreement, a difference of ±10% is considered to be an acceptable level by most laboratories. In most cases, 95% of the observations need to be within the ±10% acceptance level. The variability between the TGA-CAT results performed in Malmö and Stockholm was rather extensive, but after normalization it was reduced. The results for the ETP parameter are presented in Bland-Altman plots, before and after normalization (Figure 2). Before normalization, 29% of the samples were within the ±10% cone of acceptance. Normalization of the data improved results to 41%, still far from the 95% acceptance level. The results for peak, lag time (LT) and time to peak (ttpeak) are inferior to that of the ETP parameter (Table 2).
|Table 2. Level of agreement between Malmö and Stockholm TGA-CAT parameters, before and after normalization.|
Since we do not know the exact TF concentration for the INNOVANCE ETP method, we cannot know if the difference in TF concentration had a part in the lack of correlation between the methods.
To achieve the desired number of samples for study, specimens from PWH in both Belgrade and Stockholm were used. All plasma samples were single centrifuged, thus, the chance of small amounts of platelets remaining in the plasma cannot be excluded. According to a study by Loeffen, et al. TGA-CAT results were only affected by double centrifugation when the TF concentration was 1 pM or lower. Since our TF concentrations for the TGA-CAT method were 1 pM and 0.5 pM we cannot rule out the possibility that results may have been affected by the single centrifugation.
We did not, however, see a correlation between platelet counts and ETP values (results not shown) which indicates that single centrifugation, instead of the recommended double, did not have a significant impact on the results obtained. There are numerous reports describing contact activation as a reason for the poor reproducibility of TGA-CAT results[16,24,25] and it has been proposed that CTI (corn trypsin inhibitor) should be used for blood sampling. CTI was not used in this study, given the report by Spronk, et al. stating that the addition of CTI, preventing the contact activation pathway, can only be motivated when TF concentrations are 0.5 pM or lower.
Further, we investigated the level of agreement when the same TGA method (TGA-CAT) was performed at two centers (Stockholm and Malmö). The inter-laboratory variation was decreased for all four parameters after normalization with pooled normal plasma, where the ETP results showed the highest concordance, 29% without normalization and 41% with normalization (Table 2). Even after normalization more than half of the samples did not reach the level of acceptance. The choice of centrifugation method, blood sampling tubes, and some other pre-analytical factors are of no concern when conducting agreement studies. Of crucial importance is that the characteristics of the plasma are identical for all samples at the start of the analysis. That said, some pre-analytical factors are influential, such as transportation, thawing, resuspension of reagents, pipetting, and time scheme from the end of thawing to start of analysis. In our investigation, thawing was performed identically, 37ºC for 10 minutes. However, we did not have full control of the other factors that may have contributed to the low level of agreement. Interestingly enough, the factor that may have had the greatest impact on the results is one that is out of the control of the lab technician, that is, the analyzing temperature of the measuring equipment. In a report by De Smedt, et al. the importance of pre-heating was shown, leading to a ten-minute 37ºC incubation step before the start of measurement in the latest software version for the method (version: V18.104.22.1682). A post-study service the Fluoroscan Ascent reader in Malmö showed a temperature deviation of almost three degrees below the intended and displayed 37°C. Identical measurements were performed at two other Swedish laboratories by the same service engineer using the same measuring equipment. Measurements were approximately 1ºC above and 1ºC below ours. No temperature data from Stockholm was available, but deviation from the intended assay temperature is one possible reason for our large inter-laboratory variability. These divergent measurements indicate the need for temperature calibration in laboratories participating in multicenter studies.
The choice of using 95% of the observations within ±10% as a quality standard for the whole measurement range of the TGA-CAT method could be argued. In several routine coagulation assays, a wider acceptance range is used for measurements in the outskirts of the methods measuring capacity, with acceptance ranges of up to ±15-20% in its high and/or low measurement ranges. It might be justified to use a similar approach for the TGA-CAT method.
The main study limitation is the relatively small number of samples. That might explain the poor discrimination between the disease severity groups.
To conclude, both methods correlate in an equal manner to FVIII:C in PWH but show a poor correlation with each other. When dividing the study material into disease severity groups, both methods fail to discriminate between them. The inter-center variability for TGA-CAT method showed a low level of agreement.
Earlier studies have shown that through enhanced standardization of the assay and pre-analytical factors, the inter-laboratory variability can be reduced to acceptable levels and therefore open up the possibility of conducting multi-center clinical studies.[13,14] Still, further improvement of standardization is warranted for this method.
Grant SupportJ. P. Antovic has granted unrestricted grant from Baxter and has received lecture honoraria from Stago, Siemens, Sysmex, Roche, Baxter and NovoNordisk. None of the other authors declare any conflict of interest.
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