research paper
Effect of standardization and normalization on imprecision of calibrated automated thrombography: an international multicentre study
Yesim Dargaud,1 Rodger Luddington,1 Elaine Gray,2 Claude Negrier,3 Thomas Lecompte,4 Sirak Petros,5 John Hogwood,2 Jean-Claude Bordet,3 Veronique Regnault,4 Annelie Siegemund5 and Trevor Baglin1 1
Department of Haematology, Addenbrooke’s
NHS Trust, Cambridge, 2Division of Haematology, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK, 3Laboratoire d’Hemostase, Hopital Edouard Herriot, Lyon, 4I NSERM U 734, Universite Henri Poincare, Nancy, France, and 5
Clinical Haemostaseology, University of Leipzig,
Leipzig, Germany
Received 23 April 2007; accepted for publication 11 July 2007 Correspondence: Dr T. Baglin, Department of Haematology, Addenbrooke’s NHS Trust, Cambridge CB2 2QQ, UK. E-mail:
[email protected]
Summary Calibrated automated thrombography (CAT) enables continuous measurement of thrombin generation (TG). Initial clinical studies using the CAT method showed large variability of normal values, indicating the necessity for a standardized CAT protocol. This international study assessed the intra- and inter-assay imprecision of CAT as well as the inter-centre variability of results in five European centres using locally available reagents and conditions (study 1) and a standardized protocol in which results were normalized (study 2). Samples with and without corn trypsin inhibitor from six healthy volunteers, two haemophilia patients and one protein C deficient patient were assayed. Study 1 confirmed that the use of different sources and concentrations of tissue factor (TF) and different phospholipid (PL) mixtures produced large variability in results. The second study demonstrated that, using the same source and concentration of TF, PL and the same test procedure, this variability could be significantly reduced. Normalization of results improved the inter-centre variability. The benefit of contact factor inhibition prior to TG measurement was confirmed. These results demonstrated that standardization of CAT reduces the variability of results to acceptable limits. Standardization and normalization should be considered in future clinical studies which apply TG testing to clinical decision making. Keywords: coagulation, haemostasis, quality control, thrombin.
Thrombin is central to the coagulation process but there is currently no routine laboratory test that can quantitatively measure the thrombin-forming capacity of a plasma sample. Classical clotting tests, such as the activated partial thromboplastin time and prothrombin time assess only time to initiation of clot formation and do not reflect thrombin generation (TG) entirely. Measuring TG by the sub-sampling method was cumbersome and expensive. Over the last 15 years a technique has been developed in which a fluorescent substrate is added to both platelet-poor and platelet-rich plasma samples without defibrination with the course of thrombin formation monitored in real time (Hemker et al, 2002). These technical developments of the TG assay make it potentially applicable to clinical laboratories. Recently, Luddington and Baglin (2004) demonstrated that the clinical
measurement of TG by calibrated automated thrombography (CAT) was affected by contact factor inhibition using corn trypsin inhibitor (CTI). Correlations between the TG test parameters and clinically observed bleeding in patients with haemophilia (Siegemund et al, 2003; Dargaud et al, 2005) and with rare inherited coagulation disorders (Al Dieri et al, 2002) have been published. It has also been shown that CAT is sensitive to hyper-coagulability (Regnault et al, 2004). Measurement of TG is accepted as a research tool but the variety of sources and concentrations of reagents as well as technical constraints limit the potential for clinical use. The objective of the present international multicentre pilot study was to assess the effect of normalization and standardization on the inter-laboratory variation of CAT as
ª 2007 The Authors Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 139, 303–309 doi:10.1111/j.1365-2141.2007.06785.x
Y. Dargaud et al well as intra- and inter-assay imprecision of the test in five centres with proven experience of TG measurement and who have published on the topic of TG using CAT technology.
Materials and methods Blood samples Six healthy adult volunteers (three males and three females between 26 years and 53 years of age), an adult patient with protein C deficiency at 55% and a personal history of venous thromboembolism, one adult haemophilia A [Factor (F)VIII ¼ 7 IU/dl] and one haemophilia B (FIX ¼ 4 IU/dl) patient, routinely treated in the Cambridge Addenbrooke’s Hospital Haematology Department, were also included after obtaining informed consent. Peripheral venous blood was collected into S-Monovette tubes (Sarstedt, Leicester, UK) containing 0Æ106 mol/l trisodium citrate, loaded or not with CTI 1Æ45 lmol/l (final concentration in whole blood). Antecubital venipuncture was realized using 18G needle with a light tourniquet. Following a double centrifugation at 3000 g for 15 min at room temperature, platelet poor plasma (PPP) was collected from the upper half volume of plasma supernatant and rapidly frozen at )80C. The absence of platelets and leucocytes in PPP samples was checked with a Coulter Gen-STM Hematology Analyser (Beckman Coulter Inc., Fullerton, CA, USA). PPP was always prepared within 30 min of venipuncture. To minimize contact activation, polypropylene tubes and pipette tips were used throughout. All samples were prepared in the same preanalytical conditions in the Department of Haematology, Addenbrooke’s NHS Trust, Cambridge, UK. Frozen samples were sent to the four other centres participating in this study (Division of Haematology, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK; Laboratoire d’Hemostase, Hopital Edouard Herriot, Lyon, France; and INSERM U 734, Universite Henri Poincare, Nancy, France; Clinical Hemostaseology, University of Leipzig, Leipzig, Germany).
Design of the study The present study comprised two parts. In the first part, the inter-laboratory variation of the CAT results was assessed using ‘locally available’ reagents in each centre e.g. tissue factor (TF), phospholipids (PL), buffers and thrombin substrate. In the second part, the inter-centre variation, intra- and interassay imprecision of the CAT method were assessed by using a standardized protocol and after normalization of results against the mean of the normal plasma samples.
Calibrated automated measurement of thrombin generation All centres participating in the study measured TG according to the method described by Hemker et al (2002), in 304
a Fluoroscan Ascent fluorometer (Thermolab systems OY, Helsinki, Finland) equipped with a dispenser. Fluorescence intensity was detected at wavelengths 390 nm (excitation filter) and 460 nm (emission filter). Briefly, 80 ll of PPP was dispensed into round-bottomed 96 well-microtitre plates. 20 ll of a mixture containing TF and PL was added to the plasma sample. The starting reagent (20 ll per well) contained fluorogenic substrate and CaCl2. A dedicated software program, Thrombinoscope (Thrombinoscope bv, Maastricht, The Netherlands) enabled the calculation of thrombin activity against the calibrator (Thrombinoscope bv, Maastricht, The Netherlands) and displayed thrombin activity with time.
Study 1 Thrombin generating capacity was assessed in 18 PPP samples (nine with CTI and nine without CTI) using local reagents and conditions. Each sample was measured using five determinations in each single run over 3 d (three independent measurements) and using separate aliquots on each of the 3 d. Each centre specified the details of reagents used in their study, including respective final reagent concentrations (TF, PL vesicles, working buffer, starting solution buffer, thrombin substrate and calibrator), version of the Thrombinoscope software and the number and level of experience of the operators. The molar concentration of Innovin and Thromborel S (Dade Behring) are not declared by the manufacturer. Therefore, each centre determined the concentrations of TF specified for Innovin and Thromborel S using their local method for TF measurement.
Study 2 Thrombin generating capacity was measured by CAT in separate aliquots of the same 18 samples using the supplied TF, PL, substrate and buffers, as noted above e.g. using five determinations in each single run using two wells for the calibrator and five wells for the samples over 3 d. All reagents and buffers required for TG measurement were centrally prepared in the Department of Haematology, Addenbrooke’s NHS Trust, Cambridge, UK and frozen components were shipped to the four other centres participating in this study. Intra- and inter-assay coefficients of variation (CV%) were calculated. Thrombin generation was monitored using only the half microtitre plate for each experiment. Total reading time was 90 min for all experiments and reading frequency was systematically established at every 15 s interval.
CAT reagents used in the standardized protocol Recombinant human TF, Innovin, was obtained from Dade Behring (Marburg, Germany) and used at a final concentration of 1Æ5 pmol/l. TF concentration was determined using
ª 2007 The Authors Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 139, 303–309
897 (205) 2175 (184) 304 (43) 334 (51)
1312 (343)
2629 (204) 409 (87) 521 (122)
Mean N1–N6 (mean ± SD) Protein C: 55% FIX: 4 IU/dl FVIII: 7 IU/dl Reagents Tissue factor (final concentration) Phospholipids (final concentration) Thrombin substrate (final concentration) Thrombinoscope Microtitre plates
ª 2007 The Authors Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 139, 303–309 V3.0.0.25 Greiner U 650101
V3.0.0.25 Greiner U650201
1005 (85) 209 (37) 180 (31)
803 (150)
+CTI
V3.0.0.25 Greiner U 650101
Innovin–Dade Behring (1 pmol/l) PC 60-PS 20-PE 20 mol% (4 lmol/l) Z-Gly-Gly-Arg-AMC Bachem (2Æ5 mmol/l)
1130 (36) 222 (30) 202 (26)
889 (152)
)CTI
Centre 3
N1–N indicate the healthy normal subjects. PC, phosphatidylcholine; FIX, factor IX; FVIII, factor VIII; PE, phosphatidylethanolamine; PS, phosphatidylserine.
Innovin–Dade Behring (5 pmol/l) PC20-PS13-PE45-PA22 mol% (4 lmol/l) Z-Gly-Gly-Arg-AMC Bachem (2Æ5 mmol/l)
3036 (222) 2169 (138) 1764 (173)
2909 (462)
+CTI
Thromborel S-Dade Behring (2Æ5 pmol/l) PC 60-PS 20-PE 20 mol% (4 lmol/l) Z-Gly-Gly-Arg-AMC Bachem (2Æ5 mmol/l)
2963 (260) 2202 (251) 1695 (84)
2954 (535)
)CTI
)CTI
Sample
+CTI
Centre 2
Centre 1
892 (155) 28 (13) 63 (5)
494 (133)
+CTI
V2.106 Greiner U650204
Innovin–Dade Behring (0Æ5 pmol/l) PC 60-PS 20-PE 20 mol% (4 lmol/l) Z-Gly-Gly-Arg-AMC Bachem (2Æ5 mmol/l)
1635 (153) 56 (17) 114 (22)
1194 (325)
)CTI
Centre 4
1546 (181) 1163 (121) 566 (121)
810 (224)
+CTI
V.3.0.0.29 Greiner U650201
Innovin–Dade Behring (0Æ56 nmol/l) Pathromtin SL–Dade Behring diluted at 1:2500 Z-Gly-Gly-Arg-AMC Bachem (1Æ96 mmol/l)
1602 (179) 1163 (118) 668 (81)
768 (194)
)CTI
Centre 5
39 109 99
62
)CTI
51 115 118
83
+CTI
Inter-centre CV%
Table I. Study 1 – comparison of endogenous thrombin potential (ETP) values from each sample with or without corn trypsin inhibitor (CTI) and inter-centre coefficients of variation (CV) values obtained using locally available reagents and protocols. ETP values shown as mean of n ¼ 15 (standard deviation; SD) for each tested sample.
Intra- and Inter-Assay Imprecision of CAT
305
Y. Dargaud et al the Actichrome TF activity assay (American Diagnostica Inc., Greenwich, CT, USA). The PL vesicles used at a final concentration of 4 lmol/l, were obtained from Avanti Polar Lipids (AL, USA) and consisted of 20 mol% phosphatidylserine, 20 mol% phosphatidylethanolamine and 60 mol% phosphatidylcholine and were prepared by an extrusion method based upon that of Falls et al (2000). Briefly, PL were combined and the solvent removed by evaporation at 45C under N2. The lipids were than resuspended in TBS (20 mmol/l Tris, 150 mmol/l NaCl, pH 7Æ4) and extruded 29 times through a 0Æ1 lm polycarbonate filter (Glen Creston Ltd, Stanmore, UK). HEPES-buffered saline contained 20 mmol/l HEPES (Sigma Aldrich, Poole, UK), 140 mmol/l NaCl and 5 mg/ml of bovine serum albumin (BSA) (Sigma Aldrich, Poole, UK), pH 7Æ35. This buffer was stored at )20C until use. A fresh mixture of fluorogenic substrate and CaCl2 was prepared before each experiment. Fluorogenic substrate, Z-Gly-Gly-Arg-AMC, was obtained from Bachem (Bubendorf, Switzerland). The mixture of 2Æ5 mmol/l fluorogenic substrate and 0Æ1 mol/l CaCl2 was prepared using buffer containing 20 mmol/l HEPES and 60 mg/ml of BSA, pH 7Æ35. The Calibrator with the activity of 600 nmol/l human thrombin was obtained from Thrombinoscope BV (Maastricht, The Netherlands). CTI was purchased from Cambridge Bioscience (Cambridge, UK). Polypropylene round-bottomed Greiner microtitre plates, available in each centre, were used.
Data analysis Thrombin generation test results as well as the raw data of experiments were collected for statistical analyses. Statistical analysis was performed using the Graph Pad Instat 3Æ0 software package (San Diego, California, USA). The mean, standard deviation and coefficient of variation (CV %) were calculated for each centre. The inhibitory effect of CTI was evaluated using Wilcoxon test. A P-value of
