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Reference intervals for thromboelastometry with the ROTEM®delta in cats
Research in Veterinary Science 100 (2015) 271–276
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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Reference intervals for thromboelastometry with the ROTEM® delta in cats E. Döderlein, R. Mischke * Small Animal Clinic, University of Veterinary Medicine, Bünteweg 9, D-30559 Hannover, Germany
A R T I C L E
I N F O
Article history: Received 22 February 2014 Accepted 1 March 2015 Keywords: Reference values Viscoelastic technique Haemostasis Precision Feline blood
A B S T R A C T
This study aimed to assess precision of viscoelastic measurements of feline blood using the ROTEM® delta analyser and to establish reference intervals. Intra-assay-variability was evaluated by analysing samples of two cats in quadruplicate. Reference intervals were established based on 55 clinically healthy European shorthair cats including different sexes and age groups. Analyses were performed without activation and after activation with different reagents (kaolin, in-tem, ex-tem). For the majority of parameters, coefficient of variation was <10%. The activating reagent containing tissue factor (ex-tem) produced the shortest clotting times (reference interval: 44.0–98.7 s) and highest maximum lyses. Reference values of many parameters revealed a wide inter-individual variation. Only sporadically, differences between the individual age groups were found. In conclusion, analysis of feline blood using the ROTEM analyser showed acceptable reproducibility. The established reference intervals may be a useful orientation for measurements of feline blood using the ROTEM® delta analyser. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Analysis of global haemostatic function in citrated blood using the viscoelastic technique allows an assessment of different phases of the haemostatic process including the process of clot initiation, formation and stability and subsequent fibrinolysis. The viscoelastic test principle delivers an overall evaluation of cellular and plasmatic components of haemostasis and, in contrast to standard plasma-based coagulation tests such as prothrombin time (PT) and activated partial thromboplastin time (aPTT), provides information on the thrombus stability (Hall et al., 2012; Luddington, 2005; McMichael and Smith, 2011). It seems to be superior to coagulation tests to detect hyper- and hypocoagubility states (Donahue and Otto, 2005; Moldal et al., 2012; Wagg et al., 2009). Two different analysers are currently used in the human and veterinary medicine: the ROTEM® delta analyser (Tem Innovations GmbH, Munich, Germany) and the TEG® 5000 Hemostasis Analyser (Haemoscope, Niles, IL, USA). In both analysers a small blood sample is placed into a preheated cup (37 °C) and a pin is immersed. In the ROTEM device, the pin oscillates with the cup remaining stationary (thromboelastometry, TEM), while in the TEG® 5000 Hemostasis Analyser, the cup rotates relative to the pin (thrombelastography,
* Corresponding author. Small Animal Clinic, University of Veterinary Medicine, Bünteweg 9, D-30559 Hannover, Germany. Tel.: +49 511 953 6200; fax: +49 511 953 6204. E-mail address: [email protected] (R. Mischke). http://dx.doi.org/10.1016/j.rvsc.2015.03.005 0034-5288/© 2015 Elsevier Ltd. All rights reserved.
TEG). As blood begins to clot the viscoelastic changes within the sample restrict increasingly the rotation of the pin. The kinetic of changes of the rotation of the pin (ROTEM) or the transmitted rotation from the cup to the pin (TEG) is detected mechanically and the typical ROTEM/TEG curves and numerical parameters are generated by a computer (Luddington, 2005; McMichael and Smith, 2011). ROTEM/TEG has gained wide use in the human clinical medicine settings including point-of-care test of whole blood for perioperative haemostatic monitoring, optimisation of bloodproduct selection and usage (e.g., cardiac surgery, liver transplantation) (Cammerer et al., 2003; Innerhofer et al., 2004; Kang et al., 1985; Luddington, 2005; Roullet et al., 2010), and monitoring of anticoagulant therapy (Coppell et al., 2006; Lee et al., 1980; Zmuda et al., 2000). During the last years, TEM/TEG also achieved increasing acceptance and application as a diagnostic test in the veterinary medicine (Donahue and Otto, 2005; Kol and Borjesson, 2010; Luddington, 2005; McMichael and Smith, 2011). In cats, studies on the influence of unfractionated and low molecular weight heparin (Alwood et al., 2007), and surgery (Moldal et al., 2012) on viscoelastic technology have been published. In addition, the technique was used for the detection of hypercoagulable states possibly leading to pulmonary thromboembolism (Goggs et al., 2009). In all these studies, the TEG® 5000 Hemostasis Analyser was used. Reference values for TEG variables in cats have been established for this analyser (Alwood et al., 2004, 2007; Banerjee et al., 2011; Hall et al., 2012; Marschner et al., 2010; Montgomery et al., 2008).
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In contrast, in the accessible literature, no reference values for feline blood using the ROTEM® delta analyser could be found. Therefore, the primary objective of the present study was to establish reference values for viscoelastic measurement of feline citrated blood using the ROTEM® delta analyser and to determine a possible influence of age and sex. A secondary aim was to detect the precision of measurements of feline sample material using this device. 2. Materials and methods 2.1. Animals In total, 57 clinically healthy adult European Shorthair cats were used in this study. The health status was defined based on normal clinical examination, unchanged haematological and serum biochemical profiles (including platelet count, haematocrit, alanine aminotransferase and glutamate dehydrogenase activities, and blood plasma concentrations of bilirubin, urea, creatinine, cholesterol, glucose, total protein, albumin, calcium, potassium, sodium, and chloride). In addition, a routine haemostatic profile including PT, aPTT and thrombin time (TT) was performed. Only cats were included, whose test results were within the laboratory-specific reference range. PT was measured with Thromborel® S (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany), aPTT with C.K. Prest® (Diagnostica Stago S.A.S., Asnières sur Seine, France) and TT with test thrombin (final thrombin concentration: 2 IU/ml; Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). Coagulation assays were performed automatically (Amax Destiny Plus, Tcoag Deutschland GmbH, Germany) in accordance with the manufacturer’s instructions. Food was withdrawn 12 hours (h) before blood samples were collected, but animals had free access to water. The majority of cats (in total 44 including 42/55 cats which were used to establish reference intervals and two cats used to assess precision) were owned by the Institute for Parasitology, University of Veterinary Medicine Hannover, where they are retained in groups of 6–10 animals for experimental purposes. The cats of this population originate from a wide variety of sources. The remaining 13 cats were provided by private owners. The 55 cats which were used to establish reference intervals were aged between 6.5 and 152 months with a median of 48 months and their bodyweight ranged from 2.5 to 7.8 kg (median 3.9 kg). The sex distribution was 5 sexually intact males, 18 castrated males, 16 sexually intact females and 16 spayed females. Cats were differentiated into four age groups (1 [6–12 months]: n = 8; 2 [>12–48 months]: n = 18; 3 [>48–96 months]: n = 14; 4 [>96 months]: n = 15) and showed an inhomogeneous distribution of sexes within the groups. The remaining two cats owned by the Institute for Parasitology, which were used to assess the precision of measurements and reproducibility of ROTEM® analysis included a castrated male, aged 59 months and an intact female, aged 58 months. The private owners of the cats were informed about the study and were asked for their agreement of participation. In most cases the sample collection was combined with blood sample collection for routine health checks. The experiment was performed in accordance with the German Animal Welfare law. The study design was approved by the official animal health care officer of the university and was declared to the responsible national institution (Lower Saxony State Office for Consumer Protection and Food Safety, reference number 13A318).
ysis including haematocrit measurement and platelet count) and approx. 1.0 ml of lithium heparinate-anticoagulated blood (for blood chemistry) citrated blood was collected for TEM and coagulation analysis. Two 1.3 ml tubes containing 0.13 ml sodium citrate (Sarstedt AG & CO, Nümbrecht, Germany) were filled up to the graduation mark to achieve a mixing ratio of 9 parts of blood for one part of anticoagulant. In the two cats, whose blood was used for analysis of precision, blood samples were collected in the same way, but on three consecutive times (with intervals of approximately 2 h) during one working day one additional 1.3 ml tube of citrated blood was collected. Blood and anticoagulant were immediately and thoroughly mixed by careful rocking. Citrated blood for TEM analyses was stored at room temperature (20–25 °C) for approx. 15 minutes (min) before the analyses were started. Residual citrated blood was centrifuged two times for 10 min at 16,000 × g and room temperature to prepare platelet free plasma (PFP). The final supernatant which was used for plasmatic coagulation tests was kept in small aliquots (1 x 300 μl and 1 x 200 μl) and stored frozen at −70 °C and thawed in a water bath at 37 °C immediately before analysis. 2.3. Thromboelastometry Thromboelastometric analyses were performed from citrated whole blood using the ROTEM® delta analyser (Tem Innovations GmbH). The tests were conducted in accordance with the manufacturer’s recommendations. Immediately before the measurements which were performed approximately 15 min after the blood sample collection, the sample tubes were again gently rocked to resuspend any possible sedimentation before pipetting the blood. The automated pipette included in the instrument was used. According to the pipetting programme, 20 μl of re-calcification reagent (200 mmol/l calcium chloride solution, star-tem ® , Tem Innovations) and 20 μl of the respective activator were pipetted into the pre-warmed cup. Then, 300 μl of citrated whole blood was added. The analyses were performed after activation with three different reagents and without activation. Activating reagents included kaolin (5 g/l; C.K. Prest), in-tem® (containing ellagic acid as a contact activator; Tem Innovations) and ex-tem® (tissue factor; Tem Innovations); in tests without further activation (non-activated) 20 μl of isotonic sodium chloride solution (0.9%; B. Braun Melsungen AG, Melsungen [NaCl]) was used instead of the activating reagent. The measurement started automatically when blood was added to the cup and also stopped automatically after 2 h. Of the various parameters which are automatically provided by the instrument, the following ROTEM variables were evaluated:
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2.2. Sample collection
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After disinfecting the area of puncture, blood samples were obtained from the vena cephalica antebrachii using sterile disposable needles (0.9 × 40 mm) and only slight pressure to raise the vein. Apart from approximately 0.5 ml of EDTA blood (for haematological anal-
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coagulation time (CT), i.e., the time (in seconds) between activation of coagulation and formation of the first measureable clot (defined as time point when an amplitude of 2 millimetres [mm] is reached) which evaluates the activity of plasma coagulation factors; clot formation time (CFT) is the time (in seconds) needed to increase amplitude from 2 mm to 20 mm which correlates to initial activation of platelets and fibrin formation; alpha angle (α, expressed in degrees [°]) is given by the angle between the centre line and a tangent to the clotting curve through the point where the amplitude reaches 2 mm, which represents the kinetic of fibrin formation and crosslinking of fibrin; amplitude after 30 min (A30) is the amplitude (in mm) measured 30 min after the end of CT; maximum clot firmness (MCF) is the maximum amplitude (in mm) achieved during the test, and correlates with the maximum clot strength and depends on platelet activation, fibrin formation, and the function of FXIII;
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Table 1 Coefficients of variation (in percent) of various parameters of rotational elastometry measured by the ROTEM® delta analyser in 2 healthy cats (mean values, individual values in brackets). Parameter
Coagulation time (CT) Clot formation time (CFT) Alpha angle (α) Amplitude after 30 min (A30) Maximum clot firmness (MCF) Maximum clot elasticity (MCE) Lysis index after 30 min (LI30) Lysis index after 45 min (LI45) Lysis index after 60 min (LI60) Maximum lysis (ML)
•
• •
Activating reagent ex-tem®
in-tem®
kaolin
Non-activated
5.2 (3.0; 7.3) 7.7 (2.8; 12.6) 0.9 (0.0; 1.7) 6.3 (6.4; 6.2) 2.5 (1.4; 3.5) 6.0 (3.9; 8.1) 4.6 (4.7; 4.4) 14.7 (11.5; 17.8) 22.2 (18.1; 26.3) 28.0 (46.0; 32.0)
7.1 (7.7; 6.5) 9.2 (11.7; 6.6) 1.5 (1.0; 2.0) 2.3 (3.4; 1.2) 2.4 (3.4; 1.4) 9.2 (13.4; 5.0) 0.0 (0.0; 0.0) 0.26 (0.0; 0.51) 0.83 (1.07; 0.59) 11.2 (12.2; 10.2)
6.2 (5.5; 6.8) 13.1 (7.9; 18.3) 2.1 (0.7; 3.4) 1.4 (1.1; 1.7) 1.5 (1.1; 1.9) 5.2 (5.0; 5.4) 0.0 (0.0; 0.0) 0.25 (0.0; 0.50) 0.70 (0.54; 0.86) 6.2 (5.0; 7.4)
14.9 (16.6; 13.2) 15.4 (12.1; 18.7) 8.5 (2.2; 14.7) 4.3 (1.7; 6.9) 3.1 (1.3; 4.9) 8.1 (5.7; 10.4) 0.0 (0.0; 0.0) 0.26 (0.51; 0.0) 0.72 (0.61; 0.82) 23.0 (6.3; 42.0)
maximum clot elasticity (MCE) is a calculated value using the following formula: MCE = 100 × MCF/(100 − MCF) and converts the MCF into a value which is proportional to the elasticity; lysis indices 30, 45, 60 (LI30, LI45, LI60), the percentage of amplitude 30, 45, 60 min after the beginning of clot formation; maximum lysis (ML, maximal decrease of amplitude expressed in percent) represents the fibrinolytic activity.
Intra-assay variability was tested by performing quadruplicate measurements for each reagent (ex-tem®, in-tem®, kaolin and NaCl). In each case, a single sample tube was used for measurements with a defined activating reagent and tests were performed at the same time using the four different channels of the device to minimise possible spurious influences of sample material and storage.
2.4. Statistical analysis Data were transferred from the ROTEM device into Microsoft Excel (Microsoft Office 2007, Microsoft Corporation Redmond, USA) and statistical analysis was performed using SPSS 20.0 German (SPSS Inc., Chicago, USA). Coefficients of variation (CVs) were calculated for fourfold measurements for different ROTEM® parameters (CT, CFT, α, A30, MCF, MCE, LI30, LI45, LI60, and ML) as an indicator of precision and reproducibility of ROTEM ® analysis with different activating reagents. First, CVs were calculated for each cat by dividing standard deviation (SD) of the four results of the respective quadruplicate measurements by the mean and multiplying by 100. Finally, the mean of CVs of both cats was calculated. Data for the calculation of reference intervals were tested for normal distribution using the Kolmogorov–Smirnov test and Shapiro– Wilk test. Because part of the data in individual age groups of different parameters did not show standard normal distribution with one or both of these tests, descriptive and comparable statistics were performed with nonparametric tests. Median values were calculated and reference intervals were defined based on 2.5%- and 97.5%quantiles. Comparisons of age groups were performed using Kruskal– Wallis-test and in case of significant differences, post hoc analyses between individual groups with Mann–Whitney U test.
3. Results 3.1. Precision of measurement Coefficients of variation of most of the parameters were less than 10%. In contrast, especially ML showed remarkable imprecision when using ex-tem® and the non-activated setting which were associated with CVs of 28% and 23%, respectively (Table 1). 3.2. Reference values The reference values of all parameters of the ROTEM® delta presented an obvious inter-individual variation (Table 2). ex-tem® produced the shortest CTs and the highest ML. Statistical comparison between different age groups using Kruskal–Wallis test showed only sporadic differences for standard parameters (in-tem®: ML; kaolin and non-activated: CT; nonactivated: CFT) (Fig. 1). These results reflected that cats of age group 1 showed shorter CTs when compared with older animals and that animals of age group 2 showed lower ML in comparison with the other age groups. With the exception of ex-tem®-activated measurements, group differences were detected for different lysis indices (LI30, LI45, and LI60), but absolute differences were very low (e.g., in-tem®: LI 60: median values between 94 and 95). Graphical illustration did not reveal a relevant systematic influence of sex (for examples see Fig. 1). 4. Discussion In the present study, the ROTEM® delta analyser showed high precision for most of parameters indicating that the instrument is wellsuited to measure feline citrated blood using different activating reagents including those provided by the manufacturer. Intraassay CVs for ex-tem® and in-tem® measurements in cats assessed in the present study were similar to those reported in a previous study on human sample material (Theusinger et al., 2010). In the cited study, based on duplicate measurements, for example, CVs for CT were <15% (ex-tem® and in-tem®; own mean values 5.2 and 7.1%); for CFT <4% and <3% (own results 7.7 and 9.2%), for <3% and <6% (own
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Table 2 Reference values (median, reference interval based on 2.5% and 97.5%-quantiles) for thromboelastometry with the ROTEM® delta analyser based on 55 healthy cats. Activator
Statistical parameter*
CT [s]
CFT [s]
α [°]
A30 [mm]
MCF [mm]
MCE [mm]
LI30 [%]
LI45 [%]
LI60 [%]
ML [%]
ex-tem®
Median 2.5% 97.5% Median 2.5% 97.5% Median 2.5% 97.5% Median 2.5% 97.5%
58.0 44.0 98.7 162 107 255 152 120 207 329 181 928
55.0 34.0 92.7 58.0 36.4 213 51.0 34.8 92.0 149 52.7 506
79.0 49.3 83.0 79.0 52.4 83.0 80.0 56.9 84.0 64.0 33.4 79.0
52.0 10.4 76.3 73.0 47.1 82.0 72.0 42.8 82.0 61.0 29.4 77.7
70.0 53.1 77.7 74.0 49.5 82.0 72.0 44.9 83.0 62.0 29.7 77.7
239 113 347 278 99.1 459 259 83.0 488 161 43.1 351
79.0 14.4 100 100 96.7 100 100 94.1 100 100 96.7 100
61.0 1.0 100 98.0 89.1 100 96.0 86.7 100 99.0 88.1 100
46.0 1.0 100 94.0 83.4 100 91.0 81.4 100 95.0 80.5 100
72.0 0.4 100 16.0 1.1 26.3 22.0 6.0 30.3 18.0 2.4 32.3
in-tem®
kaolin
NaCl
* 2.5% = 2.5%-quantile; 97.5% = 97.5%-quantile. For explanations of remaining abbreviations see Table 1. s: seconds; °: degree; mm: millimetre; %: percent.
results 0.9 and 1.5%), and for MCF <3% for ex-tem®, <5% for intem ® (own results 2.5 and 2.4%). It has to be considered that measurements in duplicate may be more susceptible for possible outliers than fourfold measurements which were used in the current study.
Higher CVs were reported from different laboratories which were involved in a human multi-centre study (e.g., 15 and 29%, respectively, for the ex-tem CT and CFT), but most of results were achieved under suboptimal conditions with respect to a within-run precision analysis (Lang et al., 2005). For example, the cited data for
Fig. 1. Influence of age on selected parameters of thromboelastometric analysis with the ROTEM® delta analyser in healthy cats: Comparison between different age groups (1 [6–12 months]: n = 8; 2 [>12–48 months]: n = 18; 3 [>48–96 months]: n = 14; 4 [>96 months]: n = 15) (only parameters are presented, where Kruskal–Wallis test indicated significant differences between age groups): (a) Clotting time (CT, kaolin), (b) CT (non-activated); (c) clot formation time (CFT, non-activated); (d) maximum lysis (ML, in-tem) description of different sexes: a = sexual intact male (□), b = castrated male (■), c = sexual intact female (○), d = spayed female (●).
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measurements using ex-tem® were created based on 15 consecutive measurements of one sample in only one channel of the instrument so that the results should also be effected by storage time-induced changes. Another laboratory included in the cited study performed 12-fold parallel determinations using three samples and also three different instruments. In contrast, we tried carefully to exclude other preanalytical factors (e.g., by use of only one sample tube and parallel measurements on only one instrument). To the best of our knowledge, results of precision analyses for the instrument ROTEM® have not been published for cats so far, but within-run precision analyses in cynomolgus monkeys (Spiezia et al., 2010) and horses (Paltrinieri et al., 2008) also show an adequate to high reproducibility of results. Precision analysis results for cats (Marschner et al., 2010) and dogs (Bauer et al., 2009; Wiinberg et al., 2005) are available for the instrument TEG® 5000 Hemostasis Analyser, where tendentially higher intra-assay CVs were calculated when compared to our study. This discrepancy could indicate that the ROTEM® analyser shows a higher precision than the TEG, which may be due to the fact that the ROTEM® analyser is less prone to vibration as a consequence of a modified technology (the pin oscillates whereas the cup remains stationary). However, comparisons of results between different studies have limitations, because influencing factors other than the instrument may be variable between studies, especially regarding study design, for example, parallel or consecutive measurements, number of repetitions, etc. To the best of our knowledge, a study which directly compares precision of both instruments, which is necessary to confirm this suspicion, seems to be lacking in human and veterinary medicine so far. The differences in repeatability between different parameters including higher precision for α and MCF was also obvious in other studies (Paltrinieri et al., 2008; Spiezia et al., 2010). The low precision of lysis parameters, which is also in accordance with other reports in different species (Spiezia et al., 2010), should be considered when emphasising these parameters. Measurements in duplicate may be recommended in such patients. In general, nonactivated tests showed the highest CVs in many of the parameters. This result was also reported in another study on viscoelastic measurements in cats using a different instrument (Marschner et al., 2010) and reflects the very delicate measurement if performed without using an activating reagent. According to the proposals of the International Federation of Clinical Chemistry (IFCC) the reference values of the present study were calculated based on the 2.5%- and 97.5%-quantiles, because these non-parametric tests provide the most accurate description of the distribution of the data (Kjelgaard-Hansen and Lundorff Jensen, 2010). These reference intervals may serve as a useful orientation also for other veterinary laboratories although ideally all laboratories and/or institutions should create laboratory-specific reference values to consider specific preanalytical (e.g., blood sampling, sample tubes) and analytical influencing factors. Therefore, at least the validity of the reference intervals should be assessed by measurement of a sufficient number of controls. Age has a significant influence on different parameters of haemostasis and fibrinolysis (Attard et al., 2013; Mischke, 1994), and consequently, results of studies in humans revealed a significant influence of age on results of the ROTEM analysis (Oswald et al., 2010; Sucker et al., 2011), whereas, in contrast, no statistically significant correlation between age and ROTEM parameters was found in primates (Spiezia et al., 2010). To the best of the author’s knowledge studies on the influence of age on results of thromboelastometry in cats have not been published so far. The results of the present study on cats indicate an influence of age on selected parameters also in cats. Therefore, age-adjusted reference values may be useful, although it may not be feasible for most laboratories. The current study was unable to demonstrate a remarkable difference between male and female cats, but this was only based on
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graphical illustration. Sex is known to have an impact on several ROTEM variables of coagulation in humans (Theusinger et al., 2010). In addition Spiezia et al. (2010) found a mild (statistically nonsignificant) tendency towards hypercoagulability in female primates when compared to males when performing non-activated/native ROTEM analyses. However a previous study in dogs using the TEG® 5000 Hemostasis Analyser was also unable to find an influence of sex (Bauer et al., 2009). The reference values established in the present study for feline citrated blood using the ROTEM® delta analyser differ from those of previous studies, which evaluated reference values for viscoelastic measurements of feline blood using the TEG® 5000 Hemostasis Analyser (Alwood et al., 2004, 2007; Banerjee et al., 2011; Marschner et al., 2010; Montgomery et al., 2008). Even when focusing only on the non activated tests (to exclude reagent-dependent influences) large differences compared with previous studies cited earlier, but also between the cited studies became obvious. For example, in most cases the present study investigated shorter CT and CFT values and greater MCF and α values. Only Alwood et al. (2004) and Banerjee et al. (2011) published shorter reaction times (R; corresponding to CT) and Alwood et al. (2004) also found shorter K values (corresponding to CFT). The fact that ROTEM® delta showed shorter CT and CFT and greater MCF and α-values for feline blood than the TEG® 5000 Hemostasis Analyser is well in accordance with a previous human study which directly compared both instruments (Nielsen, 2007). According to Nielsen (2007) the most probable reason for differences between devices are different cups and pins used in both systems. Cups and pins of the ROTEM® analyser are composed of a plastic with greater surface charge, resulting in greater contact activation than that associated with the cup and pin of the TEG device. In addition, other technical factors can also markedly affect thromboelastographic data and contribute to differences between studies; for example, differences in activation between plastic sample tubes have been reported (Roche et al., 2006).
5. Conclusions In conclusion, the results of the present study indicate that analyses using the ROTEM® delta analyser deliver reproducible results and are well suited for the evaluation of haemostasis in cats. The established reference intervals may be a useful orientation for measurements of feline citrated blood using the ROTEM® delta analyser. Studies with larger numbers of healthy cats allowing to create sexmatched age groups may be useful to re-evaluate the influence of age and to perform statistical calculations regarding the influence of sex.
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Cammerer, U., Dietrich, W., Rampf, T., Braun, S.L., Richter, J.A., 2003. The predictive value of modified computerized thromboelastography and platelet function analysis for postoperative blood loss in routine cardiac surgery. Anesthesia & Analgesia 96, 51–57. Coppell, J.A., Thalheimer, U., Zambruni, A., Triantos, C.K., Riddel, A.F., Burroughs, A.K., et al., 2006. The effects of unfractionated heparin, low molecular weight heparin and danaparoid on the thromboelastogram (TEG): an in-vitro comparison of standard and heparinase-modified TEGs with conventional coagulation assays. Blood Coagulation & Fibrinolysis 17, 97–104. Donahue, S.M., Otto, C.M., 2005. Thromboelastography: a toll for measuring hypercoagulability, hypocoagulability, and fibrinolysis. Journal of Veterinary Emergency and Critical Care 15, 9–16. Goggs, R., Benigni, L., Fuentes, V.L., Chan, D.L., 2009. Pulmonary thromboembolism. Journal of Veterinary Emergency and Critical Care 19, 30–52. Hall, D.J., Rush, J.E., deLaforcade, A.M., Shaw, S.P., 2012. Kaolin-activated thromboelastography in echocardiographically normal cats. American Journal of Veterinary Research 73, 775–778. Innerhofer, P., Streif, W., Kühbacher, G., Fries, D., 2004. [Monitoring of perioperative dilutional coagulopathy using the ROTEM analyzer: basic principles and clinical examples]. Anaesthesiologie, Intensivmedizin, Notfallmedizin, Schmerztherapie: AINS 39, 739–744 (in German). Kang, Y.G., Martin, D.J., Marquez, J., Lewis, J.H., Bontempo, F.A., Shaw, B.W., et al., 1985. Intraoperative changes in blood coagulation and thrombelastographic monitoring in liver transplantation. Anesthesia & Analgesia 64, 888–896. Kjelgaard-Hansen, M., Lundorff Jensen, A., 2010. Reference intervals. In: Weiss, D.J., Wardrop, K.J. (Eds.), Schalm’s Veterinary Hematology. Wiley-Blackwell, Ames, Iowa, USA, pp. 1034–1038. Kol, A., Borjesson, D.L., 2010. Application of thrombelatography/thromboelastometry to veterinary medicine. Veterinary Clinical Pathology 39, 405–416. Lang, T., Bauters, A., Braun, S.L., Pötzsch, B., von Pape, K.-W., Kolde, H.-J., et al., 2005. Multi-centre investigation on reference ranges for ROTEM thromboelastometry. Blood Coagulation & Fibrinolysis 16, 301–310. Lee, B.Y., Taha, S., Trainor, F.S., Kavner, D., McCann, W.J., 1980. Monitoring heparin therapy with thromboelastography and activated partial thromboplastin time. World Journal of Surgery 4, 323–330. Luddington, R.J., 2005. Thrombelastography/thromboelastometry. Clinical & Laboratory Haematology 27, 81–90. Marschner, C.B., Bjørnvad, C.B., Kristensen, A.T., Wiinberg, B., 2010. Thromboelastography results on citrated whole blood from clinically healthy cats depend on modes of activation. Acta Veterinaria Scandinavica 52, 38–42. McMichael, M.A., Smith, S.A., 2011. Viscoelastic coagulation testing: technology, applications, and limitations. Veterinary Clinical Pathology 40, 140–153. Mischke, R., 1994. [Activity of coagulation factors II, V, VII and X in healthy dogs– dependence on age, sex and breed]. Berliner und Münchener Tierärztliche Wochenschrift 107, 289–294 (in German).
Moldal, E.R., Kirpensteijn, J., Kristensen, A.T., Haga, H.A., Nødtvedt, A., Eriksen, T., 2012. Evaluation of inflammatory and hemostatic surgical stress responses in male cats after castration under general anesthesia with or without local anesthesia. American Journal of Veterinary Research 73, 1824–1831. Montgomery, A., Couto, C.G., Schober, K., Vilar Saavedra, P., Westendorf, N., Iazbik, M.C., 2008. Thromboelastography in healthy cats. Research Abstracts of the 26th Annual ACVIM Forum, San Antonio, Texas, USA, June 4–7, 2008. Journal of Veterinary Internal Medicine 22, 774 (abstract 242). Nielsen, V.G., 2007. A comparison of the thrombelastograph and the ROTEM. Blood Coagulation & Fibrinolysis 18, 247–252. Oswald, E., Stalzer, B., Heitz, E., Weiss, M., Schmugge, M., Strasak, A., et al., 2010. Thromboelastometry (ROTEM) in children: age-related reference ranges and correlations with standard coagulation tests. British Journal of Anaesthesia 105, 827–835. Paltrinieri, S., Meazza, C., Giordano, A., Tunesi, C., 2008. Validation of thromboelastometry in horses. Veterinary Clinical Pathology 37, 277–285. Roche, A.M., James, M.F., Grocott, M.P., Mythen, M.G., 2006. Just scratching the surface: varied coagulation effects of polymer containers on TEG variables. European Journal of Anaesthesiology 23, 45–49. Roullet, S., Pillot, J., Freyburger, G., Biasis, M., Quinart, A., Rault, A., et al., 2010. Rotation thromboelastometry detects thrombocytopenia and hyperfibrinogenaemia during orthotopic liver transplantation. British Journal of Anaesthesiology 104, 422–428. Spiezia, L., Bertini, D., Boldrin, M., Radu, C., Bulato, C., Gavasso, S., et al., 2010. Reference values for thromboelastometry (ROTEM®) in cynomolgus monkeys (Macaca fascicularis). Thrombosis Research 126, e294–e297. Sucker, C., Tharra, K., Litmathe, J., Scharf, R.E., Zotz, R.B., 2011. Rotation thromboelastography (ROTEM) parameters are influenced by age, gender, and oral contraception. Perfusion 26, 334–340. Theusinger, O.M., Nürnberg, J., Asmis, L.M., Seifert, B., Spahn, D.R., 2010. Rotation thromboelastometry (ROTEM®) stability and reproducibility over time. European Journal of Cardio-Thoracic Surgery 37, 677–683. Wagg, C.R., Boysen, S.R., Bédard, C., 2009. Thrombelastography in dogs admitted to an intensive care unit. Veterinary Clinical Pathology 38, 453–461. Wiinberg, B., Lundorff Jensen, A., Rojkjaer, R., Johansson, P., Kjelgaard-Hansen, M., Kristensen, A.T., 2005. Validation of human recombinant tissue factor-activated thromboelastography on citrated whole blood from clinically healthy dogs. Veterinary Clinical Pathology 34, 389–393. Zmuda, K., Neofotistos, D., Ts’ao, C.H., 2000. Effects of unfractionated heparin, low-molecular-weight heparin, and heparinoid on thromboelastographic assay of blood coagulation. American Journal of Clinical Pathology 113, 725–731.
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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Reference intervals for thromboelastometry with the ROTEM® delta in cats E. Döderlein, R. Mischke * Small Animal Clinic, University of Veterinary Medicine, Bünteweg 9, D-30559 Hannover, Germany
A R T I C L E
I N F O
Article history: Received 22 February 2014 Accepted 1 March 2015 Keywords: Reference values Viscoelastic technique Haemostasis Precision Feline blood
A B S T R A C T
This study aimed to assess precision of viscoelastic measurements of feline blood using the ROTEM® delta analyser and to establish reference intervals. Intra-assay-variability was evaluated by analysing samples of two cats in quadruplicate. Reference intervals were established based on 55 clinically healthy European shorthair cats including different sexes and age groups. Analyses were performed without activation and after activation with different reagents (kaolin, in-tem, ex-tem). For the majority of parameters, coefficient of variation was <10%. The activating reagent containing tissue factor (ex-tem) produced the shortest clotting times (reference interval: 44.0–98.7 s) and highest maximum lyses. Reference values of many parameters revealed a wide inter-individual variation. Only sporadically, differences between the individual age groups were found. In conclusion, analysis of feline blood using the ROTEM analyser showed acceptable reproducibility. The established reference intervals may be a useful orientation for measurements of feline blood using the ROTEM® delta analyser. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Analysis of global haemostatic function in citrated blood using the viscoelastic technique allows an assessment of different phases of the haemostatic process including the process of clot initiation, formation and stability and subsequent fibrinolysis. The viscoelastic test principle delivers an overall evaluation of cellular and plasmatic components of haemostasis and, in contrast to standard plasma-based coagulation tests such as prothrombin time (PT) and activated partial thromboplastin time (aPTT), provides information on the thrombus stability (Hall et al., 2012; Luddington, 2005; McMichael and Smith, 2011). It seems to be superior to coagulation tests to detect hyper- and hypocoagubility states (Donahue and Otto, 2005; Moldal et al., 2012; Wagg et al., 2009). Two different analysers are currently used in the human and veterinary medicine: the ROTEM® delta analyser (Tem Innovations GmbH, Munich, Germany) and the TEG® 5000 Hemostasis Analyser (Haemoscope, Niles, IL, USA). In both analysers a small blood sample is placed into a preheated cup (37 °C) and a pin is immersed. In the ROTEM device, the pin oscillates with the cup remaining stationary (thromboelastometry, TEM), while in the TEG® 5000 Hemostasis Analyser, the cup rotates relative to the pin (thrombelastography,
* Corresponding author. Small Animal Clinic, University of Veterinary Medicine, Bünteweg 9, D-30559 Hannover, Germany. Tel.: +49 511 953 6200; fax: +49 511 953 6204. E-mail address: [email protected] (R. Mischke). http://dx.doi.org/10.1016/j.rvsc.2015.03.005 0034-5288/© 2015 Elsevier Ltd. All rights reserved.
TEG). As blood begins to clot the viscoelastic changes within the sample restrict increasingly the rotation of the pin. The kinetic of changes of the rotation of the pin (ROTEM) or the transmitted rotation from the cup to the pin (TEG) is detected mechanically and the typical ROTEM/TEG curves and numerical parameters are generated by a computer (Luddington, 2005; McMichael and Smith, 2011). ROTEM/TEG has gained wide use in the human clinical medicine settings including point-of-care test of whole blood for perioperative haemostatic monitoring, optimisation of bloodproduct selection and usage (e.g., cardiac surgery, liver transplantation) (Cammerer et al., 2003; Innerhofer et al., 2004; Kang et al., 1985; Luddington, 2005; Roullet et al., 2010), and monitoring of anticoagulant therapy (Coppell et al., 2006; Lee et al., 1980; Zmuda et al., 2000). During the last years, TEM/TEG also achieved increasing acceptance and application as a diagnostic test in the veterinary medicine (Donahue and Otto, 2005; Kol and Borjesson, 2010; Luddington, 2005; McMichael and Smith, 2011). In cats, studies on the influence of unfractionated and low molecular weight heparin (Alwood et al., 2007), and surgery (Moldal et al., 2012) on viscoelastic technology have been published. In addition, the technique was used for the detection of hypercoagulable states possibly leading to pulmonary thromboembolism (Goggs et al., 2009). In all these studies, the TEG® 5000 Hemostasis Analyser was used. Reference values for TEG variables in cats have been established for this analyser (Alwood et al., 2004, 2007; Banerjee et al., 2011; Hall et al., 2012; Marschner et al., 2010; Montgomery et al., 2008).
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In contrast, in the accessible literature, no reference values for feline blood using the ROTEM® delta analyser could be found. Therefore, the primary objective of the present study was to establish reference values for viscoelastic measurement of feline citrated blood using the ROTEM® delta analyser and to determine a possible influence of age and sex. A secondary aim was to detect the precision of measurements of feline sample material using this device. 2. Materials and methods 2.1. Animals In total, 57 clinically healthy adult European Shorthair cats were used in this study. The health status was defined based on normal clinical examination, unchanged haematological and serum biochemical profiles (including platelet count, haematocrit, alanine aminotransferase and glutamate dehydrogenase activities, and blood plasma concentrations of bilirubin, urea, creatinine, cholesterol, glucose, total protein, albumin, calcium, potassium, sodium, and chloride). In addition, a routine haemostatic profile including PT, aPTT and thrombin time (TT) was performed. Only cats were included, whose test results were within the laboratory-specific reference range. PT was measured with Thromborel® S (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany), aPTT with C.K. Prest® (Diagnostica Stago S.A.S., Asnières sur Seine, France) and TT with test thrombin (final thrombin concentration: 2 IU/ml; Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). Coagulation assays were performed automatically (Amax Destiny Plus, Tcoag Deutschland GmbH, Germany) in accordance with the manufacturer’s instructions. Food was withdrawn 12 hours (h) before blood samples were collected, but animals had free access to water. The majority of cats (in total 44 including 42/55 cats which were used to establish reference intervals and two cats used to assess precision) were owned by the Institute for Parasitology, University of Veterinary Medicine Hannover, where they are retained in groups of 6–10 animals for experimental purposes. The cats of this population originate from a wide variety of sources. The remaining 13 cats were provided by private owners. The 55 cats which were used to establish reference intervals were aged between 6.5 and 152 months with a median of 48 months and their bodyweight ranged from 2.5 to 7.8 kg (median 3.9 kg). The sex distribution was 5 sexually intact males, 18 castrated males, 16 sexually intact females and 16 spayed females. Cats were differentiated into four age groups (1 [6–12 months]: n = 8; 2 [>12–48 months]: n = 18; 3 [>48–96 months]: n = 14; 4 [>96 months]: n = 15) and showed an inhomogeneous distribution of sexes within the groups. The remaining two cats owned by the Institute for Parasitology, which were used to assess the precision of measurements and reproducibility of ROTEM® analysis included a castrated male, aged 59 months and an intact female, aged 58 months. The private owners of the cats were informed about the study and were asked for their agreement of participation. In most cases the sample collection was combined with blood sample collection for routine health checks. The experiment was performed in accordance with the German Animal Welfare law. The study design was approved by the official animal health care officer of the university and was declared to the responsible national institution (Lower Saxony State Office for Consumer Protection and Food Safety, reference number 13A318).
ysis including haematocrit measurement and platelet count) and approx. 1.0 ml of lithium heparinate-anticoagulated blood (for blood chemistry) citrated blood was collected for TEM and coagulation analysis. Two 1.3 ml tubes containing 0.13 ml sodium citrate (Sarstedt AG & CO, Nümbrecht, Germany) were filled up to the graduation mark to achieve a mixing ratio of 9 parts of blood for one part of anticoagulant. In the two cats, whose blood was used for analysis of precision, blood samples were collected in the same way, but on three consecutive times (with intervals of approximately 2 h) during one working day one additional 1.3 ml tube of citrated blood was collected. Blood and anticoagulant were immediately and thoroughly mixed by careful rocking. Citrated blood for TEM analyses was stored at room temperature (20–25 °C) for approx. 15 minutes (min) before the analyses were started. Residual citrated blood was centrifuged two times for 10 min at 16,000 × g and room temperature to prepare platelet free plasma (PFP). The final supernatant which was used for plasmatic coagulation tests was kept in small aliquots (1 x 300 μl and 1 x 200 μl) and stored frozen at −70 °C and thawed in a water bath at 37 °C immediately before analysis. 2.3. Thromboelastometry Thromboelastometric analyses were performed from citrated whole blood using the ROTEM® delta analyser (Tem Innovations GmbH). The tests were conducted in accordance with the manufacturer’s recommendations. Immediately before the measurements which were performed approximately 15 min after the blood sample collection, the sample tubes were again gently rocked to resuspend any possible sedimentation before pipetting the blood. The automated pipette included in the instrument was used. According to the pipetting programme, 20 μl of re-calcification reagent (200 mmol/l calcium chloride solution, star-tem ® , Tem Innovations) and 20 μl of the respective activator were pipetted into the pre-warmed cup. Then, 300 μl of citrated whole blood was added. The analyses were performed after activation with three different reagents and without activation. Activating reagents included kaolin (5 g/l; C.K. Prest), in-tem® (containing ellagic acid as a contact activator; Tem Innovations) and ex-tem® (tissue factor; Tem Innovations); in tests without further activation (non-activated) 20 μl of isotonic sodium chloride solution (0.9%; B. Braun Melsungen AG, Melsungen [NaCl]) was used instead of the activating reagent. The measurement started automatically when blood was added to the cup and also stopped automatically after 2 h. Of the various parameters which are automatically provided by the instrument, the following ROTEM variables were evaluated:
•
• •
2.2. Sample collection
•
After disinfecting the area of puncture, blood samples were obtained from the vena cephalica antebrachii using sterile disposable needles (0.9 × 40 mm) and only slight pressure to raise the vein. Apart from approximately 0.5 ml of EDTA blood (for haematological anal-
•
coagulation time (CT), i.e., the time (in seconds) between activation of coagulation and formation of the first measureable clot (defined as time point when an amplitude of 2 millimetres [mm] is reached) which evaluates the activity of plasma coagulation factors; clot formation time (CFT) is the time (in seconds) needed to increase amplitude from 2 mm to 20 mm which correlates to initial activation of platelets and fibrin formation; alpha angle (α, expressed in degrees [°]) is given by the angle between the centre line and a tangent to the clotting curve through the point where the amplitude reaches 2 mm, which represents the kinetic of fibrin formation and crosslinking of fibrin; amplitude after 30 min (A30) is the amplitude (in mm) measured 30 min after the end of CT; maximum clot firmness (MCF) is the maximum amplitude (in mm) achieved during the test, and correlates with the maximum clot strength and depends on platelet activation, fibrin formation, and the function of FXIII;
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Table 1 Coefficients of variation (in percent) of various parameters of rotational elastometry measured by the ROTEM® delta analyser in 2 healthy cats (mean values, individual values in brackets). Parameter
Coagulation time (CT) Clot formation time (CFT) Alpha angle (α) Amplitude after 30 min (A30) Maximum clot firmness (MCF) Maximum clot elasticity (MCE) Lysis index after 30 min (LI30) Lysis index after 45 min (LI45) Lysis index after 60 min (LI60) Maximum lysis (ML)
•
• •
Activating reagent ex-tem®
in-tem®
kaolin
Non-activated
5.2 (3.0; 7.3) 7.7 (2.8; 12.6) 0.9 (0.0; 1.7) 6.3 (6.4; 6.2) 2.5 (1.4; 3.5) 6.0 (3.9; 8.1) 4.6 (4.7; 4.4) 14.7 (11.5; 17.8) 22.2 (18.1; 26.3) 28.0 (46.0; 32.0)
7.1 (7.7; 6.5) 9.2 (11.7; 6.6) 1.5 (1.0; 2.0) 2.3 (3.4; 1.2) 2.4 (3.4; 1.4) 9.2 (13.4; 5.0) 0.0 (0.0; 0.0) 0.26 (0.0; 0.51) 0.83 (1.07; 0.59) 11.2 (12.2; 10.2)
6.2 (5.5; 6.8) 13.1 (7.9; 18.3) 2.1 (0.7; 3.4) 1.4 (1.1; 1.7) 1.5 (1.1; 1.9) 5.2 (5.0; 5.4) 0.0 (0.0; 0.0) 0.25 (0.0; 0.50) 0.70 (0.54; 0.86) 6.2 (5.0; 7.4)
14.9 (16.6; 13.2) 15.4 (12.1; 18.7) 8.5 (2.2; 14.7) 4.3 (1.7; 6.9) 3.1 (1.3; 4.9) 8.1 (5.7; 10.4) 0.0 (0.0; 0.0) 0.26 (0.51; 0.0) 0.72 (0.61; 0.82) 23.0 (6.3; 42.0)
maximum clot elasticity (MCE) is a calculated value using the following formula: MCE = 100 × MCF/(100 − MCF) and converts the MCF into a value which is proportional to the elasticity; lysis indices 30, 45, 60 (LI30, LI45, LI60), the percentage of amplitude 30, 45, 60 min after the beginning of clot formation; maximum lysis (ML, maximal decrease of amplitude expressed in percent) represents the fibrinolytic activity.
Intra-assay variability was tested by performing quadruplicate measurements for each reagent (ex-tem®, in-tem®, kaolin and NaCl). In each case, a single sample tube was used for measurements with a defined activating reagent and tests were performed at the same time using the four different channels of the device to minimise possible spurious influences of sample material and storage.
2.4. Statistical analysis Data were transferred from the ROTEM device into Microsoft Excel (Microsoft Office 2007, Microsoft Corporation Redmond, USA) and statistical analysis was performed using SPSS 20.0 German (SPSS Inc., Chicago, USA). Coefficients of variation (CVs) were calculated for fourfold measurements for different ROTEM® parameters (CT, CFT, α, A30, MCF, MCE, LI30, LI45, LI60, and ML) as an indicator of precision and reproducibility of ROTEM ® analysis with different activating reagents. First, CVs were calculated for each cat by dividing standard deviation (SD) of the four results of the respective quadruplicate measurements by the mean and multiplying by 100. Finally, the mean of CVs of both cats was calculated. Data for the calculation of reference intervals were tested for normal distribution using the Kolmogorov–Smirnov test and Shapiro– Wilk test. Because part of the data in individual age groups of different parameters did not show standard normal distribution with one or both of these tests, descriptive and comparable statistics were performed with nonparametric tests. Median values were calculated and reference intervals were defined based on 2.5%- and 97.5%quantiles. Comparisons of age groups were performed using Kruskal– Wallis-test and in case of significant differences, post hoc analyses between individual groups with Mann–Whitney U test.
3. Results 3.1. Precision of measurement Coefficients of variation of most of the parameters were less than 10%. In contrast, especially ML showed remarkable imprecision when using ex-tem® and the non-activated setting which were associated with CVs of 28% and 23%, respectively (Table 1). 3.2. Reference values The reference values of all parameters of the ROTEM® delta presented an obvious inter-individual variation (Table 2). ex-tem® produced the shortest CTs and the highest ML. Statistical comparison between different age groups using Kruskal–Wallis test showed only sporadic differences for standard parameters (in-tem®: ML; kaolin and non-activated: CT; nonactivated: CFT) (Fig. 1). These results reflected that cats of age group 1 showed shorter CTs when compared with older animals and that animals of age group 2 showed lower ML in comparison with the other age groups. With the exception of ex-tem®-activated measurements, group differences were detected for different lysis indices (LI30, LI45, and LI60), but absolute differences were very low (e.g., in-tem®: LI 60: median values between 94 and 95). Graphical illustration did not reveal a relevant systematic influence of sex (for examples see Fig. 1). 4. Discussion In the present study, the ROTEM® delta analyser showed high precision for most of parameters indicating that the instrument is wellsuited to measure feline citrated blood using different activating reagents including those provided by the manufacturer. Intraassay CVs for ex-tem® and in-tem® measurements in cats assessed in the present study were similar to those reported in a previous study on human sample material (Theusinger et al., 2010). In the cited study, based on duplicate measurements, for example, CVs for CT were <15% (ex-tem® and in-tem®; own mean values 5.2 and 7.1%); for CFT <4% and <3% (own results 7.7 and 9.2%), for <3% and <6% (own
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Table 2 Reference values (median, reference interval based on 2.5% and 97.5%-quantiles) for thromboelastometry with the ROTEM® delta analyser based on 55 healthy cats. Activator
Statistical parameter*
CT [s]
CFT [s]
α [°]
A30 [mm]
MCF [mm]
MCE [mm]
LI30 [%]
LI45 [%]
LI60 [%]
ML [%]
ex-tem®
Median 2.5% 97.5% Median 2.5% 97.5% Median 2.5% 97.5% Median 2.5% 97.5%
58.0 44.0 98.7 162 107 255 152 120 207 329 181 928
55.0 34.0 92.7 58.0 36.4 213 51.0 34.8 92.0 149 52.7 506
79.0 49.3 83.0 79.0 52.4 83.0 80.0 56.9 84.0 64.0 33.4 79.0
52.0 10.4 76.3 73.0 47.1 82.0 72.0 42.8 82.0 61.0 29.4 77.7
70.0 53.1 77.7 74.0 49.5 82.0 72.0 44.9 83.0 62.0 29.7 77.7
239 113 347 278 99.1 459 259 83.0 488 161 43.1 351
79.0 14.4 100 100 96.7 100 100 94.1 100 100 96.7 100
61.0 1.0 100 98.0 89.1 100 96.0 86.7 100 99.0 88.1 100
46.0 1.0 100 94.0 83.4 100 91.0 81.4 100 95.0 80.5 100
72.0 0.4 100 16.0 1.1 26.3 22.0 6.0 30.3 18.0 2.4 32.3
in-tem®
kaolin
NaCl
* 2.5% = 2.5%-quantile; 97.5% = 97.5%-quantile. For explanations of remaining abbreviations see Table 1. s: seconds; °: degree; mm: millimetre; %: percent.
results 0.9 and 1.5%), and for MCF <3% for ex-tem®, <5% for intem ® (own results 2.5 and 2.4%). It has to be considered that measurements in duplicate may be more susceptible for possible outliers than fourfold measurements which were used in the current study.
Higher CVs were reported from different laboratories which were involved in a human multi-centre study (e.g., 15 and 29%, respectively, for the ex-tem CT and CFT), but most of results were achieved under suboptimal conditions with respect to a within-run precision analysis (Lang et al., 2005). For example, the cited data for
Fig. 1. Influence of age on selected parameters of thromboelastometric analysis with the ROTEM® delta analyser in healthy cats: Comparison between different age groups (1 [6–12 months]: n = 8; 2 [>12–48 months]: n = 18; 3 [>48–96 months]: n = 14; 4 [>96 months]: n = 15) (only parameters are presented, where Kruskal–Wallis test indicated significant differences between age groups): (a) Clotting time (CT, kaolin), (b) CT (non-activated); (c) clot formation time (CFT, non-activated); (d) maximum lysis (ML, in-tem) description of different sexes: a = sexual intact male (□), b = castrated male (■), c = sexual intact female (○), d = spayed female (●).
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measurements using ex-tem® were created based on 15 consecutive measurements of one sample in only one channel of the instrument so that the results should also be effected by storage time-induced changes. Another laboratory included in the cited study performed 12-fold parallel determinations using three samples and also three different instruments. In contrast, we tried carefully to exclude other preanalytical factors (e.g., by use of only one sample tube and parallel measurements on only one instrument). To the best of our knowledge, results of precision analyses for the instrument ROTEM® have not been published for cats so far, but within-run precision analyses in cynomolgus monkeys (Spiezia et al., 2010) and horses (Paltrinieri et al., 2008) also show an adequate to high reproducibility of results. Precision analysis results for cats (Marschner et al., 2010) and dogs (Bauer et al., 2009; Wiinberg et al., 2005) are available for the instrument TEG® 5000 Hemostasis Analyser, where tendentially higher intra-assay CVs were calculated when compared to our study. This discrepancy could indicate that the ROTEM® analyser shows a higher precision than the TEG, which may be due to the fact that the ROTEM® analyser is less prone to vibration as a consequence of a modified technology (the pin oscillates whereas the cup remains stationary). However, comparisons of results between different studies have limitations, because influencing factors other than the instrument may be variable between studies, especially regarding study design, for example, parallel or consecutive measurements, number of repetitions, etc. To the best of our knowledge, a study which directly compares precision of both instruments, which is necessary to confirm this suspicion, seems to be lacking in human and veterinary medicine so far. The differences in repeatability between different parameters including higher precision for α and MCF was also obvious in other studies (Paltrinieri et al., 2008; Spiezia et al., 2010). The low precision of lysis parameters, which is also in accordance with other reports in different species (Spiezia et al., 2010), should be considered when emphasising these parameters. Measurements in duplicate may be recommended in such patients. In general, nonactivated tests showed the highest CVs in many of the parameters. This result was also reported in another study on viscoelastic measurements in cats using a different instrument (Marschner et al., 2010) and reflects the very delicate measurement if performed without using an activating reagent. According to the proposals of the International Federation of Clinical Chemistry (IFCC) the reference values of the present study were calculated based on the 2.5%- and 97.5%-quantiles, because these non-parametric tests provide the most accurate description of the distribution of the data (Kjelgaard-Hansen and Lundorff Jensen, 2010). These reference intervals may serve as a useful orientation also for other veterinary laboratories although ideally all laboratories and/or institutions should create laboratory-specific reference values to consider specific preanalytical (e.g., blood sampling, sample tubes) and analytical influencing factors. Therefore, at least the validity of the reference intervals should be assessed by measurement of a sufficient number of controls. Age has a significant influence on different parameters of haemostasis and fibrinolysis (Attard et al., 2013; Mischke, 1994), and consequently, results of studies in humans revealed a significant influence of age on results of the ROTEM analysis (Oswald et al., 2010; Sucker et al., 2011), whereas, in contrast, no statistically significant correlation between age and ROTEM parameters was found in primates (Spiezia et al., 2010). To the best of the author’s knowledge studies on the influence of age on results of thromboelastometry in cats have not been published so far. The results of the present study on cats indicate an influence of age on selected parameters also in cats. Therefore, age-adjusted reference values may be useful, although it may not be feasible for most laboratories. The current study was unable to demonstrate a remarkable difference between male and female cats, but this was only based on
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graphical illustration. Sex is known to have an impact on several ROTEM variables of coagulation in humans (Theusinger et al., 2010). In addition Spiezia et al. (2010) found a mild (statistically nonsignificant) tendency towards hypercoagulability in female primates when compared to males when performing non-activated/native ROTEM analyses. However a previous study in dogs using the TEG® 5000 Hemostasis Analyser was also unable to find an influence of sex (Bauer et al., 2009). The reference values established in the present study for feline citrated blood using the ROTEM® delta analyser differ from those of previous studies, which evaluated reference values for viscoelastic measurements of feline blood using the TEG® 5000 Hemostasis Analyser (Alwood et al., 2004, 2007; Banerjee et al., 2011; Marschner et al., 2010; Montgomery et al., 2008). Even when focusing only on the non activated tests (to exclude reagent-dependent influences) large differences compared with previous studies cited earlier, but also between the cited studies became obvious. For example, in most cases the present study investigated shorter CT and CFT values and greater MCF and α values. Only Alwood et al. (2004) and Banerjee et al. (2011) published shorter reaction times (R; corresponding to CT) and Alwood et al. (2004) also found shorter K values (corresponding to CFT). The fact that ROTEM® delta showed shorter CT and CFT and greater MCF and α-values for feline blood than the TEG® 5000 Hemostasis Analyser is well in accordance with a previous human study which directly compared both instruments (Nielsen, 2007). According to Nielsen (2007) the most probable reason for differences between devices are different cups and pins used in both systems. Cups and pins of the ROTEM® analyser are composed of a plastic with greater surface charge, resulting in greater contact activation than that associated with the cup and pin of the TEG device. In addition, other technical factors can also markedly affect thromboelastographic data and contribute to differences between studies; for example, differences in activation between plastic sample tubes have been reported (Roche et al., 2006).
5. Conclusions In conclusion, the results of the present study indicate that analyses using the ROTEM® delta analyser deliver reproducible results and are well suited for the evaluation of haemostasis in cats. The established reference intervals may be a useful orientation for measurements of feline citrated blood using the ROTEM® delta analyser. Studies with larger numbers of healthy cats allowing to create sexmatched age groups may be useful to re-evaluate the influence of age and to perform statistical calculations regarding the influence of sex.
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