Reference values for thromboelastometry (ROTEM®) in cynomolgus monkeys (Macaca fascicularis

Thrombosis Research 126 (2010) e294–e297 Contents lists available at ScienceDirect Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s Regular Article Reference values for thromboelastometry (ROTEM®) in cynomolgus monkeys (Macaca fascicularis) Luca Spiezia a, Diana Bertini a, Massimo Boldrin b,c, Claudia Radu a, Cristiana Bulato a, Sabrina Gavasso a, Emanuele Cozzi b,d, Paolo Simioni a,⁎ a Department of Cardiologic, Thoracic, and Vascular Sciences, 2nd Chair of Internal Medicine, University of Padua Medical School, Padua, Italy CORIT (Consorzio per la Ricerca sul Trapianto d'Organi), Italy Veterinary Pathology and Hygiene Institute, Padua, Italy d Direzione Sanitaria, Padua General Hospital, Italy b c a r t i c l e i n f o Article history: Received 22 March 2010 Received in revised form 5 July 2010 Accepted 12 July 2010 Available online 11 August 2010 Keywords: Xenotransplantation Coagulation Primate Thromboelastometry (ROTEM®) Normal coagulation parameters a b s t r a c t Introduction: The imbalance in clotting homeostasis, tending towards hypercoagulation, is recognized as the real barrier to the long-term survival of porcine xenografts in pig-to-primate xenotransplatation. The present study aimed to validate in primate blood the applicability of whole blood rotation thromboelastometry, performed by ROTEM®, which evaluates the characteristics of clot formation by dynamic monitoring. Materials and Methods: ROTEM® (Pentapharm GmbH, Munich, Germany) was used to investigate native coagulation (NATEM®), the intrinsic (INTEM®) and extrinsic (EXTEM®) pathways, the function of fibrinogen (FIBTEM®), and the presence of fibrinolysis in 40 naïve cynomolgus monkeys. Using classic validation approaches, the normal thromboelastographic profile was defined and the influence of haematocrit (Hct,%), platelet count (x109/L), fibrinogen (mg/dl), and factor VIII (FVIII,%) was evaluated. Results: In all four (NATEM®, INTEM®, EXTEM®, FIBTEM®) assays considered, Clotting Time (CT, sec) and Clot Formation Time (CFT, sec) were shorter in primates than humans. Moreover, α-angle (°), Maximum Clot Firmness (MCF, mm), and MaxVel (mm/min) were also higher in primates than humans. No substantial difference was observed for Hct and platelet count between the two species. On the contrary, FVIII was higher in primates than in humans whereas, interestingly enough, fibrinogen levels were lower in monkeys than in humans. Conclusion: ROTEM® depicts a hypercoagulable profile in primates as compared to humans. Taken together these data suggest that, with regard to coagulation, xenotransplantation in cynos may represent a much more difficult situation than xenotransplantation in humans. © 2010 Elsevier Ltd. All rights reserved. Introduction Activation of the clotting cascade, fibrin deposition and microvascular thrombosis are key features of the rejection process that takes place when pig organs are transplanted into primate. Such hypercoagulable state is maintained to be the real barrier to the long-term survival of pig organs transplanted into primate [1,2]. The exact mechanisms by which coagulation activation are induced after xenotransplantation remain uncertain. Moreover, little is known about coagulation processes in primate. Most conventional tests of Abbreviations: WB, whole blood; CT, clotting time; CFT, clot formation time; MCF, maximum clot firmness; MaxVel, maximum velocity; ML, Maximum Lysis; FVIII, factor VIII; Hct, Haematocrit; SD, standard deviation; CV, coefficient of variation. ⁎ Corresponding author. Department of Cardiologic, Thoracic, and Vascular Sciences, 2nd Chair of Internal Medicine, University of Padua Medical School, Via Ospedale 105, 35100 Padua, Italy. Tel.: +39 049 8212667; fax: +39 049 8212661. E-mail address: paolo.simioni@unipd.it (P. Simioni). 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.07.016 blood coagulation explore specific stages of clotting cascade in plasma. This may represent a limitation for the study of coagulation processes, both in human as in primate, in which the interactions between plasma factors and phospholipid surfaces act together with other blood components in clot formation. One of the equipment that takes into account the simultaneous and integrated effects of different components (i.e. plasma factors, platelets, leukocytes, and red blood cells) involved in the dynamic process of clot formation and lysis is ROtation ThrombElastograM analyser (ROTEM®; Pentapharm, Munich, Germany) [3]. This system is based on a disposable measurement cell with a fixed cup in which a pin oscillates continuously from left to right, rotating through an angle of 4.75°. The rotation is detected optically and the graphic representation produced from the measurement describes the viscoelastic changes associated with fibrin polymerization. Thromboelastometry has been validated in several animal species but not in monkeys [4]. The present study aimed to detail the characteristics of the cynomolgus monkey's thromboelastogram, to validate the use of ROTEM® and to L. Spiezia et al. / Thrombosis Research 126 (2010) e294–e297 define the normal range of thromboelastometry profiles in monkeys. Results from cynomolgus monkeys were compared to samples from healthy human volunteers. Also, the potential difference in the reference ranges in males and females animals and the dependence of results on age were investigated. Finally, the influence of the haematocrit, platelet count, fibrinogen, and factor VIII on thromboelastographic profile was considered. We hypothesize a possible role of thromboelastometry in the management of transplanted primates in order to tailor the antithrombotic therapy and, consequently, reduce the rejection process. Moreover, ROTEM® could be applied in the surgical settings to guide blood product administration. Materials and methods Animals and humans All primates were managed in accordance with the Italian Animals Act (law no. 116 of 27/1/1992) and were authorized by a special decree of the Italian Ministry of Health. Animals were fasted overnight but had free access to water. The animals were group-housed in breeding groups in large open-air caged areas maintained at 22 ± 2 °C temperature, and 50 ± 20% humidity. Blood was also obtained from normal healthy human after informed consent. Coagulation studies Venous blood (2 mL from primate and 18 mL from human) was drawn by venipuncture in 3.8% sodium citrate (wt/vol). Whole blood (WB) was processed within 1 to 3 hours from collection. Platelet-poor plasma obtained after centrifugation (2000 g for 15 min.) was aliquoted, snap-frozen, and stored at − 80 °C until use. Thromboelastometry Thromboelastometry was performed from fresh WB, within 2 hours from blood draw, with the ROTEM® coagulation analyzer according to the standard protocols supplied by the manufacturer [5]. Prior to analysis, the samples were stored at room temperature. The sample tubes were gently inverted five times to re-suspend any sedimentation before pipetting the blood. Thromboelastometric tests were stopped at least after 60 min because information about hyperfibrinolysis can be satisfactorily achieved after one hour. The four standard ROTEM® assays named NATEM®, INTEM®, EXTEM®, and FIBTEM® were performed. NATEM® (Non-Activated TEM) was used to assess WB clot formation in the absence of activation of clotting cascade other than the recalcification and spontaneous contact activation. The INTEM® and EXTEM® (ellagic acid and tissue factor activation, respectively) represent assays in which the intrinsic and the extrinsic coagulation pathways are triggered, respectively. Finally, the FIBTEM® (tissue factor activation) assay was used for the assessment of the specific role of fibrinogen in clot formation following inhibition of the platelets by Cytochalasin D [6]. The ROTEM® method defines various parameters: Clotting Time (CT, sec.) the time from the beginning of the coagulation analysis until an increase in amplitude of 2 mm, which reflects the initiation phase of the clotting process. Clot Formation Time (CFT, sec.) the time between an increase in amplitude of the thrombelastogram from 2 to 20 mm. Alfa-angle (°), the tangent to the clotting curve through the 2 mm point. The CFT and alfa-angle reflect measures of the propagation phase of WB clot formation. Maximum Clot Firmness (MCF, mm) is the maximum amplitude reached in thromboelastogram. Maximum velocity (MaxVel, mm/min) the peak of the 1st derivative of the thrombelastographic clotting curve. The MCF correlates with the platelet count and function as well as with the concentration of fibrinogen [7]. Maximum Lysis (ML) represents the maximum fibrinolysis detected during the analysis. It e295 is defined as the ratio of the lowest amplitude after reaching of the MCF and the MCF. “Hypercoagulable profile” was defined as CT and CFT shorter than the healthy controls and/or MCF, MaxVel, or α-angle higher than the healthy controls [8]. Analytic imprecision was evaluated for each thromboelastometric test by repeated analysis of 2-4 of the same 20 samples chosen at random among the all primate's samples considered in the study. Haematocrit, platelet count, fibrinogen, and factor VIII Haematocrit (Hct, %) and platelet count (x109/L) were determined on an automated multiple parameter analyzer [for primate: Celldyn 3500 (Abbott, Rome, Italy); for human: Counter Sysmex XE-2100 (Dasit Spa, Milan, Italy)]. Fibrinogen (mg/dl) and factor VIII (FVIII:C, %) plasma levels were measured on a BCT-Analyser (Dade Behring, Marburg, Germany) as previously reported [9,10]. In particular, fibrinogen was measured with a modified Clauss method using a commercially available kit (Multifibren U, Siemens, Milan, Italy) according to the manufacturer instructions. Statistical analysis ROTEM® data was exported from the instrument database and analysed by EXCEL (Microsoft Corporation Redmond Washington, USA) and a statistic programme (SPSS 14.0; SPSS, Chicago, IL). The sample size calculation was based on pilot observations and the following assumptions: i) expected increase in MCF of ≥3 mm, ii) expected SD of 4.0 mm [3] iii) power = 90%, iv) alpha= 0.05. Based on these assumptions we needed two groups of 38 individuals. Data were expressed as means plus or minus standard deviation (SD). Coefficients of variation (CV) were calculated using the estimate of the pooled variance of differences between sequential readings. The reference interval was calculated as mean ± 2 SD. ROTEM® data was analysed for sex-specific differences, calculating mean and SD of all female and male monkeys separately. Student's t-Test (unpaired, two-tailed) was employed as variance analysis method with p b 0.05 considered statistically significant. For analysis of age dependence, the ROTEM® results were plotted against the age of monkeys and a linear correlation was evaluated (p b 0.05 considered statistically significant). Age and weight dependence was also analysed for males and females animals, separately. The correlation between ROTEM® parameters and Hct, platelet count, FVIII and fibrinogen was assessed using Pearson correlation coefficient (p b 0.05 considered statistically significant). Results Forty 3-10 year-old purpose-bred males (M/F 15/25, weight range 2-10 Kg) cynomolgus monkeys (Macaca fascicularis) from Mauritius (n = 2), China (n = 1) and Philippines (n = 37), consecutively referred to the Veterinary Pathology and Hygiene Institute of Padua from March 2007 to December 2007, and 50 healthy human volunteers (M/F 20/30; age range 20-60 years) were considered in the study. Descriptive statistics and histograms (data not shown) indicated mostly normal distribution of the ROTEM® results. ROTEM® parameters, reference interval and mean ± SD, in primates and humans for all four assays considered in the study (NATEM®, INTEM®, EXTEM®, and FIBTEM®) are summarized in Table 1. CT and CFT in NATEM®, INTEM®, EXTEM®, and FIBTEM®, were statistically significant shorter in primates than in humans (Student's t-Test p value b .05 for CT in EXTEM® assay and b.0001 in all other assays considered). Moreover MCF, alfa-angle, and MaxVel (mean ± SD) were statistically significant higher in monkeys than in humans (Student's t-Test p value b .0001). No statistically significant difference was found as regard ML value between monkeys and humans. The analysis of sex-specific differences in primate showed a mild hypercoagulable state in females than in males only considering e296 L. Spiezia et al. / Thrombosis Research 126 (2010) e294–e297 Table 1 ROTEM® parameters, reference interval and mean ± SD, in Cynomolgus monkeys and humans for all four assays considered in the study (NATEM®, INTEM®, EXTEM®, and FIBTEM®). Student's t-Test p value § b .05 and * b .0001. For abbreviations see test. NATEM CT (sec.) Reference interval Mean ± SD CFT (sec.) Reference interval Mean ± SD MCF (mm) Reference interval Mean ± SD Alfa-angle (°) Reference interval Mean ± SD MaxVel (mm/min) Reference interval Mean ± SD ML (%) Reference interval Mean ± SD INTEM EXTEM FIBTEM Cynomolgus Monkeys Healthy Humans Cynomolgus Monkeys Healthy Humans Cynomolgus Monkeys Healthy Humans Cynomolgus Monkeys Healthy Humans 183 – 627 461-917 100-176 125-229 33-75 40-88 31-71 25-109 405 ± 111 689 ± 114* 138 ± 19 177 ± 26* 53 ± 11 64 ± 12§ 51 ± 10 67 ± 21* 25-197 105-409 26-54 44-124 28-76 49-157 111 ± 43 257 ± 76* 40 ± 7 84 ± 20* 52 ± 12 103 ± 27* - - 51-75 36-60 60-80 49-65 5078 46-66 9-29 4-20 63 ± 6 48 ± 6* 70 ± 5 57 ± 4* 64 ± 7 56 ± 5* 19 ± 5 12 ± 4* 54-86 29-65 78-86 65-81 76-84 60-80 66-82 51-79 70 ± 8 47 ± 9* 82 ± 2 73 ± 4* 80 ± 2 70 ± 5* 74 ± 4 65 ± 7* 0-32 2-10 22-46 9-21 16-36 7-19 8-24 0-16 16 ± 8 6 ± 2* 34 ± 6 15 ± 3* 26 ± 5 13 ± 3* 16 ± 4 8 ± 4* 5-25 8-24 2-18 2-22 1-29 2-30 15 ± 5 16 ± 4 10 ± 4 12 ± 5 15 ± 7 16 ± 7 - - NATEM® assay. In particular CT and CFT were shorter in females than males (CT 399 ± 121 vs 415 ± 96 sec; CFT 106 ± 42 vs 119 ± 43 sec) and MCF, alfa-angle and, MaxVel higher in females than males (MCF 64 ± 6 vs 61 ± 6 mm; alfa-angle 71 ± 7 vs 68 ± 8°; MaxVel 17 ± 8 vs 15 ± 8 mm/min). The differences were not statistically significant (Student's t-Test p value N 0.7, in all tests considered). No statistically significant correlation between age or weight and ROTEM® parameters, in all assays considered, was found (p value 0.63 for age and 0.82 for weight). The repeatability (within-run imprecision) of the ROTEM® results was depending on the individual tests and parameters. MCF and α-angle, in all four tests considered in the study, showed the lowest variability with CV b5%. The CV of CT and CFT, in INTEM® and EXTEM® assays, were mostly in the range between 4-9%. The variation of CT and CFT in the NATEM® was higher than in the INTEM®, EXTEM®, and FIBTEM® with a CV between 7-15% . Hct (mean ± SD) in primates (36 ± 2%) was in the normal range of humans (33-42%) [Fig. 1, Panel A]. In contrast, platelet count and FVIII Fig. 1. Distribution and mean value (black bar) of Hematocrit (Panel A), Platelet count (Panel B), FVIII (Panel C), and Fibrinogen (Panel D) in primate. Gray area represents the normal distribution in human. L. Spiezia et al. / Thrombosis Research 126 (2010) e294–e297 mean plasma levels were higher in primates (350 ± 95 x109/L and 166 ± 24%, respectively) than in humans (221-319 x109/L and 92-156%, respectively) [Fig. 1, Panel B and C]. Interestingly, fibrinogen plasma levels (mean ± SD) were lower in monkeys (151 ± 33 mg/dl) than in humans (218-362 mg/dl) [Fig. 1, Panel D]. No statistically significant difference was found in primates between males and females as for Hct, Platelet count, FVIII, and fibrinogen mean levels. Moreover, no statistically significant correlation between MCF, α-angle, or MaxVel and each of Hct, platelet count, and fibrinogen was found in all assays considered in the study. Finally, an inverse correlation between FVIII plasma levels and CT in INTEM was demonstrated (data not shown). e297 animal models to human health is often questioned because of differences between species. The results of our study suggest the possible use of thromboelastometry for studying the clotting profiles in primate recipients of a xenotransplant. In this regard, preliminary data are very encouraging. Moreover, another interest application of ROTEM® could be in the surgical settings, during xenotransplantation and mainly during liver xenotransplantation. As the matter of fact in humans, ROTEM® has been used for many years as a guide to blood product and drug administration during cardiac [17,18] and hepatic surgery [19,20]. Prospective large studies are needed to better define the potential applications of ROTEM® in the surgical scenario of the xenotransplantation model. Discussion Conflict of interest statement Our study aimed to define the normal range of thromboelastometry profile in monkeys and to compare the WB clotting profile as measured by ROTEM® between primates and humans. In our study ROTEM® analyzer showed good precision, as demonstrated by low intra-assay CVs in examining coagulation profiles in primates. Possible imprecision in study results could have arisen from limitations regarding the laboratory assays used, which are standardised for human blood. In fact, different sensitivity of primate plasma (blood) to ellagic acid (INTEM®) and to tissue factor (EXTEM® and FIBTEM®) is inherent limitations of the assays used. Despite this limitation, the NATEM® test showed relevant differences between species. From a practical point of view, our data could be used as the reference values for Macaca fascicularis. Thromboelastometry showed a hypercoagulable profile in primates as compared to humans. In particular, CT and CFT values were shorter in primates than in humans. Moreover, MCF, alfa-angle, and MaxVel values were higher in monkeys than in healthy humans. No differences were found in ML between cases and controls, but NATEM®, INTEM®, EXTEM®, FIBTEM® assays are not sensitive to minor differences in fibrinolysis. The mechanisms which lead to hypercoagulability detected by ROTEM® in monkeys are still to be clarified. The analysis of sex-, age- or weight-specific features showed no statistically significant difference in different subgroups considered. Therefore specific reference ranges do not seem to be necessary. Only a slight trend towards hypercoagulation thromboelastometry profile in females as compared with males was found. These findings were in agreement with two earlier investigations conducted in humans [11,12] nevertheless other groups have found significant differences between males and females [13]. Higher levels of procoagulant plasma factors in females compared with males are known [14] but in our studies no statistically significant difference in platelet count, FVIII and fibrinogen plasma levels were found. We try to speculate on the possible effects of the main determinants that, in our experience, could influence the thromboelastographic profile in humans and mainly Hct [15], platelet count, FVIII [8] and fibrinogen. No effects seem to be related to Hct; indeed no difference as for this parameter was seen between primates and humans. It is possible to speculate that platelet count and FVIII mean plasma levels, higher in primates than humans, could partly justify the hypercoagulable profile depicted by ROTEM® in monkeys. Interestingly enough, and quite surprisingly, fibrinogen plasma levels were lower in primates than humans. Thus fibrinogen mean plasma levels do not account for the hypercoagulable thromboelastographic profile seen in primates. We excluded that Cytocalasin D does not work properly in monkeys because the differences in MCF between monkeys and humans were also confirmed after addition to blood samples of abiciximab (final concentration 111 μg/ml) both in EXTEM and FIBTEM as suggested by Lang et al. [16] (data not shown). At present, it is not clear which are the factors that may influence the hypercoagulable state as detected by ROTEM® in primate, i.e., unknown inherited thrombophilia factors, elevated microparticles plasma levels, or other. Taken together these findings suggest that, with regard to coagulation, xenotransplantation in cynos may represent a much more difficult situation than xenotransplantation in humans. 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