Over the last years, the sheep became one of the most favored species to evaluate and characterize the performance of cardiovascular prostheses1–4 and to develop and train surgical and interventional implantation techniques used or intended for use in man.2,4–6 Apart from its advantageous anatomical dimensions, another notable feature of the ovine organism is that it allows for the evaluation of tissue calcification and thus the conduction of hemo- and biocompatibility studies.1,3,7–9 In a majority of these experiments, blood samples are analyzed with the intention to monitor the surgical procedure itself, to manage an anticoagulative drug regime, or to assess hemo- and biocompatibility of cardiovascular prostheses. Although blood values are essential for the interpretation of data obtained from those studies, manuscripts reporting on data regarding “normal” ovine blood values are rare and often based on older analytical methods. Thus, the interpretation of data obtained in the setting of today’s ovine experiments often remains vague.
The aim of this study was to establish a reference list of various ovine parameters relevant for blood coagulation obtained by modern, widely used and thus, immediately available analytical methods.
Materials and Methods
The study was conducted by evaluating a cohort of 47 ewes (Deutsches Schwarzkopf-Fleischschaf). All animals were 6 months old as determined by history and dentition (body weight, 40–50 kg) and were screened for acute and chronic diseases by veterinarians before evaluation. Housed in groups in an indoor facility for at least 2 weeks, they were fed pellets, hay, and water ad libitum. Animals were treated according to the German animal protection law (Deutsches Tierschutzgesetz), and the study protocol was approved by the local authorities (Niedersaechsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit); all sheep received care in compliance with the Guide for the Care and Use of Laboratory Animals.10
Blood samples of all 47 sheep were analyzed for the following parameters: complete blood count—platelet, erythrocyte, and leukocyte cell counts (including subsets neutrophils, lymphocytes, monocytes, eosinphils, and basophils), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and MCH concentration (MCHC); global tests of coagulation—prothrombin time (Quick’s time), activated partial thromboplastin time (aPTT); and coagulation factors—fibrinogen (FB), antithrombin (AT), and von Willebrand Factor (vWF).
Blood samples were taken from the cephalic or the saphenous vein with sterile disposable cannulas (1.1 × 30 mm2). Graduated plastic Monovettes (Sarstedt, Germany) prefilled with ethylene-diaminetetraacetic acid and sodium citrate solution (0.106 mmol/L sodium, citrate:blood = 1:9) were used. Immediately after withdrawal, tubes were moved carefully, to assure complete mixing of blood and anticoagulant, and analyzed.
Cells and Cellular Components. Cell counts were determined with a blood cell counter model H1E (Bayer Diagnostics, Munich, Germany).
Global Tests of Coagulation. The prothrombin time (= thromboplastin time; Quick’s test) was determined after addition of prewarmed thromboplastin reagent (Thromborel S, from human placenta, Siemens Health care GmbH, Erlangen, Germany). The time to clot formation was detected photo-optically and was converted into percent values according to a diluted human standard plasma.
The aPTT was determined using a kaolin-based commercial reagent (Diagnostica Stago, Mannheim, Germany)11 which was preincubated with plasma samples for 180 seconds. Clotting was started with 25 mmol/L CaCl2, and fibrin formation was detected photo-optically.
Coagulation Factors. FB was determined using the method of CLAUSS (FB; Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany). As standard reagent, FB reference (Cat. No. 886-10) lyophilized citrated human plasma-containing buffer and preservative was used.
AT activity was determined using a chromogenic substrate method (COAMATIC LR AT, Chromogenix, Mailand, Italy).12,13 A lyophilized preparation of human normal plasma (AT activity of 100%; preparation of a dilution series), calibrated against an international standard, was used for quality control.
vWF antigen was determined using a commercially available latex particle-based test reagent (STA LIATEST vWF reagent; Diagnostica Stago, Mannheim, Germany). For calibration, STA vWF calibrator was used. Lot-specific values for the calibrator were determined against the international vWF standard (NIBSC 91/666).
All coagulation tests were conducted on automated systems according to the manufacturer’s instructions: Amax 400plus (FB, AT, Quick’s test, and aPTT; Trinity Biotech GmbH, Germany) or Hitachi 911 (vWF; Roche Diagnostics GmbH, Mannheim, Germany).
To assess the precision of the analytical methods used here, blood samples from three sheep underwent serial measurements (n = 10) to determine the intra-assay coefficient of variation; stored samples from one sheep were analyzed on 6 consecutive days to determined the interassay coefficient of variation.
Statistical analyses were performed using SPSS (Statistical Package for Social Sciences) for Mac, version 17.0 (SPSS Inc, Chicago, IL). Continuous data (all blood values) underwent exploratory analysis for mean, standard deviation (SD), and the 95% confidence interval. Reference ranges were defined and calculated on the basis of 2.5th and 97.5th percentiles.
Table 1 provides reference ranges for sheep (2.5th and 97.5th percentiles) compared to human reference values determined at the Department for Hematology and Hemostasis in our institution (Hannover Medical School, Hannover, Germany). Analysis of interassay and intra-assay coefficients of variation revealed adequate analytical precision as expected from analysis of human samples (Table 2).
Today, the sheep is an accepted and widely used animal model to evaluate and characterize cardiovascular prostheses. However, because of the discrepancy between the (increasing) number of those experiments and only scanty available information regarding “normal” ranges of ovine blood values, the aim of our current study was to establish a reference list of various ovine parameters relevant for blood coagulation. Here, we exclusively focused on analytical methods that are widely used and, thus, readily available in most (human) hematology laboratories. All reference ranges were established against human, commercially available standards to facilitate adaptation for routine practice.
Comparing our ovine reference ranges to human reference ranges that were established in the same laboratory with the same reagents and equipment, we found some remarkable differences between. Higher values were observed for ovine neutrophils (21,000–26,800/ml vs. 1,200–8,000/ml), lymphocytes (68,000–74,500/ml vs. 1,500–4,000/ml), basophils (1,500–2,400/ml vs. <100/ml), eosinophils (2,300–3,800/ml vs. <500/ml), and platelets (327,200–550,700/ml vs. 150,000–450,000/ml). Lower values were observed for ovine Hb (9.3–10.4 g/dl vs. 13.5–17.5 g/dl), HCT (26.0%–29.0% vs. 41.5%–50.4%), MCV (27.8–29.3 fl vs. 80–100 fl), MCH (9.9–10.5 pg vs. 28–32 pg), AT (65.2%–73.3% vs. 70.0%–120.0%), and Quick’s test (51.9%–57.6% vs. 70.0%–130.0%), respectively.
Over the past 40–50years, only a few articles have been published that focused on the evaluation of cellular and noncellular components of the ovine blood. Although most studies comprised only small animal groups (and other sheep races) and considered only a few of the parameters analyzed here, the resulting data (mainly expressed as mean ± SD) are similar.14–21 This finding supports the validity and transferability of data across sheep races and highlight some important interspecies differences between sheep and man. Furthermore, they underscore the necessity of species-specific reference values for the interpretation of hematological and hemostaseological data obtained in ovine experimental settings.
All analyses evaluated in this study were restricted to ovine blood. Thus, a direct biometric comparison between ovine and human blood reference values was not possible. However, researchers will be able to assess their findings more reliably with the help of the ovine reference list established here.
As far as we know, this is the first study reporting a range of reference values of blood counts and coagulation parameters in sheep. All analyses were conducted with modern, widely used and available methods. In view of the frequent use of sheep as experimental models and the remarkable differences between sheep and man observed here, these data contribute to the scientific basis of preclinical cardiovascular research.
This work was supported by grants of the Else Kröner-Fresenius Stiftung, Germany, and the German Research Foundation (DFG), Germany.
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