Original Research
Effect of Different Storage Conditions on Platelet Aggregation in Horse

https://doi.org/10.1016/j.jevs.2010.06.003Get rights and content

Abstract

Platelet aggregation is the most important event in the hemostatic process, but no information is available on the stability of samples for aggregation testing in horses. The aim of the present study was to evaluate the effect of different storage conditions on platelet aggregation in horses. The study was carried out on 58 healthy horses of varying breed and gender, ranging in age from 4 to 12 years. Citrated blood samples were collected from all the subjects by means of jugular venipuncture and were used for platelet aggregation measurements. Platelet-rich and platelet-poor plasma samples were prepared by centrifugation and divided into six different aliquots to assess the maximum degree of platelet aggregation and the initial velocity of platelet aggregation at the final concentrations of 1 and 0.5 μM of the aggregating agent. The first aliquot was analyzed within 1 hour after collection at room temperature (22°C), the second 6 hours after collection at 22°C, the third and fourth were refrigerated at 8°C for 6 and 24 hours, respectively, and the fifth and sixth were frozen at −20°C for 24 and 48 hours, respectively. With the help of an aggregometer, platelet responses were quantified, and one-way repeated measures analysis of variance was used to determine significant differences. Probability values of <.05 were considered to be statistically significant. Analysis of variance showed statistically significant differences on maximum degree of platelet aggregation and the initial velocity of platelet aggregation using adenosine diphosphate at final concentrations of 1 and 0.5 μM. The results of this study suggest that the storage of equine plasma for more than 6 hours at room temperature and at 8°C has a significant effect on platelet aggregation, and that the storage of plasma for 24 and 48 hours at −20°C alters platelet aggregation. In conclusion, storage conditions had a statistically significant effect on the parameters of platelet aggregation directly correlated to temperature.

Introduction

The two main components of the thrombotic process are platelet aggregation and coagulation. In particular, platelet aggregation is a crucial step in the clot-forming process that involves cell-cell and cell-substrate adhesion, and cell signaling and response, all of which occur within the moving blood.1 The role of platelets in hemostasis and arterial thrombosis involves their adherence to sites of vessel injury or ruptured atherosclerotic plaques, aggregation to form hemostatic plugs or thrombi, and acceleration of the coagulation cascade leading to the formation of thrombin.2 This phenomenon, most accurately described as platelet cohesion although more commonly referred to as platelet aggregation, was quickly identified as the most important event in hemostatic plug formation.3, 4 Blood platelets can be considered as a component of the hemostatic system whose response to an agonist can be altered by compounds formed by or stored within platelets.5 A variety of substances are significant mediators in hemostasis and thrombosis, and they promote platelet aggregation in vitro, including adenosine diphosphate (ADP), adrenaline, collagen, and arachidonic acid.6, 7, 8 It has been well established that equine platelets aggregate in response to platelet-activating factor and ADP,9 but the potential effect of pre-analytical factors, such as storage temperature and time, on the formation of platelet aggregates (platelet clumping) is largely elusive in horses. The effect of anticoagulant, temperature of storage, and blood storage time on platelet response has been evaluated in healthy and ill dogs10, 11 and in human beings.12, 13, 14, 15, 16, 17 The loss of hemostatic function with storage at 4°C for 24 hours or longer has been well documented by autologous radiolabeled studies as well as by studies performed on thrombocytopenic patients.18, 19, 20 In many clinical conditions, alterations of platelet function occur in animal diseases such as epistaxis, hematuria, and hemorrhagic diarrhea.21 In equine medicine, verminous arteritis, systemic thromboembolism associated with endotoxemia, acute laminitis, ischemic gastrointestinal disorders,22 and several drugs used, such as acetylsalicylic acid, paracetamol, dextran,23, 24, 25, 26 are capable of modifying equine platelet aggregation. Therefore, studies on platelet function in horse, as previously reported in human beings and dogs, are essential for understanding the process of hemostasis. The platelet aggregation tests allow the assessment of primary hemostasis,27 and previously, it was used in horses to evaluate the effect of physical exercise on the daily rhythm of platelet aggregation.28

The aim of the present study was to examine the effects of short-term incubation, after storage for 6 hours at room temperature (RT), and after 6 and 24 hours storage at 8°C, and the effects of freezing at −20°C for 24 and 48 hours on platelet aggregatory responses in comparison with freshly isolated platelets.

Section snippets

Materials and Methods

Blood samples were obtained from 58 healthy horses of varying breeds and gender, ranging in age from 4 to 12 years, with a mean body weight of 480 ± 50 kg. Before starting, the study horses were subjected to a complete clinical examination, including change in hemostatic mechanisms, rectal temperature, respiratory and heart rates, as well as packed cell volume (reference range, 32%–53%) and platelet count (reference range, 120–400 K/μL), and all were considered within normal limits. Only

Results

All hematological values and clotting parameters obtained from 58 healthy horses were found to be within the normal physiological range for horses29 (Table 1).

Table 2 shows the average values of the maximum degree of aggregation and the slope of platelet aggregation, expressed in their conventional units of measurement, with SD and statistical significances, obtained in the different experimental conditions. Average values of the percentage of platelet aggregation and the slope of platelet

Discussion

The results of this study suggest that the storage of equine plasma for more than 6 hours after collection at RT and at 8°C has a significant effect on platelet aggregation, and that the storage of plasma for 24 and 48 hours at −20°C alters platelet aggregation.

Many clinicians use frozen plasma for identification of disease states or other characterization of patient health. However, there is very little information available on the effect of freezing on plasma samples. Until recently, most

References (34)

  • R.M. Clemmons et al.

    Acquisition and aggregation of canine blood platelets: basic mechanisms of function and differences because of breed origin

    Am J Vet Res

    (1984)
  • W. Siess

    Molecular mechanisms of platelet activation

    Physiol Rev

    (1989)
  • G.E. Jarvis et al.

    ADP can induce aggregation of human platelets via both P2Y1 and P2T receptors

    Br J Pharmacol

    (2000)
  • K.J. Rickards et al.

    Differential effects of phosphodiesterase inhibitors on platelet activating factor (PAF)- and adenosine diphosphate (ADP)- induced equine platelet aggregation

    J Vet Pharmacol Ther

    (2003)
  • M.E. Mylonakis et al.

    Effect of anticoagulant and storage conditions on platelet size and clumping in healthy dogs

    J Vet Diagn Invest

    (2008)
  • N.A. Alarayyid et al.

    An examination of some factors which influence the stability of in vitro platelet responses

    Platelets

    (1994)
  • C.H. Ho et al.

    The influence of time of storage, temperature of storage, platelet number in platelet-rich plasma, packed cell, mean platelet volume, hemoglobin concentration, age, and sex on platelet aggregation test

    Ann Hematol

    (1995)
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