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GENERAL ARTICLES

The Effects of Test Temperature and Storage Temperature on Platelet Aggregation: A Whole Blood In Vitro Study

Department of General Anesthesiology and Intensive Care (B), Vienna Medical University, Vienna, Austria

Address correspondence and reprint requests to Sibylle A. Kozek-Langenecker, MD, Department of General Anesthesiology and Intensive Care (B), Vienna Medical University Waehringer Guertel 18-20, 1090-Vienna, Austria. Address e-mail to sibylle.kozek{at}meduniwien.ac.at.

	Abstract

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We systematically evaluated the effects of test temperature and storage temperature on platelet aggregation using flow cytometry and impedance aggregometry. Aliquots of citrated whole blood from 27 healthy adult male volunteers were stored at 37°C and 22°C. Aliquots were subjected to impedance aggregometry in response to collagen, adenosine diphosphate, ristocetin, and arachidonic acid performed at 22°C, 34°C, 37°C, and 40°C. The expression of activated fibrinogen receptor was determined on adenosine diphosphate-activated platelets at 22°C and 37°C by whole blood flow cytometry using PAC-1 for fluorescent staining. Aggregation induced by collagen, ristocetin, and arachidonic acid was not significantly different at the test temperatures of 34°C and 37°C but was significantly impaired at 22°C. In contrast, adenosine diphosphate-induced aggregation was significantly increased at both 34°C and 22°C. Hyperthermia exclusively impaired collagen-induced aggregation. Storage temperature of 22°C exclusively enhanced adenosine diphosphate- and collagen-induced aggregation compared with storage at 37°C. The binding of PAC-1 was enhanced at test temperatures below 37°C. Prewarming the antibody above 22°C significantly decreased binding. Our results suggest that mild hypothermic test conditions have no relevant effect, whereas profound hypothermia induces defects in adhesion, thromboxane generation, and activation. The pathomechanism for the increased response to adenosine diphosphate at mild and profound hypothermia remains unclear. Storage temperature considerably affects the aggregation response to the agonists adenosine diphosphate and collagen but not to arachidonic acid and ristocetin. Flow cytometry using the temperature-labile antibody PAC-1 fails to assess temperature effects on platelet aggregability.

	Introduction

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Severe inadvertent hypothermia (<34°C) correlates with mortality in trauma patients requiring massive transfusion (1). Hypothermia-induced coagulopathy has been hypothesized to result from enzymatic dysfunctions of plasmatic coagulation factors (2,3) and modified platelet activity. Reports on the effect of hypothermia per se on platelet function have been controversial; although some authors reported platelet inhibition at hypothermic conditions (4–8), others observed platelet activation (9–11). The effect of hyperthermia on platelet aggregability remains unclear.

Aside from avoiding inadvertent hypothermia and rewarming the patient, quantitative assessment of the contribution of hypothermia-induced coagulopathy to bleeding diathesis may be critical in this clinical setting. This goal can be achieved by performing a plasmatic coagulation test at the actual core temperature of hypothermic patients instead of the routine test temperature of 37°C after storage at room temperature until analysis (2,12–15). Similarly, platelet aggregometry may not reflect platelet function at clinically relevant degrees of in vivo hypothermia unless performed at the actual hypothermic temperature. Platelet aggregometers are not routinely equipped with temperature adaption. Accordingly, we modified the novel impedance platelet aggregometer Multiplate® (Dynabyte, Munich, Germany) with a heat-exchanging device to systematically evaluate the effect of storage temperature and test temperature on platelet aggregation. Specific biochemical processes of platelet function were investigated by measuring the platelet response to various agonists (collagen, adenosine diphosphate, ristocetin, and arachidonic acid). Because we gained surprising results in pilot experiments, we further investigated the effect of hypothermia in adenosine diphosphate-activated platelets at the cellular level using whole blood flow cytometry that has been recommended as a versatile tool for the analysis of the phenotypic and functional properties of platelets (16).

	Methods

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After IRB approval and informed patient consent, blood from 27 healthy adult male volunteers was investigated in an in vitro study. All participants denied taking medication within the previous 14 days. Blood for platelet aggregometry and flow cytometry was withdrawn into 2 Vacuette^TM tubes (4.5 mL; Greiner, Kremsmünster, Austria) containing 3.8% trisodium citrate (9:1 v/v) from an antecubital vein by venipuncture without stasis using a 21-gauge butterfly needle. The first 3 mL were always discarded.

During storage at 37°C for 15 min before analysis, tubes were moved slowly in order to avoid sedimentation of blood components. Platelet aggregometry was then performed using the new impedance aggregometer Multiplate® (Dynabyte, Munich, Germany). Whole blood aggregometry measures electrical impedance between electrodes immersed in whole blood. Blood is stirred using an electromagnetic stirrer (800 rpm). The attachment of platelet aggregates on the electrodes increase the impedance between them. The impedance change is transformed to arbitrary aggregation units by the system software and plotted against time. In contrast to optical aggregometry, the use of whole blood eliminates the need for centrifugation and tests platelet function under more physiological conditions. Impedance aggregometry was introduced by Cardinal and Flower in 1979 (17). The Multiplate® technique is an improvement of impedance aggregometry using a computer-controlled 5-channel device and disposable test cells with a dual sensor unit (18). By the use of disposable test cells there is no need to clean and ensure the integrity of the electrodes after each test. Platelet aggregation was determined in response to collagen (1.6 µg/mL, adenosine diphosphate (12.8 µM), ristocetin (0.48 mg/mL), and arachidonic acid (0.5 µM) using commercially available test reagents recommended for Multiplate® analysis. Pipetting was performed using an electronic pipette connected to the analyzer.

The commercially available Multiplate® instrument cannot be set to temperatures lower than 35°C. In the present study we used a special modification to determine platelet thermophysiology. Test temperatures of 22°C, 34°C, 37°C, and 40°C were established with an external heat exchanging device. Using this technique the desired temperature was ensured within 1°C.

For assessing the effect of storage temperature on platelet aggregability, one Vacuette^TM tube from each of the 7 healthy male volunteers was stored at room temperature of 22°C for 15 min until analysis, the other tube was incubated at 37°C. All samples were analyzed at the routine test temperature of 37°C.

The rationale for the choice of storage temperatures was the clinical relevance of blood storage at either 22°C or 37°C. The reason for including 34°C as a test temperature was the clinical relevance of mild hypothermic conditions in vivo; the rationale for the lowest test temperature of 22°C was the comparability to previous studies on thermophysiology (5,6,8–10,13).

To evaluate the observed increase in platelet aggregability in response to adenosine diphosphate in further detail, flow cytometric analysis was performed using a modification of the previously described method (19). Whole blood was used as the test milieu because it permits assessment of the concert of coagulation reactions and interaction with platelets and tissue-factor bearing cells. Flow cytometry is a powerful and versatile tool that can be used to yield information on the phenotypic and functional status of platelets by quantitative assessment of the physical and antigenic properties of platelets (16). Platelet activation transforms platelet membrane glycoprotein (GP) IIb-IIIa complexes to a conformational state that is competent for binding fibrinogen (20). To determine the expression of activated GP IIb-IIIa complex, aliquots were incubated with a fluorescein isothiocyanate-conjugated activation-dependent anti-human platelet GP IIb-IIIa immunoglobulin M monoclonal antibody, PAC-1^TM (Becton Dickinson Immunocytometry Systems, San Jose, CA). Citrated whole blood from 9 volunteers was diluted (1:5) in phosphate-buffered saline to inhibit contact between individual platelets and was further divided into 3 aliquots: 2 of them were stored at room temperature of 22°C; the third aliquot was stored at 37°C for 30 min until incubation with adenosine diphosphate (20 µM) for 5 min. Fluorescent staining was performed for 30 min in the dark at the temperatures of 22°C or 37°C using saturating concentrations of PAC-1 stored at 4°C (as recommended by the manufacturer) or using PAC-1 monoclonal antibody preincubated at 37°C for 30 min before staining. Before analysis, these samples were fixed in 1% paraformaldehyde.

In each experiment, cellular autofluorescence was determined, and isotype and compensation control were performed. A gate was set around the platelet population, which was identified by forward and side scatter characteristics. Mean fluorescence intensity of PAC-1 was determined on the basis of this gate. Fluorescence was measured with a FACSCalibur^TM flow cytometer at the routine test temperature of 22°C and analyzed with CellQuestPro^TM software (Becton Dickinson Immunocytometry Systems). Quantum fluorescence microbeads (Calibrite Beads^TM; Becton Dickinson Immunocytometry Systems) were used each day for standardization of instrument settings.

Data were tested for normal distribution using the Kolmogorov-Smirnov-test. The effect of temperature was assessed by using analysis of variance for repeated measures. Post hoc comparisons between controls tested at 37°C and other temperatures were made by a paired Student's t-test. The level of significance was adjusted according to Bonferroni's correction. P < 0.05 was considered statistically significant.

	Results

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Figure 1 shows the effect of test temperature on platelet aggregation induced by various agonists. Data are given as percent of control aggregation determined at 37°C. At 34°C platelet aggregation induced by collagen, ristocetin, and arachidonic acid was not significantly different versus 37°C, but it was significantly impaired at 22°C. Adenosine diphosphate-induced platelet aggregation was significantly increased at hypothermic test temperatures. Hyperthermia exclusively impaired collagen-induced platelet aggregation.

View larger version (27K): [in this window] [in a new window]   Figure 1. Effect of test temperature on platelet aggregation induced by various agonists. At 34°C platelet aggregation induced by collagen, ristocetin, and arachidonic acid was not significantly different versus 37°C, but it was significantly impaired at 22°C. Adenosine diphosphate-induced platelet aggregation was significantly increased at hypothermic test temperatures. Hyperthermia exclusively impaired collagen-induced platelet aggregation. Data are given as percentage of control aggregation determined at 37°C. Values are expressed as mean ± sem of independent experiments (n = 17) performed in duplicate determinations. *P < 0.05 versus 37°C.

Binding of PAC-1 to the platelet GP IIb-IIIa on adenosine diphosphate-stimulated platelets was significantly reduced at the staining temperature of 37°C (mean fluorescence intensity: 67 ± 22 arbitrary units) and when using prewarmed PAC-1 (74 ± 23 arbitrary units), compared with staining at room temperature using refrigerated antibody (137 ± 50 arbitrary units).

The effect of storage temperature on platelet aggregation induced by various agonists is summarized in Table 1. Data are given as absolute values of aggregation. Storage at 22°C had no significant effect on platelet aggregation induced by ristocetin and arachidonic acid when compared with storage at 37°C. In contrast, storage at 22°C significantly enhanced collagen- and adenosine diphosphate-induced aggregation.

	Discussion

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The present experiments demonstrate that mild hypothermia exerts no significant effect on platelet aggregation induced by collagen, ristocetin, and arachidonic acid, whereas profound hypothermia induces defects in adhesion (ristocetin), thromboxane generation (arachidonic acid), and activation using a strong platelet agonist (collagen) (Fig. 1). In agreement with some previous studies, our results imply that mild hypothermia (34°C) induces no platelet-inhibiting effect (3,9). However, below a critical point, platelet defects become obvious: at 33°C Wolberg et al. (4) reported a significant decrease in thrombin- and shear stress-induced aggregation and adhesion of washed platelet by microscopically measuring the size of platelet aggregates and surface coverage of a flow chamber. Wolberg et al. (4) also found a significant decreased platelet prothrombinase activity at hypothermia. Thrombelastography, which evaluates both platelet function and plasmatic enzyme function, showed prolonged reaction and coagulation times and decreased clot formation rates at reduced assay temperatures <34°C (3,12,21), as well as during active cooling of otherwise healthy patients to 32°C after induction of anesthesia but before elective intracranial surgery (22). Thrombelastographic variables indicative for platelet function as well as in vitro bleeding times remained unchanged despite a significant decrease in platelet counts (15,22). Comparable to the insensitivity for thromboxane A₂ reduction and aspirin effects (23), thrombelastography may be unsuited for the detection of hypothermia-dependent platelet abnormalities.

Interestingly, adenosine diphosphate induced a diametrically opposed response with significantly increased platelet aggregation at hypothermic test temperatures in the present study (Fig. 1). This finding is consistent with previous studies in whole blood that have found a maximal response to adenosine diphosphate at room temperatures (9,10,13) but is in contrast to another study that found inhibited adenosine diphosphate-induced aggregation in platelet-rich plasma (7). The underlying mechanism for the observed inhibited collagen-, ristocetin-, and arachidonic acid-induced aggregation and the augmented adenosine diphosphate-induced aggregation in hypothermic test conditions remains unanswered. The increased response to adenosine diphosphate may be a compensatory mechanism to promote plug formation in hypothermic-injured individuals. Arachidonic acid acts as a substrate for thromboxane A₂ synthesis that causes activation and conformational change of GP IIb-IIIa and platelet secretion. The decrease in arachidonic acid-induced platelet aggregation in profound hypothermia suggests impairment of thromboxane A₂ synthesis and function confirming earlier studies (5,6,8). Ristocetin induces aggregation predominantly via the interaction of GP Ib and von Willebrand factor. The decrease in ristocetin-induced platelet aggregation in profound hypothermia suggests impairment of von Willebrand factor function and/or reduced availability of the constituently expressed GP Ib, confirming earlier studies (8). Collagen causes thromboxane A₂ synthesis, secretion from storage granules, and aggregation predominantly secondary to the released products, adenosine diphosphate and thromboxane A₂. The decrease in collagen-induced platelet aggregation in profound hypothermia again suggests impairment of secretion and thromboxane A₂ synthesis and/or function. Adenosine diphosphate causes shape change, activation and conformational change of GP IIb-IIIa, and platelet secretion.

Increased activation of GP IIb-IIIa assessed by increased PAC-1 binding has been hypothesized as the mechanism of adenosine diphosphate-induced platelet hyperreactivity by Faraday and Rosenfeld (9). Although the monoclonal antibody PAC-1 has been proven as a reliable marker for platelet aggregability in previous studies (8,20), our results demonstrate limitations of flow cytometry using PAC-1 in assessing the effect of temperature on platelet reactivity. Although binding of PAC-1 to adenosine diphosphate-stimulated platelets was enhanced at hypothermic conditions confirming the platelet aggregation experiments and previous reports (9), prewarming the antibody alone (without platelets) to 37°C also decreased the binding. Thus, binding kinetics of PAC-1 are temperature-dependent. Moreover, it appears unlikely to explain adenosine diphosphate-induced platelet hyperreacitivty in hypothermia by increased GP IIb-IIIa availability because this receptor is also involved in arachidonic acid- and collagen-induced platelet activation. Increased adenosine diphosphate-induced platelet aggregation may rather indicate differentiated activation of intracellular signal transduction pathways exclusively used in adenosine diphosphate-stimulation. Together, the present experiments may suggest that hypothermia decreases the intracellular enzymatic function of proteinkinase C and phospholipase A₂, which are required for thromboxane A₂- and collagen-induced platelet activation, whereas adenosine diphosphate-dependent proteinkinase A and adenylate cyclase remain intact or stimulated. Enzyme activities of humoral coagulation factors have consistently been found to be reduced in profound hypothermia (2,4,24).

The effect of the test temperature on platelet aggregation depends not only on the platelet agonist used but also on the blood storage condition until platelet analysis. The present experiments on the storage of blood at 22°C with consecutive analysis at 37°C indicate a reversible nature of hypothermia-induced platelet inhibition in ristocetin-, arachidonic acid-, and collagen-activated samples (Table 1), with an overshooting response in the latter. Also the adenosine diphosphate-induced platelet hyperreactivity at hypothermia was partially reversible (Table 1). Previous studies showed that platelet abnormalities were reversible on warming (5,8,9). It is generally accepted that blood samples should be processed within approximately 15 minutes of drawing blood and samples should be stored at room temperature in the interim (16). It should be considered that platelets may have undergone functional changes during storage conditions depending on the concentration of platelet agonists present in the sample. We recommend standardizing storage temperature and storage times to increase the intra-laboratory reproducibility of platelet aggregometry. Although the response to adenosine diphosphate and collagen varies considerably with storage temperature, arachidonic acid and ristocetin can be considered as storage temperature-robust agonists (Table 1). If adenosine diphosphate and collagen are used as platelet agonist, the aggregation response of rewarmed platelets are distinguishable from those maintained at normothermic temperatures (8,9), leading to overestimated platelet function when the sample has been stored at the non-physiological temperature of 22°C when compared with 37°C. Storage in the cold cannot be recommended because it has been found to dramatically deteriorate platelet function (11).

Hyperthermia exclusively impaired collagen-induced platelet aggregation (Fig. 1). The pathophysiological consequence and clinical relevance of inhibition of this selective platelet functional in fever remains to be determined.

	Footnotes

 
Supported, in part, by the Department of General Anesthesiology and Intensive Care (B), Vienna Medical University, Vienna, Austria.

Accepted for Publication November 23, 2005.

	References

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