Annals of Clinical & Laboratory Science 33:79-85 (2003)
© 2003 Association of Clinical Scientists
Influence of Storage Temperature on the Responsiveness of Human Platelets to Agonists
Jong Weon Choi and
Soo Hwan Pai
Department of Clinical Pathology, College of Medicine, Inha University, Inchon, Korea
Address correspondence to Soo Hwan Pai, M.D., Department of Clinical Pathology, Inha University Hospital, 7-206, 3-ga, Shinheung-dong, Jung-gu, Inchon 400-103, Republic of Korea; tel 82 32 890 2502; fax 82 32 890 2529; e-mail shpaimd{at}inha.ac.kr.
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Abstract
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To investigate the effects of storage temperature on the responsiveness to agonists of human platelets prepared from stored blood, we measured the aggregability and acid-base status of platelets from 96 healthy subjects before and after storage of whole blood at 4°C and room temperature (RT) up to 48 hr. After 24 hr storage at 4°C, there were no significant differences in agonist-induced platelet aggregability, compared to fresh specimens. When blood was kept at RT for 24 hr, all of the platelet samples showed non-responsiveness (< 20% aggregability) to epinephrine and 70% (67/96) revealed impaired responsiveness (20 to 60% aggregability) to adenosine diphosphate (ADP); there were no samples that showed impaired-or non-responsiveness to collagen or ristocetin. Among the 67 samples that showed impaired responsiveness to ADP after RT storage, 62 (93%) exhibited the loss of a secondary wave of aggregation in response to ADP. After storage of blood at RT for 48 hr (pH 6.81 ± 0.06), mean values of maximal platelet aggregability to epinephrine, ADP, collagen, and ristocetin were 8%, 16%, 19%, and 70%, which were significantly lower than the corresponding mean values after storage of blood at 4°C for 48 hr (pH 7.04 ± 0.04) (ie, 66%, 69%, 102%, and 91%, p <0.01). In summary, refrigerated storage of human blood improves the stability of platelet responsiveness to agonists. Storage at RT causes platelet non-responsiveness to epinephrine and disturbs the release reaction of endogenous ADP.
(received 27 July 2002; accepted 19 September 2002)
Keywords: platelet aggregability, blood storage, epinephrine, ADP, collagen, ristocetin
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Introduction
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Transport of blood specimens to central laboratories for testing may be delayed after venipuncture. The delay in testing results in suboptimal specimen quality. Refrigerated storage of blood has been reported to improve the stability of most test results [1–3]; however, the viability and function of concentrated platelets stored for 72 hr are optimally preserved at a storage temperature of 22°C rather than 4°C [4]. The major objections to platelets stored at 4°C are their shortened life span after reinfusion, and their marked decrease in survival after only 18 hr of storage [5]. Platelets stored at 4°C are associated with an irreversible disk-to-sphere transformation. The loss of shape in platelets stored at 4°C may be a result of microtubule disassembly, which may also contribute to the decreased survival of platelets stored at 4°C [6]. On the other hand, a problem of storing platelets at 22°C is the decrease of pH. Units of platelet concentrates stored at 22°C show decreased pH from the initial value [7]. This is primarily due to lactic acid produced by platelet glycolysis and to lesser extent to accumulation of CO2 from oxidative phosphorylation [8].
Since platelets are usually stored as concentrates, the storage of platelet concentrates or thrombapheresis products in blood banks has been extensively studied [9–12]. However, a systemic evaluation of the function of platelets prepared from overnight-stored whole blood has not been performed. In the present study, we investigated how far the platelets can retain their aggregability when stored at 4°C or RT. We found no significant differences in the mean values of agonist-induced platelet aggregability between fresh specimens and samples stored at 4°C for 24 hr. We also observed that lack of responsiveness to epinephrine and loss of release reaction of endogenous ADP were developed by 24 hr storage at RT; under the same conditions, the aggregability to collagen and ristocetin was increased, compared to fresh specimen. Hence, we delineate the changes in platelet aggregability to agonists in association with acid-base status during storage.
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Materials and Methods
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Platelet counts, agonist-induced aggregability, mean platelet volume (MPV), and pH were monitored in blood samples from 96 apparently healthy humans, before and after storage of the samples at 4°C and RT. The subjects were laboratory personnel or medical students, age 25–46 yr (49 men, 47 women). None had hematologic or bleeding disorders nor had taken any medication for 
2 wk. To avoid the effect of caffeine on platelet aggregation, the subjects abstained from coffee and herbal or black tea for 48 hr. This study was approved by the Committee on Ethics of the Inha University Hospital, and informed consent was obtained from all subjects. We excluded three subjects who had acute or chronic infections, because inflammation can influence platelet aggregability.
A total of 22.5 ml of blood (4.5 ml/tube) was drawn from each subject by direct venipuncture into 5 Vacutainer tubes (Becton-Dickinson, Cedex, France) containing 0.129 M sodium citrate. Platelet counts, mean platelet volume (MPV), and platelet distribution width (PDW) of fresh or stored blood were analyzed with an electronic counter (SE 9000; Sysmex, Kobe, Japan). After the initial platelet counts, the blood samples were divided into 2 groups (fresh sample group and storage group), and the storage group was kept at RT or 4°C and reanalyzed after 24 and 48 hr. Platelet-rich plasma (PRP) was prepared just prior to running the aggregation test by centrifugation at 200 g for 20 min. The specimens stored at 4°C were allowed to equilibrate at RT for 30 min before preparing PRP. Platelet poor plasma (PPP) was made by centrifugation of PRP at 2000 g for 15 min. Platelet counts in PRP ranged from 210,000 to 430,000 platelets/µl. PRP was incubated at 37°C with stirring (1100 rpm) for 3 min prior to addition of any agonists. Aggregating agonists (Helena Laboratories, Beaumont, TX), such as epinephrine (300 µM), adenosine diphosphate (ADP; 10 µM), collagen (10 µg/ml), and ristocetin (1500 µg/ml) were used to induce platelet aggregation, which was monitored by continuously recording light transmission in a PACKS-4 aggregometer (Platelet Aggregation Chromogenic Kinetic System-4; Helena Laboratories). The final reaction volume was 500 µl, and aggregability was expressed as a percentage, based on the transmittance of light through the sample, relative to a PPP blank.
Subjects were divided into 3 groups according to their degree of responsiveness to each agonist: individuals showing a maximal platelet aggregability of <20% were considered non-responders; those with 20–60% aggregability were classified as impaired responders, and those with aggregability >60% as normal responders, which were based on the cutoff levels of our previous report [13]. Blood gas, oximetry values, and acid-base status were measured in 39 subjects using a blood gas analyzer (ABL 520; Radiometer, Copenhagen, Denmark), using citrate-anticoagulated venous blood immediately before preparing PRP (without opening the rubber cap of the sample tube, since infusion of room air may influence the acid-base status).
Statistical differences between groups were analyzed using the paired-sample t-test (fresh specimens before storage versus aged specimens after storage) or the Mann-Whitney U test (RT storage versus 4°C storage). All p values <0.01 were considered statistically significant.
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Results
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There were no significant differences in mean values of agonist-induced platelet aggregability between fresh specimens and samples stored at 4°C for 24 hr, although the refrigerated specimens showed a slight tendency toward lower values of aggregability to ADP and epinephrine agonists (Table 1
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After 24 hr storage at RT, the mean platelet aggregability responses to epinephrine and ADP were 8.2 ± 1.9% and 47.9 ± 7.3%, which were lower than those of fresh specimens (80.6 ± 5.4% and 87.4 ± 5.9%, p< 0.01, respectively); the aggregability to collagen and ristocetin was more elevated than those for the fresh specimens. Significant prolongation of the initial time delay in response to collagen was observed in the blood stored at RT for 24 hr (44.1 ± 1.5 sec) compared to that of fresh specimens (14.2 ± 6.9 sec, p <0.01). However, substantial shortening of the initial time lag in response to the collagen was noted when specimens were refrigerated for 24 hr (3.9 ± 1.7 sec, p <0.01).
In blood samples kept at RT for 24 hr, all the 96 subjects showed non-responsiveness (<20% aggregability) to epinephrine and 69.8% (67/96) of the subjects revealed impaired responsiveness (20–60% aggregability) to ADP (Table 2). Under the same conditions, no subjects showed impaired- or non-responsiveness to collagen or ristocetin. On the other hand, in specimens stored at 4°C for 24 hr, 9.4% (9/96) and 5.2% (5/96) of subjects displayed impaired responsiveness to epinephrine and ADP. Interestingly, among the 67 subjects who showed impaired responsiveness to ADP after RT storage, 62 (92.5%) exhibited a loss of secondary wave of aggregation in response to ADP. When blood was stored at 4°C, no cases showed a loss of secondary wave among subjects with impaired responsiveness to epinephrine (n = 9) or ADP (n = 5).
As shown in Table 3, the mean values of pH and carbon dioxide tension (pCO2) of venous blood samples (n = 39) stored at RT for 24 hr were 6.90 ± 0.02 and 96.2 ± 5.7 mmHg, which were significantly lower than those of the blood samples stored at 4°C for 24 hr (7.08 ± 0.03 and 72.6 ± 7.1 mmHg, p <0.01, respectively). The mean values of maximal aggregability to epinephrine, ADP, collagen, and ristocetin in the blood samples (pH 6.81; pCO2 107.4 mmHg) after 48 hr storage at RT were 7.5%, 15.6%, 18.9%, and 69.7%, respectively, which were significantly lower than those for the corresponding blood samples (pH 7.04; pCO2 75.3 mmHg) stored at 4°C for 48 hr (65.7%, 69.4%, 101.6%, and 91.2%, p <0.01, respectively).
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Table 3. Blood gas analysis and platelet aggregability in citrate-anticoagulated venous blood stored at RT or 4°C up to 48 hr.
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There were no significant changes in MPV between the blood samples stored at RT for 48 hr and the fresh specimens. However, the MPV of the blood samples stored at 4°C for 48 hr was higher than the fresh specimens or the blood samples stored at RT for 48 hr (p <0.01, respectively).
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Discussion
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In this study, we investigated the influence of storage temperature on platelet function, especially agonist-induced aggregability in the blood stored at RT or 4°C for 24 to 48 hr. We found that refrigerated storage improves the stability of responsiveness to agonists in platelets from healthy humans. We also found that RT storage for 24 hr causes non-responsiveness to epinephrine and disturbs the release reaction in response to ADP.
Our data are in general agreement with the results of Filip et al [14], who found that the levels of platelet ADP and release reaction were best preserved for 72 hr by storage of concentrates at 4°C and that platelet ADP levels and the release reaction were markedly deteriorated when the pH fell below 6.0 during storage at 22°C. Racz and Harsanyi [15] reported that in vitro properties of platelet concentrates prepared from blood stored 18 to 20 hr at 10°C were indistinguishable from freshly-isolated platelets and platelets from blood stored at 16°C.
On the other hand, other investigators [5,6,16] found that refrigerated storage of platelet deteriorates the function of platelets: both viability and function of concentrated platelets stored at 4°C were severely compromised. In contrast, in our study, there was no significant difference in platelet aggregability between the blood stored at 4°C for 24 hr and fresh specimens. The mean aggregability remained normal up to 48 hr during refrigeration. Furthermore, the aggregability to collagen and ristocetin were 10–15% higher in blood stored at 4°C for 48 hr than in fresh specimens. These results suggest that refrigerated storage of platelets up to 48 hr does not necessarily deteriorate platelet function, at least in vitro agonist-induced aggregability. These discrepancies may be due to differences in storage conditions among the studies. Unlike the studies reported previously, in the present study we measured platelet aggregability using PRP samples that were prepared from stored whole blood immediately prior to running the aggregation tests.
A biphasic aggregation response, that is, a primary and secondary wave of aggregation, is induced by ADP and epinephrine agonists. The secondary wave is produced by the action of endogenous ADP because epinephrine or ADP not only induce platelet aggregation, but also cause platelets to release endogenous ADP [17]. In our study, blood specimens stored at RT for 24 hr exhibited non-responsiveness to epinephrine and a loss of the secondary wave in response to ADP. However, no cases showed a loss of secondary wave to ADP or epinephrine when the blood samples were kept at 4°C. These results suggest that release of endogenous ADP from platelets in response to exogenous ADP is impaired during 24 hr of RT storage; thus, no secondary aggregation curves were elicited at this temperature.
Collagen induces only a single wave of platelet aggregation. This agonist induces platelet swelling or platelet shape change, from discoid to more spherical form, which is a transition that can be detected by aggregometry as a decrease in light transmittance. This is characterized by a lag period before aggregation begins. Thus, the initial lag time is normally present in response to collagen [18]. This transformation is not a prerequisite for platelet aggregation; epinephrine aggregates platelets but does not cause a shape change [19]. In our study, we evaluated platelet shape change on the basis of the aggregometer recordings and the MPV. In the blood samples stored at RT for 24 hr, no significant changes of MPV were observed; however, a marked prolongation of the lag time was noted and a marked decline of light transmittance was detected by the aggregometer when collagen was added. These results suggest that platelets keep their discoid shape stably enough to react on agonists, even when stored at RT for 24 hr, but that RT storage merely causes a delayed response to collagen.
On the other hand, after storage at 4°C for 24 hr, the lag time was completely lost, and the initial decrease in light transmittance was not observed. These results support the concept that the transition of platelet shape does not occur in response to collagen in the specimens stored at 4°C. This may be because platelets are already transformed to spheroidal shape during refrigeration. Actually, our results showed that MPV was significantly increased in the specimens stored at 4°C for 24 hr, and even more enhanced at 4°C for 48 hr, compared to fresh specimens. Interestingly, there was no significant decline in the aggregability of platelets stored at 4°C for 24 hr, by which time the platelets were presumably transformed to spheroidal shape. These results suggest that in vitro collagen-induced platelet aggregation may occur irrespective of disk-to-sphere transformation.
Metabolic activity of platelets continues during storage. Storage temperature influences pH, glucose consumption, and lactate production. When the pH reaches 6.8, platelet morphology begins to change and it changes dramatically when pH drops below 6.0, and the shape transition becomes irreversible and viability is lost [8]. In this study, we investigated the changes in pH and pCO2 in stored blood and the attendant alterations of platelet aggregability. In our study, the mean pH of blood stored at RT for 24 hr was 6.90, at which the mean aggregability to epinephrine and ADP was markedly decreased, but responsiveness to collagen and ristocetin was normal. When the mean pH reached 6.81 in the blood stored at RT for 48 hr, complete losses of the aggregability to epinephrine, ADP, and collagen were observed, but the mean aggregability to ristocetin was still normal. These results suggest that the responsiveness of platelets to ristocetin is the most resistant to the change in pH.
We tried to define parameters relevant for hemostatic evaluation of patients and not for the preparation of blood components. Because our data were obtained from specimens stored as whole blood in test tubes, not from platelet concentrates stored in plastic bags to facilitate gas exchange, we stress that the findings of the current study are of limited relevance for blood component preparation and storage. We believe that platelets can be stored at 4°C up to 48 hr without significant decrease in aggregability; we emphasize that our data pertain to platelets collected for diagnostic laboratory tests.
In conclusion, refrigerated storage improves the stability of human platelet responsiveness to agonists. Blood storage for 24 hr at RT leads to platelet non-responsiveness to epinephrine and disturbs the release reaction of platelets in response to ADP.
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Abstract
Introduction
