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Anesth Analg 2005;100:78-81
© 2005 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000140239.43116.30

PEDIATRIC ANESTHESIA

The Functional Integrity of Platelets in Volume-Reduced Platelet Concentrates

Helge Schoenfeld, MD*,{dagger}, Manfred Muhm, MD*,{ddagger}, Ulrich R. Doepfmer, MD, FRCA{dagger}, Wolfgang J. Kox, MD, PhD, FRCP{dagger}, Claudia Spies, MD{dagger}, and Hartmut Radtke, MD§

*Department of Anesthesiology, Inselspital, University Hospital of Bern, Bern, Switzerland; {dagger}Department of Anesthesiology and Intensive Care Medicine, Charité, University Medicine Berlin, Campus Charité Mitte, Berlin, Germany; {ddagger}Department of Cardiothoracic Anesthesia and Intensive Care Medicine, University of Vienna, and Department of Anesthesiology and Intensive Care Medicine, Hospital of Oberpullendorf, Austria; and §Institute of Transfusion Medicine, Charité, University Medicine Berlin, Campus Charité Mitte, Berlin, Germany

Address correspondence and reprint requests to Helge Schoenfeld, MD, Department of Anesthesiology and Intensive Care Medicine, Charité, University Medicine Berlin, Campus Charité Mitte, Humboldt-University Berlin, Schumannstrasse 20/21, D-10117 Berlin, Germany. Address e-mail to helge.schoenfeld{at}charite.de


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Premature and low-birth-weight infants usually require small-volume platelet transfusions to treat thrombocytopenia. Also, infants undergoing open-heart surgery with extracorporeal circulation and with compromised cardiac function are at risk for excessive intravascular volume. The small-volume platelet substitution can be achieved by dispensing an aliquot from the unit of a standard single-donor platelet concentrate (PC). Alternatively, there is an indication for volume reduction of PCs to maximize the number of platelets transfused in the smallest possible volume. We determined the spontaneous and induced activation of platelets before and after volume reduction in 20 consecutive single-donor-apheresis PCs. After a mean storage time of 2 days, the PCs were plasma-depleted by centrifugation. Spontaneous, adenosine diphosphate (ADP)-induced, and collagen-induced activation were determined by flow cytometry. Furthermore, ADP- and collagen-induced aggregation were measured. A total of 33.8% of platelets in standard PCs were activated spontaneously. Volume reduction of PCs led to a mild but significant increase of spontaneous activation of platelets (43.2%). Additionally, volume reduction resulted in an impaired ADP-induced aggregability of platelets, whereas collagen induction was unaffected. Transfusion of volume-reduced PCs is an effective alternative to use of standard PCs in patients at frequent risk for excessive intravascular volume, because equal volumes increase the platelet count twice as effectively.

IMPLICATIONS: Volume reduction of platelet concentrates may be indicated for thrombocytopenic pediatric patients with a risk of excessive intravascular volume. We studied the spontaneous, adenosine diphosphate-induced, and collagen-induced activation of platelets before and after volume reduction of single-donor-apheresis platelet concentrates. The procedure resulted in slightly impaired in vitro platelet function.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
In a pediatric setting, the volume of plasma frequently needs to be reduced in the platelet concentrate (PC) before its transfusion (1). Thrombocytopenic, premature, and very low-birth-weight (<1250 g) infants often require small-volume platelet transfusions to treat thrombocytopenia associated with disseminated coagulation and sepsis (2). Infants undergoing open-heart surgery with extracorporeal circulation and with compromised cardiac function are at risk for excessive intravascular volume (3–5). Small-volume platelet substitution can be achieved by dispensing an aliquot from the unit of a standard single-donor PC. Alternatively, the PC volume can be reduced by plasma depletion through centrifugation to maximize the number of platelets transfused in the smallest possible volume.

However, Rock et al. (1) suggest that additional centrifugation for plasma removal could further affect platelets that have already been altered by storage. The functional integrity of platelets in volume-reduced PCs has been studied only infrequently in vitro (1,6–11). Only one of these studies (7) looked at single-donor-apheresis PCs. The use of single-donor-apheresis PCs as opposed to pooled PCs is steadily increasing (12). None of these studies reduced the plasma volume shortly before transfusion, but volume reduction was achieved immediately after—or during—PC production, and the concentrated product was stored for several days; thereafter, function was tested. The aim of our study was to determine the spontaneous and induced activation of single-donor-apheresis PCs by flow cytometry and by induced platelet aggregation with adenosine diphosphate (ADP) and collagen before and after volume reduction. These in vitro tests measure platelet hemostatic function.


   Methods
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 Abstract
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 Methods
Results
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Single-donor-apheresis PCs were obtained from 20 consecutive healthy volunteers by automated plateletpheresis (MCS 3p; Hemonetics, Munich, Germany). Each donor was screened according to the German guidelines (13); i.e., they were free from relevant infectious and hepatic, renal, cardiovascular, or malignant diseases. The donors had no antiplatelet drug ingestion, such as aspirin or nonsteroidal antiinflammatory drugs, in the previous 10 days and no alcohol ingestion in the previous 24 h. Every donor signed consent that at the time of blood donation the donor was waiving all rights concerning the donated unit of blood. No changes were made to our clinical routine management of either donor or recipient. It was not considered necessary to inform the ethics committee, donors, or recipients of these units of platelets. The volume of PCs was 220–250 mL. All PCs were leukocyte-depleted by filtration (LRF6/10 filter; Pall, Dreieich, Germany) and stored at 22°C with constant agitation.

According to our institutional protocol, depletion of plasma in PCs was performed as soon as clinicians requested a volume-reduced PC. Immediately before centrifugation, we removed an aliquot of 3–5 mL for the in vitro tests with a sterile docking system (SCD 312; Hemonetics). We then subjected the PC to centrifugation for 4 min at 3500g and 22°C (K80 Refriged Centrifuge; MLW, Leipzig, Germany). Subsequently plasma was removed to achieve a residual volume of 90 mL. The platelet pellet was gently resuspended in this residual plasma. Concentrated platelets rested undisturbed for 30 min at room temperature. After an additional resting time of 30 min with agitation, a second sample for in vitro testing was drawn as described previously. Thereafter, volume-reduced PCs were issued for transfusion. PCs were transfused within 6 h after completion of plasma depletion.

The in vitro studies were performed before and after the plasma depletion procedure. All tests were performed within 2 h of taking the respective samples. Platelet counts and the mean platelet volume of PCs were determined by using an automated cell counter (H1 Technicon, Tarrytown, NY). For platelet-aggregation studies, platelet samples were diluted with autologous plasma to a concentration of 250 x 109/L. Aggregation response to ADP in a final concentration of 18 µM, or collagen in a final concentration of 1.9 mg/mL, was recorded on an aggregometer (Platelet Aggregation Profiler-4; MÖLAB, Hilden, Germany). Aggregation response was defined as the maximum change in light transmittance after the induction and was expressed as a percentage of platelet-poor plasma. A higher light transmittance reflects a stronger platelet aggregation.

Spontaneous, as well as ADP- and collagen-induced, platelet CD62 (P-selectin; GMP-140) expression were analyzed by flow cytometry (FACscan; Becton Dickinson, Heidelberg, Germany), as described previously (14). The expression of CD62 is a result of activation of platelets and depends on the fusion of {alpha}-granules with the surface membrane of platelets.

Statistical analysis was performed with Student’s paired t-test. Data are given as mean ± SD unless otherwise stated. A difference was considered significant when P < 0.05. There was no correction for multiple comparisons.


   Results
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 Results
Discussion
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The mean storage time of PCs (n = 20) between production and volume reduction was 2 days (range, 0–4 days). There was no difference in mean platelet volume of platelets before and after plasma depletion. Platelet counts of PCs were almost doubled in volume-reduced PCs (Table 1).


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  		Table 1. Mean Platelet Volume (MPV) and Platelet Count
 
Platelets in volume-reduced PCs showed a significant loss in ADP-induced aggregability. In contrast, there was no significant difference between standard and concentrated platelets in collagen-induced aggregation (Table 2).


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  		Table 2. Aggregation Response of Platelets Induced by Adenosine Diphosphate (ADP) and Collagen
 
Volume reduction of PCs resulted in a statistically significant increase of spontaneously activated platelets as evaluated by flow cytometry. ADP-induced activation resulted in a significantly decreased activation response of platelets after plasma depletion. Collagen induction demonstrated no difference between standard and concentrated platelets (Table 3). We detected a tendency toward increased spontaneous activation with storage, but the susceptibility to plasma depletion-induced damage did not seem to relate to the duration of storage (data not shown).


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  		Table 3. Spontaneous, Adenosine Diphosphate (ADP)-Induced, and Collagen-Induced Activation of Platelets
 

   Discussion
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 Abstract
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 Methods
 Results
 Discussion
References
 
The advantages of a reduced transfusion volume associated with volume-depleted PCs are prevention of excessive intravascular volume (10) and reduction of transfused plasma solutes, including pyrogenic cytokines (6). Premature infants and neonatal patients often require platelet transfusion for thrombocytopenia or thrombopathia. We undertook this study to determine the in vitro functional integrity of platelets in volume-reduced single-donor PCs. We reduced the plasma volume to 90 mL. Other studies examined PC units with a suspending volume of 10–60 mL (1,6,8–11,15). The major reason for using the volume of 90 mL was a possible decrease in pH in these PCs from anaerobic conditions, increased lactic acid production, and increased risk of platelet/container surface contacts (9,16). According to our institute’s practice, the platelet-suspending volume was, therefore, maximized to increase the plasma-buffer capacity while maintaining as little volume as possible. The main difference between our work and previous studies was in the production process of PCs. In most published studies examining in vitro morphological data of plasma-depleted PCs (1,6,8–11,15), PCs were prepared from each whole-blood unit with a standard volume of 50–60 mL, whereas our PCs were prepared by plateletpheresis with a standard volume of 220–250 mL.

Generally, the plasma-depletion procedure includes centrifugation, and its shear stress could result in an increase of platelet activation and a reduction of platelet aggregation in vitro (6). In our study, we nearly doubled the platelet count in volume-reduced PCs after plasma depletion. This was comparable with results from Pisciotto et al. (10).

Initially, we showed that spontaneous CD62 expression on the membrane surface was significantly increased in volume-reduced PCs. Several authors accept that platelet activation is associated with secretion of {alpha}-granules and expression of CD62 (P-selectin; GMP-140) on the platelet surface (17–19). In this study, the platelets in volume-reduced PCs showed significantly decreased ADP-induced activation after plasma depletion. No difference was found between collagen-induced activation before and after plasma depletion. Our results are similar to the findings of Rock et al. (1), who demonstrated an increased CD62 expression in volume-reduced PCs, although this change did not reach significance.

Our aggregation tests indicated that platelets in volume-reduced PCs have significantly less aggregation when exposed to ADP. Rothwell et al. (20) showed that platelets mounting 22% of the baseline aggregation function (after storage) were still able to provide adequate hemostatic function in an in vivo kidney bleeding model. No clinical studies have correlated in vitro results of aggregation tests with clinical effectiveness of platelet transfusions in actively bleeding patients or have studied the effect of transfused platelets with different aggregation responses on posttransfusion bleeding times. Collagen-induced aggregation did not differ between standard and volume-reduced PCs. Limited data are available delineating how plasma depletion affects platelet aggregability. Comparable results were shown by Zilber et al. (6). They used a combination of aggregating agents. ADP in combination with epinephrine led to a decreased aggregation response in volume-reduced PCs, whereas platelet aggregation induced by collagen and thrombin was preserved (6). In contrast, plasma-depleted PCs stored for five days exhibited synergistic aggregation with combinations of aggregating agents: ADP and collagen or ADP and ionophore A23187. Holding reduced-volume PCs for two hours at 20°C to 24°C did not cause a change in aggregation response (11). These findings suggest that collagen and thrombin are strong platelet agonists. They can overcome the platelet desensitization that results from induced platelet stimulation. Collagen is a stronger activator that is capable of aggregating partially damaged platelets. ADP, a much weaker agonist, failed to produce the same degree of {alpha}-degranulation (14).

The mechanism of the reduced aggregation and induced activation examined by cytometry may relate to shedding or internalization of platelet surface membrane ADP or epinephrine receptors (6). In our study, PCs were plasma-depleted by centrifugation at 3500g for four minutes. A short centrifugation for 2.25 minutes at 3270g yielded platelets with better storage properties than with longer centrifugation times (4.5 minutes at 2200g) (21). Exposure of platelets to physical stimuli such as centrifugation has been reported to diminish cyclic adenosine monophosphate levels (22). This effect of centrifugation could be an explanation for a lesser inhibition of platelet aggregation and granule release (23). Furthermore, ADP released from cells during centrifugation is partly responsible for the platelet activation in volume-reduced PCs (24). In summary, plasma depletion stresses platelets. It results in an impaired ADP-induced aggregability.

Previous in vivo data indicate that hemostatic function, as well as posttransfusion recovery and survival, are maintained in volume-reduced PCs, as determined by bleeding time measurement (8) and radiolabeling of platelets with chromium-51 and indium-111 and by autologous transfusion (9). A significant reduction in viability associated with increased lactate production was found only in PCs with volumes <35 mL. The PC platelet count in this study increased through volume reduction from 1.3 x 109/mL to 2.5–2.9 x 109/mL (9).

We conclude that plasma depletion is an activator of platelet function. Little is known about success after transfusion of volume-reduced platelets. Different methods for volume reduction of PCs have been published, but there is no consensus regarding optimal centrifugation rates; therefore, practice varies (4). An aliquot of a standard-apheresis PC is often transfused. Transfusion of an aliquot of volume-reduced PC doubles the transfused number of platelets compared with a standard PC. Modern platelet apheresis programs can produce volume-reduced, plasma-poor PCs. If the use of single-donor filtered-apheresis PCs is the established routine, we see an indication for planned, primarily volume-reduced PCs produced by apheresis if a patient susceptible to excessive intravascular volume is undergoing cardiac surgery. The described procedure takes approximately 1 hour 15 minutes. Thus, volume reduction of PCs is a practicable way to maximize transfused platelets.


   Footnotes
 
This research was not supported by any institution, foundation, or company. The authors have no commercial interests related to this article.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication July 9, 2004.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2005 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press