Anesth Analg 2004;99:17-20
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000116930.28250.15
CARDIOVASCULAR ANESTHESIA
Platelet Activity in Washed Platelet Concentrates
Helge Schoenfeld, MD*,
,
Manfred Muhm, MD*,
,
Ulrich Doepfmer, MD FRCA,
Aristomenis Exadaktylos, MD*, and
Hartmut Radtke, MD
*Department of Anesthesiology, University Hospital of Bern, Inselspital, Switzerland, the
Department of Anesthesiology and Intensive Care Medicine, University Hospital Charité, Berlin, Germany, the
Department of Anesthesiology and Intensive Care Medicine, Hospital of Oberpullendorf and Department of Cardiothoracic Anesthesia and Intensive Care Medicine, University of Vienna, Austria, and the
Institute of Transfusion Medicine, University Hospital Charité Berlin, Germany
Address correspondence to Helge Schoenfeld, MD, Department of Anesthesiology and Intensive Care Med, University Hospital Charité, Campus Charité Mitte, Humboldt-University Berlin, Schumannstrasse 20/21, D-10117 Berlin, Germany. Address email to helge.schoenfeld{at}charite.de
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Abstract
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Life-threatening anaphylaxis or febrile nonhemolytic transfusion reactions after transfusion of platelet concentrates (PCs) is a serious clinical problem caused by the sensitizing of recipients to plasma components, such as immunoglobulin A, or by cytokines. There is a possible indication for washing of PCs in these thrombocytopenic patients. However, only platelets that show activation after physiological stimulation are useful. We determined the spontaneous and induced activation of platelets before and after washing. We investigated 11 consecutive single-donor-apheresis PCs. After production and leukocyte-depletion the PCs were washed in 15% acid-citrate-dextrose-solution. The spontaneous and the adenosine diphosphate (ADP)-induced, as well as collagen-induced activation, were determined by flow cytometry. Additionally, ADP- and collagen-induced aggregation were measured. Unwashed platelets (16.1%) were activated spontaneously. The washing of PCs led to a threefold increase of spontaneous activation of platelets (47.4%). Because of increased spontaneous activation after washing we could demonstrate cytometrically a loss of induced activation of washed platelets. Furthermore, washing resulted in an impaired ADP-induced aggregability of platelets. These results have led us to reduce the frequency of washing of PCs in our institution, where the only current indication for washing of PCs is in patients with a history of severe nonhemolytic transfusion reactions.
IMPLICATIONS: Washing of platelets may be indicated for thrombocytopenic patients with a history of allergic or febrile reactions after transfusion. We studied the spontaneous and adenosine diphosphate- and collagen-induced activation of platelets before and after washing of single-donor apheresis platelet concentrates. Washing procedure resulted in an impaired in vitro platelet function.
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Introduction
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The use of single-donor apheresis platelets with lower levels of leukocyte contamination and prestorage leukocyte-depletion in platelet concentrates (PCs) by filtration are standard procedures and represent strategies that have been proposed to prevent initial or recurrent transfusion reactions like human leukocyte antigene-alloimmunization in thrombocytopenic patients (1–3). A major problem for physicians of thrombocytopenic patients receiving repetitive platelet transfusions are anaphylaxis and febrile nonhemolytic transfusion reactions caused by release of cytokines during storage or by the sensitizing of receivers to the plasma proteins or plasma components, like immunoglobulin (Ig) A. Febrile nonhemolytic transfusion reactions to platelet transfusions occur in approximately 20%–30% of transfusions (4). Allergic reactions to blood products can either be mild and easily managed by antihistamine application, or they can be severe and require complete removal of plasma proteins, IgA, and cytokines from the cells before transfusion (5–10). Washed platelets have been used for transfusions in oncologic patients requiring repeated platelet transfusions (5). In addition, washing also was performed for neonatal patients to remove maternal antibodies before administration of maternal platelets to infants with neonatal alloimmune thrombocytopenia (11). Thus, platelet washing has been used successfully in patients who cannot tolerate even the small quantity of plasma contained in PCs (6).
However, only platelets that show activation after physiological stimulation are useful. The indication of platelet washing is rare; PCs are expensive, and the functional integrity of washed platelets has been studied only infrequently in vitro (5–7,12–14). It has been suggested that spontaneously activated platelets cause a diminished increase in posttransfusion platelet numbers (15). The purpose of our study was to determine the spontaneous and induced activation of single-donor platelets by flow cytometry and by induced platelet aggregation with adenosine diphosphate (ADP) and collagen before and after washing. These tests are surrogate measures to evaluate platelet hemostatic function.
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Methods
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We obtained 11 consecutive PCs from normal volunteer donors by automated plateletpheresis (MCS 3p; Hemonetics, Munich, Germany). Each donor was screened according to the German guidelines (i.e., they were free from relevant infectious, 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. The volume of PCs was 220–250 mL. All PCs were leukocyte-depleted after its production by filtration (LRF6/10-filter; Pall, Dreieich, Germany) and stored at 22°C with constant agitation.
Platelets were washed manually according to our institutional standard. We diluted the PCs with 100 mL of 15% acid-citrate-dextrose (ACD)-solution with a temperature of 22°C. Platelet sedimentation was reached by centrifugation for 8 min at 3500g. After removal of supernatant plasma and a storage time of 30 min, the platelet pellet was resuspended cautiously in 200 mL 0.9% sodium chloride-solution. The washed PCs were prepared for transfusion after a further 30 min of storage. PCs were transfused within 6 h after completion of washing.
The in vitro studies were performed before and after the platelet washing procedure. The mean platelet volume (MPV) and platelet counts of the PCs were determined electronically (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 with a working concentration of 18 µM or collagen with a working concentration of 1.9 mg/mL was recorded on an aggregometer (Platelet Aggregation Profiler-4; MÖLAP, Hilden, Germany). Platelet aggregation in response to ADP is reduced after prolonged storage. Collagen is a stronger activator capable of aggregating partially damaged platelets. Aggregation response was defined as the maximum change in light transmittance after induction and expressed as 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). The expression of CD62 is a result of activation of platelets and depends on the fusion of 
-granules with the surface membrane of platelets. For sample preparation of platelet CD62 expression 60 µL of PC was diluted in 1 mL Grotemeyer-buffer-solution (KH2PO4, 9.07 g/L; Na2HPO4, 9.47 g/L; EDTA, 4.36 g/L; pH, 7.4). One sample was mixed with 1% formaldehyde for fixing platelets and evaluation of spontaneous activation and one sample without formaldehyde for determination of induced CD62 expression by agonists. Five µL of each sample were diluted with 95 µL phosphate-buffered saline (PBS) solution. After addition of 10 µL antiCD42 antibody (Becton Dickinson) for cytometric identification of platelets and 10 µL antiCD62 antibody (Becton Dickinson) for evaluation of platelet activation, incubation for 15 min, and dilution of samples with 1 mL PBS, the spontaneous activation was measured. From the sample without formaldehyde 225 µL were added to 25 µL ADP-solution and 237 µL were added to 13 µL collagen solution. After incubation for 20 min 5 µL from each mixture were diluted with 95 µL PBS and mixed with antiCD42 and antiCD62 antibodies and incubated again as described before. After dilution with PBS the ADP and collagen-induced activation, expressed in percent, was measured.
Statistical analysis was performed using 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.
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Results
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The mean storage times of PCs between production and washing was 2.5 days (range, 0–3 days). Washing of platelets was performed as soon as clinicians requested a washed PC. Before and immediately after the washing procedure we took 3 mL to 5 mL of each unit under sterile conditions. All our measurements were done within 2 h after washing procedure. The MPV of platelets increased significantly (P < 0.001) after the washing procedure (Table 1). There was no difference in platelet counts of PCs before and after washing.
Washed platelets showed a significant (P < 0.001) loss in ADP-induced aggregability (Table 2). In contrast, there was no difference between washed and unwashed platelets in collagen-induced aggregation.
As summarized in Table 3, the washing procedure of PCs resulted in a statistically significant increase of spontaneously activated platelets (P < 0.001) as evaluated by flow cytometry. Almost half the washed platelets are activated without further stimulation. ADP-induced activation demonstrated no difference between washed and unwashed platelets. Collagen-induction resulted in a significantly higher activation response after the washing procedure (P < 0.05). We detected a tendency towards increased spontaneous activation with storage, but the susceptibility to washing-induced damage did not seem to relate to duration of storage.
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Discussion
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We studied the functional integrity of manually washed single-donor apheresis platelets in vitro. In this study, platelets were washed with ACD-solution. Previous studies testing integrity, function, activation, and aggregation of washed platelets differed by the type of washing procedures, automated and manual, and the washing solutions used (5–7,13). Generally, all washing procedures include centrifugation and its shear stress could result in an increase of platelet activation and a reduction of platelet aggregation in vitro (16).
In our study, the washing procedure led to an increase of MPV. Determination of MPV can be influenced be platelet activation alone. It could be shown that MPV was increased in activated platelets (17,18). Shimizu et al. (13) found platelet swelling in washed PCs after 3 days of storage, whereas other investigators could not show a difference in platelet size before and after washing (5). Possible explanations for cell swelling could be the removal of plasma components, leading to loss of oncotic pressure in the storage medium, and the impaired Na+/K+-ATPase of Na/K pump in the platelet membrane. The resultant accumulation of Na+ inside the platelets could cause cell swelling (13,19).
Our aggregation tests indicated that washed platelets have a significantly lesser extent of aggregation when exposed to ADP. Collagen-induced aggregation did not differ between washed and unwashed platelets. One study also showed an impaired ADP-induced aggregation response in washed platelets at comparable concentrations of ADP (15 µM) (14). Vesilind et al. (12) found no difference in their aggregation tests using ADP, arachidonic acid, collagen, and epinephrine, respectively. No difference between collagen as well as collagen plus ADP-induced aggregation was found by Shimizu et al. (13).
The in vitro function of unwashed and washed platelets was tested by induced activation using a flow cytometer. Initially, we could show that the spontaneous CD62 expression on the membrane surface was tripled in washed platelets. It is commonly accepted that platelet activation is associated with secretion of granule and expression of CD62 (P-selectin, GMP-140) on the platelet surface (20,21).
In the present study, no difference was found between ADP-induced activation before and after washing procedure. The washed platelets showed a significantly increased activation response after collagen stimulation. Our results are similar to the findings of Walkowiak et al. (14). They examined washed platelets and showed an increased expression of CD62 after thrombin stimulation, whereas stimulation with ADP exhibited resistance to ADP in washed platelets. The findings suggest that collagen and thrombin are strong platelet agonists able to overcome platelet desensitization resulting from the induced platelet stimulation. ADP, a much weaker agonist, failed to produce the same degree of -degranulation.
In summary, washing strongly stresses platelets. It results in an impaired ADP-induced aggregability. Secondary to their extremely high spontaneous activation, the remaining ability for activation in washed platelets is decreased. In vivo studies could demonstrate acceptable improvements of bleeding time tests (6). Pineda et al. (6) measured bleeding times in normal subjects who received aspirin followed by transfusion of autologous unwashed 0.9% saline- or ACD-washed platelets. The authors could show that the bleeding time after aspirin ingestion was significantly prolonged in all subjects compared with the baseline. Only transfusion of unwashed and ACD-washed platelets shortened the bleeding times significantly 24 hours after aspirin ingestion (7'58" versus 3'52" and 7'23" versus 4'13", respectively). Increases in platelet counts after transfusion of washed and unwashed PCs were similar (5). Other authors described a significantly smaller increment in platelet counts for washed platelets than for unwashed platelets, but there was no evidence of increased bleeding in the recipients (9). Uptake of 111In-labeled washed platelets in liver and spleen 24 and 190 hours after transfusion was significantly more compared with unwashed platelets (6). A possible explanation could be the increased MPV. Furthermore, irregular forms and surface membrane alterations were demonstrated by electron microscopy (12).
To establish the hemostatic relevance of our in vitro study it would be necessary to conduct pre- and posttransfusion thrombelastography and possibly standardized bleeding times in patients, with similar baseline conditions, randomized to receive washed and unwashed PCs. The alternative would be to randomize actively bleeding patients to receive washed or unwashed PCs and determine the effect on the continuing blood losses. This latter approach seems to be very difficult to conduct in clinical practice.
We conclude that washing is a strong activator of in vitro platelet function. Despite acceptable success after transfusion of washed platelets, the indication for washing has to be considered very carefully. In our institution (University Hospital Charité Berlin), the practice of washing of PCs has declined. Historically, washed platelets have been used in neonatal patients to remove maternal antibodies before administration of maternal platelets. Modern platelet apheresis programs can produce volume-reduced, plasma-poor PCs for neonatal patients. If the use of single-donor filtered apheresis PCs is the established routine, we see an indication for washing of PCs only if a specific patient has a history of severe nonhemolytic transfusion reaction or anaphylaxis, as in IgA-deficient patients. Further clinical studies need to clarify the relevance of the large percentage of activated platelets in washed PCs.
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Accepted for publication December 17, 2003.
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Abstract
Introduction
