Preparation and Storage of Platelet Concentrates S. J.
S L I C H T E R ,AND
L. A. HARKER
From the Puget Sound Blood Center and the Department of Medicine, University of Washington School of Medicine, Seattle, Washington
and storage have been examined in order to characterize optimum yield, viability, and function of the resultant concentrates. The studies summarized here are reported in detail elsewhere."*12
A technique of platelet concentrate preparation and storage is presented which permits the maximum number of viable and functional platelets to be preserved for periods of 72 hours. Although the storage conditions must be followed precisely, the method is nevertheless simple to perform and does not require specialized expensive equipment. Critical factors include: 1) preparation of the platelet concentrates with an initial centrifugation of 1000 x g for 9 minutes and a second centrifugation of 3000 x g for 20 minutes (86% f 1 platelet yield), 2) a storage bag composed of either Fenwal's PL-146 or McCaw plastic, 3) constant gentle mixing, 4) a 70 ml residual plasma volume, and 5) room temperature storage (22 C f 2). In Yivo platelet recovery after 72 hours of storage at room temperature averaged 46 per cent f 3 and survival was 7.9 days f 0.3 (81% of fresh platelet viability). The function of these platelets as measured by the correlation between bleeding time and platelet count after transflsion of pooled platelets into unimmunized, aplastic thrombocytopenic recipients was as good as that of fresh platelets. Both viability and function of concentrated platelets stored at 4 C are severely compromised.
Methods Five different types of blood bags were used for collecting 450 ml of blood from normal donors: Fenwal PA-20 with the transfer pack composed of either PL-130 or PL-146 plastic, McCaw (Double Hemo-Pak, T2250, ACD-A) and Cutter (PR2370A, ACD-A, or CL-2383*, CPD). To separate the platelets from other components, a Sorvall RC-3 centrifuge with horizontal head and swinging buckets was used. The machine was calibrated frequently to insure accuracy of the time and revolutions per minute (RPM) dial settings, and a period of about two minutes was used for braking. Centrifugation time was from the onset of centrifugation until the rotator head had completely stopped. After initial centrifugation, the platelet-rich plasma (PRP) was delivered by a Fenwal plasma extractor** into the satellite bag. The P R P was recentrifuged to pack the platelets and most of the supernatant plasma was either returned to the red blood cells or transferred to a second satellite bag for cryoprecipitate processing. The preparation was not disturbed for an hour and a half after centrifugation and then the platelet pellet was resuspended in residual plasma by gentle inversion until a smooth suspension was obtained.' All procedures were carried out at room temperature (22 c 2 C). For studies of room temperature storage, the bags were placed in an air conditioned, thermostatically controlled room with the temperature maintained at 22 C =t2 C. For studies a t 4 C, either a refrigerator or walk-in cold room was used. Continuous mixing during storage was
INCREASINGDEMANDS for platelet transfusions necessitate improved efficiency in platelet preparation and storage without compromising the use of other blood components. For example, the administration of platelets as concentrates rather than as whole blood makes it possible to provide adequate numbers of platelets while allowing the remaining plasma and red blood cells to be used for other purposes. Obviously, these concentrates should contain the maximum number of viable, functional platelets. Since platelets must be available to treat emergency situations, efficient storage techniques are essential. Various aspects of platelet preparation This work was supported by National Institutes of Health grants (CA-12190, CA-10895) and contract (NIH-NHLI-70-2214). A portion of this work was conducted through the Clinical Research Center facilities of the University of Washington (NIH grants FR-37 and RP- 133):
*New plastic bag material currently being evaluated for FDA approval for licensing. **Plasma Extractor BM-I, Fenwal Laboratories, Morton Grove, 111.
8 Transfusion Jan.-Feb. 1976
Volume 16 Number I
Volume 16 Number I
PREPARATION AND STORAGE OF PLATELET CONCENTRATES
accomplished by placing the bags without stacking on open wire racks mounted on a platform rotatort adjusted to the slowest speed at which the platelets stayed in suspension (40 cycles/minute). Air was blown over the concentrates by a fan to prevent the heat of the rotator motor from increasing the temperature of the platelets. For platelet counting, the samples were collected in potassium EDTA from the original whole blood, PRP, platelet-poor plasma (PPP), and the final resuspended platelet concentrate. An electronic particle counter was used according to a previously described method' adapted from Bull et aL3 The total number of platelets in each of the sampled fractions was calculated by multiplying the platelet count by the blood or plasma volume as determined by weighing. The viability of the concentrated fresh or stored platelets was determined by in vitro labeling with r a d i o c h r ~ m i u m ~and . ~ . ~measuring ~ recovery and survival after reinfusion of autologous platelets into normal subjects. In order to include both platelet recovery and survival in the overall assessment of platelet viability, results were expressed as the area under the survival curve. A platelet viability index (PVI) was calculated by expressing the area obtained in each experimental method of platelet concentrate p r e p aration and storage as a proportion of the area obtained in the control concentrates prepared by standard techniques and injected immediately after preparation. The function of infused platelets was determined by performing standardized template bleeding times before and at frequent intervals after the transfusion of concentrates into unimmunized aplastic thrombocytopenic recipients with baseline platelet counts less than 10,00O/pl and bleeding times greater than 60 minutes. The bleeding time in minutes can be predicted by the equation: 30.5
platelet count per pl
at platelet counts between 100,000 and lO,O00/ Prolongation of the observed bleeding time beyond that expected was considered to reflect platelet dysfunction.
Results Platelet Concentrate Preparation The centrifugation variables of time and force and their effects on platelet yield in the platelettEberbach, Ann Arbor, Mich.
rich plasma and subsequently in the PC were examined in 75 1 blood donations. The highest yield of platelets in the concentrate was obtained using an initial centrifugation of 1000 x g for 9 minutes to prepare PRP and a final centrifugation of 3000 x g for 20 minutes to concentrate the platelets. This gave an average yield of 86 per cent + 1 (* 1 S.E.)S of the available whole blood platelets into the concentrate." In normal volunteers autologous reinfusions of 5'Cr-labeled concentrates gave average recoveries of 56 per cent + 3 and .survivals of 8.2 days rt 0.2, i.e.. not significantly different from control concentrates, which gave recoveries of 59 per cent 4 and survivals of 8.1 days f 0.2."
Effects of Mixing and the Plastic Bag Without mixing of the platelet concentrates during 24-hour storage, recovery averaged 23 per cent + 3, survival was 6.7 days 0.5, and PVI was 0.35 f 0.05. When mixing was continuous, platelet viability depended on the storage bag; i.e., the average PVI with Cutter CL-2383 was 1.00 =t 0.02; with Fenwal PL-146, 0.93 0.05; with 0.08; with Cutter (PR-2370A), McGaw, 0.81 0.34 0.06; and with Fenwal PL-130 it was 0.27 0.06 (Table I);
Effect of p H Changes during Storage Platelet concentrates resuspended in 20 ml of plasma in Fenwal PL-146 bags showed excessive losses in viability after 48 to 72 hour storage despite continuous mixing. Platelet recovery averaged only 10 per cent f 5, survival was 2.4 days f 1.3, and PVI was 0.05 0.02. The pH was predictably less than 6.0 at the end of storage. In contrast, such platelet concentrates stored for only 24 hours had a pH above 6.0. Additional studies indicated that the pH could be maintained above 6 during 72 hour storage at room temperature if the concentration of platelets was less than 1.7 x 10e/pl. This concentration was consistently achieved if the residual plasma volume was increased to 70 ml. Either ACD or CPD was satisfactory as the anticoagulant as shown by the findings that concentrates stored as described above for 72 hours collected in ACD had average posttransfusion recoveries in their normal donors of 40 per cent 3, survivals of 7.9 days 0.2, and PVI of 0.71 0.06 while units collected in CPD had recoveries of 46 per cent 3, survivals of 7.9 days =t0.3, and PVI of 0.81 f 0.06.
$Variances in the Results section are given as standard error.
Transfusion Jan.-Feb. 1976
SLICHTER AND HARKER Table 1. 24 Hours 22 C Platelet Concentrate Storage Storage Conditions Interval (Hours)
Plastic Bag (Material)
Fenwal (PL-130) ACD
Fenwal (P L-146) ACD
(%I 56f 3
1.oo ?r 0.02
Fenwal (P L-146) ACD
Cutter (CL-2383) CP D
55 f 2
8.1 + 0.3
Fenwal (P L-146) ACD
51 f 3
8.2 f 0.2
Cutter (PR-2370A) ACD
Fenwal (PL-130) ACD
0.34 f 0.06*
Result is significantly different f r o m unstored platelet concentrates w i t h a p value