Paired comparison of platelet concentrates prepared from platelet-rich plasma and buffy coats using a new technique with "'In and 51Cr T. KEEGAN,A. HEATON,S . HOLME,M. OWENS,E. NELSON, AND R. CARMEN Two techniques for the preparationof platelet concentrate (PC), the standard plateletrich plasma (PRP) and buffy coat (BC) methods, were compared in nine paired studies with regard to platelet harvest, white cell (WBC) contamination, and PC quality after 5 days of 22°C storage. Platelet harvest using the BC method averaged approximately 56 percent of the whole blood level (6.2 x IO'O/concentrate), which was less than the 76 percent achieved with the PRP-PC method (8.7 x 10'Yconcentrate). An additional 5 units collected into an experimental siphon bag for BC-PC processing showed improved platelet harvest (6.7 x IO'O/concentrate, or approx. 70% of whole blood). WBCs remainin in the BC-PC averaged 0.19 x lo8 per unit compared to 3.6 x 10' per unit for RP-PC. Buffy coat processing produced red cell (RBC) units with 50 percent of the WBC contamination of conventionallyprepared units (9.8 +. 6.2 x 108/unit vs. 18.9 ? 7.1 x 108/unit). The siphon bag further reduced WBC levels in the AS-3 RBC units (6.4 +. 3.7 x 108/unit).In vitro studies performed on Days 1 and 5 after collection showed no significant differences in platelet metabolic and biolo ic function or cell integrity. p-thrombo lobulin and surface glycoprotein levels, indcators of platelet activation and mem lane alteration, respectively, did not differ significantly in the PRP-PC and BC-PC; nor was lactate production higher in PRP-PC, despite the substantially higher WBC counts. Autologous in vivo platelet viability determinations were performed by using concurrent transfusion of ''' In-labeled freshly drawn platelets and "Cr-labeled stored latelets. Paired t test analysis of BC-PC versus PRP-PC indicated no significant dilerences in platelet recovery and survival after 5 days of 22°C storage in polyolefin containers. Therefore, these studies confirm the equivalence of PC quality, comparable platelet harvest with the siphon bag, and decreased WBC contamination with the BC method. TRANSFUSION 1992;32:113-120.
Abbrevlatlons: BC(s) = b u m coat@); GPlb = glycoproteln Ib; LDH = lactate dehydrogenase; PC(s) = platelet concentrate(8); PRP = platelet-rlch plasma; WBC(s) = whlte cell(s).
IN EUROPE, SEVERAL blood centers now routinely prepare platelet concentrate (PC) from buffy coat (BC),lp2 rather than using the standard United States technique of preparing PC from platelet-rich plasma (PRP).3*4Buffy coat platelet concentrate (BC-PC) offers several potential advantages. Although platelet yield is generally lower than that from PRP-derived concentrate (PRP-PC), the BC-PCs have been shown to contain a lower number of contaminating white cells ( W C S ) , ~which - ~ may result in decreased transfusion reactions and reduced potential for transfusion-induced alloimmunization. Furthermore, reduced WBC contamination is also reported to result in improved PC quality after s t ~ r a g e . ~Some . ' ~ studies have
suggested that above lo7 WCs per unit, there was a correlation between the W C concentration and pH fall caused by increased glycolysis, platelet loss of glycoprotein Ib (GPIb), and lactate dehydrogenase (LDH)relea~e.~*ll However, other studies, including one in this laboratory using filtered, WBC-reduced PRP-PC units, have not confirmed this relationship between W C contamination and loss of platelet quality.12J3 Some studies have suggested, moreover, that platelet aggregation is improved and activation is decreased in BC-PC, which could improve the hemostatic function and viability of stored platelet^.'^ To obtain more information about differences between these platelet separation techniques with regard to platelet yield, WBC contamination, and platelet quality (with an emphasis on in vivo viability), we performed paired in vivo and in vitro studies on BC- and PRP-PCs collected from the same donor on two occasions. We used "Cr to label the 5-day stored platelets. In addition, in
From the American Red Cross, Mid-Atlantic Regional Blood Sewices, and Eastern Virginia Medical School, Norfolk. Virginia, and Cutter Biological, Berkeley, California. Supported by Cutter Biological, Miles, Inc., Berkeley, California. Receivcd for publication April 5, 1991; revision received July 23, 1991, and accepted August 1, 1991.
KEEGAN ET AL.
vivo platelet viability was evaluated by using concurrent transfusion of lllIn-labeled freshly drawn platelets with 5’Cr-labeled stored platelets. Previous studies in this laboratory have indicated that there is significant intradonor variability in platelet recovery and survival studies. Comparison of the viability of stored platelets and that of freshly drawn platelets permits evaluation of small differences between test and control in paired studies. Standard CP2D/AS-3 quadruple blood bag systems were used for collection and processing, except that the BC fraction was isolated into a collection bag (Neocel, Cutter Biological, Berkeley, CA).15 This system was similar to that used by others, in which a smaller container for BC centrifugation was used to improve platelet harvest.I6 We also performed a follow-up in vitro study using a siphon container to determine if the aspiration of RBCs from the bottom of the primary pack, followed by BC separation in a small container, would allow for platelet harvest levels comparable to those of conventional PCs.
Materials and Methods Study design On two occasions at least 28 days apart, we collected 18 standard whole blood units (471 2 16 mL) from nine donors into primary blood bags containing 63 mL of CP2D. All donors met conventional criteria of the standards of the American Association of Blood Banks and were not receiving any drug therapy that might affect platelet function. All volunteers con-
Vol. 32, No. 2-1992
sented to participate in the study, which had been approved by the Institutional Review Board of the Eastern Virginia Medical School (Medical College of Hampton Roads). We prepared one-half of the first group of units donated by the PRP technique (control) and the remainder by the BC method (test) of Pietersz et al.” as modified by Cutter Biological, Miles Inc. (Berkeley, CA). The RBC units stored in additive solution (AS-3, Nutricel, Cutter Biological) were transfused to the volunteers on the day of phlebotomy. The preparation protocol was reversed for each donor with the second unit.We collected an additional 5 units from volunteer donors for in vitro studies of BC-PCs prepared by using an experimental siphon bag collection system developed by Cutter Biological (Fig. 1). Control PRP-PCs were prepared according to standardized procedures described previously.’s Previous studies in this laboratory have indicated that platelet in vivo studies are influenced by intradonor variability that hinders the resolution of small differences in posttransfusion viability. The inconsistency between donations from a single donor can be minimized by concurrently comparing stored platelets to freshly drawn cells, using T r and I1*Inoxine.
Preparation of BC-PCs (test) The standard bag configuration of units designated for processing according to the test protocol was altered before processing by sterile connection of a Neocel collection bag between the primary container and one of the polyolefin platelet storage containers (CLX, Cutter) with a sterile connection device (SCD 312, Haemonetics, Braintree, MA). We held the whole blood units at room temperature for 4 hours before centrifuging them at 2977 x g for 9 minutes at 22°C using a centrifuge with swinging bucket rotor (KR4.22, Jouan, Winchester, VA) to separate the RBCs, BC, and plasma. The plasma was expressed to within 1 cm of the BC interface into a satellite
Sterile docking after plasma removal
Bag OIiginal status Plastic material Recessing
cornponent Final component
Neocel Empty PVC Resuspended BC BC-sediment
PVC &ern ight BC hold
FIG. 1. Schcrnatic of the experimental siphon bag system for the preparation of buffy coat platelet concentrate (BC-PC) from CPZD whole blood units.
PLATELET CONCENTRATE FROM BUFFY COAT
1992-Vol. 32. No. 2
Table 1. Com~onentharvest with different methods of platelet concentrate separation ~~
CP2DPRP-PCS~ BC-PCsll BC-PCs (siphon bag)
( x lO'O/unit)
531 f 21 538 2 8 506 2 29
359 42 266 -c 18ll 262 f 20
180 & 33 236 2 197 229 2 14
66 '. 14 58 d 14 57-c 6
8.7 f 1.6 6.2 6.7 ~t 2 i.9n 1.3
* Mean f SD. t Red cells. Platelet concentrates. 5 Platelet-rich plasma-PCs. II Buffy coat-PCs. 7 Significant difference, p5.5 x l0l0/unit). Review of the standard bag studies found that the 3 units that had total platelets below the standard3' had BC fraction hematocrits of 34 to 38 percent (0.34-0.38). In Table 2, the in vivo multiple-hit recovery and survival results (mean 2 SD) are listed for both the l*lIn-labeledfreshly drawn platelets and the Tr-labeled stored platelets. The Kruskal-Wallis nonparametric statistical test showed no effect of the order of collection on any of the in vivo results. Paired r test analysis showed no significant differences in recovery or survival in the BC-PC (test) and PRP-PC (control) units. Relative recoveries and survivals, the ratio of the T r and "'In results, were not significantly different in the test and control units. The relative percentage of recoveries and survivals for each of the donors is also shown in Table 2. Shown in Fig. 3 is the paired comparison of the concurrent "'In and T r recoveries. Although there was a significant correlation between ltlIn recovery on the two occasions (r = 0.411, p