Activation during preparation and storage of platelet concentrates The Holy Grail was the chalice used at the Last Supper. The search for the Grail has occupied mankind for generations. There is another grail, however, one that has occupied researchers in transfusion medicine for decades. This one, clearly less ecclesiastical but nevertheless sought after with equal fervency, is a simple in vitro assay that will accurately and consistently predict in vivo platelet function and survival. Platelet activation and its offspring, the platelet storage lesion, are terms that encompass the myriad changes that occur during collection, preparation, and storage of platelet concentrates.' Rather than being a function of a physiologic change, as with in vivo platelet activation, activation occurs in transfusion medicine as an artifact of the storage of platelets in plastic bags for future transfusion to thrombocytopenicpatients. It is a goal of transfusion medicine to attempt to collect, prepare, and (after up to 5 days of storage) transfuse these platelets in what is essentially their original condition. From the moment platelets are collected, they are subjected to a variety of shear stresses, exposed to various pH gradients, encased in various plastic bags, and tumbled end-over-end for up to 120 hours. Eventually, they are transfused to an in vivo environment where they are expected to function as if nothing much had happened during the past week. Actually, platelets function remarkably well after such manipulation. Published data have shown that platelets stored up to 5 days correct bleeding times, stop hemorrhage, and show acceptable ~urvival.~.~ To characterize a platelet's status during its transient stay in vitro, a series of assays have been developed. These include pH; platelet count; release of cytosolic lactate dehydrogenase as a marker for platelet lysis; release of P-thromboglobulin (P-TG), platelet factor 4, or serotonin as a marker for granule release; morphology score; response to hypotonic shock (osmotic recovery); metabolic assays including lactate production and glucose consumption; and measurements of pOz, pC02, and HC03.' Recently, mean platelet volume has been suggested as another useful assay.4 The new kid on the block is the measurement of P-selectin (CD62, GMP-140, PADGEM).S.6 P-selectin is present on the internal surface of the a-granule membrane. When the platelet undergoes the release reaction, the a-granule membrane binds to the surface canalicular system, and P-selectin appears on the external platelet surface where it can be detected by flow cytometry using specific monoclonal antibodies.

Simultaneously with P-selectin expression, P-TG appears in the platelet-poor plasma. Measurement of the percentage of release of P-TG as a marker of platelet activation during collection, preparation, and storage of platelet concentrates was reported by our group in 1981.'** We found that P-TG release increased as the platelets were subjected to shear stresses induced by preparative centrifugation and subsequent resuspension. We also examined the release of P-TG in single-donor and randomdonor platelets and found that single-donor platelets showed less P-TG release, probably as a result of the lower shear forces encountered during preparation. Correlation of P-TG release with ll'In-radiolabeled 2-hour platelet recovery was calculated in our laboratory for platelets stored in one of 11 combinations of platelet storage conditions involving three plastic storage bags and four types of platelet agitators (n= 72). Analysis of the correlation coefficients for in vitro release of P-TG versus 2-hour percentage of "'In-radiolabeled recovery in normal volunteer donors showed an r value of less than 0.75 for each combination (?=0.56).9 Thus, at best, only 56 percent of the variability in 2-hour radiolabeled recovery was predicted by percentage of P-TG release; conversely, 44 percent of the variability was not predicted. Thus, other unknown factors must have been playing a significant role in determining in vivo platelet survival. Rinder et al.1° transfused platelet concentrates with known percentages of P-selectin-positive platelets to thrombocytopenic patients and examined the levels of activated platelets in those patients before and after transfusion. They found a lower-than-predictedrecovery of those activated platelets, averaging only 38 percent of the levels predicted on the basis of the assumption that P-selectin-positive and P-selectin-negative platelets would survive equally well. They concluded that platelets expressing P-selectin are preferentially cleared from the circulation after transfusion. In this issue of TRANSFUSION, Triulzi and colleagues" examined the detection and significance of P-selectin (GMP-140) expression on platelets collected by apheresis. They investigated the usefulness of measuring GMP-140 expression on apheresis platelets to determine whether this value correlated with platelet function and viability. They concluded from their data that measurement of GMP-140 may serve as a useful in vitro measurement of the quality of platelet components, and that this assay may be useful in quality control programs.



1992-Vol. 32, No. 6


Close evaluation of Fig. 2 in their paper shows that the correlation coefficient generated (r = - 0.58) was determined in large part by three points on the curve, one with about 7 percent GMP-140 expression and two with about 53 percent GMP-140 expression. Their r value for a 1-hour corrected count increment of -0.58 produced an 9 value of 0.34. This means that 34 percent of the variability of their 1-hour corrected count increment value was explained by the percentage of expression of GMP140 (P-selectin); however, 66 percent was not explained and was likely due to other factors. This degree of correlation is, not unexpectedly, similar to that found in studies performed a decade ago involving P-TG r e l e a ~ e . ~ . ~ It is not known what other factors influence in vivo survival of activated platelets. P-selectin on the surface of activated platelets has been demonstrated to function as a ligand for neutrophils and rnonocytes.l2 P-selectinpositive platelets might be preferentially removed from the circulation after transfusion, perhaps being cleared by the mononuclear-phagocyte system as bound conjugates with white cells. Rinder et al.IO examined the relationship between P-selectin expression and in vivo "'Inlabeled platelet recoveries in normal subjects. Platelets stored under standard blood bank conditions for 2 to 4 days were subsequently examined for P-selectin expression, labeled with "'In, and transfused to the autologous donors. Platelet recoveries at 1 hour demonstrated an inverse correlation with the percentage of activated platelets, that is, those expressing P-selectin (r = - 0.55; p

Activation during preparation and storage of platelet concentrates.

Activation during preparation and storage of platelet concentrates The Holy Grail was the chalice used at the Last Supper. The search for the Grail ha...
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