Effects of prestorage white cell-reduction of apheresis platelets on platelet glycoprotein Ib and von Willebrand factor G.I. GARCIA,J.E. FITZPATRICK, L.A. HOERNIG, C.C. STEWART,AND J.D. SWEENEY Twenty plateletpheresis components were harvested from 11 health donors and stored in polyolefin bags on a horizontal flatbed agitator at 22°C. Aier 24 hours, white cells were reduced in one aliquot by centrifugation while the other aliquot was stored unaltered. Samples were obtained aseptically from each of these platelets at intervals for up to 10 days, and measurements were made of platelet glycoprotein Ib (GPlb) by both flow cytometry and polyacrylamidegel electrophoresis, of ristocetininduced platelet aggregation by impedance aggregometry,and of plasma and platelet von Willebrand factor (vWF) by enzyme-linked tmmunosorbent assay. Storage of platelets under these conditionswas associated with only minor decreases in surface GPlb, intraplatelet vWF, and ristocetin-induced platelet aggregation, and no differences were observed between the white cell-reduced and nonreduced aliquots. No benefit of white cell reduction in such components before prolonged storage is evident in the vWF-platelet interaction. TRANSFUSION 1992;32:148-151. Abbreviations: GPlb = glycoproteln Ib; PA9 = phosphate-buffered saline with albumin; SDS-PAGE = sodium dodecyl sulfate-polyacrylamldegel electrophoresis; vWF = von Wlllebrand factor; WBC(s) = white cell(s).

PLATELET TRANSFUSION has an important role in supportive care in such diverse areas of patient care as cancer, transplantation, cardiac surgery, and trauma.' Platelet components are produced from single-donor apheresis procedures or from a pool of platelet concentrates derived from whole blood donations. The platelet yield of each component varies greatly, as does the degree of white cell (WBC)and red cell contamination. Thus, even at the time of production, platelet components show marked heterogeneity in cellular content. Platelets intended for transfusion are stored in autologous plasma with agitation at 22°C. Controversy surrounds the optimal means of agitation, but conventionally, these are horizontal flatbed, elliptical, or rotary agitators.* The current maximum storage period is 5 days.3 Alterations are known to occur, however, during this storage per i ~ d Platelets .~ undergo a change from discoid to spherical shape,5 and both the platelet count and pH decrease.'j Some of these changes may reflect normal aging processes for platelets, but they could also indicate storage injury effect^.^ Although there have been several studies on the nature of these changes, the clinical implications remain

Glycoprotein Ib (GPIb) is an important membrane glycoprotein; approximately 25,000 to 36,000 molecules per cell are expressed on the surface of each platelet. GPIb consists of two chains linked by disulphide bonds: GPIb a (132 m a ) , and glycoprotein Ib p (23 kDa). GPIb a contains most of the sialic acid on the platelet surface12and also is the binding site for von Willebrand factor (vWF).13 It is considered that the binding of this ligand to GPIb is the most significant event in the adhesion of platelets to exposed subendothelium and the initiation of the hemostatic plug.14 Thus, preservation of this site on stored platelets would appear to be critical, if hemostatic competence is to be retained. Several studies15-17have attempted, with conflicting results, to quantify the GPIb changes associated with storage. One study suggested the existence of an intracellular pool of GPIb, which is expressed on the surface during storage.I7 While this hypothesis may be correct, it is unclear whether the expressed protein represents intact protein or the epitope that binds vWF. The aims of this study were to characterize changes in GPIb and vWF in apheresis platelets stored in polyolefin bags and to examine the effect of reducing WBC contamination from the plateletpheresis components prior to storage.

From the Departments of Laboratory Medicine and Flow Cytometry, Roswell Park Cancer Institute, Buffalo, New York. Supported in part by a grant from Cutter Biologicals, Berkeley, California. Received for publication August 2, 1990; revision received July 17, 1991, and accepted August 1, 1991.

Materials and Methods Twenty plateletpheresis components were obtained from 11 healthy volunteer donors. Informed consent was obtained from each donor. We harvested each component with a cell separator

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149

GLYCOPROTEIN Ib IN STORED PLATELETS

and time saver with autosurge (V50, Haemonetics Corp., Braintree, MA) with ACD-A as the anticoagulant and stored them in polyolefin bags (CLX, Cutter Biologicals, Berkeley, CA). A flatbed agitator at 22°C was used for storage. On the day of harvest (Day 0), a sample was obtained aseptically. After 24 hours, the component was equally divided into two CLX bags. We reduced the WBCs in one bag by using a WBCreduction bag (Leukotrap, Cutter Labs) and centrifugation at 550 x g for 10 minutes. We measured WBC content before and after WBC reduction. The other bag was left undisturbed. Samples were obtained under sterile conditions from each bag on Days 1, 3, 5, 7, and 10. We measured platelet count, flow cytometric analysis of GPIb, and impedance aggregation response to ristocetin. On Days 5 and 10, we also obtained samples for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and vWF antigen determination in plasma and platelets.

Platelet count and flow cytometry We added an equal volume of 1 percent paraformaldehyde to the platelet sample volume, stored the sample at 4°C for 24 hours, and then washed it twice with phosphate-buffered saline with albumin (PAB). The platelet count was determined (ELT8DS, Ortho, Raritan, NJ) and PAB was added to a final platelet count of 200 x lo9 per L. To a 50-pL aliquot, we added 10 p L of a 1:3 dilution of mouse monoclonal antibody to GPIb, obtained from Accurate Chemical and Scientific Corporation (Catalog #AXL842M; Westbury, NY). This specific antibody, termed AN51, belongs to the IgG2a K class and binds to an epitope on GPIb.I8 After incubation for 15 minutes at 22"C, the sample was washed twice with PAB. We then added 10 p L of a 1:3 dilution of fluorescein-conjugated goat anti-mouse antibody (CALTAG, San Francisco, CA). After a similar incubation and wash, we processed the sample in a flow cytometer (FACScan, Becton Dickinson, Lincoln Park, NJ). We measured 2500 individual cells for each sample. Negative controls consisted of platelet preparations to which the monoclonal antibody was not added; they were processed simultaneously with the samples to correct for nonspecific antibody binding.

performed densitometry to measure the GPIb content of the gel (Quick-Scan Junior PLC, Helena Labs, Beaumont, TX).

vWF antigen determination We measured the vWF antigen levels in platelet-poor plasma and in the platelet supernatant obtained for gel electrophoresis by using an enzyme-linked immunosorbant assay (American Bioproducts, Parsippany, NJ). The standard used in this assay was calibrated against the International Reference Plasma for Factor VIIIhWF (87/718). Values for plasma were expressed as U per dL and those for platelets as U per lo9 platelets.

Statistical Tests We performed statistical analysis by the t test. An arbitrary p level of ~ 0 . 0 1was chosen as significant.

Table 1. Characteristics of plateletpheresis harvests before and after white cell (WBC) reduction* Mean Platelet yield before WBC reduction ( x 10") Platelet yield after WBC reduction ( x 10") WBC yield before WBC reduction ( x lo8) WBC yield after WBC reduction ( x loa) Harvest volume (ml)

2

SD

4.3 f 0.7

1.5 2 0.4' 12.2 -c 7.0 1.0 f 0.8 409.0 f 61.0

Yield is one-half the original yield (i.e., the contents of one (M bag) after WBC reduction.

100

1

Impedance aggregation to ristocetin We adjusted the platelet count of the sample to 200 x lo9 per L using phosphate-buffered saline and studied it in an impedance aggregometer (Chronolog Model 560, Chronolog Corp., Haverstown, PA). We added 10 pL of ristocetin (1 mgl mL), stored the resulting impedance aggregation curve in a computer file, and analyzed it as the area under the curve by using a software program (Asystant Plus, Rochester, NY).

SDS-PAGE We first standardized the platelet sample to an absolute count of 2 x lo6 platelets and then washed it twice with PAB (2000 x g for 5 minutes at 22°C). After resuspending the platelet pellet in 1 mL of PAB, we added an equal volume of 2 percent Triton x 100 (Sigma Chemical Co, St. Louis, MO). The sample was then stored immediately at -30°C. We subsequently thawed all the samples and centrifuged them (39,000 rpm for 1 hour at 4°C) to obtain a platelet pellet and supernatant. After thawing, the samples were electrophoresed by SDS-PAGE using 7 percent gels at 220 V for 30 minutes, according to the method of Laemrnli.l9 We then stained the gels with a protein stain (ElectroSep, Collaborative Research Inc, Bedford, MA), stored them in 25 percent ethanol, and

1

3

5 Days

7

10

FIG. 1. Glycoprotein Ib-positive platelets as determined by flaw cytometry. White cell-nonreduced, m,white cell-reduced, 0.

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GARCIA ET AL.

Table 2. Plasma and platelet von Willebrand factor (vWF)* Platelet vWF (U/1O0 plateletddl)

Plasma vWF (U/dL) WBC-

WBC-

WBC-

Day

nonreduced

reduced

nonreduced

0 5 10

123 141 '. 47 I36 2 44

?

45 137 -t 36 138 z 44

'Values expressed as mean

90

WBCreduced

13 f 8 12 2 5 11 2 6 13 f 5 12 ? 7

* SD.

L

70

1

I

I

I

1

3

5

1 7

1 10

Days

FIG. 2. Changes in ristocctin-induced impedance aggregation with storage (days), with arbitrary units uscd to measure the area under the

aggregation curve. White cell-nonreduced, M; white cell-reduced, A---A.

Results Table 1 shows the characteristics of the plateletpheresis components used in the study. WBC reduction resulted in a 90 percent reduction in WBC contamination and about 30 percent decrease in platelet yield. The mean volume of each component was 409 mL. Flow cytometry results are summarized in Fig. 1. GPIb expression on the platelet surface decreased with time, although the decrement was small and gradual for both WBCreduced and WBC-nonreduced groups. Statistical analysis showed no difference between the two groups (p>O.OI). Table 2 shows vWFmeasurements. The vWFantigen, measured in platelet-poor plasma and in platelet supernatant, did not show differences between WBC-reduced and WBC-nonreduced groups (p> 0.01) Densitometric analysis of GPIb in SDS-PAGE gels likewise showed no significant difference between the two groups (p>O.Ol; data not shown). The data for platelet impedance aggregation to ristocetin are shown in Fig. 2. The total area of the aggregation curve expressed in arbitrary units was used as a measure of the degree of aggregation. As can be seen, there was a progressive decrease in aggregation with storage but no significant difference between the two groups.

Discussion

Several studies have investigated the nature and importance of WBC contamination in platelet

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c~ncentrates.~O-~~ In most cases, the predominant WBCs are lymphocytes and monocytes, with about 5 percent neutrophils."." Neutrophils are considered to be detrimental to platelet storage because they disintegrate within 2 days and release enzymes that can damage or destroy platelets.26 Lymphocytes, on the other hand, have been proven to be viable for up to 10 days.= Gottschal122 showed that an inverse relationship exists between pH and WBC count, with WBC-reduced platelets having less glucose consumption and production of lactic acid. Sloand and Klein26also studied the effect of WBC contamination on plateletpheresis components. In their study, WC-reduced platelet components were compared to platelet concentrates to which either autologous neutrophils or lymphocytes were added. The components that were neutrophil-enriched had a significantly reduced aggregation response to ristocetin, as compared to that of the lymphocyte-enriched or WBC-reduced components. SDS-PAGE analysis showed decreased amounts of GPIb in the neutrophil-enriched platelet components. The authors concluded that neutrophils in platelet components cause enzymatic proteolysis of GPIb. Although that study is important in that it indicated that neutrophils are an undesirable contaminant in platelet components, most plateletpheresis concentrates already contain less than 5 percent neutrophils, and it is unclear if a further decrease in the neutrophil contamination is of any benefit for platelet viability or function. Altered expression of membrane glycoproteins unrelated to WBC content has been studied in stored platelet^.^^-^^ D h a P used SDS-PAGE to prove that there was a progressive loss of GPIb with storage. However, Taylor,27using lectins and a monoclonal antibody against GPIb, showed that there was no loss of GPIb from the platelet surface after 3 days. George29obtained similar results in studying platelets over 7 days. Michelson17 studied platelet concentrates for 5 days by using flow cytometry and SDS-PAGE techniques and concluded that there is a large intraplatelet pool of GPIb that, with storage, redistributes from intracellular pools to surface expression. Our study analyzed GPIb by techniques similar to Michelson's, although we used plateletpheresis harvests rather than platelet concentrates prepared from whole blood donors and stored in polyvinylchloride bags (PL732, Fenwal). Our flow cytometry data showed that, over a period of 10 days, GPIb-positive platelets gradually decreased. Similar results were obtained by Michelson in that a population of GPIb-negative platelets increased with storage. In our study, WBC reduction did not influence surface GPIb expression. Our data on SDS-PAGE analysis of GPIb showed no measurable change over 10 days. The decrease observed by flow cytometry was slight, however, and it is possible that SDS-PAGE is not sensitive enough to detect this

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GLYCOPROTEIN Ib IN STORED PLATELETS

1’)92--VoI. 32. No. 2

degree of change. Moreover, bands consistent with GPIb were not altered on storage in either preparation; thus, a redistribution of GPIb from an intraplatelet pool is an unlikely explanation of the good preservation of surface GPIb. Our data on ristocetin aggregation showed a gradual decrease with time (about lo%), which is in approximate proportion to the loss of GPIb from the stored platelets. Because ristocetin aggregation is a measure of both vWF and GPIb integrity and inasmuch as plasma vWFdid not change with time, (Table 2), it seems logical to conclude that the decreased ristocetin aggregation is secondary to the storage-related decrease in GPIb-positive platelets. Our study shows that only a minor change occurs in intraplatelet vWF or in surface GPIb in apheresis platelets stored in plasma. Removal of contaminating WBCs by centrifugation did not affect these minor changes. Although greater degrees of W C reduction are possible with WBC-adhesion filtersY3’it was considered that a 1 log,,, reduction in WBCs should indicate a difference, if an influence were present. It is not possible to exclude WBC-mediated changes, but we believe that our data are in agreement with those of Sloand and Klein,26who found that autologous mononuclear cells in the ranges observed in apheresis platelet components contribute little, if anything, to alterations in GPIb on storage.

151

11. Snyder EL, Koerner TA Jr, Heuey A. Platelet concentrates. In12. 13. 14.

15. 16. 17.

18. 19. 20. 21.

22.

fluence of different preparative protocols on the in vitro release reaction. Vox Sang 1982;43:71-5. Okumura T, Lombart C,Jamieson GA. Platelet glycocalicin. 11. Purification and characterization. J Biol Chem 1976;251:5950-5. Okumura T. Jameison GA. Platelet glycocalicin: a single receptor for platelet aggregation induced by thornbin or ristoceh. Thromb Res 1976:8:701-6. Bolhius PA, Sakariassen KS, Sander HJ, Bouma BN, Sixma JJ. Binding of factor VIII-von Willebrand factor to human arterial subendotheluimprecedes increased platelet adhesion and enhances platelet spreading. J Lab Clin Med 1981;97:568-76. Dhar A, Ganguly P. Altered expression of platelet surface glycoproteins during storage. Br J Haematol 1988;70:71-5. Sloand E, Klein HG. Effect of leukocytes on platelet function and composition during storage (abstract). Blood 1988;8:285a. Michelson AD, Adelman B, Barnard MR, Carroll E, Handin RI. Platelet storage results in a redistribution of glycoprotein Ib molecules. Evidence for a large intraplatelet pool of glycoprotein Ib. J Clin Invest 1988;81:1734-40. McMichael AJ, Rust NA, Pilch JR. et al. Monoclonal antibody to human platelet glycoprotein I. I. Immunological studies. Br J Haematol 1981;49:501-9. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;277:680-5. Taylor MA, Tandy NP, Fraser ID. Effect of new plastics and leukocyte contamination on in vitro storage of platelet concentrates. J Clin Pathol 1983;36:1382-6. Snyder EL, Ezekowitz MD, Malech HL. et al. In vitro characteristics and in vitroviability of platelets contained in granulocyteplatelet apheresis concentrate. Transfusion 1987;27: 10-4. Gottschall JL, Johnson VL, Rzad L, Anderson AJ, Aster RH. Importance of white blood cells in platelet storage. Vox Sang

1984;47:101-7. 23. Skinnider L, Wrobel H, McSheffrey B. The nature of the leuco-

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Acknowledgments The authors thank the following people for technical assistance: Doris Burnett, Dee Jordan, Jim Labuzetta, Eugenia Masferrer, Amy Michnik, Ed Novak, and Richard Swank.

1985;49:309-14. 24. Aisner J, Schiffer CA, Wolff JH, Wiernik PH. A standardized 25.

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Godofredo 1. Garcia, MD, Fellow, Department of Laboratoly Medicine. John E. Firzpatrick, MD. Chief, Department of Laboratory Medicine. Lynne A. Hoernig, MS, Cancer Research Scientist, Department of Laboratory Medicine. Carleton C. Stewart, PhD, Chief, Department of Flow Cytometry, Roswell Park Cancer Institute. Joseph D. Sweeney, MD, Medical Director, Transfusion Service. Department of Laboratory Medicine, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. [Reprint requests]