Brief Reports

Ultrastructural Alterations and Phagocytic Function of Cryopreserved Platelets J. I. SPECTOR, W. J. FLORA N D C. R. VALERI From the Naval Blood Research Laboratory, Boston, Massachusetts and the Armed Forces Radiobiology Research Institute. Bethesda, Maryland

Fresh human platelets and platelets cryopreserved in 4% dimethylsulfoxide were examined ultrastructurally before and af€er incubation in a suspension of latex particles. Cryopreservedplatelets had fewer discoid forms than fresh platelets. The cryopreserved platelets had many sphered plateletscontainingan increased number of vacuoles; the sphered platelets were more electron-lucent with margination and pallor of organelles. Phagocytosis of latex into vacuoles was markedly impaired in the cryopreserved platelets. The morphologic alterations suggested that the freezing, thawing, and washing procedures reduced the functional activity of platelets.

tivity of cryopreserved human platelets and found that cryopreserved platelets had markedly impaired phagocytic function when compared with fresh platelets from platelet-rich plasma (PRP) or platelet concentrates (PC). Materials and Methods

of Plutelet Concentrate From ten healthy volunteers, 20 to 30 years of age, 450 ml of blood were collected in 63 ml of HUMAN PLATELETS remain intact during citrate-phosphate-dextrose(CPD) anticoagulant. The blood was centrifuged at 4500 X g for three freeze-preservation with dimethylsulfoxide minutes at 22 ? 2 C. The platelet-rich plasma (DMSO), but undergo significant ultrastructural alteration after thawing and ~ a s h i n g . ~ (PRP) was expressed, and the platelets were concentrated by centrifugation at 4500 x g for five Previous reports from our laboratory deminutes. The concentrated platelets were stored scribed abnormalities in platelet aggregaundisturbed at 22 C for one hour in 30 ml of plasma to prevent the clumping that sometimes tion, release reactions, nucleotide content, occurs upon resuspension.ll An additional 20 ml oxygen consumption, and activity of plateof autologous CPD-plasma was added to the let factors 3 and 4 in human platelets after platelet concentrate to facilitate resuspension.

freeze-preservation with 4 or 5% DMSO at 2 to 3 C per minute, storage for up to ten months at -80 C, and washing.''.18 Lewis et al.9 recently demonstrated through ultrastructural study the uptake and digestion of polystyrene latex particles by fresh human platelets. We used latex particles to investigate the phagocytic ac-

Received for publication April 11, 1978; accepted May 24, 1978. Supported by the U.S. Navy (Naval Medical Research and Development Command Research Task No. M0095-PN-001.0040) and the Defense Nuclear Agency (MF10103). The opinions or assertions contained herein are those of the authors, and are not to be construed as official or reflecting the views of the Navy Department or Naval Service at large.

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Preparation

Freeze-Preservution Technique

The platelets were freeze-preserved as previously described."*'" To achieve a final concentration of 4% DMSO, 50 ml of 8% V/V DMSO in plasma was added to the platelet concentrate over five minutes. The platelet-DMSO-plasma mixture was transferred to a polyolefin plastic bag* and frozen to -80 C at approximately 2 to 3 C per minute. Forty-eight hours after freezing, the platelets were thawed with agitation by immersion in a 37 C water bath, transferred to a 300 ml transfer pack, and diluted with 100 ml of 1.7% DMSO in plasma and 20 ml of acid-citrate-dextrose (ACD, NIH, Formula A) anticoagulant. After centrifugation at 4500 x g for five minutes, the supernatant fluid was re-

*

Union Carbide, New York, NY.

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moved and the platelets were resuspended in 30 ml of autologous plasma. Platelet Phagocytosis of Latex Particles Each of the blood products, fresh plateletrich plasma, fresh platelet concentrates, and previously frozen washed platelet concentrates, was warmed in a 37 C water bath for 10 minutes before testing for platelet phagocytosis of latex particles. About 50 microliters of a 0.5% latex suspension in distilled water (0.087 micron average diameter, styrene butadiene)t were then added to each 1 ml of platelet suspension. The final concentration of latex in the suspension was 0.02%. The tubes were gently agitated to mix the contents and were returned to the 37 C water bath for 60 minutes of incubation. Platelet Morphology By Electron Microscopy Platelet suspensions were fixed by the dropwise addition of an equal volume of 4.5% glutaraldehyde buffered with 0.05 N Na cacodylate containing 10% sucrose over several minutes. Fixation was allowed to continue in suspension for I5 to 30 minutes at 22 C, after which specimens were centrifuged at 3,000 rpm for 10 minutes to pellet the platelets. Fixation was continued for an additional 30 minutes at 22 C after pellet formation. Fixative was decanted and gently replaced with 0.05 N Na cacodylate buffer containing 10% sucrose, and the specimens were stored at 4 C. After treatment overnight with 1% OsO, in 0.05 N Na cacodylate containing 10% sucrose at 4 C, specimens were dehydrated and embedded in Epon 812 by standard techniques for examination by electron microscopy. Ultra-thin sections were cut on a Reichert OmU3 ultramicrotome, doubly stained with uranyl acetate and lead citrate, and examined in either a Siemens Elmiskop I or a Philips EM400 HMG electron microscope.

Results Platelet Ultrastructure Discoid and sphered forms were present in the fresh PRP in two subjects and in the fresh platelet concentrates in four subjects (Fig. 1). Approximately 40 to 50 per cent of the platelets were discoid. These were more electron-dense with tightly arranged cytoplasm and organelles than the sphered platelets which were less elect Dow Diagnostics, Midland, MI.

Transfusion May-June 1979

tron-dense and more loosely arranged, with occasional irregular pseudopods. Cryopreserved platelets showed sphering in approximately 50 to 60 per cent of the cells and vacuolization, increased variability of organelle morphology with very little in the way of filaments or tubules, margination of organelle remnants in extremely electron-lucent platelet remnants, and greatly increased amounts of cell debris (Fig. 2). Platelets with normal structural integrity were found adjacent to exploded remnants. Granules and mitochondria appeared more electron-lucent in some of the cryopreserved platelets than in fresh platelets, implying a loss of content to the cytoplasm or extracellular milieu. Platelet Phagocytosis In the PRP in two subjects and in the fresh platelet concentrates in four subjects, the addition of latex resulted in large accumulations of latex particles adherent to the platelet membrane and latex particles in cytoplasmic vacuoles (Fig. 3). Frozen washed platelets demonstrated greater interplatelet adhesion than did fresh platelets but, in general, suspension of platelets was good. Organelles were well preserved, especially granule and microvesicle integrity. In four studies, the addition of latex resulted in markedly impaired phagocytosis in cryopreserved platelets compared with fresh platelets. There was occasional phagocytosis of latex particles in the well preserved platelets, with virtually no latex particles in deteriorating ones (Fig. 4). Some of the normal-appearing platelets had phagocytized clumps of latex particles. Centrifugation of the platelet rich plasma to prepare platelet concentrates produced a greater number of sphered platelets in platelet concentrates than in platelet-rich plasma, but phagocytic activity was not significantly different.

Discussion

Crowley et ~ 1determined . ~ by ultrastructural visualization that platelets frozen with 6% DMSO at 2 to 3 C per minute and stored at -80 C had more morphologic damage than fresh platelets. It was suggested that adverse local conditions in certain areas of the freezing container may damage a portion of the platelet popu-

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FIG.1. Top, electron micrograph from a specimen of fresh platelet concentrate. Most platelets were of good discoid morphology, displayed moderate cytoplasmic electron density, and contained the standard component of subcellular organelles. t = circumferential tubule bundle (edge cut), m = mitochondria, g = granules, c = canalicular system elements and e = exterior fuzzy coat (original magnification x 15,500). FIG. 2. Bottom, electron micrograph from a specimen of platelets from a frozen, thawed, washed platelet concentrate. Many of the cryopreserved platelets were sphered, exhibited less electron density of the cytoplasm, were internally disorganized, and only a rim of marginated organelles remained. Some platelets retained normal morphology (center and bottom of figure). Cellular debris was present in these specimens, and the freeze-preserved platelets appeared to engulf the debris (top center of figure) (original magnification x 15,400).

lation while sparing others. Our previous investigation on the physiologic activity of platelets cryopreserved with 4 or 5% DMSO

at 2 to 3 C per minute and stored at -80 C showed a significant decrease in intracellular nucleotide content and diminished re-

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Transfusion May-June 1979

FIG.3. Top, electron micrograph from a specimen of fresh platelet concentrate to which latex particles had been added. Platelets were generally well-preserved and were observed in active phagocytosis. Latex was found adherent to the platelet membrane, in the invaginations of the platelet surface, and within the platelet cytoplasm in vacuoles of various sizes (arrows) (original magnification X

15,900). FIG.4. Borrorn, elec-

tron micrograph from a specimen of cryopreserved platelets exposed to latex particles. Only those platelets which appear morphologically similar to normal platelets are observed to participate actively in phagocytosis of the latex (arrows) (original magnification x 10,100).

lease of nucleotide following thrombin challenge, a decrease in platelet factor 4 activity, decreased oxygen consumption,

and diminished platelet aggregation with ADP, epinephrine, and Radiotagged cryopreserved platelets in the circu-

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CRYOPRESERVED PLATELETS

lation indicated that 35 per cent of the radioactivity was recovered two hours after infusion compared with 60 per cent for fresh platelets. The long-term survival was linear, with an eight-day lifespan for both cryopreserved and fresh platelets. About 70 per cent of the platelets were recovered after the freeze-thaw-wash procedure. In the study reported here, we used the ~ phagoprocedure of Lewis et ~ 1 to . assess cytic activity to investigate further the platelet defect associated with cryopreservation. Many investigators have reported uptake of polystyrene latex particles, bacteria, viruses, antigen-antibody complexes, thorotrast, ferritin, silicone dioxide, and fat particles by platelets in vitro. 2,5*7,10*12-15.19 Behnke' maintains that these particles were not localized within vacuoles of the platelet but were actually membrane invaginations rather than cytoplasmic vacuoles. The recent studies by Lewis et ~ 1 have . ~ demonstrated clearly the uptake of latex particles into electron-opaque phagosomes. Lewis et uL9 as well as other^^,^,^ have suggested that two distinct processes occur when platelets are challenged by latex particles. There is adhesion of particles to platelet surfaces and sequestration within cisternae of the open-channel system by a process which requires basal metabolic activity, and formation of phagocytic vacuoles which requires an increase in glucose metabolism. Our present study corroborates prior observations from our laboratory that cryopreserved platelets undergo significant ultrastructural damage as a result of the freeze-thaw-wash p r o ~ e s s In . ~ the present study using 4% DMSO for cryopreservation, 40 to 50 per cent of platelets were discoid in shape compared to 60 per cent re. ~ 6% DMSO, ported by Crowley ef ~ 1 using and 40 to 60 per cent reported by Schiffer et ~ 1 . using ' ~ 5% DMSO. Our results show greater impairment of phagocytosis of latex particles in platelets cryopreserved in 4%

DMSO than in fresh PRP or fresh platelet concentrates. Further, the impairment correlates with increased numbers of morphologically abnormal platelets. The morphologic abnormalities in the frozen washed platelets are associated with both reversible and irreversible damage, and studies are in progress to determine which if any of the morphologic abnormalities are restored after transfusion. The implication of these results with regard to transfusion of cryopreserved platelets in clinical settings remains to be determined. References 1. Behnke, 0.:Electron microscopic observations on the membrane systems of the rat blood platelet. Anat. Rec. 158:121, 1%7. 2. Clawson, C. C.: Platelet interaction with bacteria. 111. Ultrastructure. Am. J. Pathol. 70:449, 1973. 3. Cooper, I. A,, P. Cochrane, B. G. Firkin, and K. J. Pinkard: Platelet metabolism during the interiorization of two different types of particulate matter. Thromb. Diath. Haemorrh. 27:263, 1972. 4. Crowley, J. P., A. Rene, and C. R. Valeri: Changes in platelet shape and structure after freeze preservation. Blood 44599, 1974. 5 . Davis, R. B.: Electron microscopic changes in blood platelets induced by bacterial lipopolysaccharide. Exp. Molec. Pathol. 5559, 1%6. 6. DeJesus, M., Jr., S. Fikrig, and T. Detwiler: Phagocytosis-stimulated nitroblue tetrazolium reduction by platelets. J. Lab. Clin. Med. SO: 117, 1972. 7. Hovig, T.: The ultrastructural basis of platelet function. I n : Platelets: Production, Function, Transfusion, and Storage. M. G. Baldini and S. Ebbe, Eds. New York, Grune and Stratton, 1974, p. 221. 8. Kuramoto, A., M. Steiner, and M. G. Baldini: Metabolic basis of platelet phagocytosis. Biochem. Biophys. Acta 201:471, 1970. 9. Lewis, J. C., J. E. Maldonado, and K. G. Mann: Phagocytosis in human platelets: Localization of acid phosphatase-positive phagosomes, following latex uptake. Blood 47:833, 1976. 10. Mant, M. J., and B. G. Firkin: Uptake of latex and thorotrast by human platelets in vitro: Effect of various chemicals demonstrating differing mechanisms and metabolic requirements. Br. J. Haematol. 22383. 1972.

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11. Mourad, N.: A simple method for obtaining platelet concentrates free of aggregates. Transfusion 8:48,1%8. 12. Movat, H. Z., J. F. Mustard, N. S. Taichman, and T. Uriuhara: Platelet aggregation and release of ADP, serotonin and histamine associated with phagocytosis of antigen-antibody complexes. Roc. SOC. Exp. Biol. Med. 120 232, 1965. 13. , W. J. Weiser, M. F. Glynn, and J. F. Mustard: Platelet phagocytosis and aggregation. J. Cell. Biol. 27531, 1%5. 14. Mustard, J. F., and M. A. Packham: Platelet phagocytosis, Ser. Haematol. 1: 168, 1%8. IS. Packham, M. A., and J. F. Mustard: Platelet reactions. Semin. Haematol. 8 3 0 , 1971. 16. Schiffer, C. A., J. Aisner, and P. H. Wiernik: Frozen autologous platelet transfusion for patients with leukemia. Blood 50309, 1977. 17. Spector, J. I., J. A. Yarmala, L. D. Marchionni. C. P. Emerson, and C. R. Valeri: Viability and function of platelets frozen at 2 to 3 C per

Transfusion May-June 1979

minute with 4 or 5 per cent DMSO and stored at -80 C for 8 months. Transfusion 128, 1977. , E. M. Skrabut, and C. R. Valeri: Oxy18. gen consumption, platelet aggregation and release reactions in platelets freeze-preserved with dimethylsulfoxide. Transfusion 1 7 9 , 1977. 19. Tait, J., and A. R. Elvidge: Effect upon platelets and on blood coagulation of injecting foreign particles into the blood stream. J. Physiol. 62: 129. 1976.

Jesse I. Spector, LCDR, MC, USNR, Research Medical Officer, Naval Blood Research Laboratory, 615 Albany Street, Boston, Massachusetts 021 18. William J. Flor, LT, MSC, USN, Director, Electron Microscopy Laboratory, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20014. C. Robert Valeri, C A R , MC, USNR, Officer in Charge, Naval Blood Research Laboratory.

Ultrastructural alterations and phagocytic function of cryopreserved platelets.

Brief Reports Ultrastructural Alterations and Phagocytic Function of Cryopreserved Platelets J. I. SPECTOR, W. J. FLORA N D C. R. VALERI From the Nav...
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