Platelet preservation problems

(~IIYOB1OLOGI" Vol. 5, No. 1, 1968

PLATELET PRESERVATION PROBLEMS~':t ~p FIIANK H. GAI:IDNLR

Hematology Resfar('h Laboratory, Prcsbgleriatt-U~gversity of Pcm~syIva~ffo Medical Center, 51 North 39th Strec(, Philadelphia, Pen~s'glua~tia 19104

Fifteen years have elapsed since we began to transfuse platclets by dircei~ silcone syringe technique to control thronfl)ocytopenic blee(ling. ~a Ib was obvious immediately that patients wii,h throm )oe) t,osls were the most: valuable donors to limK, the volume infused, and '~n excellent recovery of eirculat, ing platelets could be obtained in thrombocytol~enie reeipient.s by t,his teclnlique. These early studic~ also demonstrated that, wllole l)lood collected in ph}sLlc ba~,s with aeld-mL~at.-dextlo._e, formula A (ACI)-A), ant, i~ , s as ' - did the direct, silcone syringe coagulant, allowed similar yietds of l) latdct~ technique. ~a From calculated ymhL in the recipient, whole blood t,lan,.syringe or antieoaguhmt) allowed about ~,0% of the transfusions (,_~lcone s" .~ fused platelets to circulate in thron~bocyiopenie, recipients. ]nit, inl centrifugabioll to prepare platelet-2'ieh plasma causes a ZO/c loss of pla;~elets. \Vhile only a few platelet, s are lost further in the prel)arat.ioll of ]n platemt~ concentrates, these manipulations altcl and decle, ,.c vi'a t)]htv. '" the recipient with platelet~ concentrqtcs we can ext)ec~ to~obt~ain only 50 to 5~',,c~.H, of the platelets from the initial whole blood cotlecI~ion (Fig. 1 ) . Despite the variation of gravity centrifuge speeds and temperatures, most, lal)or'ttories have h.ad to a c c e p t this loss in collection. 5Ioi'e,recently with a modification of the Cohn centrifuge, ~9 wc have been atgle to obtain about. 60% of the plateleL, from whole blood prior t.o i]~fus]on. However, t,his teehniqtm does allow the platclcts to be concentrated with one eentrifugation from wh9Ie blood. I-lenee, t,he yield is silnilar to the result; with two L

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can improve platelet, yield from each whole blood donation as well as shorten the time interval in preparation of p]ateleg eoneent~rates. The introduc,tion of excess sodium eit~rate to eontxol plateleL aggregation during eentrifugation has been useful to elimimlte ethylenedianfinetetraacetate antieoagulanfl from platcle~ colleet~ions and to improve recovery of circulating platelets in the recipient,, a Investigators interested in platelet~ p~esm~.at;mn should attempt, to develop techniques for,blood collections in acceptable anticoagulants. Proposals to alter the aeeeptalole standards for anticoagulants eouhl hinder the use o.f platelets as :eomponenfi therapy in the blood banks. The use of additional ACD anticoagulant to platelet-rieh plasma eolleegions decreases ~he clumping of platele~s and al.lows higher yields in the recipient,. In order to expedite ~he understand!n'g of plateleb life span and to

evaluate platelet preservation techniques, isotopic labels have been used in * Presented at, the Third Cryopreservation Conference convened during the Annual Meeting of the Soemt5 for Cryobiology, Washington, D. C., Augusl, 7-9, 1967, sponsored in par~ by the Office of Naval I:teseareh, Contract ONR3700. Some of the studies reported herein were supp6rted by Grant I-IE-11047-02 from the U. S. Pubt{c Health Service. 42

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Fro. 1. Direct transfusion offers best recove.ry yield from whole blood. Almos~ half of the plate, lets from the blood donation are losL in current methods to prepare platelet voneontraie. Further loss of viability follows freezing procedures with glycerol or D M S O . Ch,n't eompile(l from references 6, 8. 13, and 15.

the 1)as~ 10 years. A variety of labels have been tried, pa~ as Na2P3-o04 , is an i~trinsie 151atelet label that could only be derived from' donors with polyoythenlia vora treated wi~,h this source of l~radlat~on.- pa2 as diisopr0pylphosphate ( I ) F P :r-') has been uscd widery but~ has been questioned as a. reprodt]cible lat)e] because of early elut,ion and persisteng labeling by reutilization of the p3~ into pla~le~, ~r, phospholipids.T, ~,, ~7 5-t-lydroxytrypt.amine (5=HT-C~4) h'ts reutilizat, ion by other platelets following infusion which prevents assessment, of the life span) 4, ~s Selenomethionine (SeT~)is an intrinsic platelog label assoeiat, ed with labeling of all blood components tll~t h.ts eonfusecl the life span interpretation. ~ IZi~dioac~ive sodium chromate (Cr ~) has proved to be the most successful plat.eleg m a r k e r for reproducibility. ~ Cr 5~ is an extrinisie label t.hat does require increased manilmlation of platelets. Since most, studies of preservation have ~used this isotope, we can aeeepg this preparato13~ trauma as a common defect •mmng itlvestigations2 ~, ~: The ]imited number of available thrombocytopenie recipients availalfle for evaluation of platelet preservation has prevont, ed direct transfusion studies for any procedure. The use of l)lastie-. equipmeng has been helpful to allow efficient plasmaphoresis of donors for platelet, concentrates. W i t h o u t doubt we are learning •tbout the variet,y of plastic available. While t.he'elosed systems assure sterility, there is Iitt,le information about the influence of plastic elution o~a prolo~ged storage (or exposure) with platelet concentrates. There are limit'ttions in temperature tolerance, :md valuable contributions will ~be forthcoming as we obtain plastic equipment that. can be maintained wighout, b r e a k a g e below - 8 0 ° C . This information will be necessary to plan for ln;O~]onged storage. Preservat.ion. studies of platelet concentrates by freezing have used glycerol and dimet.hyl sulfoxide ( D M S 0 ) . 4, G, ,,, ao While our laborato~5" had an initial enthusia,-_'m for DMSO, the toxic reactions and limit, ed use allowed in man directed our attention to glycerol. The long-standing acceptance of glycerol-treated red cells has indicated safety and no untoward metabolic responses. Our laboratory initially reported gratifying ~e~po~ ses in ~he "

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F. It. GARDNEI~

Fro. 2. Biofreezcr for temperature control during freezing o f glycerol platelet con, cent.rate: T l m arrow points t o t h e chilling :chamber on top of. l:he cascade t y p e coolant resei~Vqir,.The temperature deelinationis regulated by t h e automatic recorder at.. the:side Of tlie fi-.aehine.

preservation of frozen canine platelets, 4 However/these empiric results were not duplicated easily in mar..., It was soon apparent; thi~t,::human plat, elets were more suseept~ible t,o-glyeerol concentrations and :tliermal Variations during eiiillin~gl Through a~long period o f lielpful eoll~.l$oration with Mr, C}iarles Harris, a controlled freezing unit~ was develo!~ed by my associate, Dr: Pi Cohen, (Fig. 2)'.-Such instrument.ation has allowed: careful monitoring so tlm~:platelet e0neentrates in 12% gtyeerol -can be:frozen in th(~, coolant at 1°C deeremm)t until::-80~C. T h e chilling SCheduie can b e p r o g r a m m e d automatically through this temperature-:range, (~: This_machinery has been used in an experimental fashion. We anticipate:that 5 to 10 u i l i t s - c a n b e frozen Simult:aneously for efficient.blood bank appliea, tion. * Known as a Biofreezer, produced by Harris Manufacturing Co., 308 River Street, Cambridge, Mass. 02139.

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FIc. 3. ],;ffect of 12c{ glycerol on platelel~ concentrate. Prior to freezing glycerol has •llIm'e~l t,lu, platelet m e m b r a n e to decrease the initiate recovery of circulat.ing isotopeh~h(,l(M pl~,~(,l~ts in t,h(~ recilqonl.. There is a. further loss of viability after freezing and t lvtwing. Tim cxi,ec,i<,d recove13" of h't,sh Cr~Mabeled platelets is plotted for comparison. J~t-drawn from r(,fol'ene~, 6.

Plau,let, exposure to glycerol is not, innocuous. Glycerol alflers the platelet. meml)r,'me prior Io freezing and decreases the initial recovery of circulating t~l'ttelets in the rccipi~mt (Fi~. 3). The, platelets that l;olerate this initial insult, have a !mnnal lifo span. The platelet, memSrane damage is related to the, coneentrat.ion of glycerol used. Through a long series of clinical studies ])r. Cohc,n (tt'ten~fine(t that a 12% final concentration of glycerol provided the. })est viability with least damage, t.o the plat, clot before and after freezi~}g. Higher concentrations, i.e., 18 to 24%, were associated with ~narked sl~orteuing of lift; simon a,~d a decreased initial eirculat~ing yield of platelets. Careful osmotic butth~g to lessen the final platelet concentration of glycerol })efore infusion has bet:J~ an important: step to assure maximum yield in the recipient.~; The recipient, e'tn expect, 30% of the glycerol frozen-thawed platelet to ~'ireulate ,q~d eontrilmte to l~emostx~sis. This is an adequate yield, bug a more eflieient sys~.em is nee.tied. This technique has the acceptance of a preservat,ion agent, glycerol, that has been used over a period of years. Standard p]astie blood banking equipment, has been utilized for these studies. With careful laboratory supervision polyvinyl chloride plastic will not shatter at low temperatures. Nevertheless, possible leakage and unknown fissures indicate that major efforts must be directed toward improvement in plastic design before adoption in a national blood program. To control the variables, the great, majority of g'lyeerol studies have been done in normal volunteers.with isotopic labeled platelets. While ~,vo have been encouraged with the reported values, we a r t disturbed t h a t the Cr 5~ label m a y be less firmly bound to the frozen-thawed 1.~latelet2 -~ ]~Ienee, there have been discrepancies in the comparison of numerical counting and isotope label in thromboeytopetaie recipients. The numerical yield has been higher than the radioactive yield (Fig. 4). In the short interval of the pl'ttelet life span, no significant, physiological isotope etution is apparenL

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Fro. 4. Comparative char~ of numerical and isotope yields in a thronibocytolmnic reeipien~ following infusion (a',",'ow) or glycerol frozen-thawed plateh.,ts. The initial yield of'eireulating platelets by C : " estimation was low (15%) compared to the numerie~fl response: "~Also there is a more rapid loss of circulating isotope to suggest~ that, the Cr a label may not remain firmly bound to the platelet membrmm. Redrawn from reference 12.

The interpre.tation of plate]et yields in [hronfl)ocytopenic recipients by numerical counting and isotope dilution has not been settled completely. The ultimate value of glycerol-preserved platelets must: be established by fl~eir usefulness in dlinical trials to control thrombocytopenic bleeding. Platele~ preservation with D= iSO has been limited to a large exten~ by our meager information regarding chronic toxigi~y of the drug. Djerassi and his as~ocmtes "~ " have continued to e~aluate ~ ~ plaScelets frozm~ m a final coneentration of 5% DMSO and 5% glucose) ° A. cooling rate of 1°C per rain from 0°C to lowest,, possible temperatures w)th liquid nitrogen as the coolant was used. As with the glycerol-frozen platelets, thawing was not, monitored closely but clone rapidly in a 50°C water bath. The data suggests that the yield in recipient is about a third of the response obtained with fi'esh plakelet transfusion s. 9 (Fig. 1). This preservation technique has nob required removal of the DMSO prior to infusion as noted above witlr glycerol. It" has the merit of more rapid preparation of ~he frozen unit.

Z~ 4"~r PLAT LLE [' P R E S E R V A T I O N P R O B L E M S

47

Unfortunately, we do not have specific data as to the circulating yield (reeovery) of the thawed platele~.s or the life span in a recipient. Since ib will be important, to define methodology for a national blood program, my laboratory had initiated a comparative study between glycerol and DMSO to determine tlm maximal recovery of each type of frozen-thawed platelet. SUMMARY

During the pasg decade efforts to preserve platelets have indicated that storage at 4°C will nob maint.-dn functional integrity. The current, ant,icoagulants are acceptable with the proposed modificat, ion of ACD solut,ions. Plastic equilnnent has been of value to ~repare platelet, eoncent,rates rapi~tly. We need to have better plastic materials for the projected temperatufts used for storage..'3Vhile *,he Cr '~ platelgt label has been of great, value to evaluate metho~lology, further data are required to determine if isotope lal)eling of frozen platelets can be used to assess hemostasis. The *,we preservatives current,ly used for freezing need comparative studies to define acceptance in a national program. At, the prc~ent time preserva~.ion s~udies continue to be done in an empiric fasl~ion. Only when we underst~and platelet, membrane funct, ions more completely can we define the cD~ophysiology of this blood component. ItEFERENCES 1. Aas, K. A., and Gardner, F. H. Survival of blood plate'lets labeled with ChromiumSL J. Clin. Invest., :17: 1257-1268, 1958. 2. Adeison, E., Rheingold, J. J., and Crosby, W. H. Studies of platelet survival by tagging ill vivo with P~'-'.J. Lab. Clin. Med., 50: 570-576, 1957. 3. AsWr, F¢. I[., and Jamll, J. H. Platelet sequestration ill man. I. Methods. J. Chn. Invest., ~.3: 843-855, 1964. 4. Co]wn, P., and Gardner, P. I-I. Platelet preservation. II. Preservation of canine platolet c.oneentrates by freezing in solutions of glycerol plasma. J. Clin. Invest., /~1: 10-19. 1962. 5. Cotwn. P.. Cdoley, M. H., and Gardner, IV. I-t. The use of selenomethionine (Se TM) as a label for e'mine and human platelets. J. Clin. Invest., ~.~: 1036-1037, 1965. 6. Cohen, P., and Gardner, F. If. Pl./kelet preservation. IV. Preservation of human phtt,eh~t cmlcentrates by controlled slow freezing in a glycerol medium. New :Eng. J. Med.. ,, ~ o.~,. 1 4 0 0 - 1 4 0 7 , 1966. 7. CoOnoy, ]). P., 'llld Smith, ]3. A. The use of di-isopropylfhmrophospha~e "~" (DIVP'~") for ,letermination of the platelet life-span. Clin. IRes., 1,5: 99, 1965. 8. I)ierassi. I. The role of platelet, administration in ~ blood transfusion service. Transfusion, 6: 55-61, 1966. 9. Djerassi, l., and Farter, S. Cor~trol and prevention of hemorrhage: Platelet transfusion. Cancer Res.. 25: 1499-1503, 1965. 10. D ierassi, I., Farter, S., Roy, A., and Cavins, J. Preparation and in r i v e circulation of humau platelets preserved with combined dimethylsulfoxide and dextrose. Transfusion, 6: ,572-576, 1966. 11. Ebbe, S., Baldini, M., and Donovan, J. Comparative sl,udies of platelet survival by different methods in ~he rabbit. Blood, 25: 548-566, 1965. 12. Gardner, F. I-T,, and Cohen, P. Platelel, life span. Transfusion, 6: 23-31, 1966. 13. Gardner, Iv. ~{[., t-lowell, D., and Hirseh, E. O. Platelet, transfusions u~ilJzing plastic equipment.. J. Lab. Clin. Med., q3: 196-207, 1954. 14. }Ieyssel, ]~. M. Determination of human platelet survival utilizing C~Mabeled serotonin. J. Clin. Invest.,/~0: 2134-2142, 1961. 15. PIirseh, E. 0., and Gardner, F. I-I. The transfusion of human blood platetets. J. Lab. Olin. Med., ,~19: 556-569, 1952. 16. Leeksma, C. H. W., and Cohen, J. A. Determination of the lifespan of human blood platelets using labelled diisopropylfluorophosphonate. J. Clin. Invest., 85: 964-969, 1956.

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