24. A membrane-stabilizing effect of sugars in red cell-suspending media and its effect in reducing freezing hemolysis

24. A membrane-stabilizing effect of sugars in red cell-suspending media and its effect in reducing freezing hemolysis

" A I~ MEETING AN" 'NU In +t. l)r(eviousstlldy (l+ap:ttz an(l I,ttyet, 1967) ()li |ilt' ery(q)rote('iivt, effects ()f variOllS ('()ll('ell',r:l)im)s ...

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In +t. l)r(eviousstlldy (l+ap:ttz an(l I,ttyet, 1967) ()li |ilt' ery(q)rote('iivt, effects ()f variOllS ('()ll('ell',r:l)im)s ¢)t" ~ly("(-'rol a n d - ( t i m ( . ( h y l ,:qfll'oxi(h.' ([)NIS()) o)t l)lo()(l, vce (.)l)s(vrv(,d thtt~/: I') lil)(:m slm¢ freezing', ihere was :t |)ro~rc'ssiv(' inereas,..r in • ,~, q( q Im)l ('e)loll wil h •I))(., l(.. a. .I eoneen ir;l.l ira} • ') ) )q-roll r-i1)i(t, l'rcezi)L,_,', lhoro was an i n('rease in hemolysis wil h increasing (.()llc(mlralions ut) to 4 5t glye,,.,r()l an(l :-I .,,i I)SIS(), t)lli, a .~r'l(h~',~l decrease in . wiili. . ]fi~her . . h(,molvq-~ c()n(:('i)irali(ms. '"111., r,,~(, re, ,;,~ ..... '~)'( d 1lint: lhe a(Idili()l{ ()f very hi.(.zh s l l l l s ,.11,~,._( ('()l'l¢'('llll':iliOl)S. o f l-u"))olr;)li).)~ proleetive ,',~(.Idil iv(,s', siwh ,'is gly('erol (.)r I ) ] I S ( ) , iiih.ibiis ()r re(hl('('s i}W l'()rnmiic)t) of ititr:welltflar ice. To 4u,ek ilii.-: l~()ssilfilil3;, an (,lect.rml mierostol)( st~uly was m;.t(.le of (he ex'teni c)f i("(' form;.iliol)t in r'tl)idly c(,)le(t e)'vltlr~)cvt~ susl)en..~i-l)lalin~)nl ;il .--1-t()°(.?. :l'ht" r(.'l)lic:is showed tllglt I ) i ) ) 11)(~ sl~('cin)(;'i)s l).)zcn "'~" ' ill 1 ~): gly('('r()l )}l( ' "II' 'W{>I'(~ tar~e in)r:)- and cxtraedhflnr ",e 1(... m : , s s e s "2) i~l tile,, s!~och))ous (,ou)ainin~-t .xt ~13"c('I'Ol l[t(.'r(' \vtls a l s o illll';i- all(] (.'Xl.l'fie(.' Iu.[~il" i(.'.0., li/il l t l ( ' s i Z e ' o f lilt' i'(. ( . l ) ; I riich:,s w:ls cmisider"ll)ly )'e(lilc~,d ;-~i l[l("t'O \VtlS Ill) (!Vi([('ll('(" o f aliV i('O ill ihost' (':lSt's in which lhe c r v l h r ~ ) ( ' v l o s Wt'FO sllsl)(:,lMe~l in S.i:i ~t glyeerol. These r(>sulis indieale (li:;ii. ihe "id(lition of t ti~h (~OlIC(-'llll';iliOllS of glycerol l;)rt'venls the .['orlll:ili(tll of it(.', i.e., I)orIllils vilr}fication, t:illd i}iev ex])]ain why ll(F/rlV colill)h;io, r(?(,ovorv of t?rvii/rt)('Vl(,s is ot.)lained t l l l d e r lheso eondilions.

23. Hvdroxypropyl S t a r c h as a n E x i r a c c l i u l a r C r y o p h y l a c t i c A g e n t . C: KxoRpp, P. (~I. KA8:

.1-[i SPE:NCI:,'I/, "x" A~'I)


(Veterans Administr:ltioi~ H-osl)i('ll and T h e l.Sniversitv of M i c h i g a n Medieal Cen(er, A n n A rbor, M i c h i g a n 4S105). D u r i n g early 1967 the a u t h o r s inyestigated the effectiveness of h y d r o x y e t h y [ Slareh ( H E S ) -as a.n extraeeltular cryoI)hyl:telic agent, for whole blood and blood produels. T h e sueeessful eomplelion of ):lie in vitro qnd al~imal studies wilh H E S p r o m p t e d us to s t u d y related substances. Aeeordingly, hvdroxvprol)vl ,-,.taleht~, ..,ent ro 1.,sl~,


,9"" _;);).

con(ri!)ii(ed 1:)3" Nation'~l S1nrell and Ctletni(:,M Corl)oraliol), were ieste(l. P:iramelers wcare Men(iettl t(.) the.sl udies l)orformed on l.)h:)od l)roiecied l)v h y d r o x y e t hyl starch nnd by p o l y v i f l y l p y r r o l i ditl(,, liamel3", ll)e total recovery of r e d blood cells, sil)lino siabilily, :tIM Slll)eFll~l[tlll(:. ])];l'Slll,fl h(,mo~lobin .. e,:)ncet~tration. S..ex~r,(l ,",, Imtehes .. of hvdroxvprol)Vl, ..l,t,~ ' r,(..h of varying..._ vis('osilv, and (l('~re(_'s of sul.)sli)li(ic))) were s'ludied, T h e fin:if c()n(,(,ntrnlion of h y d r o x y e l h y l stnreh which ap])(~;.t I'(!"(1 {0 ~ i v e l]l(~ l:)(,sl, I l'O~e(.l ioli i() t]I(., Fed ('e.l]s

w;).~ t>elween 12 d by n resislanee to IWlmtOtiic hemolysis. Since red cell tneml)ratm inlegrity also del)ends on lhe pres(,ntx: of an electrolyte in eoneentrntion equal to 0.09 M (0.535{) NCI, (tie full effe.e( of sugars in nllevi.~tin~ hyl)otonie hemolysis is only demonst ra'ole in (Ira l)r(:.'s(.'nee of q ]:)(",n,:,trating salt. 1:Ie(l cells sll.Sl-)e))ded in 0.12 .'~t sucrose, lactose, m a n n i tot, or sorbitol l)hls 0.1 a~ Xt-I,CI do not 10se eilher hemo~lobi)~ or po1:~ssimn nlihou,.z,h 1he osmolie pressure of (he s~lgars is only half (hal: required to p r e v e n t hmnolvsis in s:lli, soluiioTls. T h e use of such a hypo-osmolal solution when freezing glyeerolaie(:l cells m e a n s thai lower concentral ions of the osmotic b,)ffer are l)rc)d~ced by free/,ing 'lnd eell hemoly'sis is s u b s t n n t i a l l y reduced. It

SUl. portcd b y the..Bm'eml of M e d i c i n e and Sur"~




gory, Nax'y l)epartrnent, I~.esearch Tqsk No. MR005.02-0008. The opinions or assertions em~tained herein "i,:e the t.ll~al.t, )'" -, ', olle8 of the authors and are , , {y the not to be const.rued as official or rcth.etin,., views of the N a v y Deimrt,mc, nl, or the naval service at, large.


A N'NI~rAI. 31 l.,l,,q I N (_, A 1~,'.
2 5 . T h e P,-e.~(.-.rvafi()n of E , ' y t h r o e y t e E n zyme Aetiuritied prep.'.tr;~tions be" ~ 'll]+tI" ,}ltIVC!(I ill ;t :~11 III[tIII]PF w]~en f r o z e n n n d t h a w e d with or withoul H E S . .Th:~s, u n d e r I}~(, (.on(titi(.)ns used, HI!'S preserves key gtyc-olytic enzym(:'.,: in v i a b l e ery'throeytes d u r i n g st()rttge ,~,, liquid nitrogen telll[)oratllros...[ :.m:re+ults .also demonstr'~te the I)ossibilily of t)reserving e r y t h roc\'tes w i l h r:.~re genetic de~eets for l)rolon~e(t l~eriods, att()win~ m o r e ('omt)lele m mlgsis of tim abnormality. L(~,.

t+ )







2 6 . T h e F r e e z i n g o f H u m a n R e d Cell M e m l ) r a n e s with R e f e r e n c e to A d e n o s i n e T r i . l)hosphalase A . e l i v i t v . I. TaI{1¢H:t~:~ AND A/~Trlt,)'. W. ]~.OWlC (.The N e w Y o r k Blood Cen[er, N e w York, N e w Y o r k ) . Freezin~ a n d t h a w i n g h a s . b e e n foun(/ 1o cause -~ m a r k e d . i n e r e n s e in aetivi43: c)f a dt:,nosine l.rit)hos-

)t r i l l ]

ph:~l:~se (A ~ t. asrl in t',d cell t~tenlbr:~nt,s, \vl/t're.ts osnw)*i¢' l , , n m l y s i s d(,'s not..

i}H' {'tlll(lilit)ll.-< 1)1" (rp(.,z+

ing t l~:)t in(lut'(, lhes(, r h a n a e s in r(',I ,(,I1-men> }.)t"tl}( ' .'\'l']):tSf~ gt('liVilV

It} lllttl('rsl:)tl(l

})+q.l+'F 111(!

m('clmnis))~ ( ) f frc,(,zi))~ ,lame)m}, ~l(,m))ran(,s wet(, l)r(,l)ar(,(/ ),\" c)tw-st<,p osmoth, h<,n,)ivsis ()I" hu))~:m re, l cells in l)h()sl>Im)e "~mllcr "' ()i I)II 5.,'<. :i('(,ordit)~ t<) l)<)~l-e (.,t :tl. (-\rch l'~i~)+.i)vn), l")i(.)phys., ]q'r). 119, I.,,,:) .:\TI s(' a<,tivi)v w a s )ne:~s,.)r,.-.d I)v inor,_,anie l-)h(,s}flmt(., l n'()~h,qio)1 fr(m) .-\'I'I >, ;,cv,))'(lijt,_, to l)(~s+ ,,t : ) I . (,I. 1)'"~(,l . (.."h('t t .+ v,~.+, > "." 1-(,,)()'. I ~)(~i)~. ~ I(,))) l,)ra n,, st).q)e))sio ns w e r e fr()z+,~) })\" hn.qu.'rsion iut~> l):(t]!s e~)<)le(] It) V;tl'i()!ls l()'~," [Pll.lp('r:l!~lr('S. It \t.'~tS l'Olll](l 17h:t[ lII1lX{lltlIlll



:\ [ t : ! S 2


ti(m (.)('c~z'r('(], wlt('~t ).,'~,.'~)ranes ,.w'r(' fr()ze)~ dir r ( q l v i~ li(t+ti(l nitro~'(.,n at -,-!.q)r'+('. N():.~&litiol)~t] clmzlee i)l :\"l']~a.#c.aet}'cil\" w:~s ~)l:.-e)'ve(l qfter r('l~r.:tte(.t .(ret'zh~,~ +~)--.l'..)(;"(7~ an(i t}ittWiI!.L". lgreezin,~ t()--.5'+(: restll,,(,(l in only slight ATt':(,~(. ~t{.'liVg.tlD)ll.





for 6() t~)ill ,qll(t







1{"1(')=(.], t ll(e ';t~'~t't'(' Ol" :t(.')iVat it')ll ()11 ! !t:t\+,'ill~ ',V;iS

the s¢~),,' :as wh(m- fr()zr)) +o .-;7.)'~(7 al(.)+!e. Wlu,)t tlI('II.I}H',~t!I('S WP['(. > ("O()~(~<1 ~i~) V;+triotlS l(.)~,+," l(,l'l]])(,r~iJill'{'& ,ql ~.t (+ttlllS[:llll

('l')t}{ill( r


[[1(~ .:t('li'U:tll(~[I

of A'l'l).'tse did m., )" differ m.rk(>div ttlll()tl~ th+" .
--l!.+)ti°(. '. I.) w a s f(-)tm(l, Jmw(?v(,r, tlmt tl)t' t'zl,.-:~er t h e t'ot)lin,a n i l e tim .IlH.I.IT(.~ :wt)valit:m. \'al'iO~S ('ryol)rol(~olrvt: ;)tldJ!ive8 w(,re J't)tlll~| I.o j)r(:\'~'l)). aeti v a t m n of ltt(?ll)l)r~.tl]P ?~.'l" }'):1,50. |;'l:()lil [ JI('~S(' r('it llIH.V ~)0 C()ItCI(I(~O(~ ttP.),[ l'gi[+C o f fr(,rzin~ iS :I lltor(' iltl]'){')t'lttill " - ' ) " Ill " Ill(? a p t i v : t l i o t l (.)t • l,.ltl.<, n~(,mbr:~).., ATI?-~,-.'e /'ICIiViIV t[');tll 1hr.,. g t ~ c ) l l l l e t fqlt|)(,r:t l.ll r(~ a I l ;liIt(,d. T h e ,~(~liva)io:n of :~..1 "~'~'~ t :tS(}" lilt|\" [)(.+ t",'.ll+lS:i]lV


¢>o.mwoted w i t h

~.hc c-haI.L,,,,es in cettim~ ;~,)~(), eh'r-

])Ol'lll¢.~al)ilitv arm (.,o)1 ..UllHtttIlI, h('Itl()Jysia t h a t o ( ' c u r w } l o l l i{,([ ('(']18 ,1I • '(,.. fruz('n w i l h o u ! lhe


protection (~f , t d d m (.-_,.

27. P r e s e r v a t i o n of Ly,upht)cytes at - 1 9 6 ° C in Eti~vlene Glycol.:" C:n..~;iLZ~ F. HU(;GINS AND SH1NJI Yt'ASA (Departn~et:tt of Surgery, H a r v a r d M e d i c a l School, and tire Surgical Low T e l n f ) e r a t u r e l.."ni(, G e n e r a l ~°This w o r k was s t l p p o r t e d in p a r t I.)v U n i t e d grates Public I:[ealt.h Service, Contract 1'1t,t3-65103+1 from the Nationa.l Cancer Insdtut.e and by Contra.et N00014-66-CO-244 from the D e p a r t m e n t of the Navy, Office of NavM ].lesenrch.