EFiCECTS ()F F I I E E Z I N G AND T I I A W I N G SERU5I ANI) PI,ASNIA ()N SEI~ECTEi) Q U A N T I T A T I V E R E C O V E R I E S *

DEAN F. DAVIES, M.D., Ph.D.

Like other powerful lnethods of energy transfer such as radiation and fire, cryogenic teml)eraturcs ean be used for either preserving or destroying biological tissues. Ii/ ( 'ryosurgery the ability to destroy tissues selectively ~ometimes faces the prol)h,na of lmrt, ial or temporary damage to surrounding tissues. Sinfilarly, :~tteml)ts to l)resc'rve biological materials at, low tempera- tures have generally })t,en accompanietl by "in il~abilit.y to restore the tissue em~ll)l~'tely lo its origixml state.

I~ ibis era of instaz~t me(licille, fascination with the potential benefits of l~reserving tissues, wllole organs, and org-misms frmn tlm most. minute to mall ]~imself has leapt, forwar(t. It is ilJ this setting that some of the sim- t~le,,,t l,:iz~(ts of ext)erime~ts have twen carried out, on the simplest of all bio- logical syslems, I~loo~l sermn.

It, l~as 1)¢,en o~ir l)elicf that close exnmination of the quanti tat ive chem- ical et~angcs wl~iet~ take !~lace duri~g the freezing and t, hawi~g process will in the lon~ run cm~t|'il;ute most, toward preservat, ion of viable t.issues and orgn)~s. A few ('xa~nplcs fro)n the literature of praet~ieal problems tha~ arise i)) the l)reservation of t)1oo~l serum will l)e cited. This will be followed by a discussion of the methods wl~ieh we have used for preservation of speeimens of ~(,)'~n} at liquid ~it)'oge~ teml)('nttu)'es over a period of 4 years and our results.

PIIOBLEMS ]'~IIE~ENTEI) IN TIlE LITERATURE

A1~tihcmophilic F(tctor (AHF, I'III)

One practical l)rol~lem is offered l)v the preservation of the antihemophilie factor I factor VI I I ) . Blood banks have found that ant, ihemophilie factor is llof eollsistently found in satisfaet, ory amounts when bank blood is stored. Ii, has lwcol~e eommm~ practice to freeze fresh plasma in order to avoid the ineon~,'m~icnee of Sl)eeial donors each time it, is required. This frozen fi'esh ptasm:t should tw in plentiful sut)ply for t.rea~men~ of hemophilia. However, there is l~meh disagreemmlt about the st.ability of factor V I I I upon freezing storage. Ordinarily a uJ~it of plasma requires abou~ 4 hrs for freezing in a freezer ttl~ - 3 0 ° C . During this time there is variable loss of factor V I I I act, ivity. ~4 This finding that factor VI I I is less stable in large volumes of 100 to 300 ml of AC1) plasma when stored has been confirmed. When small aliquots of eitrate plasma are stored at. - 3 0 ° C t~here is ,~o signifieanl~ loss of factor VI [ I aet, ivi~y over at, least, 17 months. Alghough rat,-e of freezing seems to be the important variable in the difference bet,ween t.he freezing

*Presented at the Third C13"opreservation Conference convened during the Annual Meeting of the Society for Cryobiology, Washington, D. C., August, 7-9, 1967. Spon- sored in part by the Office of Naval l{esearch, Contract ON1R3700. This stud3; was also SUpl~orted in pnrt by a grant from the American Cancer Somety (L-31) to the School of Public Health and Administrative Medicine, College of Physicians and Surgeons, Colurnl)ia University, New York, New York.

87

~qS 1). F. I).\V1E,q

of large units and small ali(luots, the ini'erence is based on indirect evi- (]('lice."

Another factor is temperature "of storage. 13~'itten ml(t others e gave the euteetie lmint, of l)la~m.a as - 2 3 ° C , s t a t ing that complete freezing does not occur at - 20°C . 4t, this temlwrature, labile components are exposed io a markedly altered environment. A teml~evature of - 3 0 ° ( ' or below is prefera- ble. Weaver ar, d others '4 found little correlation between the eom.entration of factor VII I and the duration of storage pro" se. Nour-El~lin 'a showed that. repeated thawing and freezing of plasma caused marked ve(luc;tion itl the qe- tivitv_~ of l~la-m,t' ~ " factor VIII as well as factors V and IX and ~lenlmlstrate~I nicely that fnetor VI1 }~as its highest eone(,ntr'~tion at the beginni~g of the thawing t~vocess and its lowest em~centration in the melte~t Sel'~,tnl ~tt,' tlW t'n(t of the thawing ln'ocess. They conclude that, for maxin~aI activity t)f f:tetor ),:Ill, fresh plas~m shoul(1 be lmole(l, ral)i,tly frozen, and store,/ at --30 to - 4 0 ° C .

,qcrt~m Protcfi~s

~[ay and l .eoneJ ' in stmlying the scrologic-tl shelf-life of seruln t~roleins, were able to lyophilize samples that, when freshly dissolved, were sero- logically indistinguishable from cmltrols whicl, had nor ln'en lyoplxilized, ~mmely, wet lmste samt)les and components frmn flesh whole hm,mn selum. Because the prel)aration of the lmman -,*lotmlin l l-I(';'(~i} and human serum ) / ~ . -

albumin (H~.-X) was by the (.ohn l~rocc(lure, results on these isolated l)ro- rein samples may not l)e comtmrttl)le to what might lw exl)eetc(t in whole serum %" plasma. When stored at - 2 0 . 5 , an(t 20°(_; for up to 15 months, no losses in human serum a'lbumin were detected.

- O " In the case of huma~l 7 ..,lolmlin (HGG) no losses oceurre(l at 5 or 9n°(' However, at 20 a~ld 37°(3 almosl tlalf of the activity of r-globuli,~ ~ - , % 1 % _ e . .

~ ' ' ,-1 a stcriliza- was lost. iz) 6 months. Ihcs~ proteins were store~l a.~e)ticnlly fter tion } , )x filtration.

Lipoproleirts

Over 20 years ago E . . I . Cohn said. "For certain lipol)rotei~s---frcezing appears to weaken the a t tachment between lipid and l)rotein." ~

Ten ),ears qgo Loveloek ~° showed that t3-1ipoprotcins begin denatura- tion. at - 3 0 ° C and t.hat they are denatured most rapidly between - 3 0 and -40qC.

A thorough' review of the literature on freezing of serum or plasma has not revealed any report in wlfich .~tea~ a~e of prosthetic groups such as those of lipoproteins and gly('oproteins have been studied as functions of rates of freezing, duration and temper ' t ture of storage, and rate of thawin~. Never- theless, tllere is circumstantial evidence thai the weak association forces of such proteins can be b~:oken by the freeze-thaw process, a, :~, ~o

Enzymes

There are practical reasons ~or studying tl~e stabil ity of enzymes in plasma and serum s',tored at different, tempc~'atures. The half-life for plasma proteins is measureablc in h o u ~ or days. For this reason it is of interest

l{l,:t't )XI,:IIII,:S ( )1,' 1:11( )ZI :N-TI [ . \WI,:I) ,'-;1,:11!.'~1 8 9

Lo know .~l)mlt tlle s talf i l i ty of c ~ z y l n ( , s 'in ~,itro. It. is al,,o ot' in terest fro]~l th(, st:~n(tl)oi~zt of storag(, or mltilil~g fur slll)s('(luc~t use. ,Iulll" ~neasuvcd tl~e "~etivitv of cigl~t e, nzvmes in 1)lasm't :tn(l serum "~fter store(go for varvin,," lengt, lls of l ime ~tt, (liffen,nt /elnlWl'atures. Tempe r a t u r e s use,l included 38 99 4. an(t - 2 0 ° C . The stutlv wa.., l i lni ted to 8 (lays. Sterile,," L D H at - 2 0 , 4. a~l(l 22°(? lla(t essent ia l ly the sanle results" about a tl~ir~l of the ac t iv i ty was lest. l)uri2Jg an S-flay in te rva l the otlmr s(,vc,~ enzymes were (lllite sLal)le at --20o(_ ̀ anti for tl~e most talt,)' ' at 4°(22 in al~ unfrozen state. Those st'tbl(, :tt these t empera tu res ill('luded 1)seudocholinestcr:lse, g lu tamie oxalo ' leci ic t r ansami~asv ((;(Yi't ,, ' "' . • , l u t amac pvruvie t ransami~lase ( G P T ) , and alkali~le l)hospl~at:tse. Acid l)hosl)hatase, a t 4°C lost some of its act,ivity })etweczz t}m Is( an,] 8t}~ la'~. ,J~zu] ..s c. - ' results o~ s t ab i l i t y of L D H are in agree- ~nm~t, witt~ those of Kreutz( 'r : ' for t(.mlL~,ttu~c~" ~ , ' . - ' ,s l)elow 4 °C. t{owever.. Kveutzer foun(l l,l)l-I to be very stal)le for about, 10 dftys at roon'l t empera- t l l r ( , .

5Iosloy ahd (-loodwin~'-' s tudied the loss of serum g lu tamic pyruv ie (,tans- ' . ,, ,. o. ot ]).5' the aminnsc (,.'5(IP'I'I 1)v tc~nl)erqt~re al~(l , turntion of ._tma~e, but, n .

ra te of t'rt'ezing or thawi~g. "Fl~ey discovered n-taxi,hal loss of ac t iv i ty in 7 to 8 (l:tvs in an o rd ina ry freezer which var ied from -1;.) to - -30°C. A ~(_f~- er:ttOr gave l)(,tl(,r results, 1)ut, th(, best results were ob ta ined in a low tem- l)(,vatuvc fr(,ezev })('tw('e~t - -40 ~t~([ - 5 0 ° C , where the loss of aeti\ ' i t ,y ob- served nftt ' r (53 d:tys eoul(l have })em~ within the exl)eriment:~l error.

,lol~n.-ton au(t I-luff v fom~(1 eholi~estm'nse ac t iv i ty to fall hetwee~ 7 and 61 c' "a .~ " f t c r l'reczin,,"' ~n(l st~tte(l tha t the enzyme w~ts st, able for " severa l

~ ~ 0 w(,ol,:s at 0 to a (.. ]~el'so~) ~ ol~st'rve~l (leami(latio~) of il~sulin dissolved in :) x 10 - a 5t H C L

wh('~ t'roz('n to -15°(.2. T]~o a s , u m ( , d eXl)l:tnation offered was tha t the acid becomes gr(Satly concen t ra ted as the wa te r freezes out and prol)ably remains l a r g d y u~frozen even a t - 1 5 ° C .

Some ()f these refere~c(,s are cited s imply to demons t r a t e ~;ha~: the s ta te of the .~rt,.is still young even for such s imple syst.ems as serum. Some of the variabl(: re,-.'ults m a y not be related to freezing temperat :ures or ~ate~ ai~ all trot. in var ia t ions in (eeln~iques for se.l)aration of cells from serum. Crow- ley 4 found tha t the lex e~ of GO.[ , G P £ , and L D H in serum is re la ted di- rec t ly to the dura t ion of em~tact between ('ells and serum. The in te rva l between the d rawing of tim blood and the sel):tration of the cells is crit;ical.

The effect, of ra te of freezing and ra te of thawing on loss of act, iv i ty of isolate(l enzymes deserves comment . An extensive stud?" of the faetors re- sponsible for loss of activit .y on preservat, ion of isolated enzymes 2)y freez- ing has t)ecn made t)y Chilson and others in I(.~pla'~ s." D.boratory. a The resul ts may nOt. be d i rec t ly t r a n s l a t a b l e t.o o ther sys tems .-:ueh as scream and plasma, but. they do provide leads for fu r ther studies. T h e difference in the Joss of lact, ie dehydrogenase and of triosephosphat, e dehydrogenase under differing ra tes of freezing and thawing were h ighly significant,. :For exam- ple, when freezing took l)laee over a period of 2 hrs in a freezer at, - 2 0 ° C (slow freeze) , the percentage of remaining aet, iv i ty of L D H was half as grea~ as /hat, fom~d for a fast, freeze per formed in .Dry Ice and methanol . Fast, ' freeze preserved aetivit .y bet~ter whe ther t;hawing was fast, or slow.

90 .D.F. DAVIES

.qimilarly. where fr<'ezi~g rate was heht constant, a slow thaw (saml~lc stau(l- ing at room teml)erature) yiel,te(t about two-tl~iv~ts the activity found (turing a qui('k thaw (shaking under run~i)~g cold tap water).

As exl)lanation for tl~ese commonly experienec~t phe~omena, it. is suggeste(l that the most crucial moment of freezing tu~(l of thawing is rca('h(,(t at the euteetie point. Depending on the mixture the eo~nposition of tlw liq~ti(t phase at the vuteetie point can be greatly (liffcrcnt from its composition a( roo~ temperature. Similarly, the pH can change ~narkedly aeeor, ling to the orig- innl (,omt)ositio)~ of the mixture nn~t the solubility of caeh eonu')on('nt :tt tt~o euteetie poi~t.

",'4 " X " } Rl,~, I.~. "I ~'I'Vl)Y

The stu,di~,~ ~o he reported include the seeo~l phase of studies eonduete(I over a period of 4 years. It wqs c~)nsi(tere(t essential io (lete)'~ni~e whether se]eete~t eo~nponcl~ts of seru2~ (,ou|(] be q u a n t i t : t l i v e ] y rveovere(t a f t e r a fast. freeze to --196°(~ ttll<[ a fas t tllav,'. T h e set'el/([ 1Klrt)ose was to s tu( ly the

I

effect of (luratio~l of stor'tge in a iiqui(t nitrogen refriger'ttov on (tuantit'a- t ire r e c o v e r 3 " of the serum compo~2ents.

Results on the first ph:tse of the exlwrimm~t have t)een n.l)orte(l and published.'; Brietty. tIw first phase consists.el of two sets of t,xpcrimvnt:,.

It was first dctermin(,d that heat-scaling of 11/..,-('c dm)'~l ,,la~s ampules containing serum di(1 not affect the ._tablll ~ of selecte(l m~zv~fh, s an~t me- tabolites therein. In the ~(c)n(l~,,( set of experiments the rqte of frc'ezi)~g of serum in these ampules o~ imme)'sio~l into liqui(I )litrogol~ wt~s ))]e~lsure¢l })v a eopt;er-constantttl~ thermoeouple. The tmnpcrature ' ~lrops '." ~° g~o~ ~vm'a ge of 340°(; per rain or 57°C in 10 see a n d is ealh,(l "fa,-t freeze." ())'~ :rvmox'in,,.~ the specimens from the liqui~t nitrogen r('frige)'ato)" ztn~l t}mwing i~ a 3 7 0 ( ; `

water bath, the thawing rate was a.) v. t)er rain. 4 or 5 rain of wl,ich were at or above 0°C.

Total protein, glucose, urea, lae u. acid dehwtroge)~ase tLDII ) 1)hospho- hexose -~ " " ~..ommase (PI-tI), glutamie oxaloaeetie transaminase (G()T), an~t alkaline phosphatase were selected for study.

Rapid freezing to liquid ~itrogen temperatures followed by thawing ") "~1, within 2½ hrs (PHI, G ( 7 , and LDH) aml 9(5 hrs (urea, total protein,

glt~eose, and alkaline phosphatase) resulted in highly s:~tisfaetory recovery of initial eoneent, rations measured. For no component, was the change in the mean values obtained after tlaawing for four sub,ieets each more than 4.1%. The reeovery of urea averaged only 0.55% above its init.ial value.

. ~ ' " c r - , The second phase of the study was on the effects of duration of &o~ae, e and is reported herein. One to three aliquots of each of four blood serums were placed in 1.2-ml hard glass ampules which were then heat-sealed with a

_ - 1 ' t? ' I(al~lenburg ampule sealer. Ampules were l lae.d in one of six posit, ions in aluminum ~aek,.'. -.~ designed for the purpose. The racks were inserted into card- board proteetive sleeves and placed in a ea~:ister of a Linde 35-liter liquid nitrogen refrigerator. At intervals of 4 to 6 weeks evaporated liquid nitro-

r gen was replaced, usually in amom~t,_ of 15 to 25 liters at a time. It. is estimated t]~at the rate of consumption of the refrigerator when

no~ being opened except for measuring or addition of liquid nitrogen was al)proximately 18.5 liters per mm)th. Beeause of the low cost of liquid nitro-

" ' - l .S O F 1, I { O Z 1 , N q'] " : ) l i E C { } V E I t l " " ~ " " " I . \ \ \ L 1 S E I { I ' M 91

T A I J L E 1

ACT1 V] T1E8 OF I } tl O,%P I1 {)IIEX{ :BIB I ,%{ )M l,:It.% .'¢lg A N I} (.'~ I~U TAM I{3 (')XA I.OACETIC, "I'RA NB.~ Xi I N A SB

IN ~EI{I ;M I{EFOIiI,I AND AVI 'ER S T O R A G E 1N ~]*~ALEI) A M P U L E S tX LIQUI1} NITI{O( IEN I;[EFRIGEI{ATOI{. "b'{}R ;'~ Y E A R S ANI} 4 tXIONTtlS*

Subject

. . . . . . . . . . . . . . . . . . I

1 2 3 4

M e a n

Before

2(.). 3 2-I. 7 1{i.7 1S. 3

Ptl I

Af ter

30. {; 27,0 16,5

, 20.B I

Change Before

/ (

+ 4. -13 19.0 + 9 . 3 1 19.7 - -1 .19 14.3

+ 1 2 . 5 17.8

¢ •

6.26

GOT

After

41.1 50. {} 34.3 4S.5

C - - " . . . . .

Change

+110 +151 + 2 1 0 + 1 7 8

+ 1B3

* Measurome.~ ls {'arried out, t h r o u g h Ihe {,.{n~rlesy of l ) r . M o r i o n 1(. 5cl~wqrtz, S loan l ( e t t e r i u g l n s l i t u t e , New York , N, Y.

gcn t]~{.' lnaintenttnce cost of tlle systeln over long periods of time is quite low, l)elwndi~lg ol~ the ~..',,,'-,,",,fl~ic..~.,,~ area and method of supply, it, will usuall D t}e less t:ha~ 15 cents p{'r {lay.

PHI o~d 6'OT

As before, lfl~ost}hohexosc isomer'lse ~,nd glutamie oxaloaPeti{, t ransam- inase (teterminatio~s were carried out in the laboratory o1" the Divisiol] of Enzymology :tn{t :'kletabolism, Sloan Kettering Insti tute for Ca~leer Re- se,'~.rell, lln{ler the sut}ervision of I)r. Morton K. Sehwa]'tz. Specimens wcrc l}laeed in tim liqui{1 nitrogen rofrigePator June 18, 1963. and were re- merest October 25. 1966, for analysis. They had linen i]mnersed either in li{luid ~it.rogell at - 196°C or in its v:q}or 1)e]ow - 1 3 0 ° C for more thtin 3 years an{t 4 months. (_.,h{.~nl(,tl methods were "Is previously repor{e{1. Thaw- ing was earrie{l out by removing the aml)ules from the refrigerator and, within 2 rain, immersing the]n in a 37°C water l)ath. Thawing was em~]plete i~ al}proximat{'ly 5 rain. The tot:ll thawing process averaged more than 28°(3 per nfi]], }}ut the bulk of time for the thawing process was that re{luired for {.orJversion from ice to liquid phase.

Table 1 shows tl!e enzyme aetivities obt:dned on fresh unfrozen blood serum from four subieets compared with those obtained in the same labora- tory by the sam{,, lnethocts after quick freezh}g, storage for 3 years and 4: months below - 1 3 0 ° C and a quick thaw. For P H I the mean change for four subieets was 6.26% higlmr than the initial v-due. For GOT,-there was a marked rise in each serum ravaging from 116 to 210% increase. Prior {o storage each value was within the normal range. After over 3 years each value was al)ove the normal rqnge.

LD H

The initial analyses and t}egiuning of storage at cryogenic ~emperatures of serums for lactic ddD;drogenase determination were carried out on July 10, 1963, in the laboratory of Dr. Natha~l Kaplan a~ Brandeis Universi W The procedures for quick freezing and s[orage in the liquid nitrogen refrig,- crater were the same as deseril.)e~l above. The thawing; and fiual analyses

9-t I). F. I).\VII ' ;S

llr(,scilt stul ly an~t its llrctl(,c('ssor, 'i lactic ~l(,hv(lrog'(,llas(, ali(l l)liosl)llollexosc i so l l l e r : l so rt,( 'ovt, rit,.,, w('r(' ( 'xcclh' l i t w i io t l lor C, XlmS('tl to t(,li ipt, r l t tur( 's l>elow - -130°O for 2V_, l lrs or 3'71 years. A l t l i oug l l no g lu ta l l l i c oxalo ' iect ie t rans- :tirl inas(, ac ' t iv i tv was lost :ts a resul t of ral)i(1 " ~ " " <~ tit.( Z l l l , anti rapi(I l [ ,htt\vi l lg i l l 1.963, foF ]'O:tSOllS llOt VC[ ull~h'rstoo(l resul ts show~'(l Illl /tVOl'ag(' of 163% inel'oast, aftel' ox'er 3 years at liq~lid nitrogoli tc'lnlmratail 'cs. Sill('c it is inl-

"' "' i) ,,~ ' s such it p o s s i l ) l e to hol(I a l l the \ , t i l , t I(. c o n , . t a l i { o v e r . l ) er iod , it, is llO('t'sSttl'y

to ae('(,l)t, th,., evi(tcll('c its oll ly l)VeSUllll)tiv( ' that tl~(, loss rc'latc's to tliv , h l , ' a -

t i on o f stora<,'e

I {( 'sul ts on SOI'Lllll whie l i t l l t ( l t ' l 'Wt ' l / t IWO ( lu ick fr(,('zc,,-, one stow thaw, one rap id t l iaw, t i l l ( | ~lll i l l l t ' r \ 'a t of 1 to 3 days at I'O(111] tt ' l l l l)(' l ' l itl.l i 't 's ;11"O l l ] O r t >

f l i f l i cu l t to i l l l (T i ) ret . T l l c s l t t i s faotory l'O('ovcq'il's of glucose l ind tot.al l>ro - teill s t rol l f f lv -tl<,'oost t l l a t I!i(' eXil'a ll '{illsilion froll] soli, l lo li(llii(l 1)hasc and l/:tei,: (li(l not affect t l i t i i l t ( 'g r i tv c)f t i l t ' nlolec'ul(,s. T l l c ('xt)t, r i l , . lcl l t wouhl t )e/ i r ro l )c t i i io l i tlll~tCl' V t l l ' y i l l g coilct i t i0ns c)f t inw, tCl i '~ l ierat t i r l ' , : t l l / l l ' l t lc o f

t.tqill)t, ra t t i l ' ( j c i la l igo , 37o sat is factory (.onelusi()ils with I't'Sl)CCt tO efl'(','t ~)f ]Ollgtt'rill ]itlili(I

llit, rogoll storage I1.1'(' l)OS.--it)Ic regar i l i l ig l ] le ~[('Cl'('as(' i l l I l l '( ' l t i l l i l l : t lk l i l i l i ( ' l)]lo<pll,itasos ' aft('i" 4 v('al'S.. Tim fac,t their 70 to 80% of t.h(, ( ' () l l l t ) () l l ( ' l i ts Wt'I'(' rc( 'overo(l wouhl ~tl,,'<)t'.~t t l la( l l l l l l t ' l ' ll]Ol'l' c; ir( ' t ' i l l lv colitroli(,cl (,oll(litioil.-- nlucl t 1)otter l'C, covt, rh's shoul(1 ])c ln)ssil)l( ,.

The 1)est evi( lcnco olT(,r(,(1 I)v t i l ls stu(tv of ti lt:" ;t.ltVlillta<,._;t' of Ii<lui,l ni t rogei i rc, fr igerat iol~ over t l ighcr t t ' l l l l l t ' l ' t t t , tll't'.<5.' is ,'-COil i l l ~i. ( 'Ol l l l ) : t l ' iSoIl O1

, I t lu l 's l ' ( 'Covory 7 O[ SOl'till] lact ic <l(,}ly,.lrogl,nasc ( [ , I ' l l [ I t i f t t ' r 1 ;tl l[ l S (lily.-. at, - 2 0 ° C \vitl~ those of the auf.llor. A f te r 1 clay ,It, ll 1 lOSt 34.-i (< tlf I l ) I t ac- • a ' l ' ~i +

t ivit , y all(t '~c." o,c lilort, duri l lg tile fol lowing we('k. ,Jtitl] {.onclu<l(,ll thlit I , I ) I 1 i s " ~ '1 ' . unst , i )1o even :It - -20° ( ; . ' l{{.,t'()\'t,l'\" f roln tcmlwr~tturc 's t)clow - - 1 3 0 ° C in this study, liowcver, wer(, ,;xc(:.llc~lt after los- tlinli 3 hrs :is well ~ts after 31/,i years.

~I'MMAR'i

Blood sel'uin l~reserved i l l a l i( lui(t l l i t roz( 'n r { ' f r igcra tor at te in l ) ( ' ra lurcs be low - 1 3 0 ° C for 31/~ years yie](lc<t h iz l l l y sa t i s fac to ry rc(,ovoric.-, of lact ic dehydrogenase and ]) l iospl iohexosc isol'llCl'aSC. For l'('aSOllS l lot l)rt 'seilt. ly

• '' i' n l i i l a s e a f t ( ' r t i l t 1.11]dol'stoo(l l 'oeovery of 801"tllI1 gltltlIll]iC OXlIIodcc[ C Iral~sa kame interval was excessively high attt:ough rapid freezing anti rapid th:lw- ing in earlier experiments had not ~nat, oria!ly aft'coted the re('ov(:ry.

In spite of the stress('s of a slow draw followed 1)v exposure to room temperatmres for 1 to 3 days, serums in se,ale~l , a m p u l e s kel)t, for 4 years

• ' l c e o , l i r a of in a l i qu id l l i t rogcn l c f l l ~ c l < t o i y i e l d e d highly, s a t i s f a c t o r y '~ ' v e " : ' g lucose a n d to ta l lWotein b u t not, of a l k a l i n e l )hosphat , ase or urea .

REFEI{EXCES

1. l:lo.rsori, 8. A. Doami( la/ ion of insul in dur ing sioi'ag(' iri froz('n sial(.'. Dial)otc's, 15: 875-$79, 1966.

9. ] } r i l l f ,n . A., and (Irove-I{asniusson, M. Siahil i ly of factor V I I I iri the frozen slate. Transfusion, 6: 230-733, 1000.

• 3. Ch ibon , O. P., Coslello, 1~. A.. and t{al)hln, N. I). Efl'o('ls o[ fr(,(,zing oil (,iizvinos. Fed. Prec., 2.1 : No. 2, Pt. I I I , SuplJl., 55-65, 1005.

llI,2('()VI';I{II'L"; ()1'" I"I>,OZI';X-TII.\WICI) S E l l t ' M 95

• I. C()Im, IC. ,]., l ' re l ,a ra t ion an(l I,rOl)er(ies of serum an(l l)lastna: Pro(t,ins IV. J. . \ incr . Cl~(,lll. So('., a,b': 459-,175. 1946.

5. Crowh'y , 1,. V. Var ia t ions in levels ()f (rans ' tminusos and lav(w (.lehydrogenase in Imnk I)1oo(t. (: l in. (,_',lJenl., S: 626-629 , 1(.)62.

6. I)avi(,s, I). 1;. Qu:tnt i ta( ive rc,(.overy of sele('I(,(t ( 'onlpononls of s¢,rum afIer rapid fr('(,zing a~(1 ralfi(l (hawing. Fed. l)roc., 2.}: No. 2, P t . I i i , Suppl. 15, 249-252, 1965.

7. 3()hns/()n, l) . 1"., and ttuff, W. C. ,';(at~iliiy of cholinestoras~: in frozen ],Ia.-_'nm, Clin. ( ' l ) em. I I : 72(.)-7:12, 1(.)65.

S. ,Jtn~l, P. ,q(al)ili(y of lda,'-)m) enzym('s (luring storage,. Clin. Ch,"l,~., l d : 416.422, 1(.)67.

9. Kr(,ulz(,r. II. 11., and l:(,nnis, \V. II. /-I, Imvtic dolLv(h'ogenase iso(,nzymt,s in l)lood s~.run~ afl~.r s(~)r'~g(, 31 (liI'f~,r~,n~ t(:mp(,raiur(.s. Clin. Chim. A(.la, 9: 64-68, 1964.

I0. ],ov('h)(-k. ,J. IC. "FI~(. (l(,natur'~lion of liI~i(l-l,roI(,in ('OmldeX('S as 't ¢'ztt)S(' of (Imnagc I)y fr,,('zin~. Pro('. R().v. Nov., or. B, 1.~, ,~.,-.b-),:,, 1957.

11. ~l:ly, I)., an(! l~(,on¢,. C . . \ . ~erol()~ival "M~(,lf-lif(," of serun~ pro(( ' ins un(ler var ious ('on(ti(~on.-- ()f s(()r:)~(., l'~an.,,a.-, Acad . S,.i., t;(;: 771-777 , 1963.

12. ,,X,l()sh,y, ,l. W.. awl (;o()<lwix~. I/. F. Sqid)ilily of se rum-glu lamie pyruvi( ' t r ansaminase a('t~viiv ()n :~(,r:t~('. T(,( .hnol. B~fll. ( ]l(,gi,-,(. 5'Io(1. Te( 'hno l . ) , ;3.5: 183-187, 1965.

13. N()~r-I';lrt~n I"..\nl)lm(,m()l)hil~c ~lot)ulm in frozon plasma. Na tu re , 109: 187, 1963. 1.t. \V~,:,v(.r, l> , . . \ , an(t Imn~d(,ll. 1~. I). ,-knl~lwn~,)l)lfih(' fa(.lor (AI t I : ) .~tal)llil.v in fres]~

fr()z(,~ t)l()o(t ))ank l) las~a. TransfusIon, (;: 224-229, 19(i6.

"l']lc 'ttltll()r ack)lowle~lge.~ witll gratitude ttw adviee and assis-. tune~, of l)r:. ()sear Bo~tansky and ~Iorton Setlwartz and their as,-.istallce ;tt tlw Slo;tn K('ttering llls~,i(.ute for Cancer Rese'treh in Nc,w Y()rk (it3", of I)r. Natllm~ O. J(aplan m~d meml)ers of his .-taft ~tt ]gr:~ll~l~,is l_'~iversity, \V~tli, llmll, 51assachusetts; and of I)rs. :\ll)crt $)~liit) ~tnd 1)el~l)sey B. Mo:'rison, l 'n ivers i ty of Ten- lles,~('e, .%lex)~l)llis.