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66. Mechanism of hemolysis of erythrocytes by freezing at near-zero temperatures
ANNUAL MFETING half the .plasma decreased hemolysis by about half t.ha,t,, value, ()vet" the same range. Apparently, the pl'ot~eiliS (:)]7 plasma, are among the. factors responsible for injury 1,3' rapi([ freezing. 64. Relationship lmtween E l e c t r o l y t e - C o n c e n tration and R e d Cell Hemolysis. Ha[toLt) T. M).:R'r.~ta~ (Tissuc'. Preserw~tion Research Division, N~.~tionul~ Naval Medical. Cente.r, Bethesda, M a)'yl a,nd ). Th(., he~nolysis of truman r e d cells following freezing and tha,wing al:q)ears in. general ..t0 be p r o p e r ' t,ionn.l (.o. the sohlte eoneenta'ati/m produced.by the d(::hydr, tion of l'reezing, regardh:ss of whetlter the cellsre'(; suspende(l in eh.'.etroly.te or none.!eetrolyte. l.,',i'en, though ceils may. be.fi'ozen in nonelectrolyte solutions, the hyl)othesis tha(, injury resuI(,s fi'om eie-ctrolyte c o n c e n t r a t i o n is nob tested sin('e the intrade, llular c hmt.,'olyte is e0nc(mtrated to an 0smolaril,y sirnilnr to that, of the extrac(dh.flar solu(,ion.. Tiffs p a p e r reports .(,he results of fre0zing sl~u,lies COml:)arJngla varie(,y of solut.es, including some electrolytes which l:,enetrn, Le the red cell m e m b r a n e . . I t , has .been ,lenmnslrn.ted (:.hal, the hemolysis eusl.omarily seen. is.noL relai,e(t to the in.traeelhflar elecirolyte concentration per se but is proportional only to the (:oneentra(i(m of extr'tcell.t~lar, n0ndiffusiMe solute. SU,l:)l)ort,ing evidence is submitted.for "t m o , M wtu(.h 1)roI)oses that, the con(:enti'at, ion of .extr'Lcellular solute ('reatos a pressure gradient;, across tim m(~l.nbrane, whicli in turn leads i:o .the leakage of solute into t~l~e cell, ren,lering it.pot~entially bypertonie. In the absence of su('h a, gradien(;, very !figh solute (including eleetrolyle) concentrations fnil to be hemolytic. Sensitization to tl/ermal shock Ires niso t.)ee~_tshown (.o .be related to the.'develol)~nent of a twessure gradienl, across the metal)ran(:. 65. T h e Freezing. of t h e .Last Fraeiion o f Fr('ezable water and its Freezing R a t e as Factors i n . Ilemolysis27 B.. LUYI-tT ANI). C. .KROI~X),;R (Amerit~.a.n l, oundatmn for J3iologieal I{,esea rch ,- Madison, Wisconsin ). Blood frozen r(-:latively slowly to tempera.tm'es a b o v e - - 3 ° C does not lyse. F r e e z i n g to. temperaLures f r o m - - 3 ° C to , 8 ° 0
and exposure
to gh (L,.e ..:"
(:,elni)era(tlres. f9 r some. 30 rain. resu]l; in gradua.lly incr(::as.ing hemolysis (Luyet; and /~lenz, 1951). At - 3 ~ C . s o m e 80% of the.w_-';te.r is. transformed int~ iec and ',tt. - 8 ° 0 some 92% Lo's(Luyet,. and .c,:~,ehmtdt,,, " !3o0) .Phe red cells are then injm'ed whe~ the fi,t(.t)..m fi'om 80. to. a,)% freezes. On the othe.r h'm(l, Mood frozen rapMly ( s u c h as thai,, cont,ained in capillary • " -tubes' n.~mc~,.,.ed "~ , ',:":" lit bot,~s . ' ,at . tempera,t.ures from --20°(; to , o u ..;) urtdergoes a decreasing "lysis " at; gladuallx-" fas(;m" ~OOItIlgS " '" (Gehcnio; Rapatz, and IAwet,, 1963); exposing .
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such ral,i(lly frozen blood for long t i m e s to the s'IIll(} (.eml)eratures increases the lysis (Luyet and Gehcnio, 1955)• This behavior suggest.s tha(; the reason for the "eryol)roI.(,etive. act.ion" of ral)id ('ooling is that, not enough time. is allowed for the freezing of the last fra.etion of water. To test this relationship qtmntitt.ttively, we determined by mierodilatometxy and reeorde(l in motion pictures the rate of ice. formation during the freezing at varioust.(mq)e):atures of blood in capillary tubes. T h e resul(s of ~t first mries of such determinations show t.lu).(. Ihe..time t.aken for the formation of the a.tnotinL of ice readily detectable by dilatometry d(,ereases from a minute o," 2 at - 4 ° C to less than a. second at -80°C, These values are very low in comparison with the times of exposure to those temperai.ur(~S r(,l)ort, ed to result, in hemolysis. The diserepan(.ies may imlica(e that time-.consuming aftereffects of fr(:ezing are involved in hemolysis; we may als0 have t:)e(.,.n unable to detect tl~e fi'eezing of (he l,ust f:'netioa of freezable wgtter. The in(luiry is being extended to Mood contaitiingl.)rotc,.ei.ive agOll {S, 66. Mechanism o f .H(:molysis o f Erythroeytes l)y Freezing at Near-Zero Temperatures. ToKIo NI,:I (The. Institute Of Low Temperat..ure Scmnee, ~ " ItoMca i do (.rm'v c,,""',>I.v,.. i Sa pporo,~' " Japan' ). Wlmn. nmmmalian blood is.frozen iu a range of temlmrtttm'cs deere'tsing .from - - 2 ° 0 to approxi'matelv.. --Ifi"C~ and thawed,. hemolysis Occurs with a)l in('rease fi'om 0 io 100%; In order t0.eluci(laCe t h e t]~I(!CI|ttI.IISII'), " ' " . Of hemolysis . . . . . . bv freezing a.l~ thc,..e ,¢., n(:ar-ze.l'o (.empera.ttlres, microscopic observation and m e a s u r ( , m o n t of hemolysis using ral)bit, blood ~t.t(. mad(-., in the pi'esent experiment. Observa.tions with a sp(..ci,d r, •,. ,o ~ re"i" ,(..3.omi(.los,=ol.e veal ed. flmt some of erythrocyl:es were-hem01yzed ns (;he. temPerature decreased .durh~g the fi'eezing process, while others w e r e Jminolyzed upon rewarming. T h i s f i n d i n g ~ e m s to have a close relation t0 hemolysisresulting froth exposure t0 strong sohlt.ions an(1 transfer to dilu(~e solutSons: l-n regar~l to. t,he eai,se 0f..freezing damage to e ry ~hre ey tes,. h owe v.e.r, o th e r data obtai ne..d from a l)oint of View. of morphology a n d p h y s i o l o g y offer a c.eriam op!)osltmn.• t~o.&e salt,.mlur35.#heory. I~ ~eomS likely thai; .mechanical effect i is:'-rgther importan(, as a cause of ¢iell damage oecun'ing during the freezing process down to --10°C. 67. Survival o f Hmnan. B l o o d i5"ozen.by the Low Glycerol.Liquid Nitrogen R a p i d F r e e z e
:~N.D Aa~-mx Ki-n;L..',~I,m( T l m New York Blood
X
z~Sul~p0rted by. research .flmds .from Unim~Carbide Corpor~:dion. and the ~N"a(:aonal' Heart Institute
and exposure
to gh (L,.e ..:"
(:,elni)era(tlres. f9 r some. 30 rain. resu]l; in gradua.lly incr(::as.ing hemolysis (Luyet; and /~lenz, 1951). At - 3 ~ C . s o m e 80% of the.w_-';te.r is. transformed int~ iec and ',tt. - 8 ° 0 some 92% Lo's(Luyet,. and .c,:~,ehmtdt,,, " !3o0) .Phe red cells are then injm'ed whe~ the fi,t(.t)..m fi'om 80. to. a,)% freezes. On the othe.r h'm(l, Mood frozen rapMly ( s u c h as thai,, cont,ained in capillary • " -tubes' n.~mc~,.,.ed "~ , ',:":" lit bot,~s . ' ,at . tempera,t.ures from --20°(; to , o u ..;) urtdergoes a decreasing "lysis " at; gladuallx-" fas(;m" ~OOItIlgS " '" (Gehcnio; Rapatz, and IAwet,, 1963); exposing .
r ;¢
"
'
~r',.O[~
ABS'I ' " "RA ' , C'1",~ ,~
379
such ral,i(lly frozen blood for long t i m e s to the s'IIll(} (.eml)eratures increases the lysis (Luyet and Gehcnio, 1955)• This behavior suggest.s tha(; the reason for the "eryol)roI.(,etive. act.ion" of ral)id ('ooling is that, not enough time. is allowed for the freezing of the last fra.etion of water. To test this relationship qtmntitt.ttively, we determined by mierodilatometxy and reeorde(l in motion pictures the rate of ice. formation during the freezing at varioust.(mq)e):atures of blood in capillary tubes. T h e resul(s of ~t first mries of such determinations show t.lu).(. Ihe..time t.aken for the formation of the a.tnotinL of ice readily detectable by dilatometry d(,ereases from a minute o," 2 at - 4 ° C to less than a. second at -80°C, These values are very low in comparison with the times of exposure to those temperai.ur(~S r(,l)ort, ed to result, in hemolysis. The diserepan(.ies may imlica(e that time-.consuming aftereffects of fr(:ezing are involved in hemolysis; we may als0 have t:)e(.,.n unable to detect tl~e fi'eezing of (he l,ust f:'netioa of freezable wgtter. The in(luiry is being extended to Mood contaitiingl.)rotc,.ei.ive agOll {S, 66. Mechanism o f .H(:molysis o f Erythroeytes l)y Freezing at Near-Zero Temperatures. ToKIo NI,:I (The. Institute Of Low Temperat..ure Scmnee, ~ " ItoMca i do (.rm'v c,,""',>I.v,.. i Sa pporo,~' " Japan' ). Wlmn. nmmmalian blood is.frozen iu a range of temlmrtttm'cs deere'tsing .from - - 2 ° 0 to approxi'matelv.. --Ifi"C~ and thawed,. hemolysis Occurs with a)l in('rease fi'om 0 io 100%; In order t0.eluci(laCe t h e t]~I(!CI|ttI.IISII'), " ' " . Of hemolysis . . . . . . bv freezing a.l~ thc,..e ,¢., n(:ar-ze.l'o (.empera.ttlres, microscopic observation and m e a s u r ( , m o n t of hemolysis using ral)bit, blood ~t.t(. mad(-., in the pi'esent experiment. Observa.tions with a sp(..ci,d r, •,. ,o ~ re"i" ,(..3.omi(.los,=ol.e veal ed. flmt some of erythrocyl:es were-hem01yzed ns (;he. temPerature decreased .durh~g the fi'eezing process, while others w e r e Jminolyzed upon rewarming. T h i s f i n d i n g ~ e m s to have a close relation t0 hemolysisresulting froth exposure t0 strong sohlt.ions an(1 transfer to dilu(~e solutSons: l-n regar~l to. t,he eai,se 0f..freezing damage to e ry ~hre ey tes,. h owe v.e.r, o th e r data obtai ne..d from a l)oint of View. of morphology a n d p h y s i o l o g y offer a c.eriam op!)osltmn.• t~o.&e salt,.mlur35.#heory. I~ ~eomS likely thai; .mechanical effect i is:'-rgther importan(, as a cause of ¢iell damage oecun'ing during the freezing process down to --10°C. 67. Survival o f Hmnan. B l o o d i5"ozen.by the Low Glycerol.Liquid Nitrogen R a p i d F r e e z e
:~N.D Aa~-mx Ki-n;L..',~I,m( T l m New York Blood
X
z~Sul~p0rted by. research .flmds .from Unim~Carbide Corpor~:dion. and the ~N"a(:aonal' Heart Institute