(SANITIZED)RUSSIAN AND ENGLISH-LANGUAGE ARTICLE BY DR VINOGRAD-FINKEL ON DEEP-FREEZING OF BLOOD/REFLECTION OF WORK AND CHANGES IN TECHNIQUES.(SANITIZED)

• Document Type: CREST
• Collection: General CIA Records
• Document Number (FOIA) /ESDN (CREST): CIA-RDP80T00246A025000020001-1
• Release Decision: RIPPUB
• Original Classification: C
• Document Page Count: 47
• Document Creation Date: December 27, 2016
• Document Release Date: April 28, 2014
• Sequence Number: 1
• Publication Date: June 17, 1964
• Content Type: REPORT

Declassified in Part - Sanitized CopY-Wp?rOV'easfor Release 2014/04/28: CIA-RDP80T00246A025000020001-1
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Declassified in Part - Sanitized Copy Approved for Release 2014/04/28: CIA-RDP80T00246A025000020001-1
STAT
1963
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Declassified in Part - Sanitized Copy Approved for Release 2014/04/28: CIA-RDP80T00246A025000020001-1
Declassified in Part - Sanitized Copy Approved for Release 2014/04/28: CIA-RDP80T00246A025000020001-1
K c8edeatuo tutmatnezteti mcypitaAa
AIM noaytteHaSi MailHoro KOMn.neicra wypHa.na
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IloanHcHasi nmaTa Ha no.nyro,nme
. py6. 80 Kon.
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xo3ax, B ytie6Hbix aarieemislx H ripeAdlemisix.
PeaKu,HH }KypHaziow H H3,11,aTeJ1bCTB0 noLlf1HCKy Ha
AcypHatua He npmmaioT.
WYPHAJibl B PO3HINHY10 11M:0/1007 HE Ho( TsTlAIOT.
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11PHMEHEHHE raYBOK0110 XOJ1OAA
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0 3aM0paAHBaHHH mpoemix WieTOR 'Sea HX pa3pyweHHH B Te4CHHe Hecxonbloix
1.31, 47].
3
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X yseaHtlemno ee npoHHuaemocTH H na6yxamno. IlpH nepexone Taxa! Kamm cuosa
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pu, kax, Hanpumep, noapemmeHHe 3113HMaTHMeCKHX H Apyria 6KTHBHUX cc-re aA(1000A
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uecce xpHcTaa.no06pa30aat4Hn. 31mcnepHmeliTanbHo ycTaHosaeHo [38, 65], 4TO KpacTaAA006-
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Ba14m0 otietib me.niatx Hptictaa.rioh H H corposotKrlaeTCH 60Jibu1HM H3a,nevermeM BoAbl H3
4
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,pacTHopa K ero rHnepHoHneHrpanHeh. 1103TOMy yCHAHH HurrexmaTediefi H36e>Ham OH,
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7
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HbIX 3pHTp011111-0B paBHHIlest 87 94 B oHTeitHepe Taxort Ate eMKOCTM
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Declassified in Part - Sanitized Copy Approved for Release 2014/04/28 : CIA-RDP80T00246A0250000200011
Declassified in Part -
Sanitized Copy Approved for Release 2014/04/28: CIA-RDP80T00246A025000020001-1
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- Declassified in Part - Sanitized Copy Approved for Release 2014/04/28 : CIA-RDP801-0074RAn9CnnrInOrlf1/-14 4
? Declassified in Part - Sanitized Copy Approved for Release 2014/04/28 : CIA-RDP80T00246A025000020001-1
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Tp01114TOB, 1103BOJIHIOLt1HA Ilp0H313041HTb 6bicrpo c c06.rn0aeHnem crepittmHo-
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riamH, H3 KoTopbIX tIOCJIC ueurpiRivri4poaaHmt MCOKHO acenrflow 3aKpbi-
TbIM cn0co60m OTAeJIHTb Na,acroR H J106BBHTb ma3m03ametonolumit pac-
itop. B 3TOM pacraope 3pHrpowpritylo B38eCb MOX(140 HeCK0AbK0 AHal
XpaHHTb B npHroiwom AAR nepealmatmsa COCTOS1HHR rip n 4-6?.
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MbIX 3TB111013.
TaKHM o6pa3om, nprn3eileinib1ri B Hacrosnuem coo6wemm MarepHaa
110KB3bIBBeT BO3MON poBa .11. H. World Refrigeration, 1960, v. II, p. 65.-13. B141.1 o?
p a A-4) Keah O. P., FHH36 y p r 4). F., 41eiioposa .11. 11. H up. Proceedings
sth congress Europ. Soc. Haematology. Basel, 1962, p. ? 533. ? 14. 13 II norp a AA) 14 B-
e h 0 I' IX congress Internat. Soc. Blood Transfusion. Abstracts, p. 26.-15, r H H 3.
y p r 41 I". Te3t1C14 38-ro naeAyma .Vmeitoro coBera Ilenrpa.mitoro HH-Ta reMa-
U RepeARBaHRH KpOBH. M., 1959, crp. 39. ? 16. Tpae BC it itIt 3. R. Liccueuo-
-,AHRR r.riyooKomy ox.1.1*,,teinno flpOTMIL/183Mbl. J1HCC, AOKT. .11., 1946'.? 17. rpaen-
K Nh 3. 1.Ycnexn (Trip. riitos., 1948, T. 25. B. 2. crp. 185.? 18. Kaua6yx B H. H.
ran AC, 1958, B. 2/5. crp. 217.-19. KayX4RWBHAH 3. H. Teaticht uoaa. 38-ro.
\ Ma ,Vtreitoro cows 1..tenrpa.1I.itoro HH-Ta remar000riiii H riepeuitsaints KpOBH. M.,
9AP9 crp. 40.-41.- 20. Kayx?iiiianti.lit 3. IL, Bitliorpau-41anxe.ris
hi6ypr 41. r H up. B ,a0CTH)KeHliR H B Rp0H3BOACTBe H ripHMeHeHHH
.o..toua B napolliosi xoasificrse. M., 1960, c'Tp 341.-21. Klicenes A. E. B KH,:
..ongress International de transfusion Sanguine. Paris, 1955, p. 779. ?22. ,/I 03 H H a-
.1 IHHCKR It .?1 K. 1.43BeCTHR ECTeCTB. Baysnioro HH-Ta HM. JIecraOra, 1952, T. 25,
Afp I. f-Tp 23. .TIoaitita-JlositticKsii .11. K. B KH.: ,LI,OCTHIKeHHH H 38/01114
lipon T H R flpHMeHeHHH xonoua B Hap0.3HOM X03RIICTBe. M., 1960, crp. 332. --
24. 11 ('K p4) BC K H It R. H. B RH. Cospememibie npo6uemm remaronoriat H nepeuttaantm
isposa M., 1952, B. 26, crp. 143.-25, floxposcicit It TT. H., BilitoitypoBa F. n.
ram Ar, 1953, B. 28, crp. 75. ? 26. PaaymoBa .11. ./1., Kyupa W o B a C. H. Tench!
aos.1 3$-to nueityma WI-tenor? coaera Llelirpambrinro HH-Ta remaTO.ROTHH H nepenintantni
Bposu. M., 1959, crp. 41. ? 27. 4) euopo B a $1. H. Tam 'Be, crp. 42. ?28. (I) e ()-
oval! 3. H. KoucempoBakite KpOBH not remneparypax HInKe 0?C, ,E(HCC. aHA.
1960 ?29. 41 eu opoB H. H. H up, B KH.: MeXaHH3Mbl naroiornitectots peatatini,
.1.. 1'445, B. 7-8, crp, 122;, 136. ?130. Bricks M., Bessis M., C. R. Soc.. Biol.,
149, p. 875. ? 31. Haynes L. L., Tullis J. L., Pyle H. M., J.A.M,A.,
196c. \ ;73. p. 1657. ? 32. HU ggins C. E., IX Congress of the International Soc,.
of Blood Transfusion. Abstracts, p. 25.-33. Ketchel M. M., Tullis J. L. et al..
.A.M.A., 1958, v. 168. p. 404.-34. Genenio P. M., Lu yet B. J. B Procedings
.f the 6th Congress of the International of Blood Transfusion. Basel, 1958, p. 330.
15 Lovelock J. E., Biochim. biophys. Acta, 1953, v. 10, p. 414,-36. L o v e-
' o k J. E., Proc roy. Soc. B., , 1957, v. 147, p. 427.-37. Lovelock J. E.,
Bishop A1. W. H., Nature, 1959, v. 183, p, 1394.-38. Luyet B. J., Biodynatnica,
1919, v. 6, p. 207.-39. Lu yet B. J., Proc. roy. Soc. B., 1957, v. 147, p, 434. ---
40. Meryman H T.. K a f ig E.. Proc. Soc. exp. Biol. (N. V.), 1955. v. 90. p. 587.-
41. Mer y ma n 1.1. T.. Fl 0 1111110 Worth J. W. B Proceedings of the 6th
Congress of the International Society of Blood Transfusion. Basel, 1968, p.
42 Merym a.n II. T Proc. roN Soc. B., 1957, v. 147, p. 452. --43. Moll i son P. L.,
Sloviter it A., Lancet, 1952, %. 2, p. 501 -44. Mollison P. L. B htt.: 5 con?
gris International de transfusion sanguine. Paris, 1955, p. 759.-45. Jones N. C. H
Mo Ilison P. L., Robinson M. A., Proc. roy. Soc. B., 1957. v, 147, p. 476. ?
46. Parkes A. S., Proc. roy. Soc. B., 1957, V. 147, p. 424.-47. Pyle H. M., H a y-
n e s L. L., et al., IX congress Internat. Soc. of Blood Transfusion. Abstracts, p. 22.-
48. Rinfret A. P.. Doebbler G. F., Cowley C. W., Proceedings 8th Congress
of the International Society of Blood Transfusion. Basel, 1962, p, 439,-49, R I n-
f re t A. P., Cowley .C. W., Doebbler G. F. et al., IX congress. Intern. Soc. of
Blood Transf. Abstracts. 1962, p. 27.-50. Sloviter H. A., Ra v din R. G. B
Proceedings 7th Congress of the International Society of Blood Transfusion. Basel,
959, p. 70.-51. Sl o% itet H. A., Am. J. med. Set., 1956, v. 231, p. 437,--
52. Smith A. U., Lancet, 1950, 1r, 2, p. 910.-53. I d em, Nature, 1958, v. 182, p, 911, ?
54. Sproul M.. T., S loon M. H., Papers in Dedication P, H. Andersen Birth-
day Published by Munkaard. Copenhagen, 1957. -- 55. S t r ii m i a M. M., Co1-
w el 1 L. S., S trumi a P. V. B Proceedings of the 8th Congress of the Inter-
16
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Declassified in Part - Sanitized Copy Approved for Release 2014/04/28: CIA-RDP80T00246A025000020001-1
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Problemy Gematologii i Perelivaniya Smati
(Problems of Hematology and Blood Transfomiim)
Vol. 5, (entire issue pp. 3-16) 1963
[Inside cover is a notice to subscribers about subscriptions]
USE_OF DEEP-FREEZING FOR THE PROLONGED PRESERVATION OF BLOOD IN THE
FROZEN STATE
By: Prof. F. R. Viograd-Finke, Assoc. Prof., A. E. Kiselev,
F. G. Ginsburg, L. I. Federova, E. I. Kaukhchishvili
Institute of Hematolvgy and Blood Transfusion of the Order of Lenin
(Director, Assoc. Prof. A. E. Kiselev), Ministry of Health
of the USSR
STAT
At the present time the development of methods for preserving blood
in the Pprzen state occupies a central place in the field of blood
conservation. For many years living cells, erythrocytes in part-
icular, could not be maintained following freezing and thawing.
The great practical importance of this problem lies in the fact that
owing to the complete suppression of metabolism at subeero temperatures
it is possible to keep the cell viable for long periods (many months
and even years). This cannot be accomplished at positive temper-
atures.
The advances made in this field open up, even now, great possibilities
for a significant extension of the period over which blood can be pre-
served. It has been established that erythrocytes that were kept
(in glycerol) in the frozen state for a period of many months and
even several years, after thawing remain basically intact and retain
their physiological properties [31,47].
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When work on this problem was begun small quantities of blood were
frozen, and the authors proposed this method for the prolonged
storage of standard erythrocytes necessary for determining rare blood
groups[30,44].
In a number of papers it was pointed out 01,47] that this method
makes it possible to preserve blood of rare groups for long periods
for transfusion or for preparing heparinized blood in advance for
extra-corporeal circulation.
It is also thouiht that prolonged preservation of blood at sub-zero
temperatures becomes of very great iOportance in creating blood re-
serves in special conditions [49150157)6
We believe that the preservation of blood in a frozen state should
gain wide use in the practical work of blood service institutions.
In the first place, this is necessary for use in transfusions of
erythrocytes remaining after the preparation of dry plasma, the period
of storage of which at 4-6? is limited only to 3-4 weeks. Under
these circumstances it was possible to preserve blood of rare groups
and Ah-negative blood.
I. I. Fedorov and Smith L29,521 were the first to report that it is
possible to freeze and thaw erythrocytes without considerable im-
pairment to them. Several authors have reported the use of erythro-
cytes for transfusion after storage of several months at -10, -200
L2,315-7,21,27,28,431441; there are also data on the freezing of
blood cells without damage to them for periods of soTeral years [31,47].
Advances in the field of blood preservation by freezing have sub-
stantially aided modern biology in the study of the stability of
living cells and other biological objects (up to the level of entire
organisms) to the action of cold. This new branch of biology is
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called cryobiology. Cella and tissue. have been found to exhibit a
great variety of reactions to the action of cold and it was found that
negative temperatures as low as -105o do not bring about death of cells
and tissues under certain conditions. Following such a high degree of
cooling they remain viable, they 'take" in transplants, and exhibit
growth in tissue cultures. In addition, significant advances have
been made in tests with deep freezing and revival following warming of
entire animal organisms. These experiments include the freezing of
insects end other living organisms at temperatures down to -1900
[18,22,23]. In 1957 Smith and associates [46,53J showed that hamsters
frozen for an hour at .50, survive following warming by means of dia-
thermy and artificial respiration and they completely regain their
function. In connection with this it was noted that only those animals
survive in which the conversion of water to ice crystals did not exceed
50% of the total amount of fluid in the organism. Rabbits and certain
primates (salago crassicaudatus) did not endure such freezing: in spite
of the restoration of cardic activity and voluntary movements immed-
iately following warming, the animals died soon thereafter. This in-
dicates that it is more difficult to reach compatibility of the life
of an organism with a considerable degree of freezing of water, than
to preserve the viability of isolated cella and tissues under these
conditions.
These results necessitated the review of many previously dominant
concepts in the scientific literature on the boundaries of life and
the problem of amabiosis, the causes of the damaging action of deep
freezing, and on the basis of this it befase necessary to study the
conditions under which it is possible to maintain the viability of
frozen live cells.
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It was was found possible to protect erythrocytes from destruction only
after studying the causes of the injurious action of the freezing
process. In this respect there were numerous investigations of the
biochemical and biophysical changes occurring in cells and the nature
of the crystallization of water in blood subjected to various degrees
of cooling L11 24,26,35,39,42). The majority of investigators believe
that the damage to erythrocytes is the result of two phenomena: the
trauma by ice crystals and the effect of hypentoncentrated salt
solutions formed intra-cellularly and extra-cellularly in the remain-
ing living substance upon conversion of water to ice. Under these
conditions erythrocytes undergo progressive dehydration along with
an increase in the osmotic gradient between their internal medium
and their external medium.
Lovelock L.36.) observed the denaturation of lipoprotein complexes in
the presence of hyper-concentrated salt solutions. Be also found
that in addition to gross damage during the period of growth of ice
crystals, freezing also destroys the molecular bonds in the living
cella and most of all in membranes. In a medium with a high ionic
strength obtained by increasing the salt Concentration, the binding
components of the phospholipids of the cell membrane are weakened
which results in increased permeability and swelling. Upon transfer
of such a cell back into a physiological medium, a slow lysis sets
in upon thawing.
Other not yet known factors may also play a role la the destruction
of cells, such as the damage to enzymatic and other active systems
of living cells Lited , but the dominant reason is the extreme extraction
of water during the process of crystal formations It has been estab-
lished ex2erimentally L38,55J that crystallization and, consequently,
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its hareul action is observed most of all in the -3 -40? sone.
For this reason this temperature range is called critical or danger-
ous. The period during which the burzen erythrocytes are in this
zone in an environment of hyper-concentrated salt solution affects
their stability. Luyet [38] showed that when the critical temperature
zone is passed over in a period of several milliseconds no lethal
effect is found: he succeeded in preserving the morphology of erythro-
cytes by very rapid freezing of blood in a thin layer (on a metal plate)
at -196?. When this period becomes longer, for example, when cooling
a large amount of blood under the same conditions, without using pro-
tective substances the majority of erythrocytes are destroyed. The
period that the cell spends in the critical zone is el:Rally important
during the thawing of the blood.
These biophysical data served as the basis of the now-proven position
that the rates of freezing and thawing play an important role in pre-
serving the integrity of erythrocytes, the water phase of which is
present primarily in the free state and is readily transformed to ice
crystals upon cooling.
It was found that slow freezing is accompanied by extra-cellular forma-
tion of large ice crystals which do not necessarily rupture the cella
owing to their position in the canals between the crystalline lattice
of the forming ice. But the resulting dehydration and concentration
of salts have a more powerful destructive effect on the cell than the
extracellular crystals. By using substances that strongly bind water
it is possible to prevent its transformation into ice crystals or to
interfere Kith their growth in size. On the other hand, rapid freezing
leads to the formation of very fine crystals and is not accompanied by
a large extraction of water from the solution and does not render it
hyper-concentrated. Therefore,in trying to avoid factors that result in
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damage the investigators directed their efforts toward both binding
the free water by adding various substances to the blood, such as
glycerol, sugars, colloidal substances and ethyl alcohol, as well as
toward increasing the rate of cooling and thawing.
The various modern approaches to the solution of the problem of pro-
longed preservation of blood by freezing have also been based on the
above theoretical premises.
In the USSR attempts have been made to preserve blood at negative
temperatures without the
The preserving solutions
for this purpose make it
formation of crystals - in
with protecting substances
possible to preserve blood
-80? and somewhat lower in the liquid state without
the liquid state.
proposed especially
at temperatures of
hemolysis, so that
it remains suitable for transfusion on the average for 100 days C1,3,
4,7,9,21J.
However, it was not possible to preserve blood in the liquid phase
without heaolysis for longer periods. Preservation of blood for many
months or even years necessitates complete suppression of metabolism
in the cells and cooling to a complete solidification.
At the present time two concepts are Ling widely uSed in the solution
of the problem of the storage of blood in the solid frozen state. One
of these is the preservation of erythrocytes with high concentrations
of glycerol (up to 50%) at moderately low temperatures C-80?, -120?j.
This so-called slow freezing, developed about ten years ago, has been
subjected to
discussed in
with the use
detailed experimental studies and it has been widely
the literature 01,43,45147.51,57]. Clinical experience
of such blood is accumulating.
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In recent years [32,37] they began to study the use of dimethylsul-
foxide which is similar to glycerol in terms of the mechanism of its
protective action. The protective action of glycerol on the cell
lies in preventing the formation of large ice crystals outside or within
the cells owing to its especially strong ability to bind free water and
to penetrate into erythrocytes. In this manner, glyerol lowers the
degree of hyper-concentration of salts and makes it possible to extend
the period that erythrocytes can exist in the dangerous temperature
zone without damage to them. Therefore, the cells can be frosom
slowly in the presence of glycerol. Thus a flask or plastic bag with
500 ml. of a mixture of erythrocytes and glycerol placed in a refriger-
ator reaches this temperature (-80?) only after seven 4ours [31]4 This
method makes it possible to preserve the frozen erythrocytes for many
months with insignificant (from 2 to 10%) damage to them following
thawing. However, this method is not yet readily accessible for
clinical use due to theouftersome nature of the extraction of high
concentrations of glycerol causing sharply expressed hypertonia in the
cells and due to the need for special apparatus [45].
For the treatment under sterile conditions of glycerolized blood with
several solutions of successively decreasing concentrations of glycerol
anu salts there have been build in the U.S.A. costly and very ineffic-
ient fractionators, the so-called Cohn centrifuges [31,33], Even with
these fractionators the washing out of glycerol takes a great deal of
time. The defrosted erythrocytes cannot be transfused without washing
out the glycerol, because upon transferring them into the isotonic
medium of the blood stream they are rapidly destroyed. During the
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washing out procedure an additional 1088 of a certain portion of the
erythrocytes occurs aa a result of their lysis. According to recent
data [57] the loss of erythrocytes following their storage with
glycerol and washing amounts on the average to about 20%.
The otter concept is the super-highspeed cooling(requiring not more
than several minutes) to ultra-low temperatures without using glycerol.
At this rate of cooling the time spent by blood in the unfavorable
critical temperature zone is greatly reduced, i.e., conditions are
created under which the period of transition of the erythrocytes through
the dangerous temperature zone will be less than that necessary for
damage to occur to the cells in this zone. This method is more prom-
ising and it does not require the lengthy treatment of blood after
thawing, since in the protective solutions that are also required in
this method one can do without glycerol.
This method directed at obtaining crystal-free freezing as a result
of high rates of cooling has been used as the basis of the method of
preserving blood at negative temperatures worked out recently at the
Central Institute of Hematology and Blood Transfusion.
Rapid freezing of blood is favored by a number of American authors
E4o,41,48 49,55,58J. These authors, as well as the authors of this
paper, used high concentrations of carbohydrates and colloids in the
protective solutions. Working along the line of replaiing in these
solutions glycerol whieh is impractical to Use, by other water-binding
substances, we have been using the above substancts right from the
start of the studies on the freezing of blood [6/8. 10,12,14].
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We are presenting below our experience in connection with the use of
deep-freezing in the ultra-highspeed freezing of blood, its long-term
storage and subsequent clinical use. We have worked on the problem
of rapid freezing of such quantities of blood that are practically
applicable to blood transfusion (250-500 $1.). The possibility of
preserving erythrocytes in an intact condition, after rapid freezing
in liquid
was noted
by other
nitrogen of minute quantities of blood (around 0.2 ml.),
by Luyet as early as 1949 [38]. Later it was confirmed
authors
spraying of blood
[25J, and also in studies in connection with the
into liquid nitrogen L12,19,40,41]. However, in
connection with Obtaining undamaged erythrocytes with rapid freezing
of large volumes of blood required in transfusion, it was necessary
to search for other conditions for achieving rapid cooling. Simul-
taneously it was necessary to develop more effective solutions for
protecting the frozen cells from destruction.
It would be undoubtedly efficient to achieve such a rapid cooling of
blood which completely excludes the formation of crystals and results
in glassy solidification, i.e., vitrification [16,17,34]. It is
thought theoretically possible to achieve vitrification by spraying
very small blood droplets directly into liquid nitrogen (at -1960)
since the result is instantaneous cooling (up to 100 per second).
It is also thought that by storing the vitrified blood at the sane
temperature it is possible to preserve it without recrystallization
for an indefinitely long period, i.e., without damage to the cells
a
(rapid thawing is again/necessary condition). The experiments we
carried out in this direction [12,20] showed that the spraying of
blood directly into liquid nitrogen can serve as one of the ways of
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vitrifying large amounts of blood. However, such an open freezing
and thawing (by immersion of blood granules into a warm physiological
? solution) does not guarantee the sterility of the blood destined for
transfusion purposes. Therefore, the problem was to work out a method
of super-highspeed freezing of blood placed in a closed vessel. The
latter must maintain the sterility of the blood upon contact of the
container with the cooling and the regenerating media.
Going to the closed method of freezing in a container posed new prob-
lems, the major one being the creation of conditions for the rapid
elimination of heat from the container containing the blood. The
difficulty lay in the fact that the cooling of large amounts of blood
proceeds at a much slower rate than the cooling of blood droplets.
In working out a method of super-highspeed freezing of blood placed
in the container, we took into account the following factors affecting
the rate of elimination of heat: 1) material of which the container is
made, 2) boiling point and other properties of the cooling medium, 3)
geometric shape of the container and thickness of the water layer (these
factors were related to the ratio of the external surface of the con-
tainer to the volume of blood), 4) the properties of substances used
in the protective solutions, 5) the varying stability of blood to the
action of low temperatures.
The first four conditions are constant, but the last one does, as a
rule, depend on the peculiar properties of the blood of the donor.
This frequently explains the variatins in the results obtained
fol.,owing recovery of frozen blood, as noted hy many authors.
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After testing various materials we selected alutinum, and made aluminum
uontainers of different capacities keeping in mind the important* of
geometric parameters that promote the elimination of heat.
In testing these containers we judged the advantages offered by then
through the degree of recovery of erythrocytes subjected to freezing
in them, using as a criterion for this the extent of hemolysis deter-
mined in a definite sample of thawed and centrifuged blood. The freez-
ing was brought about by immersing the container with the blood, mixed
half and half with the protective solution, liquid nitrogen. Solidi-
fication of the blood was completed in 1-3 minutes, depending on the
volume of blood and the shape of the container. In thawing, the con-
tainer was rapi-lly transferred to a water bath at 43-45?. The period
of thawing of the blood corresponded approximately to the period of
freezing.
The following is a discussion of the mechanism of elimination of heat
with rapid freezing of the blood in conjunction with different com-
ponents added and the selection of the container.
At the moment of immersion of the container in liquid nitrogen, inten-
sive eliminAtion of heat is taking place from the surface of the con-
tainer containing the blood and there Wale a warming up of the ad-
jacent layers of nitrogen, as a result of which nitrogen begins to
undergo violent boiling. Observations indicate that bubbles of nitrogen
gas arising on the walla of the container rapidly increase in volume,
break away and float to the surface of the liquid nitrogenp new particles
of liquid nitrogen occupy the place freed in this manner and come into
contact with the container. This process is repeated until the
temperatures of the nitrogen an the container areekaalized.
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At this time the process of elimination of heat is completed thereby
servin as an indication of the completion of the process of freezing.
Under these conditions the cooling medium is not liquid nitrogen any
more but a peculiar gas-liquid mixture which considerably impairs the
conditions of the elimination of heat. Nevertheless, the use of liquid
nitrogen remains the single convenient method of achieving 'lipid freez-
ing of blood.
During freezing, the heat directed from the central layer of the ob-
ject (blood) overcomes the thermal resistance of the following elements:
a) a layer of liquid blood, b) frozen layers of blood, c) walls of the
container, d) gas-liquid mixture. Therefore, in selecting the optimal
container from a number of containers tested an important role is
played by the determination of the coefficient of heat transfer (K).
The numerical significance of this factor may be demonstrated by using
the well-known heat transfer equation
Q, = K.F. 4t (1)
where Q amount of heat transferred per unit time; r . surface of heat
transfer (readily determined in experiments); At - difference bbbleeen
the temperature of blood and the coolih2; medium (a known quantity). The
only factor that affects the value of (4, is the coefficient of heat trans-
fer, K.
The amount of heat (Q) that is to be removed can be determined from
the equation
= G[C ?(t -) + n (2)
2 t3
where 0 - weight of blood sample (in kg.); C1 - specific heat of non-
frozen blood (in kcal/kg ?C); c2 - specific heat in frozen blood (in
kcal/kg. C); t1 - initial temperature of blood; t2 - temperature at the
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beginning of freezing of blood; t3 - final temperature of frozen
blood; VILo- weight of ice in a kilogram of frozen water (in kg/kg);
R - latent heat of ice formation (in kcal/kg.).
By substituting the value obtained for from equation (2) and all
the remaining terms into equation (1), one can determine K, i.e., the
the overall coefficient of heat transfer (kcal/M2 co per hour).
However, in selecting the shape of the container not only the deter-
mination of the overall coefficient of heat transfer is important, but
heat
also that of the value of the coefficient ofkimmisaion (a2) from the
wall of the container to the liquid nitrogen (since the intensity
of movement of the nitrogen gas-liquid mixture depends on the shape of
the container). This coefficient can be determined using the value
of K calculated above from the following formula (in the case of
a flat-walled container)
1
(3)
1 1
- 1 )2
A
a 1
1\2
.11?1=1010.?
2
where a1 - coefficient of heat transfer from liquid to frozen dbod;
1 - total thickness of frozen layers (measured from the center of
the cross section of the container to the periphery); Ni - coefficient
of thermal conductivity of the frozen layers of blood; s)2- wall thick-
ness of container; ,>\2 - coefficient of thermal conductivity of the
container; a - coefficient of heat emission from the wall of the
container to the liquid nitrogen. From here
21_ +6'2
1 Ni -Ai
1
a2
1 1
--- or
.L
A2
1
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Therefore,
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m2 ms
1 1 2,2
1
7
o1 12
:\2
kcal/m20C per hour [4i. From the values obtained for K or m2 for
the containers tested, we chose those that provided the highest values
for these coefficients. From the containers selected in this manner
the best results in the recovery of erythrocytes following freezing
and thawing were obtained with containers with a built-up surface
in which the ratio of surfaces of elimination of heat to the volume
of frozen blood ( -) was the highest. These may be cylindrical,
V
tubular, or flat rectangular vessels (Fig. 1).
In numerous experiments we established that rapid freezing of blood
in containers with a built-up surface was always accompanied by a
considerably better recovery of erythrocytes after thawing than freez-
ing in smooth containers (Tables 1 and 2)
Figure 1 - Aluminum blood-c
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It is is seen from Table 1 that in the majority of cases in freezing
small amounts of blood (up to 40 ml.) in containers with a built-
up surface (we used cotrugated surfaces in our studies), independent
of the shape of the container, its cross section, and the volume of
blood, the percentage recovery of erythrocytes was high (from 90 to
95% after thawing blood in solutions No. 112 and 113. In the same
Table 1
INFLUENCE OF THE SURFACE ON THE RECOVERY OF ERTTHROCYTES
(in %) FOLLOWING FREEZING
!Volume of
blood So,ution no. 111 !Solution No. 112
Container frozen
(in al. )
Auilt-upt Smooth Buillt-up'Smooth .Built-up
Solution No. 11,
Surface
moth
Circular !
container 30 88 1 65 90 70 92 81
" 30 84 65 91 68 92 76
30 82 70 90 8,
30 81 62 93 72
Tube 15 ,94 81 93 53 93 78
11 15 88 77 95 60 92 78
11 15 93 70 95 62
4o 91 45 87 35 92 40
90 1 40
11 40
11
11
r1
containers with smooth surfaces the percentage recovery of erythrocytes
was most frequently 60-78% and even lower.
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Table 2
MEGENERATION OF ERYTHROCYTES FROZEN IN CONTAINERS WITH A BUILT-UP
SURFACE AT DIFFERENT VALUES OF THE S RATIO
V
Container
Shape
Gage size
(in mm.)
(thickness) of
frozen la er
Volume
in
ml.
10
10
10
100
100
100
100
30
30
30
30
Corrugated
tube
It
Circular
corrugated
cylinder
ft
10
10
10
2
2
2
2
2
2
2
2
Calculat-
ed value
of
.S
V
iRegeneration of erythro-
icytes (in 34)
Soluti
n Number
i 111
12
4.2
92
92.5
4.2
94
92.5
4.2
93
92.5
2.8
91
94
2.8
90
91
2.8
86
93
2.8
87
90
2.6
93
91
2.6
92 88
2.6
88 88
2.6
84 88
This difference in the amount of regenerated erythrocytes was regularly
observed in all our tests (Fig. 2).
Figure 2. Importance of corrugated surface of container.
Hollow circles - corrugated surface; dark circles
smooth. Ordinates: per cent recovery of erythrocytes;
abscissas - period of exposure (in hours) to preserving
solution No. 112 and 113 (Volume 150-200 ml.).
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In Table 2 there are presented data that favor containers with a
large calculated value of the ratio of the surface of elimination of
heat to the volume of frozen blood, Joe*, S since the elimination
V
of heat from the frozen blood proceeds at a greater sate in them.
In order to adapt the method to the requirements of practice (blood
volumes used in clinical work, simplicity, convenience of freezing
and storage) we made a study of the optimal value of the cross
section and size of the container. A comparison of various cross
sections with the same form and capacity Of the container showed
the best results are obtained in a container of 2 etm cross section,
since it provides for a faster elimination of heat.
Thus in experiments using blood from the same donor in a 100 ml0
container with a 2 mm. cross section the percent recovery of erythro-
cytes was 87-94. In a container of the same capacity with a 5 mm.
cross section only 62-87% of the erythrocytes were recovered (in
the first case blood froze in 25 seconds, in the second in 45 seconds)*
However, in order to make a convenient container with a large capacity
(300-500 ml.) it was necessary to increase the cross section of the
container tO 15..20 mm. which resulted in an increase in the thickness
of the layer to be frozen and consequently in a decrease in the rate
of cooling of the blood. This frequently resulted in a reduction of
the percent recovery of erythrocytes. Therefore, measures were taken
to improve the technique of freezing and thawing. These included the
stirring of the blood tswinging or Shaking of the container) upon
immersion in the nitrogen and especially in the water bath. This
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speeds up the emission of heat since the blood is divided inside the
wontainer into thinner films stratified onto the layers near the wall
during freezing. This measure did result in a certain improvement
but a more significant influence is exerted on the preservation of
cells in the case of freezing a thick layer of blood by the nature and
amount of substances added to the blood for protecting the cells.
For the super-highspeed freezing of large volumes of blood we used
various versions of solutions worked out on the basis of the composi-
tion of solution No. il which we proposed earlier for spraying olood
into liquid nitrogen L20_1. This solution included glucose (final
concentration 4%), one of the disaccharides-P.Sucrose, lactose (final
concentration 2%), or mannitollcolloids (dextran or albumin), sodium
citrate (0.4%), and sodium bromide (0.1%). The improvement of the
solution involved an increase in the concentration of the carbo-
hydrates (solution No. 141). A higher content of sugars, glucose and
especially disaccharides Xsucrose, lactose), not permeating the
erythrocytes and binding water in the extra-cellular space, preventS
the transformation of water to ice cpystals. Accordingly, the addi-
tion of large concentrations of sugars extends the period, that the
cells may spend in the dangerous temperature zone without injury, to
several tens of seconds, at the same time that without sugars the
cooling of blood in this zone must occur in a fraction of a second
in order to obtain intact cells.
We also studied the effect of the addition to the solution of in-
creased concentrations of colloidal preparations - dextran (molecular
weight 60,000-90,000), polyvinylpyrrolidone (molecular weight 15,000-
25,000). The effect of the addition of these colloidal preparations,
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also based on bindin,-; free water, is the result of reducing the
amount o'f ice formed, therefore the concentration of the dis-
solved substances in the tiny channels between the ice crystals where
the erythrocytes are located does not reach levels that are harmful
to the membrane. It is also known that colloids, due to adsorption
on the surface of the cells, protect the readily permeable membrane
of the erythrocytes*
However, we have not yet established whether their protective action
is better than that of the carbohydrates. It is necessary to con-
tinue the investigations using similar substances, One should also
make mention of the requirment that the introduction into the organ-
ism of these high molecular weight subStances mixed with blood had
to be harmless, inasmuch as they may form complexes with plasma
proteins. The physiological significance of the complex formation
is the subject of detailed study, and their Use as an additive to
frozen blood cannot as yet be recommended for large-scale clinical
practice.
Therefore sugars still remain the most proven and harmlesOrepara-
tions for clinical use. The effectiveness of the addition to frozen
blood of each of the sugars used has been invariably observed in our
experiments, but the optimal indices of recovery of erythrocytes
following freezing (up to 93-95%) were obtained by combined use of
glucose and sucrose or lactose (Fig. 3).
In addition, the regeneration of erythrocytes is influenced on the
one hand by the volume of frozen blood, and on tha other by the
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SD
70
-19-
r. ...... ais *rata ?.???
%ftft"..........."'"?"".?ft.oft ammo.
? "'Nor
10 JO
Pilc. 3. 3Hil4CNtIC ,106118/1eHHIll STACNOAOH,
11a ocif a6ctoicc o6berbi Npoan (2 Ma); Ha 001 094w-
war npoupoor DOCCTINpeNeHNIII PN?poUJIToa I ? p lICTIDOp
Ii: I - pacrftop .141 II, N 1incro31; -- paorsop. .1*
H r.mmoF I pactnop Ng I I, C :I/SATO:ion N MAMMA .
Figure 3 - Importance of adding carbohydrates
Abscissas p volume of blood (in ml.);
ordinates - per cent recovery of erythrocytes;
1 - solution No. 112; 2 - solution No. 111 and
lactose; 3 - solution No. 111 and glucose;
4 - solution No. 112 with lactose and glucose.
difference in the stability of the erythrocytes to the action of
cold in different donors. This influence could be equalized by the
addition to the solution, outside of sugars, of a small amount of
glycerol, up to 6% (solution No. 11). This can beat be done upon
freezing the erythrocytes remaining after separation of the plasma.
In Figure 4 there are shown the results of an experiment using the
blood and erythrocytes of the same donor, and in Figure 5 the
results of many tests of freezing the erythrocytes (in solutions
No0112 Th and 113) of various donors. e addition of such a small
amount of glycerol does not necessitate, after thawing of the
erythrocytes, subsequent lengthy treatment in order to remove
glycerol from the cells after removal of the sample (?).
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90
80
70
60
50
-20-
30-60 f10-%.:0 2001.50
Pitc. 4. 3liagefine ao5a?Aeliti? pac?
112 HCOOALIHNX (5-6" 0
.11HtleCTB rnimepulia,
lia oca a6cu.14cc - 061,eubt lo 114 ig%11
OpAilHaT - flpOUCHT SOCCTil NORM eti ViniTOO-
LUITOR: I pacnop N? II g; 2 -- paciaop
Nit Kppaam
TWO KR kpoeb.
Figure 4 - Importance of addition to solution No. 112
of small amounts (5-.6%) of glzfcerol.
Abscissas - volumes (in ml.); ordinates - per cent
recovery of erythrocytes; 1 - solution No. 112;
2 - solution No. 113; circles - erythrocytes;
dots - blood.
Fi;ure 5
?
KV
N 41
? tat a ?
iA ff .,
At. t
Ami A
IV . 4e 6
1
?A
we A A ? 4.
10 6
0
60 e
4C0
40
?
JI
J0-100 110 100 .1100
PHC. 5. BOCCTIMOBACHHC 4pirrpouirrom rIpli pa:3?6i oli-bema
9pmrponwritoli macclil
Ha OCR 66c11.acc ? 061.emai (a am); NA tyck twimiar ni))tteaT Hoc
CiMMOMACHNI iptITpORMTOB; IMOKKU pmeranp 112: rpcyrnmemmam
p8CTROp M
Rotovery of erythrocyteg using different volumes of
erythrocytest
Abscissas - volume (in ml.); ordinates - per cent
recovery of erythrocytes; circles - solution No. 112;
- solution No. 11
3'
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These figures iniicate that following ultra-highspeed freezing and
taawing of erythrocytes, the main portion of the cells remains
unharmed* Nearly analogous data were obtained by freezing whole
blood in the same large volumes*
In Table 3 there are given data on the recovery of erythrocytes follow-
ing freezing and thawing of different volumes of erythrocytes and whole
blood with protecting solution No* 113.
The indices of recovery of erythrocytes we obtained with super-high-
speed freezing in a container are close to those published by other
authors L48, 56].
Since in defrosted blood there is usually a mixture of free hemo-
globin and stroma which is a contraindication for transfusion, we
believe that for the time being it is not advisable to subject whole
blood to treeing. It is better to store erythrocytes in the frozen
state for clinical use which contains twice the number of cells in
the same volume, whereby it is possible to use erythrocytes left over
after the preparation of plasma.
The morpholo&ical properties and the physiological integrity of eryth-
rocytes subjected to freezing and thawing underwent little change.
Experimental transfusions of defrosted erythrocytes gave good results*
Before transfusion of the liquid portion containing hypertonic solu-
tion No. 1130 the small impurity of free hemoglobin and stroma are
removed* For this reason the erythrocytes are previously diluted with
a series of solutions (citrate-lactose-salt) of different osmotic
concentrations, they are centrifuged once, the sample CO is removed
and replaced (to the normal hematocrit volume) by an iso-osmotic
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?
sucrose-salt solution or by homologous plasma. After such treatment
erythrocytes become osmotically stable for introduction into the
blood stream.
Table 3
RECOVERY OF ERYTHROCYTES (IN PER CENT) AFTER FREEZING (AVERA3E
OF 160 EXPERIMENTS)
Medium
Volume in 11*
75-100
150-200
200-500
Erythrocytes
91
88
86
Whole blood
93
92
87
For clinical use we worked out a method of preparing erythrocytes
that makes their rapid and sferiletreatment possible without the need
for a bloodi.fractionating apparatus, For this purpose we use double
plastic bags, from which, after centrifuging, it is possible to
aseptically separate the sample (2) by the closed method and to add
the plasma-replacing solution. In this ssi,Adon the erythrocyte.
suspension can be stored for several days in a state suitable for
transfusion at 4-6?.
In Figure 6 there is shown the scheme of the aseptic preparation for
transfusion of defrosted erythrocytes, showing the sequence of the
various stages.
This material presented in this paper indicates that it is possible
to solve the practical problem of the super-highspeed freezini; of
large volumes of erythrocytes and blood for prolonged storage.
To achieve this we built several aluminum containers having optimal
geometric parameters, in which, when immersed in nitrogen blood
freezes in 1-2 minutes. We also developed special protective solu-
tions for whole blood as well as for erythrocytes. Securing the
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4??????????.....
?
Pm. 6. Cxema acer1Tpiecao4 noararoexm pa3mopomiemmoit xposm,
I 34xpbtrwil nepesox paaropomexmon x3secx MI KOHTelHepli I riaactiorrino
14e I/ nocaexosareaxxoe tiodasnetiXe K vpWrpoiorritoll Xxiecit las6ismin-
11U4x pactimpbet /// nepesox laxprovid cnocotiosi XxAttcli P esoftArMill Me
-
Mix tiorAP xewrpmbyruposiumst IV ? Ao6asnemite x spierpoluitaii ItAi1li0311111V
pacreopm: I- - kox-reiixep c xpossio; 2 ? c xposhio; 3? ayeme
metaxa, upeAwrimaxemxhie Ann 3axpk1rofo otimtexxx mlitXXOCTII 0? SpirrObsurror
neitipti.yrupunaxxx; 4 36a6notio1llie pactaopkit 6 -- 11/1$31110Salie1114110-
mmil paCTgoll.
Figure 6 - Scheme of asceptic prepar ition of defrosted blood*
closed transfer of defrosted suspension from container
to plastic bags; II - successive addition to the
erytnrocyte.suspension of diluting solutions; III -
closed transfer of saLaple (7) to the free bag after
Centrifuging; IV- addition to the erythrocytes of
plasma-replacin solution: 1 - contaiaer with blood;
2 - bag wit'A blood; 3 - empty bags for closed separa-
tion of liqui frn: erythrocytes after centrifuging;
4 - diluting solution; 5 - plasms-replacing solution*
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-24.
sterility and the physiological competancy makes these suitable for
clinical use.
In this paper we have not discussed the practical realization of
prolonged storage of frozen blood and the clinical use of this blood.
These problems are the subject of separate reports. We wish to note
only that the successful conducting of processes or rapid freezing
solves only half of the problem facing us. In order to avoid damage
to the cells during prolonged storage as a result of possible re-
crystallization, it is also necessary to store frozen erythrocytes
at ultra-10w temperatures. We observed that the storage of blood
at temperatures near -1960 (in liquid nitrogen) involves almost no
furtder harm to erythrocytes. This is also indicated by theoretical
considerations.
At the present time it is necessary to solve the technical problems
related to building special equipment for the prolonged storage of
blood in the frozen state at ultrairlow temperatures.
REFERENCES
10 A. D. Belyakov, Reports to the Thirtietn Plenum of the Scientific
Council of the Central Institute of Hematology and Blood
Transfusion, M., 1955, p. 4.
2. A. D. Belyakov, Current Problems in Hematology and Blood
Transfusion, M., 1955, No. 31, p. 46.
3. A. D. Belyakov, Probl. Gematol., 1956, No. 1$ p. 35.
40 A. D. Belyakov, Current Problems in Blood Transfusion, L., 1957,
No. 5. P ? 51.
5. A. D. Belyakov, Vestn, Khir., 1958, No. 10, p. 11.
6, F. R. Vinograd-Finkel, F. I. Ginzburg, E. I. Kaukhchishvili, et al.,
Reports to the Thirtp.fifth Plenum of the Scientific Council
of the Central Institute of Hematology and Blood Transfusion M.,
1956, p. 1
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-25-
7. F. R. Vinograd-Finkel, FO G. Ginzburg, La I. Federova, et al.,
Probl. Germatol., 1958, No, 1, p. 27.
8, F. R. Vinograd-Finkel, F. G. Ginzburg, L. 1. Federova, et al.,
Advances and Problems in the Production and Use of Re-
frigeration in the National Economy, M., 1960, p. 338.
9. F. R. Vinograd-Finkel, Reports to the Thirtieth Plenum of the
Central Institute of Hematology and Blood Transfusion,
M. 1959, p. 37.
10, F. R. Vinograd-Finkel, F. G. Ginzburg, L. I. Pederova, Current
Problems in Blood Transfusion, L., 1959, No, 7, p. 87.
110 F. R. Vinog.rad-Finkel, L. L. Razumova, 6. N. Kudryashova,
Biofizika, 1960, Vol. 5, No. 2, pe 229.
12, F. A. Vinograd-Finkel, F. G. Ginsburg, L. I. Pederov, world
Refrigeration, 1960, V. 11, p. 650
13, F. R. Vinograd-Finkel, F. G. Ginsburg, L. I. Federov, Proceed-
ings 8th Congress Europ. Soc. Hematology, Basel, 19620
Pe 533*
14. F. R. Vinograd-Finkel, IX Congress International Soc6 Blood
Transfusion, Abstracts, p. 26.
15. F. Go Ginzburg, Reports to the Thirty-eighth Plenum of the
Scientific Council of the Institute of Hematology and
Blood Transfusion, M., 1959, p? 39.
16. E. Ya, Graevskii, An Investigation of the Deep Cooling of
Protoplasm. Doctoral Dissertation, L. 19460
17. E. Ya. 0raevskii, Uspekhi Sovr. biol., 1948, Vol. 25, No. 2,
p. 185.
18. N. I. Kalabukhov, ibid., 1958, No. 2/5, p. 217.
19.
U. I. Kaukhchishvili, see ref. 15, pp. 40-410
20. E. I. Kaukhchishvili, F. R. Vinograd-Finkel, F. 3. Ginzburg,
et al., Advances and Problems in the Production and Use of
Reargeration in the National Economy, M., 1960, p. 341.
21. L. B. Kiselev, Vth Congress International de transfusion
Sanguine, Paris, 1955, p. 779.
22. L. K. Lozina-Lozinskii, Izve6tia Estestv. Nauchnoc.m in-ta im.
tesgafta, 1952, Vol. 22, No. 1, p. 3.
23. L. K. Lozina-LoxinsKii, Advances and Problems is the Production
and Use of Refrigeration in the National Lconomy, M., 1960,
p. 332.
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-26-
24. P. I. Pokrovskii, Current Problems in Hematology and Blood
Transfusion? ko, 1952, No. 26, p. 143.
25, P. I. Pokrovskii, G. P. Vinokurova, ibid., 1953, No. 28, p. 75.
26. L. L. Raxumova, S. N. Kudriashova, Riports to the Thirty-eighth
Plenum of the Scientific Counci.? of the Central Institute
of Hematology and Blood Transfusion. M., 1959, p. 41.
27. L. I. redorova, ibid., p. 42.
28. L. I. Fedorova, Conservation of Blood at Temperatures Below 0oC.
Cand. Dia., M., 1960.
29. I. I. Fedorov, et al., Mechanism of Pathological Reactions., L.,
1945, Chapter 7-43, P. 122, 136,
30. M. Bricka, Beasts M., C. R. SOC. Biol., 1955, V.1/12, p. 875.
31, L. L. Haynes, J. L. Tullis, H. M. Pyle., J.A.M.A., 1960, V. 171,
p. 1657.
32, C. E. Huggins, IX Congress of the International Soc. of Blood
Transfusion. Abstracts, p. 25.
33. M. M. Xetchel, J. L. Tullis, et al., J.A.M.A., 1958, VP 168, p. 404.
34, P. M. Genenio, B. J. Luyet, ProdIftdings of the 6th Congress of the
International Blood Transfusion, Basel, 1958, p. 330.
35* J. E. Lovelock, Biochim. biophys. Acta, 1953, V. 10, p. 414.
36, J. E. Lovelock, Proc. roy. Soc. B., 1957, V. 147, p. 427.
37. .16d:4-Love1ock, M. W. H. BiThop, Nature, 1959, V. 112, p0 1394.
38. B. J. Luyet, Biodynamica, 1949, V. 6, p. 207,
390 B. J. Luyet, Proc. roy. Soc. Be, 1957, V.1121. p. 434.
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420 ft. T. Meryman Proc. roy. Soc., B., 1957, V.112Z, p. 452.
43? P. L. Hollisov, H. A, Oloviter, Lance 1952,iV. 2, p. 501.
44, P. Li Mollison, 5th congres International de transfusion sanguine,
Paris, 1955. p. 759.
450 No CO H. Jones, P. L. Mollison, M. A. Robinson,Proc. roy. 60c. B.
1957, V. 147, p. 476.
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4.21.
46. A. S. Parkes, Proc. roy. Soc. B., 1957, V. p. 424.
47. H. M. Pyle, L. L. Haynes, et al., IX congress Internat. Soc. of
Blood Transfusion. Abstracts p. 22.
48. A. Pe Rinfret, G. F. Doebbler, C. U. Cowley, Proceedings 8th
Congress of the International Society of Blood Transfusion.
Basel, 1962, p. 439.
49. A. P. Rinfret, C. W. Cowley, G. F. Doebbler, et al., IX congress.
Intern. Soc. of Blood Transfusion. Abstracts, 1962, p. 27.
50.
H. A. Sloviter, R. G. Ravdin, Proceedings 7th Congrees of the
International Society of Blood Transfusion, &wall/ 1959,
p. 70.
51. H. A. Sloviter, An. J. med. Sci., 1956, V. ga, p. 437.
52. A. U. Smith, Lancet, 1950, V. 2, p. 910.
53. Ibid., Nature, 1958, V. 182, p. 911.
54. M. T. Sproul, M. H. Sloon, Papers in Dedication P. H. Anderson
Birthday Published by Munkaard. Copenhagen, 1957.
55. K. M. Strumia, L. S. Colwell, P. V. Strumia, Proceedings of the
8th Congress of the Interautional Society of Blood
Transfusion. Basel, 1962, p. 453.
56. M. M. Strusia, P. V. Strumia, L. S. Colvell, et al., IX congress
Intern. Soc. Blood Transfusion Abstracts f?g2, Mexico, 1962.
p. 24.
57. J. B. Tullis, Proceedings of the 7th Congress of the International
Society of Blood Transfusion. Basal, 1959, p. 45.
58. J. Tullis, M. T. Sproul, L. L. Haynes, Proceedings of the 8th
Congress of the International Society of Blood Transfusion,
Basel, 1962, p. 447.
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STAT
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