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WHOLEBLOODPRESERVATION; A PROBLEMIN GENERALPHYSIOLOGY.' AN IN VITRO ANALYSIS OF THE

PROBLEMOF BLOODSTORAGE

By ARTHURK. PARPART, JOHNR. GREGG,PHILIP B. LORENZ, ETHEL R.PARPART, ANDAURIN M. CHASE

(From the Physiological Laboratory, Princeton University, Princeton, N. J. and the MarineBiological Laboratory, Woods Hole, Mass.)

(Received for publication August 31, 1946)

A study of the storage of whole blood presentsnumerous problems in the field of cellular physi-ology. An understanding and solution of theseproblems can be of great aid in prolonging the via-bility of red cells of stored blood.

A major part of the problem centers around ex-perimental studies on the influence of a number ofenvironmental factors on survival. The more im-portant factors are: temperature, range and opti-mum; degree of dilution; tonicity of the diluent;composition of the diluent; ion balance, mainte-nance of the colloidal osmotic pressure; pHrange and optimum; buffer systems; glucose andrate of glycolysis; acid metabolite formation.

There have been many studies of the effect ofsome of these environmental factors on bloodstorage. In most cases, however, they have beenstudied as isolated, incomplete events by methodsthat frequently did not allow proper judgment ofthe state of preservation of the red cells. Morerecently,. several excellent studies along theselines have appeared (Loutit [1]).

It is essential that an in vitro study of the bloodpreservation be accomplished by a thorough exami-nation of a number of carefully controlled experi-mental criteria. Wehave found that the correla-tion obtained from the following in vitro methodspermits one to arrive at a reasonable judgment ofthe state of preservation of the stored red cells andto predict the in vivo survival of such cells. Suchpredictions have been confirmed by in vivo tests(Gibson, et al [2] ).

IN VITRO METHODSEMPLOYEDIN THESE STUDIES

1. The degree of spontaneous hemolysis, obtained spec-trophotometrically on the plasma after' thorough re-

1 The work described in this paper was done under acontract recommended by the Committee on Medical Re-search, between the Office of Scientific Research and De-velopment and Princeton University.

suspension of the cells and subsequent separation in anair turbine (Chase, et al [3]).

2. The mean and spread of the osmotic resistance, de-termined by an experimental analysis of the full curve ofosmotic resistance, in buffered salt solution at fixed pHand temperature (Parpart, et al [4]).

3. Rate of loss of potassium from the red cells, obtainedby potassium analysis on red cells and plasma (Weichsel-baum, Somogyi, and Rusk [5]).

4. The rate of glycolysis, as determined by the rate ofdisappearance of glucose from the stored blood (Hoffman[6]).

5. Rate and amount of lactic acid formation (Barkerand Summerson [7]).

6. Changes in hydrogen ion concentration of the plasma,by glass electrode measurements.

7. Hematocrit and cell volume changes, determined byan accurate (standard deviation of 0.86 per cent) airturbine method (Parpart and Ballentine [8]).

8. Initial total hemoglobin, methemoglobin formation andcell counts, methods of Chase, et al (3) and Parpart (9).

9. Rate of exchange of anions across the membrane ofthe red cells, measured photoelectrically (Parpart [10]).

10. Rate of passage of non-toxic organic molecules intothe red cells, measured photoelectrically (Parpart [10]).

All of the data reported in this paper were obtained onblood samples collected by venipuncture from healthy hu-man males (18 to 40 years of age).

RESULTS

Appropriate correlations between these criteriahave served as a satisfactory index of the state ofpreservation obtained in the various procedures wehave applied to stored blood. The results of thesestudies will be outlined under separate headings,though it is important to realize that this separa-tion is purely for convenience. Only full cog-nizance of the interaction of the various variablesstudied will allow for proper comprehension of thefactors involved in blood storage.

1. Optimd temperature for preservation.

Studies have been made on whole blood samplesstored at different temperatures under a variety of

641

A. K. PARPART, J. R. GREGG, P. B. LORENZ, E. R. PARPART, AND A. M. CHASE

-A~ . - -ability of the red cells to retain their normal po-SYMBOL STORED tassium content. Four complete repetitions of

* 15 X these experiments gave the same results.20 , x 22 0 It may be seen from Figures 1, 2, and 3, that the

El 36 optimum temperatures for survival of the humanred cell lie between 40 and 90 C.; with an optimalat about 70 C. + 10. This has been confirmed bythe in vivo studies of Gibson, et al (2).

These experimental conditions do not represent15 the best we have found for human red cell survival,

but they do illustrate the optimal temperature con-ditions for such survival.

Figure 4 presents data on survival of red cells

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FIG. 1. EFFECT OF TEMPERATUREON THE SPONTANEOUSHEMOLYSISOF STOREDBLOODee

experimental conditions (e.g. diluted, defibrinated,citrated, normal, and added glucose). 0

Figures 1, 2, and 3 illustrate the typical influ-/

ence of temperature on the survival of stored hu- 4man blood. These data were obtained on 270 ml. 7e

of blood collected in a bottle containing 30 ml. of E03.25 per cent Na,8C8HO0-2 H20 (0. 11 M). Thisblood was then diluted to 70 per cent of normal = .whole blood by the addition of an isosmotic solu- Gtion (5.4 per cent) of glucose and within 30minutes of collection stored at the temperature in-dicated in the figures. 051 5 2

Figure 1 gives the rate of spontaneous hemolysisTeprue Cwith respect to temperature and length of storage;Tmperure0CFigure 2 presents the data on the mean osmotic FIG. 2. EFFECT OF TEMPERATUREON THE MEANOSMOTICresistance and Figure 3 illustrates the effect on the RESISTANCE

642

HUMANBLOODSTORAGE

under a variety of storage conditions at 2 differenttemperatures. One of these temperatures is inthe optimal range (50 C.) while the other is welloutside this range (230). Figure 4 quite clearlyshows that while temperature is a variable in thesurvival of stored red cells (one of prime impor-tance as shown in Figures 1, 2, 3), and one whoseeffect is consistently in the same direction irrespec-tive of large variations in the composition of the en-vironment of stored red cells, nevertheless the ef-fect of temperature may be overshadowed -by theenvironmental composition. Curves a and b(Figure 4) contrast the rate of spontaneous he-molysis in the case of 12 collections of carefully de-fibrinated blood stored at 230 (a) and 6 collectionsstored at 50 (b) respectively. These blood sampleshad nothing else done to them except defibrination.It may be observed that the effect of temperatureis present but its action is slight.

Compare these curves a and b with curves cand d (Figure 4). The latter 2 curves were ob-tained in 5 collections of blood according to themethod of De Gowin, et al. (11) and stored at23° and 50 respectively. In this case the effect

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FIG. 4. SPONTANEOUSHEMOLYSIS AT 2 TEMPERATURESIN A VARIETY OF SOLUTIONS

of temperature is exceedingly striking and couldhave been deduced from the effect of temperatureon the rate of penetration of glucose into humanred cells, since the citrated bloods stored by DeGowin's method have been diluted to 40 per centof their original concentration with an isosmoticsolution of a compound that penetrates the redcells (5.4 per cent glucose). Such penetration ofglucose leads to an abnormal increase in -volumeof the red cells (Figure 8).

One may also contrast curves e and f with aand b (Figure 4). The only difference otherthan temperature between the stored bloods repre-sented in these 4 curves is the addition of sterile,solid glucose to give a final concentration of 0.5per cent added glucose to the blood samples ofcurves e and f. These latter curves are the aver-age for 6 and 9 individual blood collections. Com-parison of curve e with f shows a very markedeffect of temperature, over and above the strikinglybetter survival of the red cells to which glucosehas been added.

Spontaneous hemolysis with time of storage for90 parts of blood collected into 10 parts of anisosmotic Na8C6H507 -2 H20 (0.11 M) solution

643


is shown in curve g (Figure 4). The better,though still poor, preservation under these con-ditions as compared with defibrinated blood storedat 5° C. (curve b) is primarily associated with thedilution of the blood and not with any effect of theadded citrate. This point will be taken up laterunder the following section on the effect of dilu-tion.

Figure 4 is intended solely to illustrate furthereffects of temperature on spontaneous hemolysis.It is not intended, however, to indicate that anyof the methods of preservation illustrated in it isbetter than any* other when considered from thepoint of view of in vivo survival. The other invitro criteria that have been used on all of the sam-ples of stored blood in Figure 4 indicate that noneof these methods of preservation would give satis-factory in vivo survival expectancy.

2. The degree of dilution and preservation.

A review of the extensive literature on bloodpreservation reveals that a great variety of dilu-tions of the whole blood have been used. This hasvaried from 10 per cent whole blood through agreat many intermediate degrees of dilution up toundiluted blood. In most cases in the literature,the use of a particular dilution, whether 10, 40,50, 80, 90 per cent of whole blood or whole blooditself, has been purely arbitrary with no supportingdata for a particular dilution. Further, the tonicityand glucose content of the dilution medium havevaried along with the degree of dilution. The workdescribed below was an attempt to correlate thesurvival of red cells with the degree of dilution ofthe stored blood.

In addition to the immediate problem of the

Percent of whole blood.FIG. 5. DILUTION OF WHOLEBLOODWITH PLASMAAND THE SPONTANEOUS

HEMOLYSISDURING STORAGE

644

HUMANBLOODSTORAGE

degree of dilution of stored blood on the survivalof the red cells, there is a practical aspect of thisproblem in relation to transfusion. If it is neces-sary to increase the available hemoglobin of a pa-tient, minimum dilution of the blood would be ad-visable. On the other hand the transfusion mayalso be used to combat dehydration, to supply glu-cose or salts, or plasma proteins, in which eventhigh dilution of the red cells might be desirable.The answer to this must hinge upon a knowledgeof what dilution is best for the survival of storedred cells.

An obvious perfect diluent might be plasma.Wehave run 5 experiments in which the donor'sblood was diluted by varying amounts with itsown plasma. A typical set of such data is givenin Figure 5. The blood used in this experimentwas collected by venipuncture into a bottle con-taining enough solid Na3C8O7,2 H20 to give afinal citrate content of 400 mgm. per 100 ml. ofblood. A portion of the blood sample was trans-ferred to sterile centrifuge tubes and citratedplasma obtained from it. This citrated plasmawas then used to dilute the original blood by theamount indicated on the abscissa of Figure 5.Each of these diluted blood samples had 500 mgm.of glucose added to them and they were stored atthe optimum temperature, 70 C. One of theseblood samples was concentrated centrifugally andenough plasma removed to give a final concentra-tion of 145 per cent relative to the whole bloodconcentration which was designated as 100 percent. The blood of 3 other individuals had beenstored and examined periodically in this way.Exactly similar results were obtained with onlyminor fluctuations in the absolute values.

The data contained in Figure 5 show quiteclearly that the optimum dilution range for redcell preservation, with these conditions, lies be-tween 80 per cent and 60 per cent of the wholeblood concentration. It may also be noted thatthe optimum shifts from close to 80 per cent to-ward 60 per cent as the time of storage increases.On the average, blood which has been diluted to70 per cent of its original concentration might beexpected to give the best preservations of its redcells.

The question arises as to whether other typesof diluents have an analogous effect. The datain Figure 6 illustrate this. Curve a (Figure 6) is

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a plot of the degree of spontaneous hemolysis inblood stored for 48 days, at 70 C., under the follow-ing conditions. Five parts of blood were collectedinto 1 part of a 3.2 per cent solution ofNa8C6H5O7 2 H20, cold (50 C.). A portion ofthis blood had 500 mgm. per cent (mgm. per 100ml.) of glucose added, was stored at 70 C. and wasexamined at weekly intervals. This appears as83.3 per cent of whole blood in curve a. Aliquotsof the rest of this citrated blood were diluted withan isosmotic solution of glucose (5.4 per cent)to give the percentage concentrations of curve a.All were stored at 70 C. and examined at weeklyintervals. The data in curve a (Figure 6) arethose for the forty-eighth day of storage. Theyshow that the optimum dilution lies between 60 and40 per cent of the whole blood concentration andthat a dilution which gives 40 per cent of the wholeblood concentration is close to the borderline whichmay lead to rapid hemolysis. The latter is thepreservative solution used by De Gowin, et al ( 11 ).The fact that survival of red cells in this solutionis very poor when judged from the other in vitrocriteria we have used has been previously men-tioned under Figure 4, curve d.

645

5


Curve b (Figure 6) contains data for blood col-lected into solid citrate (see page 645) while curvec gives data for defibrinated blood. These bloodsamples were diluted with a sterile isotonic saltsolution of the following composition: NaCl, 0.90per cent; KCl, 0.042 per cent; NaHCO3, 0.024per cent; NaH2PO4, 0.05 per cent. They had 500andl1,000 mgm. per cent of glucose added respec-tively and were stored at 7° C. The curves repre-sent the degree of spontaneous hemolysis occur-ring at 35 and 21 days of storage respectively. Theshapes of these curves and of curve a did not alterappreciably with time of storage, i.e. after the firstweek, though their position on the ordinate al-tered. It will be shown in section 5 that the differ-ence in glucose concentration of these 2 samplesdoes not account for the survival differences.

Curves b and c also show an optimum dilu-tion and this occurs at about 80 and 90 per centof whole blood. The effect is slight until bloodconcentrations of 60 per cent or lower are exam-ined. It is obvious that dilutions of 60 per centor greater are definitely deleterious when a bal-anced salt solution is used as the diluent. It isagain noteworthy that defibrinated blood does notsurvive as well as citrated blood.

Figures 5 and 6 are convincing evidence of thefact that earlier experiments in the literature onblood preservation which failed to take account ofthe effect of dilution on survival of the red cellsmust be considered with respect to the actual dilu-tion that was used. The foregoing experiments,combined with an analysis of data from a numberof others, have led us to the conclusion that thesurvival time of stored red cells is increased whenthe blood is diluted. The data in Figures 5 and 6would further indicate that for a variety of diluentsa dilution consisting of 70 parts of blood and 30parts of diluent (70 per cent whole blood) mightbe expected to give optimum survival.

Even this value of 70 per cent must be em-ployed with caution, as the data of Figure 7 willshow. In this case blood was collected into aminimum amount of citrate and diluted with a 6 percent gelatine solution prepared in 0.9 per cent NaClat pH 7.2.2 The days of storage are indicated on

2This diluent was supplied through the courtesy ofDr. D. Tourtellotte, and is a product of Knox GelatineProtein Products, Inc.

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FIG. 7. DILUTION WITH GELATIN

the figure. Through 24 days of storage the sur-vival was good at all dilutions and excellent at 40per cent. At later periods of storage the survivalwas poor, but it is evident that the greater dilutions(40 and 20 per cent of whole blood concentration)gave better protection.

3. Tonicity, chemical composition and colloidalosmotic pressure of the diluent.

The diluent of historical interest is an isosmoticsolution (5.4 per cent) of glucose (Rous andTurner [12]). They diluted whole blood as fol-

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646

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HUMANBLOOD STORAGE

lows: 30 parts blood, 20 parts of 3.8 per centNa3C8H507 5 H20 and 50 parts of a 5.4 per centglucose solution. This has been used more re-cently at a 40 per cent concentration by De Gowinand his coworkers (11).

Those who have used this isosmotic glucose solu-tion as a diluent for human red cells have ignoredthe fundamental fact that the human red cell isreadily permeable to glucose. Thus, human bloodcollected by the methods of Rous and Turner orof De Gowin would be expected to show a markedincrease in the volume of its red cells. That thisis the case is shown in Figure 8, curve a, whichexpresses the relation between the red cell volumeand the degree of dilution of the whole blood, un-der conditions where whole citrated blood wasdiluted with isosmotic glucose solution. Themeasurements given are for the seventh and forty-eighth days of storage at 7° C.

Contrasted with the very marked increase in vol-ume of curve a are the much smaller red cell vol-ume changes which occur during the storage ofcitrated blood which has been diluted with Ringer-Locke solution (calcium-free). The latter are

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represented in Figure 8, curves b and c, and arethe data for the first and forty-ninth days of stor-age at 7° C. These diluted blood samples had500 mgm. of added glucose.

The pronounced swelling of red cells dilutedunder the conditions of De Gowin or of Rous-Turner is the result of the penetration of the glu-cose. These red cells would continue to swell tothe hemolytic volume were it not for the fact thatthey are being subjected to a shrinking effectbrought on in part by the loss of chloride ions fromthe cells (Jacobs and Parpart [13]). An addi-tional and more important factor in checking theswelling of human red cells diluted with isosmoticglucose solution is the rapid loss of potassium fromthese cells. Our data on potassium ion loss fromthe red cells stored under these conditions indi-cate that 50 per cent of the diffusion equilibrium isattained between the fifth and eighth days of stor-age, while the outward diffusion of potassium iscomplete after 10 to 20 days.

The balance between these several factors act-ing on the cell volume is a delicate one and needsonly a slight change in environmental conditions(e.g. temperature rise) to cause a rapid rate ofspontaneous hemolysis by the red cells of bloodthus diluted, see curve c (Figure 4). Humanblood stored under conditions where it is dilutedto 40 or 30 per cent of its original concentrationwith an isosmotic glucose solution would be ex-pected to show very poor in vivo survival of itsred cells (Gibson [2]).

The question has been frequently raised as towhether it is important to maintain the normal col-loidal osmotic pressure of the fluid surrounding thestored red cells. In an earlier section of this paperwe have demonstrated that dilution of whole bloodto 70 per cent of normal affords a better environ-ment for the survival of the red cells. This wastrue irrespective of whether the diluent was (1)plasma, (2) a 6 per cent gelatine in isotonic NaClsolution, (3) a balanced salt solution, (4) anisosmotic glucose solution, or (5) an isotonicphosphate solution which when added to the bloodestablished an initial pH of 7.1 (Figures 5, 7, 6,and 9).

Diluents (1) and (2) above had normal colloidalosmotic pressure while the other 3 diluents re-duced the colloidal osmotic pressure of the plasmaof the stored blood to 70 per cent of normal. The

100 80 60 40 20Percent of whole blood.

FIG. 8. DILUTION ANDREDCELL VOLUME

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rate of spontaneous hemolysis in these variousdiluents, however, did not differ greatly and whatdifferences there are certainly cannot be ascribedto this factor. In fact, the survival of the red cellsin stored blood diluted to 70 per cent of wholeblood concentration with the phosphate solution(Figure 9) is much better than that in plasmaor gelatine solution judged from our in vitro dataand the in vivo data of Gibson (2).

It should be pointed out that the differencesthat do exist are outside the limits of variation ofindividual blood samples. This is also illustratedby Figure 9, where the degree of spontaneous he-molysis is plotted against the days of storage.The 8 individual blood samples represented inFigure 9 were from data taken at random over a

period of 1% years on citrated human blood sam-

ples stored at 7° C. and diluted as follows: 70parts of whole citrated blood (4 mgm. Na,,C6H5O72 H2O per ml. of blood) and 30 parts of a solu-tion containing 60 volumes of 0.11 MNa2 HPO4and 40 volumes of 0.11 M NaH2PO4. The ini-tial pH of blood thus diluted is 7.1 and during 50days of storage drops to pH 6.7.

Another series of tests on the influence of thecolloidal osmotic pressure of the plasma on redcell survival were run on 4 different samples ofhuman blood. The blood samples were collectedinto citrate (in amount indicated above), dividedinto 2 equal volumes, transferred to sterile cen-

trifuge tubes, spun, and the major portion of theplasma removed aseptically. The plasma of 1

aliquot of cells was exactly replaced by a solutionrecommended by Gibson, viz.:

NaC6H507 2H20 480 mgm.Glucose 1,080 mgm.NaCi 590 mgm.Conc. HCl (Sp. G. 1.19) 0.25 ml.

made up to 100 ml. with pyrogen-free water.

The other aliquot had its plasma replaced by thesame solution except that 3 grams of plasma pro-

tein- lipid Fraction IV-3, 4 Run S301, Cohn (14),were added to the 100 ml. of solution before re-

placing the plasma.In the case of all 4 blood samples thus treated

there was good survival over 45 days storage, butthe difference slightly favored the solution withoutthe added Fraction IV. In brief, the colloidal os-

motic pressure of the medium surrounding humanred cells does not appear to be a factor in theirin vitro survival.

4. pH range and optimum, buffering.The influence of pH on the survival of stored

red cells has not been consistently investigated.Recently Loutit (1) has gathered together dataon a number 6f samples of blood stored in acidcitrate-dextrose that indicate that blood dilutedwith this solution and having an initial pH of 7.1to 7.3 shows a better survival of its red cells.

In a report, dated October, 1943, to the BloodSubstitutes Committee of the Committee on Medi-cal Research we showed the Very marked influenceof pH on the survival of the red cells of storedhuman blood. Our studies of the influence of pH

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on in vitro survival have been carried out underconditions where a minimum shift in the pH ofstored blood was attained during long periods ofstorage. The citrate buffer system has no buffercapacity over the pH range for maximum survivalof stored red cells (Figure 10). The rate of gly-colysis of blood and the rapid formation of lacticacid, even at the optimum temperature (70 C.), isso high that over long storage periods blood sam-ples will show marked decreases in pH unless ade-quately buffered.

We, therefore, turned to the phosphate buffersystem and studied survival of the red cells at vari-ous hydrogen ion concentrations. These bloodsamples were followed over long time periods un-der conditions where a minimum decrease in pHwith time of storage occurred. Citrated blood wasdiluted with isotonic (0.11 M) phosphate buffersolutions in the proportion of 30 parts of buffer to70 parts of whole blood. In all cases these storedbloods had 0.5 per cent added glucose and storagewas at 70 C. Thus, a number of blood sampleshaving an initial pH between 6.0 and 7.5 wereprepared and red cell survival determined at fre-quent intervals. Seven individual blood sampleswere run in this manner; the typical result of the

effect of pH is shown in Figure 10 where the percent of spontaneous hemolysis is plotted againstthe pH of the blood samples at the fourteenth,twenty-eighth, and forty-sixth days of storage.

Figure 10 demonstrates that the optimum pH forprolonged storage of human blood lies between pH6.7 and 7.0. It may also be noted that during thefirst 2 weeks of storage there is slight effect of pHover the range pH 6.5 to 7.5. Longer storage pe-riods, however, show a marked influence of pH.Thus storage for 4 and 7 weeks clearly requiresa pH between 6.7 and 7.0 if optimum preservationof the red cells is to be attained.

Another striking action of pH on the survival ofthe red cells of stored blood is on the ability ofthe red cells to retain their normally high potas-sium content. This effect is illustrated in Figure11, where the per cent of the original amount of,potassium in the red cell which has diffused outof the cell at various times of storage is plottedagainst the pH of storage. In this experimentthe blood was buffered by phosphate solutions asdescribed above and 0.5 per cent glucose addedand stored at 70 C.

Figure 11 illustrates that human red cells storedat a pH between 6.7 and 7.0 will lose their potas-

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sium at a much less rapid rate than at a pH higheror lower than this optimum range. Even after 4weeks of storage at pH 6.8 the potassium of thered cells has diffused to only 60 per cent of equilib-rium.

The studies thus far dealt with have employedthe phosphate system. The results outlined aredue primarily to the pH and not to specific actionof the phosphate itself. This has been establishedby experiments involving storage of human bloodin maleic, in maleic-succinate, and in glycyl-gly-cine buffer systems, under conditions otherwiseidentical for the phosphate system. The data ob-tained for these 3 systems as concern the optimumpH range for red cell survival are the same as thatfor the phosphate system, though the absolutemagnitude at the optimum pH for cell survivaldoes vary with the buffer used (Table I).

TABLE I

Comparison of the per cent of spontaneous hemolysis inhuman blood after 4 weeks' storage at 7° C. in

the presence of different buffer systemsEach blood sample was composed of a mixture of 30

parts isotonic buffer solution and 70 parts whole (citrated)blood. The initial pH of each was 7.10 to 7.20 and thepH on the fourth week of storage is given. Each bloodsample had 0.5 per cent glucose added.

Sponta- OriginalpH after neous glucose

Buffer system 4 weeks' hemolysis utilizedstorage after by fourth

4 weeks week

per cent per cent

NaH2PO4/Na2HPO4 6.82 0.5 20Glycyl-glycine/Na glycyl-

glycine 6.75 0.7 18Maleic-succinic acids/Na

maleate-Na succinate 6.95 7.0 13Maleic acids/Na maleate 6.82 15.0 0None 6.94 1.8 36

In this respect, Table I shows that the phos-phate system was better than the other buffers.It is thus certain that in addition to the pH effecton survival the phosphate itself markedly increasessurvival. The glycyl-glycine buffer is also ex-

ceptionally effective in prolonging red cell sur-

vival of stored blood (Table I).Table I also shows that red cell survival in the

maleic and maleic-succinate systems is poor even

at the optimum pH. This effect, however, is prob-ably associated with the low or completely in-hibited glycolytic rate in the presence of these lat-ter buffer systems (Table I).

Citrated blood, diluted to 70 per cent of wholeblood with its own plasma and with 0.5 per centadded glucose, stored 4 weeks at 70 C., but with nobuffer system added is included in Table I. Itmay be seen that while the degree of spontaneoushemolysis is low at this time, it is, however, con-siderably greater than in the blood with phosphateor glycyl-glycine buffer system (see also Figure5).

From the foregoing it would appear that thephosphate buffer solutions which have on mixingwith blood an initial pH between 7.1 and 7.3 anda pH after 4 weeks of storage at about 6.9 (with0.5 per cent added glucose and storage at 7° C.)are exceptionally fine for increasing the survivalperiod of the red cells of stored blood of man. Thequestion remains as to what might be the minimalconcentration of phosphate buffer to add to bloodfor optimal or good preservation of the red cells.

To answer this question, whole citrated humanblood was diluted with varying amounts of phos-phate buffer (Table II). The degree of dilution

TABLE II

Phos- Bal- pBlood Citrated hate danced Added pH pHsample blood buffer salt glucose O days 50 days

mixture solutionstrg soae

no. ml. ml. ml. mgm.397 70 30 0 500 7.29 6.74398 70 20 10 500 7.35 6.78399 70 10 20 500 7.48 6.86400 70 3 27 500 7.54 6.90401 70 0 30 500 7.59 7.03

(70 per cent whole blood) was maintained con-stant (Table II), by using a calcium and phos-phate-free Ringer-Dale solution. The glucoseadded to each blood sample was 0.5 per cent andall bloods were stored at 7° C. The data in TableII are expressed in terms of 100 ml. of blood plusdiluent. The phosphate buffer mixture used con-sisted of 60 parts of 0.11 M Na2HPO4 and 40parts of 0.11 MNaH2PO4.

The degree of spontaneous hemolysis which oc-curred in these stored samples after 14, 36, and50 days of storage is illustrated in Figure 12. The"mean osmotic resistance" initially and at the samestorage times is given in Figure 13.

As noted before, the presence or absence of thephosphate buffer has little or no effect during thefirst 2 weeks of storage (Figure 12). However,

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FIG. 12. EFFECT OF PHOSPHATE CONCENTRATION ONSPONTANEOUSHEMOLYSIS

when one examines the mean osmotic resistancecurves (Figure 13) one observes that even after2 weeks of storage the bloods stored with no, or

a small amount of, phosphate buffer would bepoor risks in transfusion and this becomes more

striking on the fifth and seventh weeks of storage.Blood stored with 0.0044 M NaH2PO4 and

0.0066 M Na2HPO4 per liter of blood and with0.5 per cent added glucose, 30 per cent dilution andat 7° C., has its red cells well preserved over 5 weeksof storage. Blood stored under these conditionsand injected in 500 ml. amounts would increasethe normal plasma phosphate level by only 40per cent.

5. Optimal Glucose Content.Glucose added to stored blood increases the red

cell survival. This is a fact upon which all ex-periments on blood preservation have concurred.No other sugar has been observed to have a com-parable effect.

Surprisingly no one has investigated the effectof varying the glucose concentration while keep-ing other possible variables as constant as possible.We therefore performed a series of experimentson the effect of glucose concentration on red cellsurvival of stored human blood. Several seriescompared the survival in whole blood to whichvarying amounts of glucose were added. Bloodwas collected directly into solid citrate in the pro-portion of 400 mgm. Na3C5H7O6'2 H20 to each100 ml. of blood. To aliquots of this citrated blood,amounts of glucose varying from 0 to 6 per centwere added and the survival followed over a 7-week period. The glucose was added on the basis

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tion and buffering against pH change. The re-sults as far as the minimal effective amount ofglucose to add to stored blood, viz. 0.5 per cent,are quite conclusive. Glucose penetrates the hu-man red cell and, hence, its osmotic effects may beneglected under the conditions of these experi-ments.

Completely analogous results have been obtainedon citrated blood diluted to 70 per cent with phos-phate buffer at the optimal pH and stored withand without added glucose. After 42 days ofstorage these blood samples showed 8 per centspontaneous hemolysis when no glucose was addedand 1.8 per cent in the presence of 0.5 per centadded glucose. These data contrast only in themagnitude of the hemolysis with those for 42 days'storage in Figure 14 but are parallel as to the ac-tion of glucose. They show again the importanceof dilution and buffering. In fact, blood storedunder these latter conditions is in better state for

2 3 4Percent Glucose Added.

FIG. 14. OPTIMAL GLUCOSECONCENTRATIONFORSTORAGE

of grams of glucose per 100 ml. of stored blood,e.g. 1 per cent means 1 gram per 100 ml. blood.All bloods were stored at 70 C. The results ofsuch experiments are illustrated in Figures 14, 15,and 16.

Data on the effect of added glucose on the degreeof spontaneous hemolysis occurring during storageare given in Figure 14. The data in this figureshow quite clearly that even a low total concentra-tion (0.25 per cent) of added glucose aids in thered cell survival for 33 days of storage when com-pared with the cells of blood stored with no addedglucose. From 33 to 42 days of storage, however,this low concentration becomes insufficient. Itmay also be noted in Figure 14 that amounts ofglucose, including 0.5 per cent and greater, in-crease to a considerable degree the red cell survivalalthough there is no cumulative effect of thegreater amounts of glucose. It must be empha-sized that these experiments were designed to varyonly 1 factor, namely, glucose concentration.This, of course, precluded the possibility of study-ing survival under the optimal conditions of dilu-

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FIG. 15. EFFECT OF ADDEDGLUCOSECONCENTRATIONONTHE MEANOSMOTICRESISTANCE

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FIG. 16. EFFECT OF ADDED GLUCOSEON THE Loss OFPOTASSIUM

transfusion after 42 days of storage than undiluted,unbuffered blood was at 22 days, regardless ofadded glucose.

Figure 15 brings out the noteworthy fact thathigh concentrations of added glucose, 1 per centand higher, lead to a rapid decrease in the osmoticresistance of the stored red cells. These experi-mental observations are obviously the resultant ofthe effect of the increased concentration of glucosein the cells stored with this high amount of addedglucose and the consequent swelling this wouldproduce in the hypotonic saline solution in whichthe osmotic resistance was measured. They dogive point to the fact, however, that such red cellswhen transfused would undergo rapid osmotic he-molysis, thus rendering them useless for suchpurposes. This danger point lies between 1 and 3per cent added glucose (Figure 15).

The slight, but noticeable effect of added glu-cose on the retention of potassium by the storedred cells is illustrated in Figure 16 and may becontrasted with the good retention of potassiumwhen the blood is diluted and buffered to the opti-mumpH (Figure 11).

We have studied the preservative action of a

number of sugars in comparison with glucose. Inall of these studies the particular sugar was com-

pared with identical conditions for survival withadded glucose. The sugars used were xylose, lac-tose, fructose, maltose, sucrose. None of thesesugars gave as good survival of the red cells ofstored blood as compared with cells stored with

added glucose. In all cases except xylose the redcell survival was better than under parallel experi-ments where no sugar was added. Xylose gaveno better survival than control blood with no addedsugar.

SUMMARY

An analysis has been made of a number of fac-tors concerned in prolonging the life of the redcells of stored human blood.

Blood collected and stored under the followingconditions has been found to give excellent sur-vival of its red cells over a period of at least 6 weeks:Seventy parts of blood collected into 10 parts of 4per cent Na3CH5O7-2 H20 and 20 parts of aphosphate buffer solution (composed of 60 vol-umes 0.11 MNa2HPO4and 40 volumes of 0.11 MNaH2PO4); 500 mgm. per cent of glucose isadded and the blood is stored at 70 C.

Blood thus stored will be under the optimal con-ditions of (1) pH (initially 7.2 and 6.8 after 6weeks), (2) good buffering, (3) dilution (70 percent), (4) glucose concentration (500 mgm. percent), (5) temperature (70 C.).

BIBLIOGRAPHY

1. Loutit, J. F., Factors influencing the preservation ofstored red cells. J. Path. & Bact., 1945, 57, 325.

2. Gibson, J. G., 2nd, Evans, R. D., Aub, J. C., Sack, T.,and Peacock, W. C., The post-transfusion survivalof preserved human erythrocytes stored as wholeblood or in resuspension, after removal of plasma,by means of two isotopes of radioactive iron. J.Clin. Invest., 1947, 26, 715.

3. Chase, A. M., Lorenz, P. B., Parpart, A. K., andGregg, J. R., A method for simultaneous determina-tion of methemoglobin-oxyhemoglobin ratio and percent hemolysis in stored blood. J. Lab. & Clin.Med., 1944, 29, 99.

4. Parpart, A. K., Lorenz, P. B., Parpart, E. R., Gregg,J. R., and Chase, A. M., The osmotic resistance(fragility) of human red cells. J. Clin. Invest.,1947, 26, 636.

5. Weichselbaum, T. E., Somogyi, M., and Rusk, H .A.,A method for the determination of small amountsof potassium. J. Biol. Chem., 1940, 132, 343.

6. Hoffman, W. S., A rapid photoelectric method forthe determination of glucose in blood and urine.J. Biol. Chem., 1937, 120, 51.

7. Barker, S. B., and Summerson, W. H., The colori-metric determination of lactic acid in biologicalmaterial. J. Biol. Chem., 1941, 138, 535.

8. Parpart, A. K., and Ballentine, R., Hematocrit deter-mination of relative cell volume. Science, 1943,98, 545.

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9. Parpart, A. K., Is osmotic hemolysis an all-or-nonephenomenon? Biol. Bull., 1931, 61, 500.

10. Parpart, A. K., The permeability of the mamalianerythrocyte to deuterium oxide (heavy water). J.

Cell. & Comp. Physiol., 1935, 7, 153.11. De Gowin, E. L., Harris, J. E., and Plass, E. D.,

Studies on preserved human blood. J. A. M. A.,1940, 114, 850.

12. Rous, P., and Turner, J. R., The preservation of liv-

ing red blood cells in vitro. J. Exper. Med., 1916,23, 219.

13. Jacobs, M. H., and Parpart, A. K., Osmotic propertiesof the erythrocyte. Biol. Bull., 1933, 65, 512.

14. Cohn, E. J., Oncley, J. L., Strong, L. E., Hughes, W.L., Jr., and Armstrong, S. H., Jr., Chemical, clini-cal and immunological studies on the products ofhuman plasma fractionation. J. Clin. Invest., 1944,23, 417.

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