TRANSFUSION FLUIDSby

J. P. Bull, M.D.Medical Research Council Industrial Injuries and Burns Research Unit, Birmingham

Accident Hospital

TRANSFUSIONS MAY BE given for a number of purposes, for instance tosupply cells in aplastic anaemia, to provide clotting factors in haemophilia,or even to attempt to change the personality of the recipient as in the earlyexperiments on transfusion in the 17th century. Our concern here iswith transfusion for the maintenance of circulatory volume.

Whole bloodWhere whole blood has been lost, the obvious choice for a replacement

fluid is blood itself. The history of blood transfusion is that of the variousattempts to perform this replacement safely. From the beginning of thiscentury the ABO groups have been recognized and in the last 30 yearsmany other blood group antibodies have been identified. For the purposesof blood volume replacement ABO and Rhesus groups remain much themost important. For an account of the relevance of the other moresubtle factors, one cannot do better than recommend Mollison's bookBlood Transfusion in Clinical Medicine.

For emergency transfusion it is often important to balance the risk oftransfusion reaction against the risk of delay. Sevitt has described apractical scheme giving the appropriate procedure corresponding to differ-ent degrees of urgency. If immediate transfusion is required, the first fewbottles must be Group 0 blood, preferably Rh negative. By the timethese bottles have been given it should be possible to continue with bloodof correct ABO and Rhesus type. If a delay of 15 minutes is practicable,ABO grouping is possible, and often Rhesus typing also. Homologousblood can be used initially followed by blood which also has been cross-matched. If, as may well be the case, a delay of up to three-quarters of anhour is possible, full blood grouping, Rhesus typing and cross-matchingtests can be performed. Other necessary precautions to ensure that onlysafe blood is given have also been described (Sevitt, 1959).

So far we have been assuming that what the bottle contains is equivalentto normal circulating blood. To what extent is this true? The donorwill have been screened for disease and anaemia; 420 ml. of the blood willhave been mixed with 120 ml. of Acid Citrate Dextrose (ACD) solution.Thus only four-fifths of the reputed pint is actually blood. ACD solutioncontains citrate to combine with calcium and so prevent clotting; the acidand dextrose are present so as to favour survival of the red cells. Thecitrate is present as the sodium salt so the final sodium concentration isnot grossly abnormal. Potassium concentration, however, may be quite

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high, since during storage potassium leaves the red cells and is then insolution in the plasma. Unless, however, the recipient already has a highserum potassium, there seems to be little risk in transfusing bottled blood,though levels of about 12 mEq./litre are not uncommon in the fluid astransfused. Whether citrate is innocuous has been disputed. There isno evidence of damage in transfusions of moderate volume given topreviously fit persons. Citrate is a normal intermediate of metabolismand is ultimately broken down to carbon dioxide. There is normally ablood level of about 1 mg./100 ml. The level in plasma oftransfused bloodmay, however, be more than 100 times this and, after massive transfusion,it is possible for dangerous levels to be produced. Much of the toxiceffect of citrate is attributable to its causing hypocalcaemia, but there issome evidence that citrate itself can damage the heart, apart from itseffects on calcium. All these ill effects can, however, be prevented bygiving extra calcium with blood transfusion; 10 ml. of 10 per cent. calciumgluconate per litre of transfused blood has been recommended for thispurpose. Even without this precaution, large transfusions have oftenbeen given without any evidence of ill effects. The liver is a main site ofbreakdown of citrate and therefore particular caution should be exercisedwhen giving large transfusions of bottled blood to persons with known orsuspected liver damage.

There is a further possibility that the acidity of the ACD mixture mayalso cause trouble. Gibson et al. (1956) suggested that the high acidity(pH c. 5.0) of the ACD solution can cause damage to the first part of theblood donation as it runs into the ACD anticoagulant, though it is notcertain how important this effect may be in practice. A potentially moreserious clinical implication has been pointed out recently. The pH ofstored blood, initially about 7.1, falls to 6.6 during storage. This impliesthat the buffer system of plasma and cells has been overwhelmed by acombination of the citric acid already present in ACD solution and theother organic acids (chiefly lactic acid) produced during storage. Withtransfusions of moderate amounts the recipient's buffering capacity isnormally fully adequate to correct the acidity and as the citrate is meta-bolized this itself tends to raise the pH. Both processes require that carbondioxide can be lost freely. However, there may be patients with a com-bination of severe blood loss and an acidosis-particularly if this is ofrespiratory origin-for whom the further addition of acid in the trans-fusion may be a severe burden. A case in which this appears to havehappened has been described by Bradfield (1963).

Having mentioned these various hazards it is perhaps necessary toemphasize again that in the presence of good laboratory facilities andproper supervision, blood transfusion remains a surprisingly safe procedureeven when sufficient is given to correct the very large blood loss which mayoccur in multiple injuries.

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Alternative methods of storing blood have been suggested. Apart fromvariants upon the ACD mixture, such as the addition of materials likeadenosine which favour longer survival of red cells, preservation at verylow temperatures is a feasible alternative. During the last 10 years it hasbeen shown that many living cells can survive storage at -40° C. or below,if precautions are taken to prevent damage during the processes offreezingand thawing. There are two main methods. Slow cooling and mainten-ance at -40' to -78° C. is satisfactory if red cells are suspended in glycerol.Mollison et al. (1952) and Hughes-Jones et al. (1957) have shown that redcells stored in this way can give good survival on subsequent transfusion,though there remain some difficulties in applying these methods on a largescale and with the speed often required for emergency transfusion.Another method which has recently been developed to an advancedstage is the rapid freezing of blood to the temperature of liquid nitrogen.This can be done without the addition of glycerol, and offers the possi-bility of convenient long-term storage combined with speedy availability.

PlasmaBy analogy with the use of whole blood when blood has been lost, it is

rational to transfuse plasma when plasma has been lost. Burns are thecommonest condition where plasma loss predominates; the fluid whichleaves the circulation into blisters and tissues and which is lost from thesurface as exudate is similar to plasma in composition and contains all themain plasma protein fractions. Material commonly available is freeze-dried small pool plasma. As implied above, red cells have limited viabilitywhen stored in ACD. After 14 days' storage about 85 per cent. of cellssurvive in the circulation of the recipient 24 hours after transfusion. It istherefore the usual practice to withdraw the blood from the bank soonafter this time. The plasma of such blood can be separated and salvaged;it still contains the blood group antibodies but by mixing together theplasmas of blood of different groups the resulting pool does not containenough antibody to be troublesome, and such pooled plasma cannormally be given without regard to the blood group of the recipient.

The freeze-dried plasma still contains the electrolytes from the originalstored blood and so has rather a higher sodium concentration and a muchhigher potassium and citrate level than present in the circulation. Theproblem of acidity is not so severe since plasma is not so heavily bufferedas whole blood. When reconstituted as recommended with sterile waterthe solution contains 5 per cent. plasma protein. Electrophoresis showsthat the main fractions of this protein are fairly normal, but the lipo-proteins are damaged and do not go properly into solution; this largelyaccounts for the cloudy appearance common in reconstituted plasma.Plasma of this type will give good circulatory volume replacement and itseffect in patients with burns is immediately shown by the expected fall ofhaematocrit. If plasma is given when whole blood has been lost, the

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haematocrit will fall below normal corresponding to the dilution of redcells. In animal experiments the response to such replacement may besimilar to that obtained with whole blood, but most clinicians who havetried both in patients feel sure that the red cells of whole blood with theiroxygen-carrying power give blood transfusion a great advantage overplasma transfusion as a treatment for blood loss. If plasma is made up inmore concentrated form, circulatory volume expansion occurs in propor-tion to the amount of plasma protein transfused, water being attractedinto the circulation until protein levels return to normal.

The risk of hepatitis is often cited as a serious contra-indication to theuse of plasma. Fear of this dates largely from wartime, when plasmaprepared from extremely large pools was processed in America and used inthe Armed Forces. Some batches of this plasma gave rise to a highincidence of hepatitis, often with high mortality. It is understandable thatrecollections of such experiences should breed caution, but experience inrecent years has been much more favourable. The most recently pub-lished survey in this country (Medical Research Council, 1954) showed anincidence of less than 1 per cent. of plasma jaundice, and very slightmortality. We have continued to use plasma extensively in the Birming-ham Burns Unit, and have not found hepatitis to be a serious clinicalproblem. The reason for the current safety is probably that the smallpools minimize the risk of dissemination of infection from a donor carry-ing the disease. The present screening of blood donors is also probablymuch more efficient than that used in the emergency conditions of wartime.As might be expected, blood transfusion itself also carries a slight risk oftransmitting hepatitis, estimated at about 0.1 per cent.

As mentioned previously, the blood group of the recipient need notusually be taken into account when reconstituted pooled plasma is given.We have recently found evidence, however, that when very large volumes ofplasma are given for severe burns the quantities of A antibody given in thetransfusion may be sufficient to cause haemolysis to the patient's cells if heis of Group A or AB. It is probably advisable, therefore, not to give morethan, say, two plasma volumes of pooled plasma to such patients.Albumin, homologous plasma or whole blood would be a satisfactoryalternative (Topley et al., 1963).

SerumIn the early days of transfusion, serum was often given as an alternative

to plasma, the main differences between them being that serum contains nofibrinogen and no anticoagulant. Recently, dried plasma has become moreplentiful as a by-product of the Blood Transfusion Service, and serum hasfallen largely out of use. Special donations of blood need to be collectedfor serum preparation; it is then supplied in freeze-dried form and can beused in place of plasma. As at present prepared, the reconstituted

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solution contains about 7 per cent. protein, so that a standard solution israther more effective in maintaining circulatory volume and plasmaprotein levels than is the standard plasma preparation.

AlbuminWith the development of modern methods of protein separation, it is

now feasible to use solutions of pure albumin as an alternative to othertransfusion fluids. Such solutions are relatively expensive but have theadvantage of being clear solutions, free from anticoagulant materials andcontaining whatever electrolytes may be desired. They do not transmithepatitis and can also be given at high concentration (e.g., 25 per cent.solution), so economizing in the transport of sterile water and giving a largevolume expansion for a small transfusion. Albumin shares with plasmaand serum the disadvantage of adding nothing to the oxygen-carryingcapacity of the blood, so that its ideal role is for situations where no redcells have been lost. In burns and other conditions where there is adisproportionate loss of albumin there is a special case for the use ofalbumin solutions in therapy. If used alone for burns, and given in largequantities, there is danger of excessive dilution of other plasma proteinfractions, in particular of the gamma-globulins which may be neededfor defence against infection. The use of alternate bottles of plasmaand albumin for treatment of burns seems a good compromise.

Plasma substitutesThe search for suitable substitutes for plasma has continued since

World War I, when gelatin and gum arabic were tried for this purpose.If one could find a colloid of the approximate molecular size of plasmaprotein which was not itself toxic or otherwise harmful, and which slowlydisappeared subsequently from the circulation, it would clearly have a rolein economizing in the need for blood donors. An aspect of this which hasstimulated much recent research is the requirement for transfusion fluidssuitable for stock piling for use in a major emergency such as an atomicdisaster.

Normal plasma proteins provide a guide to the molecular size necessaryto give the desired retention in the circulation. Albumin (MW 69,000)leaves at a moderate rate; 6 lipoprotein (MW 1.3 million) hardly leavesat all. The ideal molecular weight for a plasma substitute must lie some-where in this range of size. A practical aim is retention in the circulationof at least 50 per cent. of the material for 24 hours and complete ultimateelimination by either excretion or metabolism. Eligible materials havebeen found among proteins (e.g., gelatin and globin), synthetic plastics(e.g., polyvinyl pyrrolidone) and carbohydrates (e.g., gum acacia, dextran).

Gelatin was the first substance to be tried and has the advantage ofavailability. It can be metabolized and it is not antigenic. However,there are difficulties in preparation and handling, degradation occurs on

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heating and the smaller material is lost rapidly from the circulation;solutions of molecules of sufficient size to be retained also become solidin the cold. As a solution for maintenance of circulatory volume for a fewhours-perhaps until definitive transfusion can be given-gelatiln has avalid role (Ravdin, 1952). Attempts have been made to improve thefluidity and one such modified gelatin solution (MW 34,000) was investi-gated in Korea; about 10 per cent. was found to be retained after 24 hours,but this temporary effect was thought to be useful during evacuation ofcasualties (Frawley et al., 1955). Other gelatin preparations have recentlybeen tried in Germany and Switzerland.

The protein globin, available by hydrolysis of the haemoglobin fromtime-expired blood, has also been suggested as a plasma volume expander.It can be prepared in solution in saline, a 4.1 per cent. solution having thecolloid osmotic pressure of plasma. It appears to be safe but, since themolecular size is only 34,000 and there is rapid breakdown, retention in thecirculation is brief. However, the support of circulatory volume has beenclaimed to be useful. Very little of the globin appears in the urine,virtually all being metabolized, so contributing to the input of first classprotein. Since many conditions which require treatment by transfusionalso require extra protein input, administration of globin provides areasonable combination of the two functions (Strumia, 1951) and makesuse of human protein which would otherwise be discarded.

Polyvinyl pyrrolidone (PVP) was developed in Germany during the lastwar (Hecht and Weese, 1943) and can be prepared in a range of molecularsizes. It is the one widely used plasma substitute which is almost certainlynot metabolized. Correspondingly, ranges of molecular size which areretained adequately in the circulation are also liable to be stored in-definitely, for instance in the reticulo-endothelial system, and there havebeen reports that prolonged storage after transfusion can be carcinogenic.Material of molecular weight c. 35,000 appears to have been found satis-factory for temporary maintenance of circulatory volume. A largeproportion of the infused material is excreted in the urine within a fewhours. Recently a type ofPVP has been found useful as a stabilizer in thepreservation of red cells by rapid freezing.

Of the carbohydrates, hydrolysed gum acacia was introduced as a plasmasubstitute during the first World War. As obtained in the form of avegetable gum this consists of a complex of hexose and pentose sugars anduronic acid. The exact structure has not been determined. By acidhydrolysis the gum is reduced in molecular size for use as an infusion withsaline. It was demonstrated to have value in replacing circulatory volume(Bayliss, 1919), but subsequent studies showed that storage and liverdamage might ensue. Possibly if it had been investigated and improved bymodern chemical techniques, gum acacia might have been developed as a

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satisfactory plasma substitute, but the source of its raw material, a naturalproduct subject to variations in quality and contamination, would remaina serious disadvantage.

Dextran is a polysaccharide consisting of long chains of glucose unitsproduced by fermentation of a sucrose medium by Leuconiostocmesenteroides. Native dextran has a molecular weight of many millions,but it can be hydrolysed to molecules of a range of sizes correspondingto those of plasma proteins. By fractional precipitation a series of fairlyhomogeneous fractions can be obtained. The use of this material fortransfusion was suggested in England before the war and was independentlydeveloped in Sweden. Initially several different strains of Leuconostocwere used which produced different degrees of branching in the dextran.The more highly branched dextrans were found to cause more unwantedreactions and in recent years all manufacturers, at least in Westerncountries, have used the strain B.512, which produces an almost linearmolecule associated with the lowest reaction rates. Replacement ofcirculatory volume has been demonstrated in many studies, the persistenceof plasma volume expansion depending on molecular size (see Squire et al.,1955). In clinical use untoward reactions have been rare. Allergicreactions have, however, been described when several different types ofdextran have been given experimentally to volunteers. Reactions includeflushing, pyrexia and vomiting and some have been shown to be associatedwith a fall of plasma volume. Another effect, again mainly troublesomein experimental rather than clinical situations, has been a tendency fordextran to cause prolongation of bleeding time. This we suspected in ourearlier trials of dextran for burns. It has been further extensively studiedin the United States; fairly large volumes of dextran must be given but theeffect is not simply that of dilution since similar quantities of albumin donot cause prolonged bleeding. The mode of action is uncertain; the effectis at a maximum several hours after the infusion and has been thought to bedue to interference with platelets (Langdell et al., 1958). Studies in dogshave shown prolonged bleeding time, low platelet count and reducedprothrombin and fibrinogen when severe haemorrhages have beenreplaced with dextran, PVP or pectin solutions. Modified fluid gelatincaused less marked disturbances, and unmodified gelatin was free from illeffects (Behrmann and Hartmann, 1959).

Since the first clinical trials of dextran it has been known that sedi-mentation rate was raised by the larger dextran molecules. For a givenmolecular size the elevation of E.S.R. rises sharply with increasing con-centration (Hardwicke et al., 1950). Though transfusion of large amountsof dextran was known to raise the E.S.R., this was thought for the purposeof the British specification to be acceptable in return for the sustainedplasma volume expansion. The Swedish and American specificationsprovide for a lower average molecular weight and hence shorter mainten-

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ance of volume and more rapid excretion. Swedish workers also claimthat dextran of relatively small molecular weight can offset the raisedE.S.R. due to larger dextran and its supposed ill effects (Gelin, 1956).This small molecular material is now marketed as a treatment for" sludging" and has also proved valuable as a priming fluid in work onextra-corporeal circulation. The material has a mean molecular sizeof about 40,000 and is therefore fairly rapidly lost from the circulation.Since it is given at 10 per cent. concentration rapid infusion causes a briefextra expansion while the hyperoncotic solutions draw water into thecirculation and before both the dextran and the water are lost again intothe tissues or into the urine.

Recently Stalker (1961) has repeated some of the Swedish experimentsand has confirmed that large molecular weight dextran can cause" sludging " and microscopic lesions of the liver and heart. These lesionscan be seen in rabbits as areas of necrosis and cellular infiltration if thetissues are inspected a few days after infusion. If the animals are allowedto survive the tissues return to normal appearance. The reversibility ofthese changes may account for their having been overlooked in otherexperiments. There still remains the question as to their significance inman, but it seems reasonable to re-examine the specification of clinicaldextran to see whether the possibility of such effects can be minimizedwhile still retaining the advantages of sustained support of circulatoryvolume; experiments on this are now in progress.

SUMMARY1. Blood transfusion remains the treatment of choice for replacement

of blood loss. With proper precautions to ensure compatibility, storedcitrated blood is usually safe though there are certain special hazards tobe kept in mind when large volumes are given quickly.

2. Where plasma loss predominates, reconstituted dried pooled plasmais rational treatment. There are certain differences from fresh plasmawhich may sometimes be important.

3. Of the available plasma substitutes, the value of dextran has beenmost fully established.

4. Recent developments include more prolonged storage of blood atlow temperatures, the availability of pure plasma protein fractions andpreparations of dextran giving a variety of effects depending upon theirdiffering molecular sizes.

REFERENCESBAYLISS, W. M. (1919) Spec. Rep. Ser. Med Res. Coun. (Loud.), 25.BEHRMANN, V. G., and HARTMANN, F. W. (1959) J. lab. Invest. 4, 190.BRADFIELD, W. J. D. (1963). In the press.FRAWLEY, J. P., ARTZ, C. P., and HOWARD, J. M. (1955) Surgery, 37, 384.GELIN, L-E. (1956) Acta chir. scand., Suppl. 210.GIBSON, J. G., MURPHY, W. P., SCHEITLIN, W. A., and REES, S. B. (1956) Amer. J. clin.

Path. 26, 855.

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HARDWICKE, J., RICKETTS, C. R., and SQUIRE, J. R. (1950) Nature, 166, 988.HECHT, G., and WEESE, H. (1943) Munch. med. Wschr. 90, 1 1.HUGHES-JONES, N. C., MOLLISON, P. L., and ROBINSON, M. A. (1957) Proc. Roy. Soc., B.

147, 476.LANGDELL, R. D., ADELSON, E., FURTH, F. W., and CROSBY, W. H. (1958) J. Amer. med.

Ass. 162, 346.MEDICAL RESEARCH COUNCIL (1954) Lancet, 1, 1328.MOLLISON, P. L., (1961) Blood Transfusion in Clinical Medicine. 3rd edition. Oxford,

Blackwell.SLOVITER, H. A., and CHAPLIN, H. (1952) Lancet, 2, 501.

RAVDIN, I. S. (1952) J. Amer. med. Ass. 52, 10.SEVITT, S. (1959) in Modern Trends in Accident Surgery and Medicine. London,

Butterworths.SQUIRE, J. R., BULL, J. P., MAYCOCK, W. d'A., RICKETTS, C. R. (1955) Dexlran, its

Properties and Use in Medicine. Oxford, Blackwell.STALKER, A. L. (1961) A microcirculatory study of the effects of dextran. M.D. Thesis,

Aberdeen.STRUMIA, M. M. (1951) in Proc. Conf. Burns, Nat. Acad. Sci., Washington.TOPLEY, E., BULL, J. P., MAYCOCK, W. d'A., MOURANT, A. E., and PARKIN, D. (1963)

J. clin. Path. 16, 79.

Contribution from Mr. B. N. CatchpoleA problem which concerns us is that of the management of those who

have lost a great deal of blood, e.g. the patient who has ruptured his aortaor has had a massive blood loss into his gut. We know that these patientsare likely to have a developing impairment of their carbohydrate meta-bolism and a marked trend towards acidosis. We treat them with massiveblood transfusion. As we have heard, the pH of stored blood is 6.8 oreven lower, and it is loaded with citrate; both of these features have to becorrected by the patient. The fact that most of our patients may be ableto deal with this situation should not blind us to the problem; some dieand perhaps these have died a " metabolic death " which could have beenprevented. What part, if any, have solutions of sodium bicarbonate andT.H.A.M. (trishydroxymethylaminomethane) to play in the treatmentof this condition?

Reply by Dr. BullI would first like to emphasize that so far as I am aware the acidifying

effect of large blood transfusions rarely causes trouble.I think that sodium bicarbonate should be satisfactory in many circum-

stances, including those you mention. Two limitations are:(1) the extra sodium load introduced may be undesirable;(2) the neutralising action of sodium bicarbonate involves evolution of

Co2.In the particular circumstance I mentioned, of severe blood loss combinedwith respiratory embarrassment, it may be difficult for the patient to getrid of this extra CO2.T.H.A.M. might be a useful alternative to sodium bicarbonate, but I

have no experience of its use for this condition. Have other membersof the audience any suggestions on this point?

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Contribution by Dr. H. J. BrennanIn the 1930s I carried out an investigation into the effects of haemor-

rhage on a considerable series of neurosurgical patients. Among otherobservations red cell counts and blood volume estimations were madebefore operation, serial red cell counts every 10 minutes during its progressand blood volume estimations and measurements of external blood losspost-operatively. At that time it was not the practice to put up dripsroutinely before operation and to replace blood as it was lost. Strangeas it may sound nowadays this was not done until the patient had alreadylost considerable amounts of blood, often as much as 1 or I4 litres.Obviously these observations can never be repeated clinically! In thosepatients who had lost an appreciable amount of blood without replacementone of the constant findings was that after allowing for those lost externallyvery considerable numbers of R.B.Cs.-on the average some 20 per cent.of those originally circulating-were " missing" from the circulation atthe end of operation. I suggested that they had been by-passed and werestationary in innumerable capillary loops-the term " sludging" had notthen been coined. I found that a transfusion of plasma, given whilewaiting for blood to arrive, not only improved the patient's circulation andblood pressure but reversed the fall in cell count which had proceededsteadily during the operation, and actually resulted in an appreciable risein cell count-not, as might have been expected, a further fall from furtherdilution. Presumably this indicated that many of these missing red cellshad been washed back into the circulation. This led me to advocateplasma transfusions in the early treatment of haemorrhage as a form ofauto-transfusion. It is possible that a low molecular fraction dextranwould be even more successful in bringing about this effect, and I wonderif Dr. Bull has any information bearing on this point.

Reply by Dr. BullI agree that stagnation of cells may well occur as you suggest. My

colleagues also have sometimes found unexpected rises of red cell volumeafter transfusion in patients in whom serial measurements were made.The suggested mobilization of red cells could probably occur with anyinfusion which improves local circulation, and I agree that it may be partof the response to Rheomacrodex.

Contribution from Professor ShackmanIt is possible, in theory at least, to exchange hydrogen ions in extra-

corporeal blood by means of haemodialysis in a manner similar to thatemployed in the treatment of patients with acidosis of renal failure. Asmall artificial kidney of the Minicoil type may be used for dialysis ofbottles of stored blood before they are given to a patient. Of course,anti-coagulation during haemodialysis and subsequent neutralization ofthe anti-coagulants would be required.

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