TRANSFUSION FLUIDS

by

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 to supply cells in aplastic anaemia, to provide clotting factors in haemophilia, or even to attempt to change the personality of the recipient as in the early experiments on transfusion in the 17th century. Our concern here is with transfusion for the maintenance of circulatory volume.
Whole blood Where whole blood has been lost, the obvious choice for a replacement fluid is blood itself. The history of blood transfusion is that of the various attempts to perform this replacement safely. From the beginning of this century the ABO groups have been recognized and in the last 30 years many other blood group antibodies have been identified. For the purposes of blood volume replacement ABO and Rhesus groups remain much the most important. For an account of the relevance of the other more subtle factors, one cannot do better than recommend Mollison's book Blood Transfusion in Clinical Medicine.

For emergency transfusion it is often important to balance the risk of transfusion reaction against the risk of delay. Sevitt has described a practical scheme giving the appropriate procedure corresponding to different degrees of urgency. If immediate transfusion is required, the first few bottles must be Group 0 blood, preferably Rh negative. By the time these bottles have been given it should be possible to continue with blood of correct ABO and Rhesus type. If a delay of 15 minutes is practicable, ABO grouping is possible, and often Rhesus typing also. Homologous blood can be used initially followed by blood which also has been crossmatched. If, as may well be the case, a delay of up to three-quarters of an hour is possible, full blood grouping, Rhesus typing and cross-matching tests can be performed. Other necessary precautions to ensure that only safe blood is given have also been described (Sevitt, 1959).

So far we have been assuming that what the bottle contains is equivalent to normal circulating blood. To what extent is this true? The donor will have been screened for disease and anaemia; 420 ml. of the blood will have been mixed with 120 ml. of Acid Citrate Dextrose (ACD) solution. Thus only four-fifths of the reputed pint is actually blood. ACD solution contains citrate to combine with calcium and so prevent clotting; the acid and dextrose are present so as to favour survival of the red cells. The citrate is present as the sodium salt so the final sodium concentration is not grossly abnormal. Potassium concentration, however, may be quite 175

J. P. BULL

high, since during storage potassium leaves the red cells and is then in solution in the plasma. Unless, however, the recipient already has a high serum potassium, there seems to be little risk in transfusing bottled blood, though levels of about 12 mEq./litre are not uncommon in the fluid as transfused. Whether citrate is innocuous has been disputed. There is no evidence of damage in transfusions of moderate volume given to previously fit persons. Citrate is a normal intermediate of metabolism and is ultimately broken down to carbon dioxide. There is normally a blood level of about 1 mg./100 ml. The level in plasma oftransfused blood may, however, be more than 100 times this and, after massive transfusion, it is possible for dangerous levels to be produced. Much of the toxic effect of citrate is attributable to its causing hypocalcaemia, but there is some evidence that citrate itself can damage the heart, apart from its effects on calcium. All these ill effects can, however, be prevented by giving extra calcium with blood transfusion; 10 ml. of 10 per cent. calcium gluconate per litre of transfused blood has been recommended for this purpose. Even without this precaution, large transfusions have often been given without any evidence of ill effects. The liver is a main site of breakdown of citrate and therefore particular caution should be exercised when giving large transfusions of bottled blood to persons with known or suspected liver damage.
There is a further possibility that the acidity of the ACD mixture may also 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 the blood donation as it runs into the ACD anticoagulant, though it is not certain how important this effect may be in practice. A potentially more serious clinical implication has been pointed out recently. The pH of stored blood, initially about 7.1, falls to 6.6 during storage. This implies that the buffer system of plasma and cells has been overwhelmed by a combination of the citric acid already present in ACD solution and the other organic acids (chiefly lactic acid) produced during storage. With transfusions of moderate amounts the recipient's buffering capacity is normally fully adequate to correct the acidity and as the citrate is metabolized this itself tends to raise the pH. Both processes require that carbon dioxide can be lost freely. However, there may be patients with a combination of severe blood loss and an acidosis-particularly if this is of respiratory origin-for whom the further addition of acid in the transfusion may be a severe burden. A case in which this appears to have happened has been described by Bradfield (1963).

Having mentioned these various hazards it is perhaps necessary to emphasize again that in the presence of good laboratory facilities and proper supervision, blood transfusion remains a surprisingly safe procedure even when sufficient is given to correct the very large blood loss which may occur in multiple injuries. 176

TRANSFUSION FLUIDS

Alternative methods of storing blood have been suggested. Apart from variants upon the ACD mixture, such as the addition of materials like adenosine which favour longer survival of red cells, preservation at very low temperatures is a feasible alternative. During the last 10 years it has been shown that many living cells can survive storage at -40 C. or below, if precautions are taken to prevent damage during the processes of freezing and thawing. There are two main methods. Slow cooling and maintenance 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 red cells stored in this way can give good survival on subsequent transfusion, though there remain some difficulties in applying these methods on a large scale and with the speed often required for emergency transfusion. Another method which has recently been developed to an advanced stage is the rapid freezing of blood to the temperature of liquid nitrogen. This can be done without the addition of glycerol, and offers the possibility of convenient long-term storage combined with speedy availability.
Plasma By 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 the commonest condition where plasma loss predominates; the fluid which leaves the circulation into blisters and tissues and which is lost from the surface as exudate is similar to plasma in composition and contains all the main plasma protein fractions. Material commonly available is freezedried small pool plasma. As implied above, red cells have limited viability when stored in ACD. After 14 days' storage about 85 per cent. of cells survive in the circulation of the recipient 24 hours after transfusion. It is therefore the usual practice to withdraw the blood from the bank soon after this time. The plasma of such blood can be separated and salvaged; it still contains the blood group antibodies but by mixing together the plasmas of blood of different groups the resulting pool does not contain enough antibody to be troublesome, and such pooled plasma can normally be given without regard to the blood group of the recipient.

The freeze-dried plasma still contains the electrolytes from the original stored blood and so has rather a higher sodium concentration and a much higher potassium and citrate level than present in the circulation. The problem of acidity is not so severe since plasma is not so heavily buffered as whole blood. When reconstituted as recommended with sterile water the solution contains 5 per cent. plasma protein. Electrophoresis shows that the main fractions of this protein are fairly normal, but the lipoproteins are damaged and do not go properly into solution; this largely accounts for the cloudy appearance common in reconstituted plasma. Plasma of this type will give good circulatory volume replacement and its effect in patients with burns is immediately shown by the expected fall of haematocrit. If plasma is given when whole blood has been lost, the 177

J. P. BULL

haematocrit will fall below normal corresponding to the dilution of red cells. In animal experiments the response to such replacement may be similar to that obtained with whole blood, but most clinicians who have tried both in patients feel sure that the red cells of whole blood with their oxygen-carrying power give blood transfusion a great advantage over plasma transfusion as a treatment for blood loss. If plasma is made up in more concentrated form, circulatory volume expansion occurs in proportion to the amount of plasma protein transfused, water being attracted into the circulation until protein levels return to normal.
The risk of hepatitis is often cited as a serious contra-indication to the use of plasma. Fear of this dates largely from wartime, when plasma prepared from extremely large pools was processed in America and used in the Armed Forces. Some batches of this plasma gave rise to a high incidence of hepatitis, often with high mortality. It is understandable that recollections of such experiences should breed caution, but experience in recent years has been much more favourable. The most recently published survey in this country (Medical Research Council, 1954) showed an incidence of less than 1 per cent. of plasma jaundice, and very slight mortality. We have continued to use plasma extensively in the Birmingham Burns Unit, and have not found hepatitis to be a serious clinical problem. The reason for the current safety is probably that the small pools minimize the risk of dissemination of infection from a donor carrying the disease. The present screening of blood donors is also probably much more efficient than that used in the emergency conditions of wartime. As might be expected, blood transfusion itself also carries a slight risk of transmitting hepatitis, estimated at about 0.1 per cent.
As mentioned previously, the blood group of the recipient need not usually be taken into account when reconstituted pooled plasma is given. We have recently found evidence, however, that when very large volumes of plasma are given for severe burns the quantities of A antibody given in the transfusion may be sufficient to cause haemolysis to the patient's cells if he is of Group A or AB. It is probably advisable, therefore, not to give more than, say, two plasma volumes of pooled plasma to such patients. Albumin, homologous plasma or whole blood would be a satisfactory alternative (Topley et al., 1963). Serum In the early days of transfusion, serum was often given as an alternative to plasma, the main differences between them being that serum contains no fibrinogen and no anticoagulant. Recently, dried plasma has become more plentiful as a by-product of the Blood Transfusion Service, and serum has fallen largely out of use. Special donations of blood need to be collected for serum preparation; it is then supplied in freeze-dried form and can be used in place of plasma. As at present prepared, the reconstituted 178

TRANSFUSION FLUIDS

solution contains about 7 per cent. protein, so that a standard solution is rather more effective in maintaining circulatory volume and plasma protein levels than is the standard plasma preparation. Albumin With the development of modern methods of protein separation, it is now feasible to use solutions of pure albumin as an alternative to other transfusion fluids. Such solutions are relatively expensive but have the advantage of being clear solutions, free from anticoagulant materials and containing whatever electrolytes may be desired. They do not transmit hepatitis 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 large volume expansion for a small transfusion. Albumin shares with plasma and serum the disadvantage of adding nothing to the oxygen-carrying capacity of the blood, so that its ideal role is for situations where no red cells have been lost. In burns and other conditions where there is a disproportionate loss of albumin there is a special case for the use of albumin solutions in therapy. If used alone for burns, and given in large quantities, there is danger of excessive dilution of other plasma protein fractions, in particular of the gamma-globulins which may be needed for defence against infection. The use of alternate bottles of plasma and albumin for treatment of burns seems a good compromise. Plasma substitutes The 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 plasma protein which was not itself toxic or otherwise harmful, and which slowly disappeared subsequently from the circulation, it would clearly have a role in economizing in the need for blood donors. An aspect of this which has stimulated much recent research is the requirement for transfusion fluids suitable for stock piling for use in a major emergency such as an atomic disaster. Normal plasma proteins provide a guide to the molecular size necessary to give the desired retention in the circulation. Albumin (MW 69,000) leaves at a moderate rate; 6 lipoprotein (MW 1.3 million) hardly leaves at all. The ideal molecular weight for a plasma substitute must lie somewhere in this range of size. A practical aim is retention in the circulation of at least 50 per cent. of the material for 24 hours and complete ultimate elimination by either excretion or metabolism. Eligible materials have been 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 of availability. It can be metabolized and it is not antigenic. However, there are difficulties in preparation and handling, degradation occurs on 179

J. P. BULL

heating and the smaller material is lost rapidly from the circulation; solutions of molecules of sufficient size to be retained also become solid in the cold. As a solution for maintenance of circulatory volume for a few hours-perhaps until definitive transfusion can be given-gelatiln has a valid role (Ravdin, 1952). Attempts have been made to improve the fluidity and one such modified gelatin solution (MW 34,000) was investigated 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 of casualties (Frawley et al., 1955). Other gelatin preparations have recently been tried in Germany and Switzerland.
The protein globin, available by hydrolysis of the haemoglobin from time-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 the colloid osmotic pressure of plasma. It appears to be safe but, since the molecular size is only 34,000 and there is rapid breakdown, retention in the circulation is brief. However, the support of circulatory volume has been claimed to be useful. Very little of the globin appears in the urine, virtually all being metabolized, so contributing to the input of first class protein. Since many conditions which require treatment by transfusion also require extra protein input, administration of globin provides a reasonable combination of the two functions (Strumia, 1951) and makes use of human protein which would otherwise be discarded.

Polyvinyl pyrrolidone (PVP) was developed in Germany during the last war (Hecht and Weese, 1943) and can be prepared in a range of molecular sizes. It is the one widely used plasma substitute which is almost certainly not metabolized. Correspondingly, ranges of molecular size which are retained adequately in the circulation are also liable to be stored indefinitely, for instance in the reticulo-endothelial system, and there have been reports that prolonged storage after transfusion can be carcinogenic. Material of molecular weight c. 35,000 appears to have been found satisfactory for temporary maintenance of circulatory volume. A large proportion of the infused material is excreted in the urine within a few hours. Recently a type of PVP has been found useful as a stabilizer in the

preservation of red cells by rapid freezing.

Of the carbohydrates, hydrolysed gum acacia was introduced as a plasma substitute during the first World War. As obtained in the form of a vegetable gum this consists of a complex of hexose and pentose sugars and uronic acid. The exact structure has not been determined. By acid hydrolysis the gum is reduced in molecular size for use as an infusion with saline. It was demonstrated to have value in replacing circulatory volume (Bayliss, 1919), but subsequent studies showed that storage and liver damage might ensue. Possibly if it had been investigated and improved by modern chemical techniques, gum acacia might have been developed as a 180

TRANSFUSION FLUIDS

satisfactory plasma substitute, but the source of its raw material, a natural product subject to variations in quality and contamination, would remain a serious disadvantage.
Dextran is a polysaccharide consisting of long chains of glucose units produced by fermentation of a sucrose medium by Leuconiostoc mesenteroides. Native dextran has a molecular weight of many millions, but it can be hydrolysed to molecules of a range of sizes corresponding to those of plasma proteins. By fractional precipitation a series of fairly homogeneous fractions can be obtained. The use of this material for transfusion was suggested in England before the war and was independently developed in Sweden. Initially several different strains of Leuconostoc were used which produced different degrees of branching in the dextran. The more highly branched dextrans were found to cause more unwanted reactions and in recent years all manufacturers, at least in Western countries, have used the strain B.512, which produces an almost linear molecule associated with the lowest reaction rates. Replacement of circulatory volume has been demonstrated in many studies, the persistence of plasma volume expansion depending on molecular size (see Squire et al., 1955). In clinical use untoward reactions have been rare. Allergic reactions have, however, been described when several different types of dextran have been given experimentally to volunteers. Reactions include flushing, pyrexia and vomiting and some have been shown to be associated with a fall of plasma volume. Another effect, again mainly troublesome in experimental rather than clinical situations, has been a tendency for dextran to cause prolongation of bleeding time. This we suspected in our earlier trials of dextran for burns. It has been further extensively studied in the United States; fairly large volumes of dextran must be given but the effect is not simply that of dilution since similar quantities of albumin do not cause prolonged bleeding. The mode of action is uncertain; the effect is at a maximum several hours after the infusion and has been thought to be due to interference with platelets (Langdell et al., 1958). Studies in dogs have shown prolonged bleeding time, low platelet count and reduced prothrombin and fibrinogen when severe haemorrhages have been replaced with dextran, PVP or pectin solutions. Modified fluid gelatin caused less marked disturbances, and unmodified gelatin was free from ill effects (Behrmann and Hartmann, 1959).

Since the first clinical trials of dextran it has been known that sedimentation rate was raised by the larger dextran molecules. For a given molecular size the elevation of E.S.R. rises sharply with increasing concentration (Hardwicke et al., 1950). Though transfusion of large amounts of dextran was known to raise the E.S.R., this was thought for the purpose of the British specification to be acceptable in return for the sustained plasma volume expansion. The Swedish and American specifications provide for a lower average molecular weight and hence shorter mainten181

J. P. BULL

ance of volume and more rapid excretion. Swedish workers also claim that dextran of relatively small molecular weight can offset the raised E.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 on extra-corporeal circulation. The material has a mean molecular size of 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 brief extra expansion while the hyperoncotic solutions draw water into the circulation and before both the dextran and the water are lost again into the tissues or into the urine. Recently Stalker (1961) has repeated some of the Swedish experiments and has confirmed that large molecular weight dextran can cause " sludging " and microscopic lesions of the liver and heart. These lesions can be seen in rabbits as areas of necrosis and cellular infiltration if the tissues are inspected a few days after infusion. If the animals are allowed to survive the tissues return to normal appearance. The reversibility of these changes may account for their having been overlooked in other experiments. There still remains the question as to their significance in man, but it seems reasonable to re-examine the specification of clinical dextran to see whether the possibility of such effects can be minimized while still retaining the advantages of sustained support of circulatory volume; experiments on this are now in progress.

SUMMARY 1. Blood transfusion remains the treatment of choice for replacement of blood loss. With proper precautions to ensure compatibility, stored citrated blood is usually safe though there are certain special hazards to be kept in mind when large volumes are given quickly. 2. Where plasma loss predominates, reconstituted dried pooled plasma is rational treatment. There are certain differences from fresh plasma which may sometimes be important. 3. Of the available plasma substitutes, the value of dextran has been most fully established. 4. Recent developments include more prolonged storage of blood at low temperatures, the availability of pure plasma protein fractions and preparations of dextran giving a variety of effects depending upon their

differing molecular sizes.

REFERENCES BAYLISS, 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.

182

TRANSFUSION FLUIDS

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,
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.

Aberdeen.

Contribution from Mr. B. N. Catchpole A 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 aorta or has had a massive blood loss into his gut. We know that these patients are likely to have a developing impairment of their carbohydrate metabolism and a marked trend towards acidosis. We treat them with massive blood transfusion. As we have heard, the pH of stored blood is 6.8 or even lower, and it is loaded with citrate; both of these features have to be corrected by the patient. The fact that most of our patients may be able to deal with this situation should not blind us to the problem; some die and perhaps these have died a " metabolic death " which could have been prevented. What part, if any, have solutions of sodium bicarbonate and T.H.A.M. (trishydroxymethylaminomethane) to play in the treatment of this condition? Reply by Dr. Bull I 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 circumstances, 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 combined with respiratory embarrassment, it may be difficult for the patient to get rid 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 members of the audience any suggestions on this point? 183

J. P. BULL

Contribution by Dr. H. J. Brennan In the 1930s I carried out an investigation into the effects of haemorrhage on a considerable series of neurosurgical patients. Among other observations red cell counts and blood volume estimations were made before operation, serial red cell counts every 10 minutes during its progress and blood volume estimations and measurements of external blood loss post-operatively. At that time it was not the practice to put up drips routinely before operation and to replace blood as it was lost. Strange as it may sound nowadays this was not done until the patient had already lost considerable amounts of blood, often as much as 1 or I4 litres. Obviously these observations can never be repeated clinically! In those patients who had lost an appreciable amount of blood without replacement one of the constant findings was that after allowing for those lost externally very considerable numbers of R.B.Cs.-on the average some 20 per cent. of those originally circulating-were " missing" from the circulation at the end of operation. I suggested that they had been by-passed and were stationary in innumerable capillary loops-the term " sludging" had not then been coined. I found that a transfusion of plasma, given while waiting for blood to arrive, not only improved the patient's circulation and blood pressure but reversed the fall in cell count which had proceeded steadily during the operation, and actually resulted in an appreciable rise in cell count-not, as might have been expected, a further fall from further dilution. Presumably this indicated that many of these missing red cells had been washed back into the circulation. This led me to advocate plasma transfusions in the early treatment of haemorrhage as a form of auto-transfusion. It is possible that a low molecular fraction dextran would be even more successful in bringing about this effect, and I wonder if Dr. Bull has any information bearing on this point.
Reply by Dr. Bull I agree that stagnation of cells may well occur as you suggest. My colleagues also have sometimes found unexpected rises of red cell volume after transfusion in patients in whom serial measurements were made. The suggested mobilization of red cells could probably occur with any infusion which improves local circulation, and I agree that it may be part of the response to Rheomacrodex.

Contribution from Professor Shackman It is possible, in theory at least, to exchange hydrogen ions in extracorporeal blood by means of haemodialysis in a manner similar to that employed in the treatment of patients with acidosis of renal failure. A small artificial kidney of the Minicoil type may be used for dialysis of bottles of stored blood before they are given to a patient. Of course, anti-coagulation during haemodialysis and subsequent neutralization of the anti-coagulants would be required. 184