Testing for Blood Groups
Reference to the table of blood reactions given on p. 71 will show that in order to discover the blood group of any individual it is only necessary to test his corpuscles against the serum of Groups II and III. These reactions may be recapitulated as follows:
(i) If he be Group I, his corpuscles will be agglutinated by the serum of Groups II and III.
(ii) If he be Group II, his corpuscles will be agglutinated by the serum of Group III only.
(iii) If he be Group III, his corpuscles will be agglutinated by the serum of Group II only.
(iv) If he be Group IV, his corpuscles will be agglutinated by neither serum.
Only the serum, therefore, collected from people known to belong to Groups II and III need be kept in stock. This can generally be obtained from the Lister Institute, and if kept sterile will retain its agglutinating properties for some months, but under no circumstances should serum more than six months old be used, since the consequences of a failure to agglutinate may be very serious. Nevertheless, the agglutinins contained in serum are very resistant to physical and chemical changes in their environment. Dried serum has been successfully used for testing purposes, and Culpepper has shown that the reactions are not interfered with by cold or by heat until actual coagulation of the serum takes place. Bacterial contamination does not affect the reactions, so that the serum is still active even when putrid. Various methods have been used for preserving the serum. Its properties are not affected by the addition of dilute cresol (1 : 250) or of chloroform.
In the absence of any stock sera, the agglutinating test may be applied directly. A few cubic centimetres of blood are taken from the patient, and the serum as soon as it has separated is tested against the corpuscles of the prospective donor. If agglutination occurs, this donor is at once excluded. If no agglutination occurs, he is either of the same group as the patient or belongs to a compatible group. Supposing that a donor actually of the same group as the patient is wanted, then the reverse test must be performed in addition, that is to say, the corpuscles of the patient must be tested against the serum of the donor. If both tests are negative, then donor and patient are proved to be of the same group. The method of direct test cannot be applied in an emergency owing to the loss of time involved; it is better, therefore, that anyone who intends to be ready to perform a blood transfusion should always have serum of Groups II and III immediately available.
The collection of stock sera is not a matter of any difficulty. With strict aseptic precautions 20 cc. of blood are withdrawn in a syringe from persons known to belong to Groups II and III; the bloods are put into a sterile test-tube and allowed to clot. As soon as the serum has separated it is drawn up into sterile glass bulbs of suitable capacity, which are sealed off at each end. The most convenient form of storage for actual use is a capillary glass tube sealed at each end. Each tube may be made to hold a single drop, which is the amount used for a test. There is then no wastage of serum, and no chance of contaminating the remaining stock. When the blood has been withdrawn and has clotted, the complete settling of the corpuscles can be hastened by the use of the centrifuge. If the serum be left in contact with the corpuscles for more than twelve hours, some auto-hæmolysis may take place, so that the serum will become tinged with hæmoglobin. It is exceedingly important that the two stock sera should not become confused, and this may easily happen unless each tube has some distinguishing mark.
The methods of testing for blood groups have been simplified by successive observers since the existence of the groups was first demonstrated in 1907. Moss used an elaborate technique such as was essential for putting a new discovery upon a secure scientific basis. In order to obtain a suspension of corpuscles, blood was drawn into a syringe containing a solution of sodium citrate to prevent clotting. The corpuscles were collected by means of the centrifuge, and were thoroughly washed twice in normal saline solution so that they were finally collected free from serum and from citrate. Serum was collected in the manner already described. A series of small tubes was then filled with equal quantities of serum and the suspension of corpuscles, and was incubated for two hours at 37·5° C. At the end of this time observations were made and again after the tubes had stood for twelve hours in an ice chest. Varying degrees of agglutination and hæmolysis were then accurately recorded, and far-reaching results were obtained.
Later workers had the advantage of using stock sera belonging to known groups, so that the number of observations to be made was very greatly reduced. Brem introduced in 1916 a method of testing in which he mixed the serum and suspension of washed corpuscles in very small quantities on a coverslip, which was inverted over an ordinary cell slide rimmed with petroleum jelly. The results could then be observed macroscopically or under the microscope, and the presence or absence of agglutination could be determined within fifteen minutes. The detection of hæmolysis by the hanging drop method requires that the cells should be incubated and observed at intervals for several hours, but it is not always easy to see the disintegrated corpuscles unless the process has taken place extensively. The diagram on p. 105 gives in a tabulated form some idea of the appearances presented by the corpuscles of the different groups when mixed with the stock sera and observed in a hanging drop under a microscope. Agglutination must be distinguished from the formation of rouleaux, which may be seen in any of the mixtures.
For scientific purposes these very careful tests are necessary, but it seems to be clear that for clinical purposes a much rougher and quicker test is adequate. In the clinical determination of blood groups it is superfluous to carry the test to the point of watching for hæmolysis, for it is upon the presence of agglutinins in the serum and the corresponding iso-agglutinins in the corpuscles that the determination of the groups depends. Further, no error is introduced by neglecting the hæmolysis, since it has been shown that hæmolysis is invariably preceded by agglutination. It is the occurrence of agglutination therefore that is of prime clinical importance. If that is excluded, hæmolysis is necessarily excluded also, and the prolonging of the test is seen to be only of academic interest. In the methods described above the corpuscles were always tested in the form of a washed suspension. This precaution was taken on the supposition that the presence of any of the serum belonging to the corpuscles might interfere with the reaction. If, however, the amount of this serum be small relatively to the amount of the test serum, then no such interference takes place.
Fig. 7.—Tabulation of Serum Reactions as seen in Hanging Drops.
The ordinary clinical method of testing may therefore be greatly simplified, and the one commonly used at the present time is as follows: A single drop of each of the stock sera is placed on two glass slides, or, better, side by side upon a white glazed tile or plate, the numbers of the groups, II and III, being written above the respective drops. The lobe of the ear of the person to be tested is then washed with ether and pricked with a sterile surgical needle. A small quantity of the blood which exudes is taken up on the end of a blunt metal or glass rod, and is intimately mixed with the drop of serum under the number II. The end of the rod is then carefully wiped clean, and a similar small quantity of blood is mixed with the drop of serum marked III. The amount of blood to be used should not be so great as to make the drop of too deep a colour, which may interfere with observation of the reaction, but it should be enough to impart to it a very definite red tint. The slide or tile is then gently rocked, so that some slight movement is imparted to the drops, which are at the same time closely watched in a good light. The agglutinating reaction is readily seen with the naked eye, especially against the white background provided by the tile. If the serum be properly active, the agglutination of the corpuscles begins to be apparent as a definite granular appearance resembling brick dust within a minute of mixing. With a little practice this appearance is easily recognized, but it must be distinguished from the appearance produced by a mechanical gravitation of the corpuscles towards the centre of the drop. If agglutination is taking place, the granulation appears simultaneously throughout the drop, and not only in the centre. With an active serum the process may proceed rapidly, so that in less than five minutes the corpuscles have been aggregated into a few irregular masses; often it stops short of this, but the drop presents, nevertheless, a coarsely granular appearance which is quite unmistakable. If no granulation can be seen at the end of five minutes, it can be assumed that the test is negative for the serum of that group, and the group of the corpuscles may be deduced upon the principles already explained.
The test carried out in this way is admittedly not susceptible of the same finesse as if it were done with the assistance of the hanging drop, the incubator, and the microscope; nevertheless, my own experience in a large number of cases has shown that, clinically, this test may be relied upon, and the same view has been expressed by other writers on the subject. Very seldom is there any doubt as to the presence or absence of agglutination. When doubt exists, it is easy to repeat the test and obtain a confirmation of the result. It may perhaps be urged that this test is quite insufficient for eliminating the slighter degrees of incompatibility which have produced serious results when the transfusion has been repeated several times. But in the cases reported, the blood that was used had not shown any agglutination even when most carefully observed under the microscope. It seems, therefore, that the results were probably due to another factor, as already suggested (see [p. 57]), which the more elaborate test failed to eliminate. The efficiency of the rapid test is therefore not invalidated. It is, nevertheless, in the present state of knowledge, a wise precaution to perform the direct test between patient and donor in addition to the group test when circumstances permit. It is essential when the patient is suffering from any form of blood disease. It is unnecessary when the transfusion is to be performed as a life-saving operation in hæmorrhage or shock.
CHAPTER VII
THE METHODS OF BLOOD TRANSFUSION
Some reference has already been made in the first chapter to the rapid development in recent years of the technique of performing a blood transfusion. The earlier operators, owing to the difficulties introduced by the coagulation of blood outside the body, were constrained to make use of some method of direct transfusion, the blood flowing directly from an artery of the donor into the patient’s veins. This has now been largely replaced by one of the methods of indirect transfusion, the blood being withdrawn from the donor into a vessel in which clotting is delayed or prevented, and then injected or allowed to run into the patient’s circulation.
Direct Transfusion.—The obvious method of performing a direct transfusion is by making an end-to-end anastomosis between an artery of the donor and a vein of the recipient. The most readily accessible artery is the radial at the wrist, and this is indeed almost the only artery that is available. The most accessible vein is the median basilic or the median cephalic at the elbow. The operation of end-to-end anastomosis, using an artery of so small a calibre as the radial artery at the wrist is usually found to be, is one of great technical difficulty; this effectually prevented transfusion from being used at all frequently. A modification has been used by Sauerbruch and others, in which the end of the radial artery is drawn into the lumen of the vein through a slit in its wall. A suture is passed through the radial artery close to its cut end, and the needle is then passed through the slit in the vein and out again through the wall of the vein an inch or so higher up. Traction on the suture then pulls the artery into the vein. The artery has meanwhile been temporarily occluded by a clip, which is removed when the artery is inside the vein, so that the blood can then flow from one to the other. This is easier to do than the anastomosis, but, in addition to the other objections to direct transfusion to be mentioned presently, the difficulty occurs of occlusion of the artery by the physiological process of inversion of its coats at the cut end. This is likely to happen before much blood has passed, so that apparent success at first is often not maintained. Sauerbruch claimed that the amount of blood that had passed could be estimated by measuring the time taken for 1 cc. of blood to flow from the artery before it was introduced into the vein; but there is no proof that the rate of flow remains constant.
If direct transfusion be desired, there can be no doubt that Crile’s method, introduced some fifteen years ago, is the best to employ. After much patient work Crile perfected a method of anastomosis which ensures that no occlusion of the vessels can take place at the site of junction. This depends on the use of a short silver tube, through which the end of the artery is threaded. The artery is then pulled back again outside the tube in the form of a cuff and fixed in position. The end of the artery has thus been made rigid, and over this the vein is pulled in its turn and fixed by a ligature. A watertight junction is thus made, and blood can flow through it without interruption—unless clotting takes place in the vessels as the result of handling and injury to their walls. This method has been extensively used in America, and it was the first to render the operation of transfusion a comparatively popular one.
Various other devices for achieving the same result have been elaborated by other workers, and attention may be drawn to those of Elsberg and Bernheim, both of which are described in the book by the latter on “Blood Transfusion.” During the war a simpler method was introduced by Colonel Andrew Fullerton, who, working at a Base Hospital in France, found that he could get good results by employing a thin rubber tube with a small silver cannula at either end. The apparatus was first coated on the inside with a thin layer of paraffin wax, in order to discourage clotting within the tube, and the cannulæ were introduced into the donor’s artery and the recipient’s vein respectively. The blood could then flow freely from one to the other. The fact that blood was being transmitted was taken to be proved by the visible pulsation of the thin rubber connecting-tube synchronously with the arterial pulsations. The disappearance of this was assumed to be evidence that clotting had occurred. This method was described by Colonel Fullerton to the surgeons working at the Casualty Clearing Stations, where blood transfusion was likely to be of most service, but it was never used extensively. The coating of the inside of the tube with paraffin is in itself an operation of some difficulty. Under conditions in which any loss of time could not be permitted, success by this method was not attained with sufficient certainty, and it was shortly afterwards replaced by the more satisfactory methods described below. The most recent work on direct transfusion has been done by J. M. Graham at Edinburgh, who has however reached the conclusion that the technique is always more difficult than that of indirect transfusion.
It can easily be seen, therefore, that all the known methods of direct blood transfusion present great technical difficulty, which renders the method unsuitable for general use. There are, in addition, certain other objections to it of an obvious nature. It is, in the first place, impossible to measure the amount of blood which has passed from the donor to the recipient. Sometimes an indication may be obtained from the evident improvement in the condition of the patient, accompanied by the signs of loss of blood in the donor. More often clotting takes place, unknown to the operator, at some point, with the result that blood ceases to pass a considerable time before the end of the operation, and the patient has consequently received very much less blood than is supposed. It has been claimed by Libman and Ottenberg that the amount of blood transferred may be estimated by weighing the donor before and after the operation. This presupposes that a very accurate weighing machine is easily available, which usually is not the case.
A second objection is the extent of the injury which is necessarily inflicted on the donor. His radial artery must be exposed through an incision of considerable length, and must be ligatured at the conclusion of the process. The operation becomes, therefore, a matter of some moment to the donor, who will be permanently scarred, and can under no circumstances be used for transfusion more than twice.
A third objection is that the transfusion cannot be done with due regard to the condition of the patient. A delicate and difficult operation has to be performed with the donor and recipient lying side by side, their arms close together. It is therefore almost imperative that both should be on operating-tables of a convenient height. Often, however, with an exsanguinated patient it is very important that he should not be moved from his bed, but as a bedside operation direct transfusion becomes difficult indeed!
A final objection is that in some people the radial artery is of very small calibre, so that when all preparations have been made, and the artery exposed, it is found to be quite impossible to proceed. Another element of uncertainty is thus introduced.
There is, therefore, little to be said in favour of direct transfusion, and much to be urged against it. This method has, indeed, in my own opinion, come to be of historical interest only. For this reason the different methods have only been very briefly described. For more detailed information, reference must be made to the various original communications, which will be found in the Bibliography.
Indirect Transfusion.—The methods of indirect transfusion may be divided into those which depend upon the use of an anticoagulant mixed with the blood and those in which the blood is given unaltered. The technique of either process is simple compared with that of direct transfusion, though any method which makes use of whole blood can never be quite as free from uncertainty or difficulty as one which introduces the use of an anticoagulant. If the blood is prevented from clotting, the chief cause of failure in performing blood transfusions is removed. With any whole-blood method of transfusion speed is exceedingly important, frequent practice is a very great advantage, and it is essential, as with direct transfusion, that the donor and recipient should be in close proximity to one another, if not actually side by side.
On the other hand, the use of an anticoagulant renders speed and frequent practice of less account. The blood can be drawn, and can then be put on one side until the best moment for giving it has arrived. Due regard may be had to the patient’s condition, since the blood can be carried about and can be given at leisure to the patient in his bed without disturbing him and almost without his knowing it. The donor, too, is not exposed to the mental shock of lying for some time side by side with a patient who may be in extremis, or may even expire during the operation.
There are, however, those who consider that the use of whole blood, instead of blood which has been chemically treated, has advantages which outweigh the possible disadvantages mentioned above. Two methods of using whole blood are, therefore, described first; the use of anticoagulants is then described in detail, and their advantages and possible dangers are enlarged upon.
Whole Blood Transfusion with Syringes.—It is obvious that, if blood can be drawn from the donor’s vein into a glass syringe and injected into the recipient so rapidly that clotting has no time to occur, then a transfusion of any quantity of blood that may be wished can be given by this simple means. The measure of the amount of blood transfused is given by the number of syringes that have been filled and emptied. This method has been successfully used by several workers, and it has the advantage that no very special apparatus is necessary. It does, however, require that several syringes, and more than one assistant, should be available, since clotting will take place in the syringes, unless they be frequently washed out. There is also the possibility that clotting may take place in the needle which is introduced into the donor’s vein, since this cannot be withdrawn and replaced for each syringeful of blood that is transferred. With practice, however, and with good assistants, the process can be done quickly enough to avoid this. Wide-bore needles with short rubber connexions are introduced into the veins of donor and recipient; if, as often happens, this is difficult to do through the skin in the case of the recipient, his vein must first be exposed through an incision and a glass or metal cannula introduced into it. The operator then fills the syringes with blood in quick succession and hands them to his first assistant, who injects the blood into the recipient. Blood is prevented from escaping from the needles when the syringes are disconnected by nipping the rubber connexions with the fingers. The first assistant passes the empty syringes to the second assistant, who washes them out with normal saline, and hands them back if needed to the operator. This can be done with six 20 cc. syringes used in rotation, possibly with only four.
The most recent description of this method has been published by J. M. Graham of Edinburgh, who has introduced an improved form of needle. This consists of a double tube; the inner tube has a needle point which is used for puncturing the vein, and can be withdrawn into the blunt outer tube when the vein has been entered. Any further wounding of the vein is thus avoided. In addition, movement of the needle-cannula is prevented by a bull-dog forceps attachment, which is clipped to the skin. Graham finds it advisable to lubricate the cannulæ and syringes with vaseline before being used. He also states that: “As the absence of clotting depends upon the rapidity with which the syringes are filled and emptied, a series of syringes should be used in strict rotation, and all trace of blood must be washed out with saline before the syringes are used again. One or two additional assistants are necessary for this method.” The disadvantages are evident, and it is not suitable for general use.
A modification of the method has been described by Unger, in which only one syringe is used. The barrel of this is cooled by an ether spray so that clotting is discouraged or prevented.
Whole Blood Transfusion with Kimpton’s Tube.—The principle of this method depends upon the use of paraffin wax as a coating for the vessel into which the blood is drawn, so that clotting is prevented or greatly delayed. The form of the vessel has been modified by different workers, but the essentials are the same in each. One form of the apparatus, known as the Kimpton-Brown tube, is illustrated in the accompanying diagram. It consists of a graduated glass cylinder, of about 700 cc. capacity, the lower end of which is drawn out into a cannula point at an acute angle with the body of the cylinder; the point is of a size convenient for introducing into a vein and its bore large enough to allow of a free flow of blood through it. Near the upper end is a side tube to which a rubber tube can be attached, and an opening at the top is closed by a rubber bung. An ordinary rubber double-bulb bellows is the only other apparatus that is needed.
Fig. 8.—Kimpton-Brown Tube
The glass vessel is first sterilized in the autoclave, and then it must be coated on the inside with a thin layer of paraffin wax. The whole success of this method depends upon this wax coating being absolutely complete right up to the tip of the cannula at the bottom. If the tiniest area of glass be left exposed in the cannula, the process will fail. The production of this perfect wax coating used to be exceedingly difficult of attainment without very frequent practice. The apparatus was first raised to exactly the right temperature; sterile, melted paraffin was then put into it, and distributed evenly over the surface, excess being allowed to run out. The apparatus was then cooled down, and could be put away in a sterile towel ready for use, great care being taken that the lumen of the cannula was patent and not blocked with excess of wax. A simplification of the process was introduced by the use of a saturated solution of wax in ether. This solution is put into the vessel, which must not be heated, and is made to run all over the surface, excess as before being allowed to escape through the lower opening. The ether quickly evaporates, leaving a very thin and perfect film of wax over the surface of the glass. As before, it must be ascertained that the lumen of the cannula is patent. The apparatus is then ready for use.
The donor and recipient need not be lying close together, but they must be in the same room. A vein is exposed in the arm of each by dissection under a local anæsthetic. The operator then picks up the vein with a pair of dissecting forceps, and makes an oblique cut into the lumen as in the diagram on p. 131. A flap is thus made which is held in the dissecting forceps in the left hand or is picked up with a fine-pointed pair of artery forceps. The Kimpton’s tube is taken in the right hand, and the point of the cannula is introduced into the vein; that part of the lumen lying opposite the flap serves as a gutter which guides the cannula directly into the lumen, so that it is introduced without any fumbling or delay. The cannula is pushed on so that its widest part engages the whole circumference of the vein, forming a joint through which blood does not leak. The cannula having been pushed well up into the vein, the forceps holding the venous flap may be let go. At the same time an assistant grips the donor’s upper arm, or some form of tourniquet of the necessary degree of tightness is applied, so that the veins become congested without obliteration of the arterial pulse. Blood now flows rapidly into the tube, and the venous pressure is always sufficient to overcome the counter-pressure of the increasing head of fluid in the tube. It is unnecessary, therefore, to produce any negative pressure within the tube with a reversed Higginson’s syringe or an exhaustion pump, which has been used by some workers. Blood is allowed to flow into the tube until the requisite amount has been obtained. The venous congestion is then released, and at the same time the tube and cannula, held at the lower end with the right hand in such manner that the index finger is free, is withdrawn from the vein. At the moment of withdrawal the end of the cannula is closed with the right index finger. To prevent hæmorrhage from the donor’s vein, a ligature previously put round it is tied by an assistant, or pressure on it is maintained with a sterile swab. The operator must now, without a moment’s delay, carry the tube filled with blood over to the recipient. An opening in his vein is made by an assistant in the same manner as already described, the finger is removed from the cannula, and its point is instantly introduced into the vein. It is now necessary to produce some degree of positive pressure in the tube to ensure that the blood shall at once begin to flow steadily into the vein. This is done with a rubber bellows, attached by an assistant to the upper side tube, and the level of the blood in the tube should at once begin to fall. Great care must be taken that the positive pressure is released before the tube is completely emptied of blood in order to avoid the obvious danger of the entry of air into the patient’s vein. When the tube is nearly empty it is withdrawn, the vein is ligatured, and the wounds in donor and recipient are sutured. The most convenient pattern of Kimpton-Brown tube holds only about 500 cc. of blood, so that if more is needed, the process must be repeated.
There is virtually only one cause of failure in transfusion by this method, and that is the occurrence of clotting in the cannula or at the bottom of the tube. If it does occur at any stage of the operation, it cannot be remedied. It may happen when the tube is nearly full; if so, the blood that has been withdrawn cannot be used. Clotting may be due to an imperfection in the paraffin coating on the glass, but if there is any delay from any cause, it may take place independently of this. The method is therefore never absolutely certain of success even in the hands of an expert, and for general use it is certainly unsuitable. It was introduced into the British Army by some of the American surgeons in 1917, and was used by the writer under the guidance of Major Alton of the Harvard Medical Unit during the first battle of Cambrai with good results. Many of the English surgeons, however, soon abandoned it as a routine method in favour of anticoagulants. There are other objections to it besides its uncertainty. A vein must be exposed by dissection in both donor and recipient, so that avoidable injury is inflicted on the former. It is not a perfectly clean method, some blood necessarily escaping at each successive stage in the process, though an expert can reduce this to a minimum. In the hands of a novice it may occasion a very bloody scene. The whole operation is one of urgency, and the best interests of donor and recipient cannot always be considered.
Modifications have been introduced, such as that of Vincent, who uses an attachment with a needle instead of the glass cannula point. This obviates some of the objections, but introduces other difficulties, such as the necessity for coating the inside of the needle with paraffin wax. The technique can certainly be acquired, and the method has rendered excellent service in the past, but it has no obvious advantages except the uncertain one of avoiding chemical treatment of the blood.
Transfusion with Anticoagulants.—It will have become evident from the descriptions of the transfusion of whole blood already given, how great a difficulty is introduced into the technique of these methods by the physiological process of clotting in blood outside the body. It is clear how much the process of transfusion would be simplified if the clotting were to be prevented. Something has already been said in the historical sketch of the various means by which this problem was attacked, and it need only be stated here that the most suitable substance for this purpose has been found to be sodium citrate. This method was introduced by Lewisohn as recently as 1915, and it soon became the method of choice among most of those who tried it.
The process of the formation of a blood clot has always been one of the great problems of physiology, and numerous theories have been propounded to explain it. The theory accepted at the present time regards the process as a complicated one depending on the presence in the blood of a number of different factors. This theoretical explanation may be represented diagrammatically as follows:
Plasma Tissues and platelets
Prothrombin Ca salts Thrombokinase
Fibrinogen Thrombin
Fibrin
The clot consists of fibrin in which blood corpuscles are entangled. It is clear that if any one of the reacting agents can be removed or rendered inert the clotting cannot take place. There is only one inorganic substance taking part in the reaction, and it is this factor that is more easily removed than any of the others. Calcium is precipitated in an insoluble form by various chemical reagents, but it is obvious that for purposes of transfusion the formation of an insoluble precipitate is not permissible. It is therefore necessary to use a substance which will form a soluble compound with the calcium and which is at the same time harmless when introduced into the circulation. The only substance which has been found at present to possess both these properties is citrate of sodium. This forms with calcium a soluble double salt, in which calcium is rendered inert. It is usually held that the calcium to be active must be present in the ionized form, but recent investigations by Vines into the rôle of calcium tend to modify slightly the accepted view of its action. He has shown that calcium is present in the blood in two forms, ionized and combined, and that both take part in the coagulation reaction. He has, in addition, demonstrated that a quantity of anticoagulant sufficient to combine with the whole of the calcium present in a given quantity of blood is not enough to prevent coagulation. It seems, therefore, that the anticoagulant acts by combining with a large organic molecule of which calcium is only one constituent, and not merely by combining with ionized calcium. The organic complex with which the calcium is associated possibly corresponds to the thrombokinase of the theory.
About the time that the use of the citrated blood was introduced by Lewisohn, some investigations upon animals were carried out by Salant and Wise in order to determine how sodium citrate was dealt with and eliminated by the body. These observers found that it very quickly disappeared from the circulation, nearly 90 per cent. of the salt having been got rid of within ten minutes of its intravenous injection. Part of the citrate is destroyed by oxidation, and the rest, 30 to 40 per cent., is eliminated by the kidneys, the urine being rendered alkaline. It was also shown that if a very large dose was given, so large that toxic symptoms resulted, the effect was rapidly obtained; but that if the toxic dose were not fatal, no remote effects followed. Its injection never resulted in any albuminuria.
Lewisohn showed by experiment on the human subject that up to 5 grammes of sodium citrate in the form of a 0·2 per cent. solution could be injected intravenously without any harmful results. It was also shown that this concentration of the salt was sufficient to prevent clotting outside the body, and that the microscopic appearance of the blood cells was not altered by the admixture of this solution.
Theoretically, therefore, the amount of citrate that should be used as an anticoagulant should be 2 grammes for 1,000 cc. of blood, or 100 cc. of 2 per cent. solution for 900 cc. of blood. In practice it is better to err on the side of safety and to use a slight excess of citrate. This amount of citrate should be used for the 750 cc. of blood which constitutes the ordinary maximum amount of blood used in a transfusion. For smaller quantities of blood the amount of citrate may be correspondingly reduced.
The use of citrated blood was introduced to the British Army in France in 1917 by Oswald Robertson, who recommended the use of a larger amount of citrate than this. His object in increasing the amount was to produce a solution which, when diluted with the correct amount of blood, would be isotonic with it. It was thought that a hypotonic solution might result in some damage to the red corpuscles by osmosis, and Robertson therefore recommended the use of 160 cc. of a 3·8 per cent. solution of citrate, which, when mixed with 750 cc. of blood, will give a solution of which the osmotic pressure equals that of 0·9 per cent. saline solution. It may be doubted, however, whether this consideration is of more than theoretical importance. There can be little doubt that in practice the effect of a slightly hypotonic solution, such as is given by the 100 cc. of 2 per cent. solution of citrate, is negligible as regards destruction of corpuscles. If, however, it be thought necessary, an isotonic solution may be produced by the addition of sodium chloride. Other considerations, as will be seen shortly, weigh in favour of giving the smaller amount of citrate. The dosage to be recommended, therefore, on practical and experimental grounds is 2 grammes of citrate in 100 cc. of water for 900 cc. of blood, or 1 gramme of citrate in 50 cc. of water for 450 cc. of blood or less. These proportions need not be observed very accurately. Latitude may be used in either direction without harming either the transfused blood or the patient.
It has been stated above that sodium citrate introduced into the circulation in small quantities, such as are sufficient for anticoagulant purposes, is non-toxic to man. In the light, however, of the extended experience of the last four years, it is seen to be possible that this statement may not be quite literally true. Probably there is an individual variation in the tolerance of different people to sodium citrate. Certainly in some cases a reaction follows the injection of citrated blood. The symptoms of this reaction are a slight headache, a rise in temperature to two or three degrees above normal, sometimes accompanied by a rigor or a sensation of chill, and an increase in the pulse rate. The effect is, however, always very transitory, lasting only two or three hours, and is never, in my own experience, attended by any symptoms which need give rise to anxiety for the patient’s welfare; nor does it in any way prejudice the therapeutic results of the transfusion.
That the reaction is caused by the citrate and not by another constituent of the transfused blood has been believed by several observers. In a case seen by the writer a slight citrate reaction occurred in a youth who acted as blood donor. The transfusion was carried out by a modification of the syringe method, which involved the injection at intervals of a syringeful of citrate solution into the donor’s circulation. The possibility that the reaction was produced by another factor was therefore not present in this instance.
Nevertheless, it must be admitted that citrate has not yet been absolutely proved to be the cause of this slight reaction in all the cases in which it occurs. Evidence has, indeed, been brought forward by Lewisohn and by Meleney to show that citrate is definitely not responsible for the reaction. The statement is made that some reaction occurs after 10 per cent. of all transfusions, and that this percentage is unaffected whether whole blood or citrated blood is used. Lewisohn has himself investigated the effects in a long series of parallel cases in which different methods were employed, and he reports that the results following the use of citrated blood were as good as with any other method. Drinker states that reactions follow the use of citrated blood slightly more often than they do that of whole blood, but this has not been confirmed. He was unable to find any impurity in the citrate that might be held responsible. It is quite possible that all the reactions observed are in reality caused by the “minor agglutinins” mentioned on p. 73. Meleney has noticed that the blood of some donors is more likely to produce a reaction than that of others; this suggests that the responsibility rests with the blood and not with the citrate. The occurrence of a toxic reaction constitutes the only real objection to the use of citrated blood that has yet been brought forward, but even this has not yet been fully substantiated; in any case, the reaction is of so little importance that it is greatly outweighed by the numerous advantages that are conferred by the use of citrate. The possibility that a citrate reaction does sometimes occur may be taken as an indication in favour of using the smaller amount recommended by Lewisohn rather than the larger dose used by Robertson. The experience of a great many observers has established the fact that citrated blood is quite as effective as whole blood in its therapeutic effects.
It is convenient to have the sodium citrate in a form ready for immediate use. I have therefore been in the habit of keeping it in the solid form in small stoppered bottles, each containing 1 gramme of the salt. These are sterilized at 130° C., and can be kept indefinitely until wanted. If 450 cc. of blood or less are to be drawn, the contents of one bottle is shaken into the transfusion flask; 50 cc. (approximately 2 oz.) of sterile warm water are added, in which the citrate will rapidly dissolve. If more than 450 cc. of blood is to be used, the contents of two bottles must be dissolved in 100 cc. or 4 ozs. of water. Alternatively a concentrated solution of citrate may be kept in sealed ampoules, but the salt is less stable in solution, and I prefer to keep it in the solid form.
The ideal method of blood transfusion seems to me to require that it shall be absolutely certain of success, that the blood shall not necessarily be injected into the patient immediately it has been drawn, so that other circumstances besides the demands of the transfusion operation can be considered, and that no injury shall be done to the donor beyond the puncturing of a vein. In addition to this, the method should be so simple and free from special apparatus that it can be easily learnt and carried out by one operator without skilled assistance. All these requirements are fulfilled by the citrate method, and a satisfactory method of performing this will next be described. As will be seen, the blood can be drawn with the minimum amount of injury to the donor; when drawn, it can be put on one side, for several hours if necessary, and then given to the patient at whatever may be judged to be the most favourable moment; the whole process can be carried out by a single operator without any assistance; and finally, but little practice is needed to make success certain every time.
The transfusion apparatus known as “Robertson’s bottle,” first described by Oswald Robertson in 1918, is the basis of most citrate methods. This could be easily improvised in a field laboratory, and was extensively used during the last year of the war. The apparatus consisted of a glass bottle of about a litre capacity, the mouth of which was closed by a rubber bung. Through the bung three glass tubes passed. One, connected by a short rubber tube with a wide-bore needle, ended about an inch from the bottom of the bottle; through this the blood flowed into the bottle. A second tube, which reached to the angle between the side and the bottom of the bottle, was connected by a rubber tube with a cannula; through this the blood was injected into the patient. The third tube reached only just beyond the bung, and to this was attached a Higginson’s syringe, by means of which either negative or positive pressure would be produced inside the bottle, according to which end of the syringe was attached.
It is unnecessary to describe this apparatus any further, for it was found by myself and others that it could be with advantage modified in the direction of simplicity. It is in the first place unnecessary in drawing the blood to create any negative pressure if a needle of a large enough bore (2 or 3 mm.) be used, and, further, it is an advantage not to have the needle attached in any way to the bottle, which, as the blood flows into it, has to be freely agitated in order to mix the blood quickly with the citrate. The needle may, therefore, be attached to a rubber tube of suitable length which hangs freely into the collecting vessel as shown in the diagram on p. 127. The third tube of “Robertson’s bottle” may be dispensed with by using a conical flask provided with a side tube to which a rubber bellows can be attached. The delivery tube is therefore the only one that need pass through the rubber bung. This tube should have an angle in it inside the flask so that its lower end reaches into the corner, and the extremity should be ground down obliquely so that, although it reaches right into the corner, it does not become occluded by too accurate contact with the surface of the vessel. By this means any wastage of blood is prevented. I have found it a very great convenience to introduce into the delivery tube just outside the flask an air-lock,[8] the value of which will be seen shortly. To the barrel of this air-lock a rubber tube with a cannula is attached. Close to the cannula is some form of clip. The whole apparatus is illustrated in the figure on p. 133, and with the help of this its use may be readily understood.
Fig. 9.—Transfusion Needle
(Actual Size)
The particular form of needle which I have been in the habit of using is shown in the figure. Its lumen has a diameter of 2 mm., and the steel tube ends off flush with the wide shoulder to which the rubber tube is attached. This avoids any recess within the needle in which clotting may begin. The point of the needle should not be too long, in order that it may not wound the opposite side of the vein when it has been introduced. For ease of introduction, however, the extremity should be very sharp and should have cutting edges. The point and edges should be touched up on a bevelled hone each time before the needle is used. The needle should be kept ready for immediate use in liquid paraffin. I have found that the most convenient way of keeping it is to put it into a test-tube containing paraffin, which is plugged with cotton-wool and sterilized at 130° C. in the hot air oven or by careful heating over a flame. In this way the needle may be kept ready for an indefinite time without any chance of its rusting. When it is taken out of the test-tube, a sterile rubber tube is slipped on to it and it is then ready for use. As an additional precaution, a small quantity of paraffin may be drawn up into the rubber tube, which is thus lubricated on the inside, but this is not absolutely necessary. The tube must be sterilized with the rest of the apparatus, as rubber is destroyed by liquid paraffin.
Fig. 10.—Drawing Blood for Transfusion
When the donor’s arm has been congested by gripping it above the elbow, or better by the application of a tourniquet[9] drawn to the requisite degree of tightness, a suitable vein, usually the median basilic, is chosen. The area of puncture is washed with ether and a very small quantity, 2 to 3 minims, of 2 per cent. novocain is introduced over the vein with a hypodermic syringe. If a larger quantity is used, the vein may become obscured, but this small amount may be dispersed by a few moments’ pressure with the finger, and is usually enough to anæsthetize the very small area of skin that is to be operated upon. A tiny cut in the skin is then made with the point of a scalpel, and the needle is pushed through into the vein. If the donor’s vein is a large one, such as is usually found in the type of donor recommended in a previous chapter, this is quite easy to do. To make it equally easy if the vein be smaller, it has been suggested by Watson that the vein may be fixed by pushing an ordinary fine sewing-needle through the skin at right angles to the line of the vein, into the vein, and out again through the skin. This needle is held with the forefinger and thumb of the left hand, while the right hand pushes the transfusion needle into the lumen of the vein just below it. When the needle is in the vein, the blood flows out rapidly through the tube which hangs into the flask containing the citrate, as illustrated. This flask is held by an assistant, who mixes the blood with the citrate by gently swinging it. If a properly adjusted tourniquet is kept on the donor’s arm while he works his forearm muscles by clasping and unclasping his hand, a flow of blood is obtained which is fast enough to prevent clotting in the needle, and indeed is quite as fast as most donors can tolerate. Blood up to 1,000 cc. may be collected in this way in ten to twenty minutes. If the vein be of a good size, it makes no difference whether the needle be inserted towards the heart or away from it. When enough blood has been collected, the tourniquet is removed, the needle is withdrawn, and pressure is maintained with a sterile swab over the site of puncture for a few minutes. No further bleeding will take place after this, and no suture is needed. The donor’s part in the operation is then finished. He should be made to lie on his back for a few hours afterwards, and given plenty of fluids, but beyond this no special precautions are necessary.
When the blood has been drawn, and has been satisfactorily mixed with the citrate, the flask may be put on one side until it is wanted, its mouth having been closed with a cotton-wool stopper. If the blood is wanted at once, the flask may be stood in a basin of warm water to keep it at body temperature. Otherwise it may be allowed to cool, and can be warmed up again when it is to be administered. The citrated blood may be kept for a considerable time without undergoing any appreciable change in its therapeutic value. It has been given twelve hours or more after being taken with the same good effects as if it had been newly drawn. During the war advantage was taken of this fact to anticipate during quiet times the necessity for many transfusions during times of stress. The blood was drawn in some quantity and kept for several hours in an ice chest, so that it was readily available during the expected battle. Recently I have administered to a woman who had been operated upon for a ruptured ectopic gestation 600 cc. of citrated blood which had been kept for twenty-seven hours at room temperature after it was drawn. The effect was in every way as satisfactory as if it had been freshly drawn, and there was no sign of any toxic reaction. So far as I know, blood had not ever been kept so long as this before being used, but there does not seem to be any objection to so doing.
When the blood is to be given, the delivery tube with the rubber bung is inserted in the flask, and the corpuscles which have gravitated to the bottom are distributed again through the fluid by gently shaking it. In administering the blood, it is very often advisable to inject it through a cannula which is tied into a vein. If the patient is a woman, it will usually be found that the veins are small and buried in fat. Also many transfusions will be given to combat the collapse due to shock and hæmorrhage, in which case the veins will be empty and the use of a cannula will be found essential. Sometimes, however, the patient will have large veins which can be readily distended; this may sometimes be encouraged by keeping the arm for half an hour beforehand in a bath of hot water. Under these circumstances the blood can be given through a needle introduced in exactly the same way as has already been described in the case of the donor. In the following account of the process it will be assumed that the use of a cannula is necessary.
When choosing a vein in the patient, the operator must be guided by circumstances. Usually the median basilic will be the most convenient, and if, in a collapsed patient, this is invisible, previous knowledge of the position of the vein must determine the site of the incision. If another operation is being done simultaneously upon the upper part of the patient’s body, it may be more convenient to use the internal saphenous vein in Scarpa’s triangle, or even one of the superficial veins about the ankle. In administering blood to an infant, several methods have been used. These are described separately at the end of the present chapter.
Fig. 11.—Transfusion Cannula
(Actual Size)
Whatever vein be chosen, the line of the incision is first infiltrated with a small quantity of a 2 per cent. solution of novocain. The vein is then dissected out, and is ligatured near the lower end of the incision. A ligature is also put loosely round the upper part. The operator now takes the barrel of the air-lock, which, together with the attached rubber tube and cannula, is filled with 0·9 per cent. saline solution, all air bubbles being carefully excluded. The tube is clipped near the cannula, so that the whole system, including the cannula, remains filled with the fluid. The form of the cannula used will depend upon the operator’s particular preference, but a type which I have found very convenient is shown in the accompanying figure. It is made of glass, and its extremity is ground down at an angle, which makes it very easy to introduce into the vein. The slight constriction near this end ensures that it can be securely tied into the vein and that no leakage round it shall occur. This is very necessary, because there is sometimes a considerable pressure to be overcome, due to venospasm in a collapsed patient, before the blood begins to flow.
An oblique cut is now made in the vein, as shown in the illustration, the cannula is introduced, and the upper ligature is tied.
Fig. 12.—Insertion of the Cannula in a Vein
The barrel of the air-lock, with its contained saline solution, is then fixed firmly on to the rubber bung, so that the nozzle of the delivery tube projects into the saline solution. Meanwhile, an assistant has fixed a rubber bellows on to the side tube of the flask; a short piece of glass tubing loosely packed with cotton-wool should be interposed between the bellows and the flask to prevent any particles of dust being blown over into the flask from the bellows, which is not sterilized. The clip near the cannula is released, and some positive pressure is produced inside the flask by means of the bellows. The citrated blood then rises in the delivery tube, and a corresponding quantity of saline solution is displaced from the air-lock into the patient’s circulation. The blood then flows from the nozzle of the delivery tube into the air-lock, and the remainder of the saline solution is driven on into the patient. Finally the blood flows steadily through the cannula, and the rate at which it is flowing can be observed in the air-lock.
The presence of this air-lock facilitates, as has been seen, the introduction of the cannula, into the vein, since there is no leakage of blood to obscure the operation. In addition, the operator can see at a glance whether the blood is flowing in properly, and can regulate the rate of flow to a nicety by varying the pressure in the flask by means of the bellows. If a very slow injection is required, the blood can even be made to run drop by drop. If the patient is suffering from acute anæmia, the blood can be pumped in rapidly, 750 cc. of blood being given in the course of twenty minutes. If, on the other hand, the patient has a plethora of fluids, such as is seen in some cases of secondary anæmia, the blood must be given very much more slowly than this, since it is dangerous rapidly to increase the blood volume. A half to three-quarters of an hour must be occupied in giving 500 cc., and even then the patient may complain of a sensation of tightness in the chest and of dyspnœa, due to embarrassment of the right heart during the transfusion. This complaint, however, is usually transient, and will disappear quickly if the injection be stopped for a few minutes.
It has been said that the lower end of the delivery tube reaches into the angle between the side and the bottom of the flask. When therefore the flask is nearly empty, it should be tilted so that very nearly the whole of the blood can be forced up the tube. When the flask is quite empty, the blood in the barrel of the air-lock must be carefully watched, and when its level has fallen to the bottom of this, the clip must be applied to the tube above the cannula. By this means no blood is wasted except the small quantity which remains in the tube below the air-lock. As soon as the tube has been clipped the cannula is withdrawn, the vein is ligatured above the opening into its lumen, and the edges of the skin incision are sutured.
Transfusions carried out in this way can be performed with uniform success. The technique is simple and straightforward at every stage, and can be easily demonstrated and learnt. It is, in addition, a perfectly clean process, and not a single drop of blood need be spilt. Any method which involves the injection of blood under pressure is open to the objection that it is possible to overlook the fact that the flask has been emptied and to kill the patient by injecting air into his veins. This can, however, only happen as the result of great carelessness on the part of the operator. The presence of the air-lock affords an additional safeguard, as it can hardly escape the operator’s notice that blood has ceased to flow from the nozzle of the delivery tube.
Fig. 13.—Injection of the Blood, showing use of Air-lock
The method may also be criticized on the ground that some damage is caused to the corpuscles of the donor’s blood by the shaking which is necessary to mix it with the citrate solution. This objection is, in my opinion, theoretical rather than practical. If, however, it be desired to avoid any such shaking, the apparatus designed by A. E. Stansfeld and described by him in 1918 may be used. This ensures that the citrate and the blood flow into the containing vessel together, so that no further mixing is needed. The apparatus is more cumbrous, more fragile, and less easy to clean and to sterilize than that described above. In the hands of an expert it will give excellent results, but its use requires some little practice, and it is therefore not so well adapted for general use.
The whole of my own apparatus, as described above, may be obtained from Messrs. Allen & Hanburys, Wigmore Street, London, W.1, who also provide a convenient box for carrying it.
Transfusion of Infants.—The technique of transfusions performed upon children over the age of about four years does not differ from that used for adults, except that less blood is to be given. The antecubital veins are much smaller and a finer cannula may have to be used, but this is the only source of trouble. The transfusion of infants and very young children may, however, be found to be much more difficult. The operation will have to be done for conditions such as melæna neonatorum, which was discussed on p. 48 of the present work, or for post-operative collapse, such as may follow an operation for congenital hypertrophic stenosis of the pylorus, for intussusception, or for some of the more extreme cases of harelip and cleft palate. In all such instances the transfusion will be a matter of some urgency. Speed and certainty will depend on previous knowledge of the best method to be employed.
In the case of melæna neonatorum treated by R. D. Laurie, which has been already referred to, a needle was introduced into one of the antecubital veins, and 20 cc. of citrated blood were injected with a syringe. This method, however, is not to be recommended, on account of its great difficulty.
The method used by Helmholtz and also by Howard depends on the introduction of a syringe needle into the superior longitudinal sinus through the anterior fontanelle. A needle two to three inches long attached to a 20 cc. syringe is inserted near the upper angle of the fontanelle at an angle of about 25° with the scalp. As the needle pierces the wall of the sinus, a sensation of resistance is experienced, similar to that given by the piercing of the dura mater in doing a lumbar puncture. Blood should then be allowed to enter the syringe in order to demonstrate that the point of the needle really is lying in the sinus. Abnormalities have occasionally been met with, in which the sinus was situated to one side of the middle line or was very much smaller than usual. The danger of injecting the blood in such a case into the brain or the subdural space need not be emphasized. Difficulty may also be caused by restlessness on the part of the child, and to overcome this Helmholtz has devised an apparatus which grips and fixes the child’s head at a suitable angle. All this, however, makes the process unnecessarily elaborate. As an alternative, Vincent has exposed one of the internal jugular veins into which he introduces a cannula. This again is a comparatively difficult operation, which may leave a permanent scar in a conspicuous place. Vincent had previously used the femoral vein, but he found this difficult to approach, and the wound was apt to become contaminated afterwards.
The method of choice is undoubtedly that used by Bruce Robertson, who has performed a much larger number of transfusions upon infants and children than any other worker in this field of surgery. He has found that the internal saphenous vein near the ankle is a vessel possessing a fairly wide lumen and thick walls even in infants, so that a needle or cannula can be introduced into it with comparative ease and rapidity. The vein must, of course, be freely exposed through an incision, but its situation removes any objection there might otherwise be to this operation. Robertson has usually employed the syringe-cannula method described earlier in the present chapter, but there is no objection to the use of an anticoagulant. The small amount of blood to be given, 15 cc. per pound of body weight, makes the use of the transfusion flask unnecessary. It is better to use a 20 cc. syringe, into which 2 cc. of a 10 per cent. solution of sodium citrate is drawn as a preliminary. The needle in the donor’s vein and the cannula in the infant’s saphena should each be provided with a rubber connexion, which can be clipped, or pinched by an assistant, when the syringe is not attached. The syringe containing the citrate is filled with blood and injected into the infant as often as may be necessary until the total amount decided upon has been given.
Robertson has used this method for complete replacement of the circulating blood in treating streptococcal septicæmia following erysipelas, and for shock in children due to burns. The infant’s blood is removed through the anterior fontanelle, while a fresh supply is injected into the saphenous vein. Complete replacement has not, so far as I know, ever been performed upon an adult, but the process is feasible, given a large enough assemblage of donors. In this way some vieillard might attempt the rejuvenation, which at present, as we are told, has only been obtained from the transplantation of “monkey glands” by Viennese professors.