Blood Transfusion

by Geoffrey Keynes

(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 of blood are withdrawn in a syringe from persons known to belong to Groups II and III; the bloods are put into a sterile test-tube and allowed to clot.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.