Lee’s Technique.—For the regular carrying out of this method one should keep on hand the sera of individuals belonging to groups 2 and 3 (Moss). To carry out the tests prepare a suspension of the donor’s red cells by dropping 2 or 3 drops of his blood into 1 cc. of citrated salt solution. Deposit a platinum loopful of standard serum 2 on a slide and emulsify in it a loopful of the donor’s red-cell suspension. A concave slide with two concavities is convenient, the serum-cell emulsion being made on the cover-glasses which are to be inverted over the vaseline ringed concavities. The agglutination can be observed with a high power magnifying glass or the ⅔-inch objective. Agglutination, when it occurs, is usually complete in five to fifteen minutes. Repeat test with serum 3. If both test sera agglutinate the donor’s cells he belongs to group one. If neither agglutinate, to group four.
Agglutination by group two serum but not by three puts the donor in group three. Agglutination by group three serum but not by group two shows a group two donor. It would seem safe to use the cells of any donor of group 4, as such cells are not agglutinated by the sera of any group. It is, however, advisable to try to obtain a donor whose blood belongs to the same group as the donee. When standard sera 2 and 3 are not on hand one may use the following emergency method of Lee:
“A small amount of blood is collected from a patient (1 cc. from the ear or finger is sufficient), and allowed to clot. The serum is then obtained. One drop of this serum is placed on a slide and mixed with a drop of suspension of blood of the donor taken into 1.5% citrate solution. (A few drops of blood are taken into approximately 10 times the amount of 1.5 citrate solution and shaken. It is very important that the blood be dropped directly into the citrate, and should not be partially coagulated.) The test will appear in a few moments, and is best examined under the microscope, where, in the event of a positive test, marked agglutination will be evident. The test will also be evident macroscopically. In the event of a negative test it is a wise precaution to raise the cover-glass, and after making sure that the serum and cells are well mixed, to examine the preparation again. The only possible source of confusion is the appearance of rouleaux of the red corpuscle, indicating a too thick emulsion. If the test is negative, transfusion may be regarded as entirely safe.”
In the absence of agglutination haemolysis never occurs. Only about one-fifth of agglutinating sera prove also haemolytic. Rarely a pernicious anaemia patient’s serum may agglutinate his own red cells. This auto-agglutination is regarded as an important test in acquired haemolytic jaundice.
Occult Blood
When the presence of blood in the faeces, gastric contents, urine or body fluids, is suspected but cannot be recognized by macroscopic or microscopic methods, it is necessary to resort to spectroscopic or chemical tests. These tests are, however, individually unsatisfactory. The spectroscopic method is not delicate, the haemin-crystal method does not give uniform results and the various color tests, although very sensitive, are given by many substances other than blood. Consequently, it may be said that, with the color tests, it is negative results that are significant, and with other than the color tests it is positive findings that are informative. Serological tests are the most satisfactory medicolegally.
Haemin Crystal Test (Teichmann).—Prepare a solution (stable) of 0.1 gm. each of KI, KBr, and KCl in 100 cc. acetic acid. Mix a few drops with some of the material on a slide, apply a cover-glass, and gently warm until bubbles begin to appear. Then cool slowly, and examine for the characteristic dark-brown crystals.
Haemochromogen Crystals (Donogány).—Mix one drop each of suspected fluid, pyridin, and 20% NaOH on slide, and let dry. If positive, radiating needles will form after several hours.
Spectroscopic Tests.—These depend upon the recognition of the characteristic absorption spectra of haemoglobin or its derivatives (Fig. 24). The degree of concentration influences their appearance, and one should start with a relatively concentrated solution, diluting cautiously until the bands are typical.