104 THE PRINCIPLES OF IMMUNOLOGY 



" Collection of the Blood. The blood is taken from the patient and the pros- 

 pective donors in a i-io mixing pipette, such as is used in counting leucocytes. 

 The pipette is rinsed beforehand with 10 per cent, sodium citrate in water; the 

 citrate solution is drawn up to the mark i ; the pipette is rapidly filled with blood 

 from a puncture of the ear or finger ; and without pause the mixture is expelled 

 into a small, narrow test-tube. There is thus obtained a citrated blood containing 

 slightly less than i per cent, of citrate. The pipettes which we have employed hold 

 only 0.25 c.c. of fluid. This much blood is easily obtained from a single puncture. 

 There is no objection to increasing the flow by pressure. Should it cease before 

 the pipette is full, the blood must be at once expelled into a test-tube, in order 

 that it may mix with the citrate and clotting be avoided. The mixture is then 

 taken up again, a new puncture made, and the pipette completely filled. After 

 each blood is obtained, the pipette is rinsed with citrate, then with distilled water, 

 then with fresh citrate, and it is ready for another blood. If several donors are 

 to be tested, two pipettefuls of citrated blood should be obtained from the patient. 

 It is best to take them from different puncture wounds, in order to avoid a pos- 

 sible clotting in the pipette. 



" Mixing. The mixing is done in pipettes with a capillary end the so-called 

 Wright pipettes obtained by drawing out glass tubing in the flame. (Fig. n.) 

 The citrated bloods are used as such, and two combinations are made of the 

 patient's blood with that of each prospective donor, a mixture containing nine 

 parts of the patient's blood to one of the donor's, and a mixture of equal parts 

 of the two. The proportions used need be only approximate. In case of 

 emergency the first of the mixtures will suffice, since by its use the most 

 dangerous possibility, namely, that the blood of the recipient might destroy that 

 of the donor, can be ruled out. Following the technic usual with Wright pipettes, 

 the capillary tube is marked, blood is drawn to the mark, and each column of 

 the blood is separated by an air bubble from the next that is drawn up. To 

 insure proper mingling, each mixture should be expelled on a slide, or Widal 

 plate, and then drawn high in the pipette, which may be sealed off in the flame 

 in case the examination is not to be made for some time. 



" Incubation. No incubation in the ordinary sense is necessary. The pipettes 

 are kept at room temperature, and readings are begun after two minutes if 

 there is need to hurry. Readings are for agglutination, and even within two 

 minutes this is plainly evident, except when the agglutinating forces are notably 

 weak. In the final choice of a donor it is safest to rely on results obtained after 

 the mixtures have stood for fifteen minutes. But the ruling out of individuals 

 with unfit blood may be begun practically at once. 



" Readings. The capillary end of each pipette is broken, a small drop of the 

 blood expressed on a slide, a large drop of normal salt solution superimposed 

 without mixing, a coverslip put on, and the preparation examined for agglutin- 

 ation under the microscope. Fresh preparations can be made at intervals if de- 

 sired. The salt solution is not absolutely necessary; but very clear pictures are 

 obtained as the blood spreads in it. When agglutination has occurred, the red 

 cells show a characteristic clumping, sometimes in small masses, often in large 

 ones that are very evident microscopically. 



" If there is no clumping in the preparations made after the mixtures have 

 stood fifteen minutes, the assumption is warranted that the bloods do not agglu- 

 tinate or hemolyze each other. But if clumping is present in the 9-1 mixture 

 and to a less degree or not at all in the i-i mixture, it is certain that the blood 

 of the patient agglutinates that of the donor, and may perhaps hemolyze it. 

 Transfusions in such cases are dangerous. Clumping in the i-i mixture with 

 little or none in the 9-1 indicates that the plasma of the prospective donor 

 agglutinates the cells of the prospective recipient. For practical purposes these 

 findings suffice. But if there is a desire to know whether both bloods contain 

 agglutinins, a 1-9 mixture should be made. If this and the 9-1 mixture show 

 large clumps, whereas the clumps are smaller when the bloods are mixed in equal 

 parts, two agglutinins must be present. Should there be only one agglutinin, 

 little clumping or none will be observed when the blood containing the agglutinin 

 is diluted with nine parts of the other blood." 



By the use of the technic indicated in the last paragraph, it is 

 possible to overcome error due to weak agglutinin content of the re- 

 cipient's blood. This we believe is of especial importance if the 

 patient has been ill for a long time. 



