Il8 , .AGGLUTINATION 



quantity comprised by each "part" or "unit" varies according to the 

 amount of blood or serum obtained from each donor and recipient. If 

 sufficient, it is best to work in drops (each drop being about 0.05 c.cm.), 

 thus mixing 3 drops of serum (0.15 c.cm.) and i drop of the red-cell emulsion 

 (0.05 c.cm.). If the serum is not sufficient, a smaller arbitrary unit may 

 be used in the form of Wright's pipettes (4 to 5 millimeters caliber) fitted 

 with rubber nipples, drawn out to a length of 2 to 3 inches, and marked off 

 about i inch from the tip with a blue pencil, this distance comprising the 

 unit. 



The tubes are placed in the incubator for three hours and then in 

 the ice box for 24 hours. Agglutination when it occurs does so rather 

 promptly, within 15 to 30 minutes after the mixtures have been made. 

 It is recognized macroscopically by the clumping of the red blood cells into 

 small floccules which later on appears like a distinct clot. A hanging drop 

 preparation of such a mixture shows the same phenomenon; usually this is 

 unnecessary as the macroscopical appearance is characteristic. If one is 

 in doubt, however, microscopical examination should be made. Hemol- 

 ysis, if pronounced, is observed in an hour or even less, but certainly after 

 the three hours' incubation; the finer grades of hemolysis are detected after 

 the tubes have remained in the ice box 12 to 24 hours. The control tubes 

 must show no agglutination or hemolysis. 



In cases where for any reason the taking of blood from the vein is not 

 allowed or impossible (as in infants), Wright's method of working with 

 small quantities (Epstein and Ottenberg) should be resorted to. 



The skin at the bed of the finger nail or of the lobe of the ear is pricked 

 deeply with a Hagedorn needle. For the red blood-cell suspension several 

 drops of blood are allowed to flow or taken up with a dropper and expelled 

 into a tube with sodium citrate solution. The red blood cells are washed 

 and diluted as before. 



For the serum, three or four Wright's capsules (see under opsonins) are 

 filled with blood which is allowed to clot, and centrifugalized. Each cap- 

 sule is nicked with a file, allowing the capsule to be broken open and the 

 serum pipetted off. 



For making the mixtures Wright's pipettes (four to five millimeters 

 caliber) fitted with rubber nipples are used. With a blue pencil an arbi- 

 trary point is marked off upon the drawn out extremity of the pipette. 

 Three volumes of serum (a bubble of air between each volume), and one 

 volume of cell suspension are drawn into the same pipette. These ingre- 

 dients are mixed by running them gently out and then drawing up again 

 into the pipette. The entire mixture is then drawn into the body of the 

 pipette and the tip is sealed in a flame. Each pipette serves as a little tube. 



The time and manner of incubation, and the observation for hemolysis 

 follow the rules mentioned above. 



