(2) It must be stressed that enzymes considerably enhance the reactions of
cold autoagglutinins; therefore, many normal serums will react with enzyme-pretreated
cells at room temperature, and in some instances demonstrate carry-over reactivity at
37C because of the presence of cold auto-agglutinins in these serums. On these
occasions, it is helpful to warm the enzyme-pretreated cells and the serum under test
separately to 37C, before mixing. This technique prevents the cold autoagglutinins
from reacting with the cells below 37C.
(3) It should also be noted that if enzyme-pretreated cells are used with
antiglobulin serum, the AHG must be shown to be free of unwanted antibody activity
(that is, anti-A, -B, -H, antispecies) that might demonstrate increased reactivity with the
enzyme-pretreated cells.
1-27. READING AND INTERPRETATION OF REACTIONS
a. lmmediately after centrifugation, observe for hemolysis. The appearance of
free hemoglobin not present in the original sample must be interpreted as a positive
reaction. A white light and a white background is optimal for the observation of
hemolysis.
b. The manner in which the RBCs are dislodged from the bottom of the tube is
critical. The tube must be shaken gently until all cells are dislodged. This is most easily
accomplished by holding the tube at a sharp angle so that the fluid "cuts" across the cell
button in a manner that permits the fluid to do most of the work in resuspending the
cells. When cells no longer adhere to the tube, it should be tilted back and forth gently,
until an even suspension of cells (or agglutinates) is observed. Overshaking can break
up fragile agglutinates, resulting in a negative reaction, while inadequate mixing may
counterfeit agglutination.
c. For maximum reproducibility, observations should be made with a consistent
light source. A microscope is sometimes useful in distinguishing rouleaux from true
agglutination, but it is not required.
d. A scheme of grading the strength of agglutination reaction in common use is
given in the following chart.
MD0846
1-36