1-24. SOURCES OF ERROR
a. False Negative Results.
(1) Inadequate washing of cells will result in neutralization of the antiglobulin
serum by trace amounts of residual globulin. A final concentration of only two mg of
lgG/ml can cause neutralization of the antiglobulin serum. Thus, the RBCs have to be
washed free of unbound IgG, until it is below this figure.
(2) Contamination with human serum will neutralize the reagent. If the
reagent dropper is contaminated with serum and replaced in the vial, the entire contents
of the vial may be neutralized. If the tube is inverted over the thumb or finger in the
washing process, serum contaminating the skin may result in neutralization.
(3) Elution of antibody from the RBCs may take place if the test procedure is
interrupted or delayed, particularly during the washing phase.
(4) The optimum temperature for reactivity of the antibody must be
maintained during incubation to achieve maximal coating of the cells.
(5) A cell suspension that is too heavy will not permit optimum coating with
the antibody; if too weak, reading agglutination may be difficult. A 2 to 5 percent
suspension of RBCs is preferred.
Test cells, test serum, and antiglobulin serum lose reactivity if improperly
(7) Some antibodies may be detected only in the presence of active
complement. Anticoagulants such as ACD, CPD, or EDTA will chelate calcium,
preventing activation of complement. Thus, the use of plasma rather than serum may
lead to a false negative reaction. Old or improperly stored serum will also have
impaired complement activity.
(8) A prozone reaction should not be a problem with licensed products.
Standardization is done by the manufacturer; directions for the test must be followed.
Antiglobulin serum may have been omitted.
(10) Undercentrifugation or overcentrifugation (the latter because of the
excessive force needed to resuspend cells).
(11) Insufficient incubation time.
The lack of agglutination of presensitized RBCs, added following
completion of a negative antiglobulin test, will demonstrate a false-negative
determination caused by 1, 2, 6, or 9 above.