II.

Washing phase
A. Decant the saline as completely as
possible between each washing, shake to
loosen and resuspend the cells completely.
B. do not cover the mouth of the test tube
with the finger or the palm of the hand
when mixing. Serum remaining on fingers
after handling specimen can inactivate the
antiglobulin reagent.
C. Use adequate volumes of saline at least
3 quarters full of saline
D. After the final wash, discard the saline
as completely as possible. Resuspend the
cells and add the appropriate amount of
antiglobulin serum, Mix well and
centrifuge.
 Source of Error
I. False Negative Results
A. Inadequate washing of cells will result
in neutralization of the globulin serum by
tracing amounts of residual globulin.
B. Contamination with human serum will
neutralize the reagent.
 C. Elution of the antibody from the red
cells may take place if the test procedure
is interrupted or delayed particularly
during the washing phase.
D. The optimum temperature for
reactivity of the antibody must be
maintained during incubation to achieve
maximum coating of the cells.
E. A cell suspension that is too heavy will
not permit optimum coating with the
antibody : if too weak, reading
agglutination may be difficult . A 2.5%
suspension of the red cell is preffered.
 F. Test cells, test serum, and antiglobulin
serum lose reactivity if improperly
stored.
G. Undercentrifugation or over
centrifugation (the latter beacause of
excessive force in resuspension of cells)
H. Incubation for too long or too short a
time.
I. Poor reading technique

J. Serum non reactive because of

deterioration of the complement.
 A. Bacterial Contamination of test cells or
septicemia in a patient may result in
agglutination.
B. Extreme reticulocytosis has been
reported to give a positive result because
of transferrin bound to reticulocytes
reacting with anti transferrin in the
antiglobulin reagents.
II. False Positive Results

C. saline stored in metal containers, or

used in equipment with metal parts,
may contain metallicions which bring
about protein coating of the red cells.
D. Improperly prepared antiglobulin
serum may contain traces of species
specific ab.
E. Extraneous antibodies in reagents
e.g. anti A or anti B in anti Rho
(D) serums.
 F. When all of the antiglobulin test
are weakly postive, the cause may
be improperly cleaned glassware or
other forms of contamination.
G. Overcentrifugation may give false
positive results.