Factors That Affect Agglutination
in Tube Testing
Immunohematology Review 436
FACTOR COMMENTS
Sensitization stage
Temperature
pH
Ionic strength
Ag/ab ratio
Incubation time
Agglutination stage
Type of ab molecule
Density of ags & location on RBC surface
Zeta potential
Attachment of ab to ag
Clinically significant abs react best at 37°C.
Most abs react at pH 5.5–8.5.
Reducing ionic strength of medium facilitates interaction of ab with ag (e.g., low ionic
strength solution [LISS]).
Too much ab can cause prozone (false neg). Optimum serum-to-cell ratio is 80:1. Usually
2 drops serum to 1 drop of 2%–5% RBCs. Follow manufacturer’s directions.
Depends on medium. Usually 10–30 min. Follow manufacturer’s directions.
Formation of ag-ab bridges between RBCs
IgM is larger, can span distance between RBCs more easily.
Affects ease of attachment of abs.
Difference in charge between neg-charged RBC surface & cloud of pos ions that surround
RBCs. Reducing zeta potential allows RBCs to move closer together (e.g., enzyme treatment
of test cells).
