b. Serology. To diagnose and classify the patient correctly, several questions
have to be answered:
QUESTION: Are the patient's cells sensitized with protein?
EXPLANATION: The patient's washed RBCSs are first tested with a
broad-spectrum antiglobulin reagent. Most commercial ant globulin
serums will detect lgG sensitization adequately, but approximately 30
percent of all AIHA cases have only complement (C3d and C4d) on their
RBCs and very rare cases have been described with only lgA or lgM on
their cells. Some commercially prepared antiglobulin serums will not
reliably detect these proteins; others, will only detect the sensitization if
the antiglobulin serum and the sensitized red cells are incubated at room
temperature for 5 to 10 minutes before centrifugation and reading. A
single negative direct antiglobulin test should not be considered definitive
evidence against a diagnosis of AIHA in a patient with suggestive signs
and symptoms. Other rare cases exist in which the number of lgG
molecules on the cells is below the threshold of the antiglobulin test and
AIHA exists in the presence of a negative direct anti-globulin test.
QUESTION: What proteins are present on the RBCs?
EXPLANATION: The RBCs are often sensitized with lgG and
complement, or IgG alone, and sometimes, by complement alone. The
presence of these proteins is best detected by performing antiglobulln
tests using monospecific antiglobulin serums, such as anti-lgG and
anticomplement. lgA and lgM are sometimes detected but they are
usually present together with IgG and/or complement.
A word of caution should be added concerning monospecific antlglobulin
reagents. At present only anti-IgG and anti-C3 are available as a licensed
reagent, for use with RBCs. Other antiserums (for example., anti-
IgA, -lgM, -C4) are readily available as precipitating reagents for use, in such
techniques, as immunoelectrophoresis, but are not so readily available for
use with RBCs. The precipitating reagents can be used as long as
they are carefully standardized and controlled, (for example, they often
contain antispecies agglutinins, which, have to be removed either by dilution,
orabsorption with nonsensltized human RBCs). The quality
assurance must be precise, as agglutination is a far more sensitive technique
than precipitation, and monospecificity by precipitation techniques, does not
ensure monospecificity by the antiglobulin test. It is preferable to obtain
reagents specifically standardized for use with RBCs.