7. Does the patient lack the antigen corresponding to the antibody?

Individuals cannot make alloantibodies to antigens that they possess in

their RBCs therefore the last step in identification studies is to test the
patients RBCs for the corresponding antigen.
A negative result is expected, providing additional evidence that the
identification is correct.
If the patients RBCs are positive for the corresponding antigen,
misidentification of the antibody or a false positive typing are most
likely the explanation
Antigen typing can also help resolve complex cases, as it may
eliminate possible specificities
It is not typical to do extended typing on all patients with antibodies,
however , judicious use of this procedure can be useful, especially in
patients who chronically receive transfusions and at risk for
alloimmunization, such patients with sickle cell disease or thalassemia

Phenotyping may be complicated when a patient has a positive DAT or has

been transfused in the last 3 months. Positive DAT is may be due to IgG
coating the RBCs. Removal of the antibody coating will be necessary to get
an accurate phenotype.
2 reagents useful in stripping antibody from the RBC surface:

Chloroquine diphosphate- washed RBCs are incubated with the

reagent at room temperature for 30 mins. to 2 hours. These cells are
washed to remove the chloroquine diphosphate. When the treated cells
yield a negative DAT, they may then be phenotyped
Acid glycine/EDTA (EGA)- rapid method for removing antibody. Kell
antigens are denatured in this method, so patient cells cannot be
reliably typed for these antigens.

Absorption method- done when coating antibody resists elution.

Phenotyping patients who recently undergone transfusion is difficult because
they show mixed field agglutination, so to resolve this one, RETICULOCYTE
TYPING is employed.
Note: positive and negative control cells should be tested with each antisera
used for antigen typing, on the day of use. Positive control should have
heterozygous antigen expression to ensure that the antiserum has the
sensitivity to detect small quantities of antigen. The negative control cell
should lack the target antigen to confirm reactivity with only the target
antigen.

Methods in performing antigen typing:

Tube method
Solid phase adherence
Gel method
Flow cytometry
PCR(polymerase chain reaction)