Rh PHENOTYPING

PRINCIPLE
The term Phenotype refers to the detectable antigens that may be demonstra-ted by typing for
them directly using the five standard anti-sera: Anti-Rho(D), anti-rh' C), Anti-rh" E), Anti-hr' c), and Anti-
hr" (e).
The term Genotype refers to the total sum of the genes present on the chromosomes,
regardless of whether or not they produce detectable products. Gene frequen-cies have been
calculated from numerous stud-ies, and the genotype can be estimated from the phenotype pattern
with a fair degree of accuracy.
The determination of most probably genotype becomes important when a prenatal patient has
become immunized to one or more of the Rh anti-gens. By estimating her genotype plus that of the
father, it is possible to predict the likelihood of the fetus carrying the antigen(s) the mother is sensitized
to, and there-fore suffering hemolytic disease of the newborn (HDN).
Determination of most probably genotype also becomes important when selecting blood to
transfuse patients with allo- or auto-antibodies in the Rh system.

SAMPLE
A small EDTA tube is preferred; a clotted sample is acceptable.

REAGENTS, EQUIPMENT, AND SUPPLIES

Reagent Anti-D (Rho)
Reagent Anti-C (rh')
Reagent Anti-E (rh")
Reagent Anti-c (hr')
Reagent Anti-e (hr")
Rh control
12 x 75 mm test tubes
test tube rack
indelible marking pen
37C incubator
serofuge
lighted agglutination viewer

PROCEDURE
Prepare a washed 3% suspension of each sample to be tested.
Label the follow-ing tubes for the D typing: D+ con-trol
D neg control
initials pt. 1 - D
initials pt. 2 - D
initials pt. 3 - D
Label a similar set of tubes for each of the other antigen typing to be done: C, c, E, and e
Place one drop of the appro-priate antisera into each tube. (Anti-D into the D tubes, anti-C into the C
tubes, etc.)
Place one drop of a cell known to be heterozygous positive for each antigen being typed into each +
control. (Example: an R1r cell for the D+ control, a r'r cell for the C+ control, etc.)
Place one drop of a cell known to be negative for the antigen being typed for into each neg. control.
Place one drop of each washed patient cells into each patient tube.
Set up a PATIENT CONTROL tube for each sample to be tested:
Add 1 drop of Rh control and 1 drop washed patient cells to each PATIENT CONTROL tube. Incubate
these tubes along with your test in step 9, below.
Shake all tubes gently to mix, and incubate 15-30 minutes at 37C.
Centrifuge the time appropriate for the albumin calib-ration of the serofuge. Because all tubes cannot fit
in the serofuge at once, leave the ones you are NOT centrifuging in the 37c waterbath.
Gently resuspend and examine macroscopically for agglutina-tion, using the lighted agglutination
viewer.
Read, grade and record reactions.
From the phenotype pattern obtained, determine the most probable genotype for each sample.

NOTES

Red cells that are coated with antibody and have a positive DAT may give false positive Rh
typing due to the albumin added to the Rh antisera. The presence of the albumin causes the zeta
potential to be reduced, so that the cells may come close enough together to agglutinate. If the cells are
already coated with antibody in vivo (not due to the reagent antisera), they may spontaneously
agglutinate in the presence of the albumin-enhanced reagents, whether they are positive or not for the
antigen being tested.

This situation is detected by the use of the patient control, which consists of patient cells and
albumin only. If there is agglutination in the control, the test should be repeated using a modified
antiserum that does not require albumin and therefore is not subject to false positives.

Note that no typing sera exists for the d phenotype. If a patient types D negative, he or she is
assumed to have the d phenotype.

Proper antigen typing require the use of known positive and negative controls to be set up and
run in parallel with each test. A positive control should contain a heterozygous expression of the
antigen being typed for, to ensure that the antiserum is picking up weaker expressions of that antigen.
A negative control may be any cell known to be negative for the antigen.

NOTE: Unexpected reactions in the controls invalidates the test.