(a) STEP 1: Place one drop of saline-reactive anti-Rho(D) in properly
(b) STEP 2: Add one drop of 2 to 5 percent saline suspension of well-washed
(c) STEP 3: Mix gently and incubate at 37C for 15 to 60 minutes,
according to manufacturer's direction.
(d) STEP 4: Centrifuge and gently resuspend to look for agglutination.
For controls, use a saline suspension of known Rh-positive cells and one of known Rh-
negative cells. Be sure the concentration is comparable to that of the test cells. Test all three
tubes, suitably labeled, at the same time.
(e) STEP 5: Interpret the results. Agglutination indicates that the cells
are Rh-positive. Du cells will not ordinarily be agglutinated, and the saline reagent
cannot be used for Du-testing. This test is done only on patient's cells; any seemingly
negative recipient should be given Rh-negative blood. Blood from a potential donor
whose cells cannot satisfactorily be tested with slide or rapid tube test anti-Rho(D)
should not be used for transfusion.
d. Rho(D)-Typing in Hemolytic Disease of the Newborn. Occasionally, an
infant's cells may be so heavily coated with maternal anti-Rho(D) that no sites are left to
react with the antiserum. Some such cells can be successfully typed with saline anti-
Rho(D) but many cannot. This should be suspected if cells with a strongly positive
antiglobulin test are negative on anti-Rho(D)-testing. To resolve this discrepancy,
antibody can be eluted from the cells at 45C (see Subcourse MD08460846 para 1-40).
Usually, enough sites will be liberated so that anti-Rho(D)-testing can be done. The
specificity of the eluate should be determined to confirm that the antibody coating the
cells is indeed antiRho(D), and also to see if there are additional antibodies present.
2-27. TESTS FOR ANTIGENS OTHER THAN Rho(D)
Both saline-reactive and slide or rapid tube test antiserums are available for the
other 4 major Rh antigens, rh'(C), rh"(E), hr'(c), and hr"(e). High-protein serums for slide
or rapid tube test are more common, and the same kind of high-protein control should
be used for these tests as for anti-Rho(D). The manufacturer's instructions should be
followed for whatever serum is used. Contamination with antiglobulin-reactive
antibodies other than those specified on the label is more common with these Rh
antibodies than with anti-Rho(D). Unless the instructions clearly indicate their suitability,
these reagents should not be used with antiglobulin serum. If a blood bank uses these
reagents only rarely, positive and negative control cells should be tested along with the