GROUP 2

EXPERIMENT NO 11: D (Rh) TYPING DIRECT, SLIDE METHOD

1. Characterize completely the Rh antigens (D,C,E,c,e) in terms of their biochemical nature and serologic
property.
The final result of gene action in RBC groups is the production of a biochemical structure; in
the Rh system it is a nonglycosylated protein. This means that there are no carbohydrates attached to the
protein.
The gene products of RHD and RHCE are remarkably similar in that both encode for proteins
composed of 417 amino acids that traverse the cell membrane 12 times and that their sequence differs
by only 44 base pairs. The gene products of RHCE, RHCe, RHce, and RHcE are even more similar. C
and c differ from one another in four amino acid positions, and one amino acid differentiates E from e.
Only small loops of the Rh proteins are exposed on the surface of the RBC and provide the
conformational requirements for the serologic differences between the Rh blood types.
In comparison with ABO and Kell (K) blood groups, A1 cells possess approximately 1.0 _ 106 A
antigens, whereas homozygous Kell cells have 6000 K sites. The greatest number of D antigen sites is
on cells of the rare Rh phenotype D--. (D-- cells carry only D antigen and completely lack Cc and Ee.)
However, of the commonly encountered Rh genotypes, R2R2 cells possess the largest number of D
antigen sites.
Summary:
Rh antigens are non-glycosylated
Rh antigens are transmembrane polypeptides and are an integral part of the RBC membrane
Highly immunogenic: Next to A and B
D>c>E>C>e
Weighs approximately 15-174 kD
Well developed in early fetal life
2. Give a listing of the Rh antigens in their different nomenclatures, and their frequency of occurrence

FREQUENCY
D: 85% Caucasians, 92% Blacks, 99% Asians
C: 68% Caucasians, 27% Blacks, 93% Asians
E: 29% Caucasians, 22% Blacks, 39% Asians
c: 80% Caucasians, 96% Blacks, 47% Asians
e: 98% Caucasians, 98% Blacks, 96% Asians

Researcher: LANUZA, Rosvan Butch P.

References: Immunohematology by Harmening

http://www.ncbi.nlm.nih.gov/books/NBK2269/
3. Give a list of the more common phenotypes and genotypes of the Rh system, including the antigens
present and their serologic reactions with the 5 antisera.
WEINER

FISHER-RACE
R1r
R1R1
rr
R1R2
R2r

DCe/dce
DCe/DCe
dce/dce
DCe/DcE
DcE/dce
R2R2

DcE/DcE

Researcher: CABOBOS, Mary Antonette L.

Reference: Modern Blood Banking and Transfusion Practices by Harmening

4. What are the causes of false positive and false negative reactions in Rh typing by the slide method?

a. Improper and inadequate washing of the red cell suspension may cause pseudoagglutination
due to the presence of serum macromolecules in the suspension. This should cause a positive
control as well.
b. The presence of strong autoagglutinins in the patient's or donor's serum may cause
agglutination. Proper washing and the control are designed to prevent or detect this problem.
c. Antibody coating of the red cell (positive DAT) can cause a false positive reaction, particularly
in the weak D test. A DAT will detect this occurrence.
d. A false negative reaction may be seen due to the "blocking phenomenon". This occurs most
commonly in HDFN due to anti-D. Since the red cell's antigen sites are heavily coated with
maternal antibody, they may not react with the antiserum.
e. False positive or negative reactions may occur in the Rh test due to many of the technical
errors..
f. RBCs that react with one manufacturers anti-D reagent but not with another may have a partial
D antigen.
Researcher: CABOBOS, Mary Antonette L.
Reference: cache:-ukZJ95UOwkJ: indianinitiative.org/wp-content/uploads/2011/06/211-RhTyping.doc+&cd=4&hl=en&ct=clnk&gl=ph
5. What is the clinical; significance or importance of Rh typing?
Transfusion Reactions
Rh antigens are highly immunogenic. The D antigen is themost immunogenic antigen outside
the ABO system. When anti-D is detected, a careful medical history will reveal RBC exposure through
pregnancy or transfusion of products containing RBCs. Circulating antibody appears within 120 days of
a primary exposure and within 2 to 7 days after a secondary exposure.
Rh-mediated hemolytic transfusion reactions, whethercaused by primary sensitization or
secondary immunization, usually result in extravascular destruction of immunoglobulin- coated RBCs.
The transfusion recipient may have an unexplained fever, a mild bilirubin elevation, and a decrease in
hemoglobin and haptoglobin. The direct antihuman globulin test is usually positive, and the antibody
screen may or may not demonstrate circulating antibody. When the direct antiglobulin test indicates that
the recipients RBCs are coated with IgG, elution studies may be helpful in defining the offending
antibody specificity. If antibody is detected in either the serum or eluate, subsequent transfusions should
lack the implicated antigen.
Hemolytic Disease of the Newborn (HDN)
HDN is briefly described here because of the historic significanceof the discovery of the Rh
system in elucidating its cause. As stated previously, anti-D was discovered in a woman after delivery
of a stillborn fetus. The mother required transfusion. The fathers blood was transfused, and the mother
subsequently experienced a severe hemolytic transfusion reaction. Levine and Stetson1 postulated that
the antibody causing the transfusion reaction also crossed the placenta and destroyed the RBCs of the
fetus, causing its death. The offending antibody was subsequently identified as anti-D.HDN caused by
Rh antibodies is often severe because the Rh antigens are well developed on fetal cells, and Rh
antibodies are primarily IgG, which readily cross the placenta. After years of research, a method was
developed to preventsusceptible (Rh0 D-negative) mothers from forming anti-D, thus preventing
Rh0(D) HDN. Rh-immune globulin, a purified preparation of IgG anti-D, is given to a D-negative
woman during pregnancy and following delivery of a D positive fetus.
Researcher: CAISO, Zenelei Grace A.
Resources: Harmening, D. M. (2012). Modern Blood Banking And Transfusion Practices (6th Ed.). F.A.
Davis, 2012.

6. Why is it necessary to run the control (with BSA) in Rh typing?

To assess the validity of the high-proteinRh typing results, a control reagent was manufactured
and had to be tested in parallel with each Rh test. If the control reacted, the test result was invalid and
had to be repeated using a different technique or reagent anti-D.
To assess the validity of the high-protein Rh typing results, a control reagent was manufactured
and had to be tested in parallel with each Rh test. If the control reacted, the test result was invalid and
had to be repeated using a different technique or reagent anti-D.
Bovine Albumin is primarily used to enhance the reactivity of blood group antibodies, either in
direct agglutination tests or indirect antiglobulin test. This fact was confounded by Cameron and
Diamond, 1945, who established that certain anti Rho (Anti-D) Sera would agglutinate Rh positive red
cells when they were suspended in albumin, however no agglutination was observed when same red
cells were suspended in Normal Saline medium.
PRINCIPLE: Bovine Albumin is frequently used as a control for Rh typing. Every blood specimen
tested by the slide, stick or modified tube method should be controlled by testing simultaneously with a
medium such as Bovine Albumin. Since cells coated with an autoagglutinin or suspended in serum
having a protein abnormality may give a false positive result in Rh typing.
Researcher: Caiso, Zenelei Grace A.
References: Harmening, D. M. (2012). Modern Blood Banking And Transfusion Practices (6th Ed.).
F.A. Davis, 2012.
Http://Jmitra.Co.In/Download/Procedure/Manual-Bovinealbumin.Pdf
Http://Webmedia.Unmc.Edu/Alliedhealth/Cls/Cls422%2010/10%20rhpro.Pdf