GROUP 2

EXPERIMENT NO 1: D (Rh) TYPING DIRECT TUBE METHOD

1. What are the advantages of the tube method of Rh typing over that of the slide?
more sensitive
adaptable to batch testing up to approximately 200 samples
It allows for fairly long incubation without drying up of the tubes contents.
clean and more hygienic
Researcher: Comtiag, Klevee A.
References:
http://www.trinitytoo.org/teachers/hayes/Docs/med2section44.pdf
https://books.google.com.ph/books?
id=D9oirqPhCpwC&pg=PA87&lpg=PA87&dq=Tube+method+of+Rh+typing+advantages&source=bl
&ots=nk8jIm8biz&sig=h5kPrTtXl11S0dkV9haW3jEO2EQ&hl=en&sa=X&ved=0ahUKEwjGrueYic3L
AhVHJ6YKHaj7DvUQ6AEIMjAE#v=onepage&q=Tube%20method%20of%20Rh%20typing
%20advantages&f=false
2. What are the types of Rh typing reagents? Describe the preparation and the use of each.
a. Saline reactive reagents
contain IgM immunoglobulin
low-protein-based and can be used to test cells that are coated with IgG antibody
cannot be used for weak D typing, lengthy incubation time
b. High-protein anti-D reagent
raw material: human plasma containing high titer D-specific antibody
potentiators of bovine serum albumin and macromolecular additives such as dextran or
polyvinylpyrrolidone are added to the source material to optimize reactivity in the standard
slide and rapid tube tests
commonly referred to as high-protein reagents
c. Chemically modified
chemically modified the anti-d molecule by breaking the disulfide bonds that maintain the
antibodys rigid shape
this allows the antibody to relax and to span the distance between RBCs in low protein medium
d. Monoclonal/ blends of monoclonal
the antibody-producing cells are hybridized with myeloma cells to increase their reproduction
rate and thereby to maximize their antibody-producing capabilities
because the D antigen appears to be a mosaic and the monoclonal Rh antibodies have a narrow
specificity, monoclonal anti-D reagents from several different clones to ensure reactivity with a
broad spectrum of Rh positive RBCs
some companies also blend anti IgM and anti IgG to maximize visualization of reactions
Researcher: Comtiag, Klevee A.
Reference: Modern Blood Banking And Transfusion Practices (6 th Edition) By Denise M. Harmening
P142
3. Characterize the Rh antibodies
Rh antibodies are:
Mostly IgG immunoglobulins and react optimally at 37 degrees celsius or after anti-globulin
testing.
Usually produced following exposure of the individuals immune system to foreign RBCs, through
either transfusion or pregnancy.
Highly immunogenic; the D antigen is the most potent.
As with most blood group antigen sensitization, IgM Rhantibodies are formed initially, followed by
a transition to IgG.
Often persist in the circulation for years.

Do not bind complement. For complement to be fixed (or the complement cascade activated), two
IgG immunoglobulins must attach to an RBC in close proximity. Rh antigens (to which the
antibody would attach) are not situated on the RBC surface this closely. Therefore, when an Rh
antibody coats the RBCs, intravascular, complement-mediated hemolysis does not occur.
RBC destruction resulting from Rh antibodies is primarily extravascular.
Rh antibodies formed by Rh-negative pregnant women do cross the placenta and may coat fetal
RBCs that carry the corresponding antigen. This results in the fetal cells testing positive by the
direct antiglobulin test.
D>c>E>C>e
Immunogenicity of common Rh antigens.

Researcher: CUEVAS, Kathrina Joie A.

Reference: Modern Blood Banking and Transfusion practices by Harmening
4. What is the clinical significance of Rh antigens?

Exposure to less than 0.1 mL of Rh-positive RBCs can stimulate antibody production in an Rhnegative person. IgG1, IgG2, IgG3, and IgG4 subclasses of Rh antibodies have been reported. IgG1
and IgG3 are of the greatest clinical significance because the reticuloendothelial system rapidly
clears RBCs coated with IgG1 and IgG3 from the circulation.
Most commonly found Rh antibodies are considered clinically significant. Therefore, antigennegative blood must be provided to any patient with a history of Rh antibody sensitization, whether
the antibody is currently demonstrable or not.

Transfusion Reactions
Rh-mediated hemolytic transfusion reactions, whether caused 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.
Hemolytic Disease of the Newborn (HDN)
Levine and Stetson 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.
Researcher: CUEVAS, Kathrina Joie A.
Reference:: Modern Blood Banking and Transfusion practices by Harmening