Benjamin Jeremiah S.

Santiago
3AMT
Blood Banking Lecture Guided Activity
A. Antiglobulin Tests
a. What is Coombs’ serum? Coombs’ cells? Give the purpose of each.
Coombs’ cells are red blood cells coated with IgG that is used to confirm a negative D
antigen test. A positive reaction on Coombs’ cells indicate the procedure was done
correctly, and a negative reaction indicates possible error.
Coombs’ serum is also known as antihuman globulin. It is made from immunizing rabbits
using human gamma globulin, the resulting serum contains antibodies for both IgG and
complement.
b. What is the importance of using monospecific and polyspecific AHG reagent?
Polyspecific AHG reagents are used in the direct antiglobulin test (DAT) for
determining either IgG or complement molecules attached to the red cell in vivo. It is
able to do so because it contains both anti-IgG and anti-C3d antibodies.
Monospecific AHG reagents are used to investigate a positive DAT test, to
determine if the patient’s red cells are sensitized by IgG, complement, or both proteins.
Doing this requires a differential DAT test prepared with monospecific AHG reagents
with individual sources of anti-IgG and anti-C3d separately.
c. Differentiate DAT and IAT, tabulated. Provide at leat 5 points of difference
Direct Antiglobulin Test (DAT) Indirect Antiglobulin Test (IAT)
Procedure only requires one stage Procedure requires two stages
Tested red cells do not need an Tested red cells need an incubation step
incubation stage
IgG attachment occurs within the IgG attachment occurs during the
patient’s body incubation step
Conditions tested for: HDFN, HTR, Conditions tested for: antibody screening
autoimmune haemolytic anemia and antibody identification panel
Determine sensitization in vivo Determine sensitization in vitro

d. Enumerate sources of false (+) and false (-) errors in antiglobulin test.
False (+) False (-)
Red cells agglutinated before washing Inadequate washing of red cells before
and adding AHG reagent adding AHG reagent
Dirty glassware Testing was delayed by failing to
immediately add AHG reagent
Overcentrifugation Failure to identify weak reactions
Low reagent activity
Failure to add AHG reagent
Undercentrifugation
Red cell suspensions outside the 2%-5%
range
B. Rh Blood Group
a. What makes Rh Blood Group system 2nd to ABO in terms of importance despite the
fact it is not the 2nd one to be discovered?
The Rh Blood group is the 2 nd most important system because it is also a leading
cause of haemolytic disease of the fetus and newborn (HDFN), especially during the
1940s. This blood groups is also highly immunogenic, meaning that it causes an immune
reaction on an Rh (-) recipient upon exposure to Rh (+) blood, therefore initial exposure
is safe, but after that the patient is expected to develop antibodies against the Rh
antigens and may cause haemolytic transfusion reactions (HTR) or HDFN. This is most
important for pregnant mothers who are Rh (-) pregnant with Rh (+) babies inherited
from the father. The baby may be fine during the first pregnancy, but the next Rh (+)
baby may experience HDFN because the mother would have developed antibodies to
the Rh antigen by then.

b. Enumerate the 5 major Rh antigens and named them in for 4 different ways. Present
this in tabulated format.
FISHER-RACE NUMERIC ISBT WEINER
D Rh1 004001 Rh0
C Rh2 004002 rh’
E Rh3 004003 rh”
c Rh4 004004 hr’
e Rh5 004005 hr”

c. What makes C different from c and E from e?

C is different because of its subsequent presence of the G antigen. The G
antigen is another Rh antigen found only in D (+) OR C (+) blood. Therefore given a
phenotype where there is no C or D antigens such as ce and cE, the G antigen would not
exist. The anti-G mimics reactions of anti-C and anti-D, and D (-) but C (+) blood may
appear to have both anti-C and anti-D when in fact it is just anti- G. Structurally, C is
different in amino acid sequence produced by the RHCE gene. C has serine residue 103
Cc, c has proline, E has proline residue number 126 Ee, and e has alanine.

d. Elaborate the main functions of Rh antigens.

The function of Rh antigens are for cation transport and membrane integrity.
For immunohematology cases, it is significant for preventing transfusion reactions like
HTR and HDFN.

e. What is a compound antigen? Give examples. Is G an example of this?

Compound antigens are additional antigen products formed when a gene codes
for an additional protein. Examples include Rh6 (ce or f), Rh7 (Ce), Rh27 (cE), Rh22 (CE).
The G antigen is not an example of a compound antigen, it is simply an antigen present
on red blood cells containing the C or D antigen.

f. Differentiate the following phenotypes: -D- (D-deletion) , Rhnull , Rhmod

The -D- phenotype demonstrates no reaction when tested with anti-E, anti-e,
anti-C, or anti-c. This is because genetic material was deleted at the RHCE gene site.
 The Rhnull phenotype has no Rh antigens through two genetic mechanisms. First,
their cells have membrane abnormalities with shorter lifespan, causing haemolytic
anemia. Individuals that lack all Rh antigens, but have produced antibodies through
immunization require Rhnull blood. Second, this phenotype can be inherited from a
regulator/amorph gene caused by a mutation in the RHAG gene (a different blood
group). The presence of RHAG is essential to the expression of Rh antigens. The
mutation occurring in the case of an Rh null phenotype results in an absence of RHD gene
and a lack of expression of the RHCE gene, making both phenotypes absent.

The Rhmod phenotype is caused by an inherited modified RHAG gene similar to

Rhnull. Red cells in this phenotype also lack most of their Rh antigen expression, and may
experience haemolytic anemia as well.

g. What is Du (weak D)? Enumerate the mechanisms of its existence. Explain its
significance in transfusion medicine.
Weak D cells may appear as if they are D (-), but may be proven otherwise after
an indirect antiglobulin test (IAT). Red cells positive only in the IAT test are labelled as
“weak D” antigens.

The weak D phenotype may be expressed through genetic means, the position
effect, and the partial D. The genetic mechanism occurs when RHD genes end up coding
for a weak D expression. This more commonly occurs in blacks as a result of the cDe
haplotype. The position effect occurs when the r’ gene is inherited in trans to the RHD
gene, and an r’ gene paired with R1 or R0 gene weakens the expression of the D antigen.
The partial D occurs when some D cells are missing parts of the complete D antigen
complex. When exposed to a complete D cell, a partial D cell will produce antibodies to
the parts of the D antigen that it lacks.

This is significant because weak D positive individuals are Rh (+) as donors, but
Rh (-) as recipients. Therefore without weak D testing, a donor may donate blood to an
Rh (-) patient thinking he/she is Rh (-), and end up causing the recipient to produce Rh
antibodies.

h. Illustrate the mechanism of HDFN due to Rh incompatibility. (You may refer to

internet sources. Just provide proper citation.)

i. What is the relationship between the LW and Rh Blood Group system?

There is a phenotypic relationship between the LW and Rh blood groups. The
antigens and antibodies of both groups are serologically similar, but genetically
unrelated. Like the anti-D, the LW antibody was also made from guinea pigs and rabbits
immunized with rhesus macaque cells. An LW antibody will have a strong reaction with
D (+) cells, a weak reaction with D (-) cells, and a negative reaction with Rh null cells.