1.

ABO Reverse Typing This test is used to detect ABO ANTIBODIES in an

individual’s serum or plasma, and is used to confirm the ABO Forward Type.
There are structures present in nature on certain bacteria and pollens which
are very similar to the A and B antigens on RBCs. Individuals will produce
potent, naturally occurring antibodies directed against the ABO antigens they
lack. The terms “non-red cell stimulated antibodies” and “iso-hemagglutinins”
are also used to describe these antibodies. The patient’s serum is mixed with
reagent group A1 cells. Agglutination indicates the presence of anti-A in the
patient’s serum. Mixing the patient’s serum with the reagent group B cells
similarly allows for the detection of anti-B in the patient’s serum. The
outcome of the serum typing (Reverse Typing) is compared with the outcome
of the cell typing (Forward Typing) to ensure the accurate ABO determination.
Any discrepancy MUST be resolved before final interpretation of the blood
group is made. Group A individuals LACK the B antigen and their serum will
agglutinate the reagent B cells due to anti-B present in their serum. Their
serum will NOT agglutinate reagent A1 cells since the A1 antigen is present
on their own cells. Group B individuals LACK the A antigen and their serum
will agglutinate the reagent A cells due to anti-A present in their serum. Group
B individual’s serum will NOT agglutinate reagent B cells. Group O
individuals LACK both A and B antigens. Their serum will agglutinate both
reagent A and reagent B cells. Group O individuals have 3 naturally occurring
antibodies: anti-A, anti-B, and anti-A,B. Group AB individuals HAVE both A
and B antigens. Their serum will NOT agglutinate reagent A or reagent B
cells. Group AB individuals do not have any naturally occurring antibodies in
their serum. These two (2) tests, forward and reverse typing, constitute the
ABO typing of an individual and are one of the “check” systems in routine
blood banking. Remember that the forward typing is indicative of the antigens
present on the individual’s RBCs, while the reverse typing is indicative of the
antibodies present in the individual’s serum or plasma. The forward type
must correlate with the reverse type. Any discrepancies must be resolved
before a final determination is made. Exercise 3 Laboratory Procedure
Manual MLAB 2431 ABO and D Typing Page 3 Helpful hint: Think about what
is “known” in the test

4.Reverse Typing

Reverse typing refers to the testing of a patient's serum for the presence of ABO
antibodies. The patient's serum is mixed with known red cells in a test tube. A
specified number of drops of patient serum are placed into each of three properly
labeled tubes. A specified number of drops of known A1cells are added to the A
tube, and a specified number of drops of known B cells are dded to the B
tube.The tubes are mixed by gently shaking, centrifuged, and observed against a
well-lit white background for the pres eofhemolysisin the supernatant fluid. The
cell button is then gently dispersed and inspected for agglutination, again using a
well-lit background. Hemolysis or agglutination is a positive reaction.
 7.The current success rate of transplant surgery and immunosuppression has
led to a demand for organs that has outstripped the supply. This has required
investigation of alternate strategies. Therefore, allotransplantation across the
ABO blood group barrier has commenced, and pig-to-human xenotransplantation
is under consideration. The first immunological barrier to both these types of
transplantation is the prevention of the antibody-mediated rejection. This rejection
is a result of natural preformed antibodies circulating in the serum of the recipient
binding to either ABO (for allo) or α-galactose (α-Gal) (for xeno) antigens
expressed on the donor tissue. These antibodies recognise antigens that are, in
both cases, carbohydrate molecules with the characteristic feature that the
nonreducing terminal carbohydrate is either a Gal or N-acetlygalactosamine
residue in an α1,3 linkage. These epitopes are synthesised by closely related
members of a single family of glycosyltransferases. This review discusses the
carbohydrate antigens, the enzymes involved in their synthesis and the
consequences of natural antibodies binding these antigens.
http://onlinelibrary.wiley.com/doi/10.1111/j.1399-0039.2006.00721.x/full

5.ABO discrepancies happen when there is no match in results between forward

and reverse grouping.

ABO discrepancies are usually technical in nature and can be simply resolved by
correctly reporting the testing and carefully checking reagents with meticulous
reading and recording of results.There are some ABO discrepancies that can
happen due to technical errors and may lead to false positive or false negative
reactions.

False positive reactions are due to;

Un-calibrated centrifuges
Contaminated reagents
Dirty tubes or glassware

alse negative reactions can be due to many causes

Failure to add serum or reagents

Use of incorrect reagents or samples
Cell suspension is too heavy or too light
Inadequate identification of samples or test tube

Group I discrepancies
These discrepancies are between forward and reverse grouping due to weak
reaction or missing antibodies.These kind of discrepancies are the most
common.The reason for the missing antibody or weak reaction is that the patient
has depressed antibody production or cannot produce the ABO antibodies.This
type of discrepancy can be seen in new born infants, elderly patients.Patients
 This type of discrepancy can be seen in new born infants, elderly patients.
Patients with lymphoma.Patients using immunosuppressive drugs.Patients with
immunodeficiency disease, BM transplant.

Resolving discrepancies

Eliminate all technical errors

Enhancing the reaction in reverse grouping
Incubate the patient’s serum with reagent cells at room temp. for 15 mins.

Group II discrepancies
These discrepancies are between forward and reverse grouping due to weak
reaction or missing antigens.This group is the least one. Can be caused by some
subgroups of A or subgroups of B or both.Also it can be present in patients with
leukaemia and hodgkin’s disease.To resolve the problem wash the patient’s cells
with saline.

Group III discrepancies

These discrepancies are between forward and reverse grouping due to protein
or plasma abnormalities.These can be caused by elevated levels of globulin from
certain diseases such as multiple myloma, hodgekin’s lymphoma. Some are
caused by (Rouleaux formation).