 Group II DISCREPANCIES: Weakly

Reacting or Missing Antigens

 Group III DISCREPANCIES: Protein or
plasma Abnormalities Resulting to
Rouleaux Formation
 Group IV DISCREPANCIES: Miscellaneous
COMMON SOURCES OF TECHNICAL ERRORS
 use a microtiter plate with 96 wells to RESULTING IN ABO DISCREPANCIES
serve as the substituted test tubes. The
microplate technique can be adapted to  Incorrect or inadequate identification of
red cell antigen testing or serum testing blood specimens, test tubes, or slides
for antibody detection. The principles  Cell Suspension either too heavy or too light
that apply to agglutination in test tubes  Clerical errors or incorrect recording of
also apply to testing in microplate result
methods.  A mix-up in samples
3. Solid-Phase Red cell Adherence  Missed observation of hemolysis
 Failure to add reagents or sample
 Failure to follow manufacturer’s instruction
 Uncalibrated centrifuge
 Overcentrifugation or undercentrifugation
 Contaminated reagents
 Warming during centrifugation
GROUP I DISCREPANCIES: WEAKLY REACTING OR
MISSING ANTIBODIES
 If the problem lies on the antibodies,
problem lies in the reverse grouping
antibodies.
Forward Reverse Grouping
Grouping
Reverse Anti-B A1 Cells B cells
 The antigen or antibody is immobilized
Grouping
to the bottom and sides of the
O O O O
microplate wells. - IgG antibodies or red
 Patient’s probable group: O (elderly patient
cell antigens adhere to the microplate
or newborn)
wells if an Ag-Ab is observed.
 The most probable reason for this
ABO DISCREPANCIES discrepancy is that the patient is too old or
too young.
 Group I DISCREPANCIES: Weakly
 For newborns, antibodies (even though
Reacting or Missing Antibodies
produced during gestations) peak at 4-6

FURIEL, M.A l SABADO. V.

 months. ABO antibodies are naturally chance of antigen-antibody
occurring but for newborns, they have reaction.
minimal amounts making weak reaction and
CAUSES OF GROUP I ANTIBODIES
may be negative in the reverse grouping.
This is why reverse grouping should not be  Newborns (the production of ABO
performed on newborns. antibodies is not detectable until 4 to 6
 For elders, ABO antibodies start to weaken. months of age)
Reverse grouping is not also the best way .  Elderly patients (the production of ABO
 Note: The absence of agglutination with antibodies is depressed)
reagent cells in the reverse type is because  Patients with a leukemia (e.g., chronic
the production of ABO antibodies can be lymphocytic leukemia) or lymphoma (e.g.,
weak or absent in the elderly. malignant lymphoma) demonstrating
 Resolution: hypogammaglobulinemia
o Check age of the patient.  Patients using immunosuppressive drugs
o Use of 2-5% RCS that yield hypogammaglobulinemia
 For optimum concentration  Patients with congenital or acquired
for the antigen- antibody agammaglobulinemia or immunodeficiency
reaction to happen. diseases
o Increase incubation time to 30  Patients with bone marrow or stem cell
minutes (not appropriate for transplantations (patients develop
newborn sample). If negative. hypogammaglobulinemia from therapy and
 This solution can be done start producing a different RBC population
only to elderly patients since from that of the transplanted bone marrow)
they have weakly reactive  Patients whose existing ABO antibodies may
antibodies. Increasing the have been diluted by plasma transfusion or
incubation time will increase exchange transfusion
the amount of antibodies by  ABO subgroups
giving more time for the  The last cases (3-6) are conditions wherein a
antigen and antibody to bind patient will have a case of
before facilitating the hypogammaglobulinemia cused by having a
antigen-antibody reaction. disease or immunosuppressive drugs
 For newborn, they have  Hypogammaglobulinemia (decreased levels
minimal antibodies. of globulins) is relevant since it causes
Increasing the incubation weakly reactive antibodies.
time will not help the reaction  Gammaglobulins are antibodies.
of antibodies.  The lower the gammaglobulins, the lower
o Lower the temperature to 4°C for 15 the antibodies.
minutes (include O cells and an  An example of immunosuppressive drug is
autocontrol) Tacrolimus. This drug is used when a patient
 This has something to do with has undergone a transplant surgery in order
potentiating or increasing the
FURIEL, M.A l SABADO. V.
 to avoid host versus graft reactions. Even o Colon cancer is diagnosed by:
though you have a donor that rematches the  Carcinoembryonic antigen
area, the organ transplanted does belong to  Fecal Occult Blood Test
him so the body attacks the foreign organ,  Formation of color blue
o Infection with bacteria P. vulgaris
thus reactions may happen.
 Another condition that leads to
 Agammaglobulinemia patients are
acquired B phenomenon
susceptible to a lot of infections because
they have very depressed and almost  Resolution:
virtually none detectable levels of o Use monoclonal anti-B clone (ES4)
gammaglobulins. o Treat RBCs using acetic anhydride
 If the patient’s plasma is diluted, levels of
antibodies are lower and weakly reactive.
GROUP II DISCREPANCIES: WEAKLY REACTING OR
MISSING ANTIGENS

 The problem lies on the forward typing

antigens.

Forward Reverse Grouping

Grouping
Anti-A Anti-B A1 Cells B cells
4+ 2+ O 4+  The patient is naturally blood group A with
 For the forward grouping, the patient is AB. But
immunodominant sugar N-acetyl-
it is not correct to look at this reaction because
galactosamine.
basically, for forward grouping, we are looking
 In this phenomenon, there is the process of
for antigens. We must take note that reactions
acetylation caused by bacterial enzymes of P.
involving ABO blood groups are very strong
vulgaris.
because ABO antibodies are IgM in nature
o Acetylation is the removal of N-acetyl of
because of their pentameric and bigger structure
the immunodominant sugar.
to easily bind on antigen. It is atypical that there
 Galactosamine is very similar to galactose
is a 2+ reaction.
(immunodominant sugar for blood type B)
 We can also observe mix field reaction.
 Galactosamine would cross react with anti-B
 Based on the reverse typing, it is blood type A.
sera that could generate weak reaction (2+) and
 Acquired B Phenomenon
mf reaction
o The most common cause of Group II
o Clumping of 3-5 cells against a sea of
discrepancy.
agglutinated cells
o Occurs when bacterial enzymes
 In a group B phenomenon, it would look like
(deacetylase) modify immunodominant
there are two blood groups because of the
blood sugar A (N-acetyl-D
acetylation of the immunodominant sugar.
galactosamine) into D-Galactosamine
 How do we resolve this?
(similar to D-galactose) which cross
o We can just perform and confirm thru
reacts with Anti-B antisera
reverse typing because the problem lies
o If the patient suffers from colon cancer

FURIEL, M.A l SABADO. V.

 in the antigen. The expected reaction is spontaneously agglutinate, independent of the
3+ or 4+ under B cells. specificity of the reagent antibody
 Patient has circulating RBCs of more than one
GROUP III DISCREPANCIES: PROTEIN OR PLASMA
ABO group due to RBC transfusion or
ABNORMALITIES RESULTING TO ROULEAUX marrow/stem cell transplant
FORMATION  Unexpected ABO isoagglutinins
 The problem now relies on both/ either  Unexpected non-ABO alloantibodies
forward and reverse typing.

Forward Reverse Grouping

Grouping
Anti-A Anti-B A1 Cells B cells
4+ 4+ 2+ 2+
 In the forward typing, the blood type of the
patient is AB and in the reverse grouping, it is O
blood type.
 Probable Group: AB
 Elevated levels of globin from certain disease
states (e.g. multiple myeloma, Waldenstrom’s
macroglobulinemia)
 Elevated levels of fibrinogen
 Plasma expanders
 Wharton’s jelly
 Resolution: Wash RBCs with saline several times

GROUP IV DISCREPANCIES: MISCELLANEOUS

Forward Reverse Grouping

Grouping
Anti-A Anti-B A1 Cells B cells
2+ 4+ 4+ 2+

 Reaction with anti-A in forward type is due to

spontaneous agglutination of antibody coated
cells; reaction with B cells in reverse type is due
to cold autoantibody (e.g., anti-I) reacting with I
antigen on B cells.
 Resolution:
o Wash patient cells with warm saline and
retest;
o Run DAT and autocontrol;
o Run antibody screen
 Cold reactive autoantibodies in which RBCs are
so heavily coated with antibody that they

FURIEL, M.A l SABADO. V.