1. As undercentrifugation or overcentrifugation could
lead to erroneous results, it is recommended that each
laboratory calibrates its own equipment and determine
the time required for achieving the desired results.
2. It is strongly recommended that as a routine quality
control measure known Rho(D) positive and Rho(D)
negative red cells be occasionally run, preferably on a
daily basis so as to control reagent performance and
3. After usage, the reagent should be immediately
recapped and replaced at 2–8°C storage.
Blood Group Testing in Microplates
The microplates consist of 12 × 8 = 96 wells. We can do 12
Anti-A O O O O O O O O O O O O
Anti-B O O O O O O O O O O O O
Anti-ABO O O O O O O O O O O O
A Cell O O O O O O O O O O O O
B Cell O O O O O O O O O O O O
O Cell O O O O O O O O O O O O
In the first 5 wells we do direct blood grouping (cell
grouping) and in remaining 3 wells we do serum grouping.
a. For all microtitration techniques, the cell suspension
required is 2% (so we prepare 2% cell suspension of
patient/donor cell and also 2% suspension of A cell,
b. Antisera used in microtiter plates are also diluted.
Roughly we dilute anti-A and anti-B seras as 1:20
dilution and anti-D as 1:10 (Dilution of antisera
depends on the titration of anti-A, anti-B and anti-D.
We select the best dilution at which the reaction occurs).
1. In the first 5 wells put one drop each of diluted (as
described above) anti-A, anti-B, anti-AB, anti -D1, and
anti-D2 (i.e. anti-D of two different companies).
Add 1 drop of patient/donor red cell suspension (2%).
2. In the next three wells (reverse grouping) put one drop
each of A cell, B cell and O cell
Add one drop of patient serum.
3. Incubate the tubes at RT for 60 minutes
In emergency cases, centrifuge the plates at 1000 rpm
for 3–4 minutes and see for agglutination.
Results: In negative reaction, the red cells trail from the
center of the well; and in positive reaction, cells remain in
the center or fall in discrete button to the bottom of well.
Direct Anti-human Globulin Test (DAT)
DAT is used to detect in vivo sensitization of red blood cells
with immunoglobulin, complement or both. A positive
DAT, with or without shortened red blood cell survival,
¾ Autoantibodies to intrinsic red blood cell antigens
¾ Alloantibodies in recipients circulation reacting with
antigens on recently transfused donor red blood cells
¾ Alloantibodies in donor plasma, plasma derivatives or
blood fractions, which react with antigens on red blood
cells of transfusion recipients
¾ Alloantibodies in maternal circulation, which cross
placenta and sensitize fetal red blood cells (HDN)
¾ Antibodies directed against certain drugs, which bind
to red blood cell membranes (e.g. Penicillin)
¾ Adsorbed proteins, including immunoglobulins, which
attach to abnormal membranes or red blood cells
modified by therapy with certain drugs, notably those of
¾ Complement components or rarely IgG bound to
red blood cells after administration of drugs such as
quinidine and phenacetin may induce drug antidrug
¾ Non-red blood cell immunoglobulins associated with
red blood cells in patient with hypergammaglobulinemia or recipients with high dose of intravenous
¾ In patient with organ transplantation, passenger
lymphocytes of donor origin produce antibodies
directed against ABO or other antigens on the
recipient’s cells, causing a positive DAT
Blood Banking (Immunohematology) 341
¾ Patients receiving ALG (anti-lymphocyte globulin) or
ATG (anti-thymocyte globulin) of animal origin may
develop a positive DAT within a few days, apparently
related to high titer heterophile antibodies in these
products and the presence of corresponding antibodies
Hemolytic Disease of the Newborn (HDN)
In HDN fetal red blood cells in vivo are sensitized with IgG
alloantibody of maternal origin thereby demonstrating a
positive DAT with cord red blood cells. The most commonly
observed HDN is due to Rho(D) incompatibility between
If the father is Rho(D) positive and the mother is Rho(D)
negative and during first pregnancy their progeny inherits
providing antigenic stimulus for the production of anti-D
antibodies. These anti-D antibodies normally will not have
any effect during the first Rh-incompatible pregnancy
unless the mother has anti-D antibodies by previous
incompatible blood transfusions.
During subsequent pregnancy, for the same couple, if
the fetus is Rho(D) positive again, the anti-D antibodies will
be activated along with the presence of anti-D antibodies
from the first pregnancy already in the circulation. Since
the IgG antibodies cross the placental barrier, these
circulating anti-D will sensitize and destroy fetal Rho(D)
positive cells. This process is demonstrated by a positive
DAT on cord red blood cells (Fig. 11.4).
A patient will demonstrate positive DAT, if serum contains
antibodies against red blood cell antigens of donor red
blood cells. Likewise antibody present in donor plasma
may also react with recipient red blood cells thereby
Other Immune Hemolytic Diseases
A positive DAT may be observed due to acquired hemolytic
anemia probably because of autoantibodies directed
against individual’s own intrinsic red blood cell antigens.
Classification of Autoimmune Hemolytic Anemia
¾ Warm autoimmune hemolytic anemia (WAIHA)
• Secondary (to conditions such as lymphoma, SLE,
¾ Cold agglutinin syndrome (CAS)
• Secondary (to conditions such as lymphoma,
Mycoplasma pneumoniae, infectious mononucleosis).
¾ Mixed type autoimmune hemolytic anemia
• Secondary (to conditions such as SLE, lymphoma).
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