Dr. Mohammed H Saiemaldahr
MED TECH DEP
Antibody Detection and Identification
The purpose of the antibody screen is to detect red blood cell
antibodies other than anti-A or anti-B.
These antibodies are called “unexpected” because only
0.3 to 2 % of the general population have positive
Once an unexpected antibody is detected, antibody
identification studies are performed to determine the
antibodies specificity and clinical significance.
All red blood cell antibodies are significance if they cause
shortened survival of antigen positive red blood cells. For
example, anti-D is a clinically significant antibody,
because it will bind to D-positive red blood cells,
resulting in immune destruction or hemolysis.
Proper detection and identification of red blood cell
antibodies is important for the selection of appropriate
blood for transfusion and in the investigation of
hemolytic disease of the new born, and immune
Antibody screening test involve testing patient’s serum
against two or three reagent red blood cell samples called
Screening cells are commercially prepared group O cell
suspensions obtained from individual donors that are
phenotype for the most commonly encountered and
clinically important red blood cell antigens.
Group O cells are used so that naturally occurring anti-A
or anti-B will not interfere with detection of unexpected
The cells are selected so that the following antigens are
present on at least one of the cell sample;
D, C, E, c, e, M N, S, s, P, Lea, Leb, K, k, Fya, Fyb, and Jkb.
An anti-gram listing the antigen makeup of each cell
provided with each lot of screening cells issued from a
manufacture. It is important that the lot number on the
screening cells matches the lot number printed-On the
anti-gram because antigen make up will vary with each lot.
The “ideal” screening cells have red cells with homozygous
expression of as many antigens as possible.
There is no requirement that screening cells contain red
cells with homozygous expression of antigens, however,
the most workers prefer that such red cells are included
in screening cells sets because many antibodies,
especially JK and M antibodies, show dosage effect and
give stronger reactions when tested against cells with
homozygous expression of their corresponding antigen.
As a result of dosage, weakly reacting antibody may not
be detected if serum samples are not tested against red
cells with homozygous expression of the their
The screening cells are available in three form
1- a single vial of no more than two donors pooled
together in one vial.
2- two vials each with a different donor.
3- 3 vials representing three different donors.
Two or three cells screening sets are required for
detection of antibodies in pre-transfusion testing.
Detection of very low levels of antibody in a
recipient’s serum is important because
transfusion of antigen-positive red cells may
result in a secondary immune response with
rapid production of antibody and subsequent
destruction of transfused red blood cells.
Red blood cells should be re-suspended by gentle
shaking or tilting the tube until the cells no longer adhere
to the sides. Agglutination is graded once the red blood
cells are re-suspended.
Agglutination reactions are routinely graded as negative
Weakly positive, and 1+ through 4+. The degree of the
positive reaction generally indicates the amount of Ab
present not its significance.
Autologous control is considered as part of the Ab
screening, it can be performed in parallel with the Ab
screen and involves testing the patient’s serum against
the patient’s red blood cells.
A positive auto-logous control is an abnormal finding
and usually means that patient has a positive direct
antiglobulin test (DAT).
Aggregation or hemolysis of test red blood cells is the
visible end point of an Ab-Ag interaction.
Test results should be read immediately after
centrifugation as delays in reading may cause elution of
antibody and false- negative test results
The first step in reading hem-agglutination reactions is
inspection of the supernatant for signs of hemolysis (red
or pink coloration).
Evaluation of the antibody screening and auto-
logous control results can provide clues and give
direction for the identification and resolution of
the Ab or Abs. The investigator should consider
the following questions;
1- In what phase (s) did the reaction (s) occur?
Low temperature: Abs of the IgM class will react best at
low temperatures and are capable of causing
agglutination of saline-suspended red blood cells
(immediate spin reading (IS). Of the commonly
encountered Abs are, anti-N, -I. and -P (IgM)
Abs of the IgG class will react best at the AHG Phase
(37°C). Of the commonly encountered Abs are; anti-Rh,
kell, Kidd, and Duffy (IgG).
Where as Lewis, M, Abs may be IgG, IgM, or a mixture of
2- Is the auto-logous control negative or positive?
A positive Ab screen and a negative auto-logous control indicate an
allo-antibodv has been detected.
A positive auto-logus control may indicate the presence of auto-
antibodies, antibody to medication or it may idiopathic
If the patient has been recently transfused, the positive
auto-logous control may be due to alloantibody coating
circulating donor red blood cells.
3- Did more than one screening cell react and if so, did
they react at the same strength and phases?
If the patient has multiple Abs, when the Abs
corresponding Ag is found on more than one screening
cell, or when the patient’s serum contains an
autoantibody, more than one screening cell will be
A single Ab specificity should be suspected when all cells
react at the same phase and strength.
Multiple Abs are most likely when cells react at different
phases and strengths and auto-antibodies are suspected
when the auto-logous control is positive.
4-Are the cells truly agglutinated or rouleaux present?
Serum from patients with multiple myeloma or who have
received high molecular weight plasma expanders
(dextran) may cause non-specific aggregation of red
blood cells known as rouleaux.
Rouleaux is not a significant finding in Ab screening
tests but it is easily confused with Ab mediated
agglutination. To differentiate between rouleaux and
1- Rouleaux cells have “a stacked coin” appearance when
2- It will not interfere with the AHG phase of testing
because the patient’s serum is washed away prior to the
addition of the AHG reagent.
3- It is observed in Ab tests containing the patient’s
serum including the auto-logous control and the reverse
4- Rouleaux is dispersed by the addition of 1 to 3 drops of
saline to the test tube.
Reagent Red Blood Cell Screening Cell
Sectional Listing of Antigens Present
F F J J L
L P I 3
D C E c e K k y y k k e S s M N AHG CC
No a b a b ea b 1 S 7
I + + 0 ++ + 0 0 + + + 0 + + + + 0 + 0 0 2+
II + 0 + +0 0 + + 0 + 0 0 + 0 + + 0 + 0 0 0 2+