1. Antibody screening tests patient serum against reagent red blood cells to detect unexpected antibodies that could destroy transfused donor cells. 2. Screening cells must contain many common antigens and include some cells with homozygous antigen expression to detect weakly reacting antibodies. 3. A positive antibody screen requires antibody identification testing to determine the antibody specificity so that antigen-negative blood can be transfused.

1. ANTIBODY SCREENING
DR NAGLAA MAKRAM
CONSULTANT CLIN ICAL PATHOLOGY
BGH

2. Antibody screens are used for:
Patients needing a transfusion
Cases of transfusion reactions
Blood and plasma donors
Pregnant women

3. Purpose
 To ensure optimal survival of transfused red
cells in recipients.

4. Routine pretransfusion testing
 ABO Group
 Rh (D) type
 Antibody screen for irregular antibodies
 IAT with donor’s RBC’s

5. Antibody screen
 Principle: Antibody screen test is done using
patient’s serum and antibody screen cells to
try to detect unexpected antibodies that are
capable of destroying transfused donor cells
in vivo.
 These antibodies are called “unexpected”
because only 0.3 to 2 % of the general
population have positive antibody screen.

6. Unexpected antibodies are a result of red cell
stimulation (transfusion, HDN)
Unexpected antibodies may be:
Clinically significant (IgG)
Not clinically significant (IgM)
Clinically Significant antibodies defined as:
One that shortens the survival of transfused red
cells
Cause Hemolytic disease of the newborn (HDN)
Hemolytic Transfusion reaction

7. Key Concepts
In blood banking, we test “knowns” with
“unknowns”
When detecting and/or identifying antibodies,
we test patient serum (unknown) with reagent
RBCs (known)
Known:Unknown:
Reagent RBCs+patient serum
Reagent antisera+patient RBCs

8. Characteristics of screen cells
 Group O cells (so that naturally occurring
anti-A or anti-B will not interfere with
detection of unexpected antibodies. )
 Known antigenic content
 They should be positive for all common blood
group antigens
 They should come from donors who are
homozygous for genes that produce antigens
showing dosage.

9. 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
corresponding antigen. (Rh, Duffy, Kidd).

10. Examples
Fya
Fyb
SCI + + 2+
SCII 0 + 4+
Fya
Fyb
SCI + 0 4+
SCII 0 + 0
If patient’s serum contains
anti-Fya
, there will be a
stronger reaction
because SCI is
homozygous for the Duffy
antigen
In this case, the person
has anti-Fyb
. The antibody
reacts weaker with SCI
(heterozygous) and
stronger with SCII
(homozygous)

11. 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.
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
.
Screening cells may also contain low incidence antigens like
V, Cw
, and Kpa
The presence of these antigens is not required for screening
cells

12. D
Fyb
Lea
Jkb
Jka
s
S
NM
k
K
e
E
c
C
B
A
Lua Fyb
Fya
Leb

13. Screening cells come with a sheet of paper called an antigram.
Screening cells are an already prepared 2-5% RBC suspension
An antigram (2 or 3 cells) will list the antigens present in each vial
A reaction to one or more cells indicates the presence of an
unexpected antibody

14.  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.

15. Reagent Red Blood Cell Screening Cell
Sectional Listing of Antigens Present
No
cell
D C E c e K k
F
ya
F
y
b
J
ka
J
k
b
L
ea
L
eb S s M N
P
1
I
S
3
7
AHG CC
I + + 0 + + + 0 0 + + + 0 + + + + 0 + 0 0 2+
II + 0 + + 0 0 + + 0 + 0 0 + 0 + + 0 + 0 0 0 2+

17. Screening Cells:
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.
single-donor vials offer increased sensitivity
Two or three cells screening sets are required for detection of antibodies
in pre-transfusion testing.

18. Antibody ID Testing
A tube is labeled for each of the panel cells
plus one tube for AC:
AC
1 2 3 4 5 6 7 8 9 10 11
 1drop of each panel cell
+
2drops of the patients serum 

19. IS Phase
Perform immediate spin (IS) and grade
agglutination; inspect for hemolysis
Record the results in the appropriate space
as shown:
2+
0
0
Last
tube

20. Auto-logous Control.
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).

21. Autocontrol
Screen
Antibody
Panel (w/AC)
If Positive
IfPositive
DAT
The AC and DAT can help in
determining whether the antibodies
are directed against the patient’s
cells or transfused cells (allo or
autoantibody).

22. Interpretation

23. Remember Landsteiner’s Rule
Individuals DO NOT make allo-
antibodies against antigens they
have

24. Multiple antibodies
Multiple antibodies may be more of a
challenge than a single antibody
Why?
–Reaction strengths can vary
–Matching the pattern is difficult

27. Grading Reactions

28. Grading Reactions.
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).

29. 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 (no agglutination).
Weakly positive, and 1+ through 4+. The degree of the positive reaction
generally indicates the amount of Ab present not its significance.

30. Interpretation
 Agglutination or hemolysis at any stage of testing is a positive
test result, indicating the need for antibody identification
studies.
 However, evaluation of the antibody screen and autologous
control results can provide clues and give direction for the
identification and resolution of the antibody or antibodies.
 The investigator should consider the following questions:
1. In what phase(s) did the reaction(s) occur?
2. Is the autologous control negative or positive?
3. Did more than one screening cell sample react, and, if so,
did they react at the same strength and phase?
4. Is hemolysis or mixed-field agglutination present?
5. Are the cells truly agglutinated, or is rouleaux present?

31. Interpretation
1. In what phase(s) did the reaction(s) occur?
Antibodies of the IgM class react best at low
temperatures and are capable of causing
agglutination of saline-suspended RBCs (immediate
spin reading).
Antibodies of the IgG class react best at the AHG phase.
Of the commonly encountered antibodies,
 anti-N. anti-I, and anti-PI are frequently IgM,
 whereas those directed against Rh. Kell. Kidd, and
Duffy antigens are usually IgG.
 Lewis and M antibodies may be IgG, IgM, or a mixture
of both.

32. Colder reacting antibodies are therefore considered
insignificant and just cause interference when
performing lab testing
The only important thing to remember concerning
cold antibodies is that they may bind complement if a
persons body temperature becomes low
 Open-heart surgery
 Hypothermia
 If a lab uses an AC with the screen and it is
positive, they may run a DAT (patient cells +
AHG) to detect in vivo coating

33. Positive Coombs

34. Negative Coombs

35. 2. Is the autologous control negative or positive?
 A positive antibody screen and a negative autologous
control indicate that an alloantibody has been detected.
 A positive autologous control may indicate the presence
of autoantibodies or antibodies to medications.
 If the patient has been recently transfused, the positive
autologous control may be caused by alloanti­body
coating circulating donor RBCs.
 Evaluation of samples with positive autologous control or
DAT re­sults is often complex and may require a lot of
time and experience on the part of the investigator.

36. 3. Did more than one screening cell sample react,
and, if so, did they react at the same strength
and phase?
 A single antibody specificity should be suspected when all
cells react at the same phase and strength.
 Multiple antibodies are most likely when cells react at
different phases and strengths.
 autoantibodies are suspected when the autologous
control is positive.

37. 4. Is hemolysis or mixed-field agglutination
present?
 Certain antibodies­such as anti­Lea
, anti­Leb
, anti P+P1
+Pk
,
and anti­Vel­are known to cause in vitro hemolysis.
 Mixed­field agglutination is associated with anti­Sda
and
Lutheran antibodies.

38. 5. Are the cells truly agglutinated, or is rouleaux
present?
 Serum from patients with altered albumin­to­globulin ratios
(e.g., patients with multiple myeloma) or who have received
high­molecular­weight plasma expanders (e.g.. dextran) may
cause nonspecific aggregation of RBCs, known as rouleaux.
 Rouleaux is not a significant finding in antibody screening
tests, but it is easily confused with antibody­mediated
agglutination.
 Knowledge of the following characteristics of rouleaux helps in
differentiation between rouleaux and agglutination:
a. Cells have a "stacked coin" appearance when viewed
microscopically.
b. Rouleaux is observed in all tests containing the patient's

39. c. Rouleaux does not interfere with the AHG phase of testing
because the patient's serum is washed away prior to the
addition of the AHG reagent.
d. Unlike agglutination, rouleaux is dispersed by the addition of 1
to 3 drops of saline to the test tube.

40. Limitations of pretransfusion testing
 Patient’s antibody is too week to be detected.
 Patient misidentification
 Hemolysis before entering the patient
 Non-hemolytic reactions
 Adverse reactions
 Transfusion Transmissible diseases
 cannot detect all such antibodiescannot detect all such antibodies.

41. Limitations
 Antigens with frequencies of less than 10 percent
(e.g., Cw
. Lu­, Kpa
) are not usually represented on
screening cells, and, as a result, their corresponding
antibodies are not detected in routine screening
tests.

42.  antibody levels decrease over time when the individual is
no longer exposed to the corresponding antigen.
 If the level of an RBC antibody drops too low, results of
antibody screening tests and crossmatches will appear
negative and may lead to transfusion of donor units that
carry the corresponding antigen.
 Re exposure to the RBC antigen will elicit a secondary
immune response, resulting in a dramatic increase in the
antibody titer and possible immunologic destruction of the
transfused RBCs.
 this is called a delayed hemolytic transfusion reaction
(DHTR) because it occurs days or weeks after the
transfusion.

43. Patient History
 GET THE HISTORY!!
– Mixed red cell populations from a previous transfusion
can remain for up to 3 months
– Patient may have come from another hospital
– Some diseases are associated with antibodies
– Some antibodies occur at a higher frequency in some
races
– Get diagnosis, age, race, etc…

44. Potentiators
 Used in antibody detection and
identification to enhance antigen­antibody
reaction
– Saline (may only enhance if incubated long
time)
– Low-ionic strength solution (LISS)…common
– Bovine serum albumin (BSA)
– Polyethylene glycol (PEG)
– Proteolytic enzymes (can destroy some antigens)

45. Potentiators
Albumin Serum/cell mixture should incubate at least
20 - 30 minutes;
doesn’t enhance warm autoantibodies
LISS Incubation time of 10 minutes;
lowers ionic strength allowing better
reaction; sensitive and quick!
PEG
Polyethylene glycol
Enhances warm autoantibodies;
does not react well with insignificant
antibodies (IgM)

46. Advantages
 Screen cells can detect antibodies more
effectively than can donor cells because they
come from donors who are homozygous.

47. Specimen requirements for
Pretransfusion testing
 Special Identification Procedures
If the patient was transfused or pregnant in the
past three months, the blood specimen used
for pretransfusion testing must be no older
than 3 days.

48. Selecting Blood for Transfusion
For a patient with clinically significant Antibodies (37o
C and
IAT(
Red cell should be tested and be negative for the
appropriate antigen
Even if Ab. Is no longer detectable to prevent a
secondary immune response
An antiglobulin cross-match is required
The absence of Ag should be confirmed with a potent
commercial antisera
FDA requires use of licensed (commercial( reagents

49. Selecting Blood for Transfusion
When rare type is needed
–High- Incidence
–Multiple antibodies frequency of random donors
negative for each antigen should be used,
–Example: serum contains anti-C, Fya
and s among
random donors
18%CNeg
34%Fya
Neg
45%SNeg

50. Selecting Blood for Transfusion
The Frequency of compatible units would be
0.18×0.34 ×0.45= 0.028
If patient is group O then 45% of random
donors are group O then 0.028 ×0.45=1.3%
of random donors would be compatible with
the patient serum.

51. Requisitions
 Signatures of blood collector & nurse
 Patient identification
 Name of the attending physician
 Birth date
 Past history
 Diagnosis
 Blood products required

52. Type and
Screen
Cross-match
and Save
vs

53. Type and Screen
 Once upon a time (in bloodland) all donors
were crossmathed by IAT, even if the
antibody screen was negative.
 In addition, for many types of surgeries a
high percentage of the donor blood that
was crossmatched and held for patients
was never used.
 This is wasteful to both the time and
money.

54. Advantages of Type and Screen
 Better use of blood donor, as it is not tied up
by being cross-matched and held
 More efficient service of patients

55. Right blood
To the
Right patient
At the
Right time

56. savesblood lives
Safe

57. Example 1
Screening
Cell
IS 37°C AHG CC*
I 0 0 0 
II 0 0 2+ ND
•IgG antibody
•Single specificity
•CC: Coombs Control Red Blood Cells
•ND: Not Done

58. Example 2
Screening Cell IS 37°C AHG CC
I 0 0 3+
II 0 2+ 3+
•IgG antibody
•Multiple specificities

59. Example 3
Screening Cell IS 37°C AHG CC
I 1+ 0 0 
II 3+ 0 0 
•IgM antibody
•Single specificity showing
dosage
Neg AHG, add
CC

60. Example 4
Screening Cell IS 37°C AHG CC
I 0 0 2+
II 0 0 2+
•IgG antibody
•Allo or autoantibody?
)don’t know without further testing(

64. Possible results
 All antibody screen cells are negative
 One or more screen cells are positive
 Screen cells negative and donor positive in
IAT phase