IMMUNOHEMTOLOGY - RBC reagents, enhancement reagents &
Lecture 7 AHG reagents
ANTIBODY SCREENING AND
- agglutination
IDENTIFICATION
a. Incubation at 37C
b. Enhancement media
DETECTION AND IDENTIFICATION OF ANTIBODY SCREENING
ANTIBODIES
1.Reagent Red Blood Cells – screening cells,
commercially prepared group O cell suspension
Routine pre transfusion testing includes
obtained from individual donors that are
1.ABO & Rh typing phenotyped for the most commonly encountered
and clinically important RBC antigens.
2.Antibody screening
3.Compatibility testing
Screening cells are available in three forms:
1.Single vial with no more than two donors
pooled together in one vial
Antibody detection
2.Two vials- each from a different donor
Investigate potential hemolytic transfusion
reactions 3.Three vials representing 3 donors.
Immune hemolytic anemia
Detect & monitor those who are at risk of HDFN ● Pooled cells are less sensitive but may
be used for the detection of antibodies
in donor units.
● Two cell or three cells screening sets
Unexpected Ab can be
are required for the detection of
Alloantibodies antibodies in pretransfusion testing.
Auto antibodies
Once an unexpected antibody is detected, Ab
Idenification studies are performed to determine
the antibody’s specificity and speculate its
clinical significance
Alloantibodies – produced in response to red
blood cells simulation through transfusion,
transplantation or pregnancy.
Ø naturally occurring antibodies
Ø clinically significant antibodies
Autoantibodies – directed against antigens 2. Enhancement reagent – solutions added to
expressed serum and cell mixtures to promote antigen-
antibody binding or agglutination.
Antibody Screen - potentiators
I.Tube method – indirect antiglobulin test --- increase the ability to detect lower levels
of antibody.
--- most widely used are
C. 22% bovine albumin – serum albumin
A. Low Ionic Strength Saline protein derived from a cow
B. Polyethylene glycol
C. Bovine albumin - allows the RBCs to
D. AHG reagent approach each other
and increase the
chances of
agglutination.
- Used in blood banking
A. Low Ionic Strength Solution – as an intensification and
contains glycine suspension medium
- increases the uptake of antibody into the - Suitable for antibody
RBC during the sensitization phase titration
- potentiate reactions between
antibodies and red cells
D. Antihuman Globulin Reagents – use to
- a solution of glycerine and albumin promote agglutination of RBCs sensitized with
IgG or complement molecules.
Coombs Control Red Blood Cells - RBCs
coated with human IgG antibody. They are
usually prepared by incubating D-positive
RBCs with potent anti-D.
--- used to ensure that AHG tests with
negative results are not false negative
3. Gel Method – The patient’s serum or
plasma specimen & screen cells are added to
a reaction chamber that sits above a gel.
- up to six chambers/gel microtubules in a
plastic card
- incubated at 37C for 15 mins – 1 hour
- centrifuge the card for 10 mins
- the gel contains IgG
- if sensitization occurred, the anti IgG will
react with the antibody-coated RBCs, resulting
in agglutination
B. Polyethylene Glycol – PEG - if no agglutination, RBCs will form a pellet at
- removes the the bottom of the microtubule.
water from the test system,
thereby concentrating any
antibodies present.
- more sensitive than
LISS, albumin, NSS
- increases rate of
detection of clinically significant antibodies
- if no sensitization occurred, the indicator cells
form a pellet in the bottom of the well.
-- RBC adherence is used as endpoint
instead of hemagglutination.
Advantages of gel method
Grading Reactions – agglutination or hemolysis
1.It is as sensitive as tube test method of the test RBCs is the visible endpoint of an
2.Consistent standardized interpretation of antigen antibody interaction.
reaction ØTest results should be evaluated immediately
3.Reactions are more stable and can be after centrifugation because delays may cause
evaluated 24 hours after they are performed false negative tests results
4.Time saving – ability to automate many of the Agglutination reactions are graded as
pipetting & reading steps Negative, weakly positive, and 1+ and 4+.
5.Hands on time required to perform the test is
reduced
Autologous control – part of antibody
screening
4. Solid Phase Adherence Method – applied
-- performed in parallel with the antibody
to blood group serology for RBC antibody
screen
detection and identification.
-- involves testing the patient’s serum against
- Immucor’s Capture – R
patient’s rbc.
- RBC antigens coat microtiter wells
- patient serum or plasma is added
LIMITATIONS
- incubation at 37C
1.It will not detect antibodies when the antibody
- If sensitization occurs, formation of diffused titer has dropped below the sensitivity for the
pattern in a well screening method employed.
2.Antibodies showing dosage may not be
detected if none of the screen cells have Neutralization – substances in the body and in
homozygous expression of the target antigen. nature that have antigenic structures similar to
RBC antigens.
- the neutralizing substance inhibits
FACTORS THAT MAY INFLUENCE THE reactions between the antibody and panel
SENSITIVITY OF THE ANTIBODY SCREEN RBCs.
Cell–to–serum ratio - uses soluble antigen to inhibit the reactivity
Temperture & Phase of Reactivity of certain antibodies in hemagglutination
assays.
Length of Incubation
pH
SOURCES OF SUBSTANCES FOR
ADDITIONAL TECHNIQUES FOR NEUTRALIZATION OF CERTAIN
RESOLVING ANTIBODY IDENTIFICATION ANTIBODIES
Antibody Source
Selected Cell Panels of Neutralizing Substance
Enzymes Anti P1
Neutralization Hydatid cyst fluid
Anti Lewis
Adsorption
plasma, serum, saliva
DAT & Elution Techniques
Antibody Titration
Adsorption – procedure to bind antibodies to
Selected Cell Panels – additional cells from the red cells in order to remove them from the
different panel plasma and better analyse the antibodies that
might remain behind.
- useful when a patient has a known
antibodies & want to know other antibodies - In the adsorption method, the antigen-
present. antibody complex is composed of solid
precipitates and is removed from the test
Enzymes – modify the RBC surface by system by centrifugation
removing sialic acid residues and by denaturing
or removing glycoproteins. - serum is tested against RBC panel
- destroy certain antigens and enhance the
expression of others
oDirect Antiglobulin Test – use to detect the in
vivo sensitization of RBCs.
Enzymes - useful in antibody identification ØRBCs are washed thoroughly
since they increase detection of some weakly ØAHG reagent is added
reactive antibodies
ØAgglutination is observed if IgG antibodies or
- it also helps separate and complement are coating the RBCs
identify multiple antibodies
ØWhen IgG Ab are detected, dissociate the
- it provides clues to the antibodies from the RBC surface to allow for
antibody identity. identification
Elution – techniques used to release,
concentrate and purify antibodies.
- the antibody freed into a solution is known
as the eluate, which may be tested against the
RBC panel to identify the antibody.
An elution may be indicated to aid in the ff:
1.Diagnosis of AIHA ( Autoimmune Hemolytic
Anemia
2.Diagnosis of HDFN
3.Identification of specificity when multiple
antibodies exist in a patient’s serum or plasma
4.Phenotyping red cells with a positive DAT
SPECIAL PROBLEMS IN ANTIBODY
IDENTIFICATION:
ØMultiple antibodies
ØAntibodies to high frequency antigens
ØAntibodies to low frequency antigens
ØCold reactive autoantibodies
ØWarm antibodies
ØAntibodies to reagents and drugs
