

ANTIHUMAN GLOBULIN TEST Later studies revealed that the complement activity was a result of
C3 and C4.

 COMB’S Test
 Based on the principle that antihuman globulins (AHGs) obtained
ANTIGLOBULIN REAGENTS
from immunized nonhuman species bind to human globulins such
as IgG or complement, either free in serum or attached to antigens POLYSPECIFIC AHG REAGENT
on red blood cells (RBCs).  It contains antibodies to human IgG and to the C3d component of
 Used to detect the presence of antibodies against circulating red human complement.
blood cells (RBCs) in the body, which then induce hemolysis.  Contains both anti-IgG and anti C-3d (complement)
 Can be used to detect RBCs sensitized with IgG alloantibodies,
IgG autoantibodies, and complement components. MONOSPECIFIC AHG REAGENT
 Sensitization can occur either in vivo or in vitro.  Contains only one antibody specificity: either IgG or antibody to
 The use of AHG to detect in vitro sensitization of RBCs is a two- specific complement components such as C3b or C3d (i.e.
stage technique referred to as the indirect antiglobulin test (IAT). anticomplement).
 In vivo sensitization is detected by a one-stage procedure called the
direct antiglobulin test (DAT). Licensed monospecific AHG reagents in common use are anti-IgG and anti-
 The IAT and DAT remain the most common procedures performed C3b-C3d.
in blood group serology.
Anti-IgG

Antihuman: antibodies against human antigens  no anti-complement activity; specific for the Fc fragment of the
Globulin: all antibody molecules are globulins gamma heavy chain of the IgG molecule.

Antihuman Globulin: an antibody directed against the Fc portion of human Anticomplement

antibodies and/or complement components.
 reactive against only the designated complement components and
contain no activity against human immunoglobulins. Monospecific
DIRECT ANTIGLOBULIN TEST anti-complement reagents are often a blend of monoclonal anti-
 Detects the presence of antibodies attached directly to the RBCs, C3b and monoclonal anti-C3d.
which takes place by washing a collected blood sample in saline to
isolate the patient’s RBCs; this procedure removes unbound
antibodies that may otherwise confound the result.

INDIRECT ANTIGLOBULIN TEST

 Used to detect unbound antibodies to RBCs, which may be present
in the patient’s serum.

HISTORY

1945
Coombs and associates described the use of the antiglobulin test for the
detection of weak and non-agglutinating Rh antibodies in serum.

1946
 Coombs and coworkers described the use of AHG to detect in vivo
sensitization of the RBCs (later called the direct antiglobulin test
[DAT]) of babies suffering from HDN.
 The first of the Kell blood group system antibodies and the
associated antigen were reported only weeks after Coombs had
described the test.

1947
Coombs and Mourant demonstrated that the antibody activity that detected Rh
antibodies was associated with the anti-gamma globulin fraction in the
reagents.

1951
Dacie presented the first indication that there might be another antibody
activity present that influences the final reaction.
PRINCIPLE OF ANTIGLOBULIN TEST
1. Antibody molecules and complement components are globulins.
2. Injecting an animal with human globulin stimulates the animal to
1957 produce antibody to the foreign protein (i.e. AHG). Serologic tests
employ a variety of AHG reagents reactive with various human
 Dacie and coworkers published data showing that the reactivity of
globulins, including anti-IgG antibody to the C3d component of
AHG to cells sensitized with warm antibodies resulted from anti-
human complement, and polyspecific reagents that contain both
gamma globulin activity, whereas non-gamma globulin component
anti-IgG and anti-C3d activity.
was shown to be beta globulin and had specificity for complement.
 AHG reacts with human globulin molecules, either bound to RBCs or free in
serum. Washed RBCs coated with human globulin are agglutinated by AHG.

DIRECT ANTIGLOBULIN TEST / DAT

 It detects in vivo sensitization of RBCs with IgG or complement
components.
 Clinical conditions that can result in in vivo coating of RBCs with
antibody or complement are:
o Hemolytic Disease of the Newborn (HDN) Maternal
antibody coating fetal RBCs.
o Hemolytic transfusion Reaction (HTR) - Recipient antibody
coating donor RBCs.
o Autoimmune and Drug -induced Hemolytic anemia (AIHA)
– Autoantibody coating individual RBCs.

The antibody must be removed from the RBCs for accurate phenotyping.
Other techniques can be used to remove antibody from patient’s RBCs.
These include the following:
INDERECT ANTIGLOBULIN TEST
1. Chloroquine diphosphate
 It determines in vitro sensitization of RBCs and is used in the
2. EDTA- glycerine
following situation:
3. Murine monoclonal antibodies.
 Detection of incomplete (non-agglutinating) antibodies to potential
donor’s RBCs (compatibility testing) or to screening cells
(antibody screen) in serum.
 Determination of RBC phenotype using known antisera (e.g. Kell
typing, Weak D testing).
 Titration of incomplete antibodies.

A positive DAT may occur without clinical manifestations of immune-

mediated hemolysis. The AABB Technical Manual states that “a positive
DAT alone is not diagnostic”. The interpretation of the significance of this
positive result requires knowledge of the patient’s diagnosis; recent drug,
pregnancy, and transfusion history; and information on the presence of
acquired or unexplained hemolytic anemia.
FACTORS AFFECTING THE ANTIGLOBULIN
Answering the following questions before investigating positive DAT for TEST
patients other than neonates will help determine what future testing is
1. Ratio of serum to cell (2:1)
appropriate:
2. Reaction medium (Saline / LISS
 Is there evidence of in vivo hemolysis?
a. Albumin
 Has the patient been transfused recently? If so, did the patient b. Low ionic strength solution (LISS)
receive blood products or components containing ABO- c. Polyethylene glycol (PEG)
incompatible plasma? 3. Temperature (should be 37C?)
 Does the patient’s serum contain unexpected antibodies? 4. Incubation time (60mins)
 Is the patient receiving any drugs? 5. Washing of red cells (3-6 times)
 Is the patient receiving antilymphocyte globulin or antithymocyte 6. Saline for washing
globulin? 7. Addition of AHG (should be added)
 Is the patient receiving intravenous immune globulin (IVIG) or 8. Centrifugation for reading (speed and time)
intravenous Rh immune globulin (IV RhIG)?
 Has the patient received a marrow or other organ transplant?

FALSE POSITIVE
1. Improper specimen (refrigerated, clotted) may cause in vitro
complement attachment.
2. Overcentrifugation and overreading.
3. Centrifugation after the incubation phase when PEG or other
positively charged polymers are used as an enhancement medium.
4. Bacterial contamination of cells or saline used in washing.
 5. Dirty glassware. A process that detects RBC antigen-antibody reactions by means of using a
6. The presence of fibrin in the test tube may mimic agglutination. chamber filled with polyacrylamide gel. The gel acts as a trap; free
7. Cells with a positive DAT will yield a positive IAT. unagglutinated RBCs form pellets in the bottom of the tube, whereas
8. Polyagglutinable cells. agglutinated RBCs are trapped in the tube for hours. Therefore, negative
9. Saline is contaminated by heavy metals or colloidal silica. reactions appear as pelletes in the bottom of the microtube, and positive
10. Using a serum sample for a DAT (use EDTA, ACD, or CPD reactions are fixed in the gel.
anticoagulated blood).
11. Samples collected in gel separator tubes may have unauthentic Types of Gel Test
complement attachment. 1. NEUTRAL
12. Complement attachment when specimens are collected from  main application: antibody screening and identification
infusion lines infusing dextrose solutions. with enzyme-treated or untreated RBCs and reverse
13. Preservative- dependent antibody directed against reagents. ABO typing.
2. SPECIFIC
FALSE NEGATIVE  use a specific reagent incorporated into the gel and are
1. Inadequate or improper washing. useful for antigen determination.
2. Failure to wash additional times when increased serum volume is 3. ANTIGLOBULIN
used.  The Gel Low ionic Antiglobulin Test (GLIAT) is a
3. Contamination of AHG extraneous protein (i.e. glove, wrong valuable application of the gel test and may be used for
dropper). the IAT or DAT. AHG is incorporated into the gel. The
4. High concentration of IgG paraproteins in test serum. detection of unexpected antibodies by GLIAT compares
5. Early dissociation of Bound IgG from RBCs due to interruption in favorably with conventional AHG methods and
testing. provides a safe, reliable, and easy-to-read AHG test.
6. Early dissociation of bound IgG from RBCs due to improper
testing temperature (i.e. saline or AHG too cold or hot).
7. AHG reagent nonreactive because of deterioration or neutralization
(improper reagent storage).
8. Excessive heat or repeated freezing and thawing of test serum.
9. Serum nonreactive because of deterioration of complement.
10. AHG reagent, test serum or enhancement medium not added.
11. Undercentrifuge or over centrifuge
12. Cell suspension either too weak or too heavy
13. Serum: cell ration not ideal.
14. Rare antibodies are present that are only detectable with
polyspecific AHG and when active.
1. complement is present.
15. Low pH of saline.
16. Inadequate incubation conditions in the IAT.

MODIFIED AND AUTOMATED ANTIGLOBULIN

TEST TECHNIQUE

Low Ionic Polybrene Technique

The technique relies on low ionic conditions to rapidly sensitize cells with
antibody. Polybrene, a potent rouleaux-forming reagent, is added to allow the
sensitized cells to approach each other and permit cross-linking by the
attached antibody. A high ionic strength is then added to reverse the rouleaux,
however if agglutination is present it will remain.

Enzyme Linked Antiglobulin Test (ELAT)

An RBC suspension is added to microtiter well and washed with saline. AHG,
which has been labeled with an enzyme, is added. The enzyme-labeled AHG
will be of IgG-sensitized RBCs. Excess antibodies are removed, and enzyme
substrate is added. The amount of color produced is measured
spectrophotometrically and is proportional to the amount of antibody present.
The optical density is usually measured at 405 nm. The number of IgG
molecules per RBC can also be determined from this.

Solid Phase Technology

It may be used for performing antiglobulin tests. Several different techniques
have been reported using either test tubes or microplates.
With the availability of microplates readers, this modification lends itself to
the introduction of semiautomation.
Direct and indirect tests can be performed using solid-phase methodology.

Gel Test