MONOSPECIFIC

COOMBS’ TEST
Usi Sukorini
Departement of Clinical Pathology & Laboratory Medicine
Faculty of Medicine, Nursing & Public Health
UGM
2021
 Coombs Test (Antiglobulin test)
 The antiglobulin test is a method of demonstrating the presence
of antibody or complement bound to red blood cell (RBC)
membranes by the use of anti-human globulin to form a visible
agglutination reaction
 This test is used to detect free and sensitized blood group
antibodies
 Y

Y Y

Coombs test detect Coombs test detect Coombs test detect

sensitized antibodies free antibodies free and sensitized
&/ complement antibodies &/
complement
 Principle of Antiglobulin test
• Antibody molecules and complement components are globulins
• Injecting an animal with human globulin stimulates the animal to produce antibody
to the foreign protein (i.e., AHG)

• Serologic tests employ a variety of AHG reagents reactive with various human
globulins, including anti-IgG antibody to the C3d component of human
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
 Types of Coombs Test

Direct Coombs Test (DCT)/

Direct Antiglobulin Test (DAT)
Coombs test
(Antiglobulin Test)
Indirect Coombs Test (ICT)/
Indirect Antiglobulin Test (IAT)
 Direct Antiglobulin Test

• Principle:
The direct antiglobulin test (DAT) detects in vivo sensitization of
RBCs with IgG or complement components
• Application
• Hemolytic disease of the fetus and newborn (HDFN)
• Hemolytic transfusion reaction (HTR)
• Autoimmune and drug-induced hemolytic anemia (AIHA)
Direct Antiglobulin Test
 Indirect Antiglobulin Test
• The IAT is performed to determine in vitro sensitization of RBCs and is used
in the following situations:
• Detection of incomplete (nonagglutinating) antibodies to potential
donor RBCs (compatibility testing) or to screening cells (antibody
screen) in serum
• Determination of RBC phenotype using known antisera
• Titration of incomplete antibodies
Indirect Antiglobulin Test
rodak
 Indication of Antiglobulin test
• The antiglobulin test, also known as a Coombs test, has high
diagnostic value in transfusion medicine
• This test is mainly used to investigate:
• hemolytic disease of the fetus and newborn (HDFN)
• autoimmune haemolytica anemia (AIHA)
• hemolytic transfusion reactions (HTRs)
 Antihuman Globulin Reagents
• When RBCs become coated with antibody, complement, or both, but
do not agglutinate in regular testing, special reagents are needed to
produce agglutination  antihuman globulin reagents
IgG

IgG
C3d

C3d
 Anti-human Globulin Reagents

Polyspecific reagents Monospecific reagents

• anti-IgG and • anti-IgG or anti-C3d
IgG
• anti-complement C3d/
C3b
IgG C3d
Polyspecific AHG can determine if RBCs
have been sensitized with IgG antibody or
Monospecific AHG reagents react only with
complement (components C3b or C3d) or
RBCs sensitized with IgG or complement
both
 Monospecific anti-IgG reagents

• Reagents labeled anti-IgG contain no anticomplement activity

• Anti-IgG reagents contain antibodies specific for the Fc fragment of the
gamma heavy chain of the IgG molecule
 Complement
• Complement is an important blood component that affects detection in
pretransfusion compatibility tests
• The content of the complement system is relatively stable in the serum of
healthy people and is irrelevant to the immunostimulatory effects of
foreign antigens
• Complement activation can play an important role in cell lysis in vivo
• Changes in the complement content and activity can occur in some diseases
• The detection of which show important clinical value for assessment of
immune status and diagnosis and treatment of disease

Wang et al., 2020

Complement Classical Pathway
(CCP)
Mechanism of hemolysis
in warm antibody
autoimmune hemolytic
anemia

Berentsen et al., 2019

 Monospecific anticomplement reagents

• Anticomplement reagents, such as anti-C3b,-C3d reagents, are reactive

against only the designated complement components and contain no
activity against human immunoglobulins
• Monospecific anticomplement reagents are often a blend of monoclonal
anti-C3b and monoclonal anti-C3d
• Monospecific anti-IgG reagents were applied in approximately 70% of
laboratories in the United States in 1998, which was increased by
approximately 19% compared with 1995 (Shulman et al., 2001)

• Certain irregular antibodies, named complement-fixing antibodies, can

only be detected in the presence of polyspecific AHG reagent and
activated complement
• This type of antibody includes anti-Jka and anti-Jkb antibodies of the Kidd
system, IgM-type anti Lea antibodies of the Lewis system and a few anti-Fy
antibodies of the Duffy system
• However, these antibodies are rarely clinically significant
Wang et al., 2020
 Anticomplement

• Some antibodies “fix”

complement components
to the RBC membrane
after complexing of the
antibody with its
corresponding antigen
 Positive polyspecific DAT
• Positive results are monitored by a DAT panel using monospecific anti-IgG
and anti-C3d to determine the specific type of protein sensitizing the cell
• In an effort to save valuable tech time, some institutions run polyspecific
and monospecific reagents at one time as well as a saline control
• In warm AIHA, including drug-induced hemolytic anemia, the RBCs may be
coated with IgG or C3d, or both

R
• Most IgM agglutinin agglutinate RBCs and cause complement sensitization
at low temperatures
• These IgMs can detach from RBCs at high temperatures or by repeated
washing, while the complement still adheres to the RBCs
• Thus, the use of polyspecific AHG reagents in serological tests will result
in a positive reaction through anti-C3d component binding to complement
on RBCs but cannot cause RBC destruction and hemolysis
• If monospecific anti-IgG AHG reagents are used instead of polyspecific
AHG reagents, occurrence of false positives will be avoided

Wang et al., 2020

 INFLUENCE OF COMPLEMENT ADHERING TO RBCS IN THE USE OF
AHG REAGENTS AND THEIR MECHANISMS

• Under in vivo and in vitro experimental conditions, complement adheres to

the RBC membrane through one or two of the following mechanisms:
• First, antibodies specifically bind to the RBC membrane to form antigen-
antibody complexes that further activate complement to adhere to the RBC
membrane
• Second, immune complexes nonspecific to RBC antigen are present in the
plasma and activate complement components non specifically adhering to
the RBC membrane

Wang et al., 2020

 Antihuman globulin reagent for Ab screening (1)

• Both serum and plasma sample can be used for antibody screening and
identification, but if the detection target is an antibody that needs to be
confirmed by activating complement, such as Kidd system antibodies, serum
samples and polyspecific anticomplement reagents are recommended

• In the test for irregular antibody screening and identification, some IgMs that are
reactive at room temperature (such as anti-Lea , anti-Leb , anti-I and anti-IH
antibodies) are considered clinically insignificant and negligible antibodies, and
thus, there is no need to provide corresponding antigen negative donor RBCs to
recipients during blood transfusion

Wang et al., 2020

 Antihuman globulin reagent for Ab screening (2)

• To avoid the insignificant positive results caused by the above antibodies,

monospecific anti-IgG reagents should be used for irregular antibody screening
and identification
• For detection of immune antibodies in patients with cold autoimmune hemolytic
anemia or cold agglutinin disease, the screening cells and patient plasma are
normally incubated at 37°C before the IAT to exclude the screening cells coated
with complement components (mostly C3d) that are activated by cold IgMs
• Using monospecific anti-IgG reagent instead multispecific AHG reagents is
helpfull

Wang et al., 2020

 Antihuman globulin reagent for Ab screening (3)

• If the use of polyspecific AHG reagents and monospecific anti-IgG reagents

exhibits positive results and negative results, respectively, the serum samples
should be incubated at 56°C for 30 minutes to inactivate complement before
irregular antibody screening and identification

Wang et al., 2020

 Antihuman globulin reagent for crossmatching (1)

• The main purpose of crossmatching is to test whether there are specific antigen-
antibody reactions between donor and recipient blood.
• In addition to crossmatching in saline medium, a crossmatching test should also be
conducted to detect IgG-type alloantibodies in AHG medium, which could adopt
the same procedure as antibody screening and identification.
• However, patients with certain diseases, such as mycoplasmal pneumonia,
infectious mononucleosis, malignant lymphoma, viral infection and autoimmune
diseases, easily produce high titer of cold agglutinin

Wang et al., 2020

 Antihuman globulin reagent for crossmatching (2)

• These cold agglutinins in the serum of the above mentioned patients can
cause RBC agglutination and bind to complement, resulting in hemolysis at
low temperatures; when the ambient temperature rises, the antibodies
detach from RBCs while complement remains on the surface of RBCs, which
results in false-positive reactions in crossmatching

Wang et al., 2020

 Antihuman globulin reagent for crossmatching (3)

• Because of the complement C3d coating on patient RBCs, which often leads to strong positive
results in direct AHG tests, RBCs should be washed with saline at 37°C to remove IgM-type
cold agglutinins.
• The interference of complement in the crossmatching test can also be avoided by using
monospecific anti-IgG reagent
• To detect the presence of IgG alloantibodies, the crossmatching temperature should be strictly
controlled at 37°C to eliminate the interference of cold agglutinins
• When using an AHG test for crossmatching, the recipient's serum and donor's RBCs should be
mixed well and incubated at 37°C for 30 minutes before detection
• When the sample is serum rather than plasma, the use of monospecific anti-IgG reagents is
preferable to polyspecific AHG reagents
Wang et al., 2020
 MONOSPECIFIC COOMBS TEST
• When positive DAT is detected by polyspecific reagent  should be
followed by DAT testing using monospecific reagent

• The use of monospecific anti-IgG reagents can avoid the false-positive

reactions caused by clinically insignificant cold antibodies, while the use of
polyspecific reagents can prevent missed detection of complement-
fixing antibodies
Evaluation of positive Direct Antiglobulin Test of human
blood samples

• The DG Gel DC Scan is used for the evaluation of positive Direct

Antiglobulin Test of human blood samples, in gel technique
• It allows differentiating red blood cells sensitized in vivo by IgG type
immunoglobulins or the complement C3d fraction
 PRINCIPLE OF THE TEST (1)

• The principle of the test is based on the gel technique described by Yves Lapierre in
1985 for detecting red blood cell agglutination reactions
• The DG Gel cards are composed of eight microtubes. Each microtube is made of a
chamber, also known as incubation chamber, at the top of a long and narrow
microtube, referred to as the column
• Buffered gel solution containing:
• a mixture of polyclonal anti-human globulin with monoclonal anti-C3d antibodies
(anti-IgG and anti-C3d)
• polyclonal anti-human globulin (anti-IgG) or monoclonal anti-C3d antibodies
(anti-C3d)
has been prefilled into the microtube of the plastic card
 PRINCIPLE OF THE TEST (2)

• The agglutination occurs when the red blood cell sensitized in vivo or in vitro by human
IgG antibodies or complement fraction react with the antibodies anti-human globulin
present in the gel solution
• The gel column acts as a filter that traps agglutinated red blood cells as they pass
through the gel column during the centrifugation of the card
• The gel column separates agglutinated red blood cells from non-agglutinated red blood
cells based on size
• Any agglutinated red blood cells are captured at the top of or along the gel column, and
non-agglutinated red blood cells reach the bottom of the microtube forming a pellet

Agglutinated RBCs

Non-agglutinated RBCs
 Material provided
• Each microtube of the DG Gel DC Scan card contains a gel in buffered medium with
preservative. The microtubes are identified by the front label of the card.
• Microtubes AHG: buffered low ionic strength solution (LISS) with polyspecific anti-
human globulin. Mixture of rabbit polyclonal anti-IgG and monoclonal anti-C3d
antibodies (IgM antibodies of murine origin)
• Microtubes IgG: rabbit polyclonal anti-IgG in buffered low ionic strength solution
(LISS).
• Microtubes C3d: monoclonal anti-C3d (IgM antibodies of murine origin, in low ionic
strength solution.
• Microtubes Ctl.: buffered solution without antibodies (control microtube).
All microtubes contain sodium azide (NaN3) as a preservative at a final concentration
of 0.09%.
 Sample collection and preparation

• Blood samples collected in EDTA or sodium citrate should be used

• Do not use grossly hemolyzed, cloudy or contaminated samples
• Samples should be tested as soon as possible
• If necessary, samples stored at 2 - 8 °C can be used up to 48 hours after their
collection
• Red blood cells from bags collected in CPD, CPDA or SAG-Mannitol can also be
used up until the expiration date indicated on the label of the bag
• If red blood cells from a bag segment are used, it is recommended that these be
washed with physiological saline solution before preparing the suspension
Procedure
 Results & Reaction Grades

• Report results as an agglutination grade, absence of agglutination or hemolysis

Terima Kasih