COOMBS¶ (ANTI-GLOBULIN) (ANTITESTS

Moderator:Moderator:Respected S.K. Sharma (Lecturer) Respected A.P. Chauhan (Tutor) Department of Haematology

Presented by:by:Rakesh Kumar B.Sc. MLT (Final Year)

NATIONAL INSTITUTE OF PARAMEDICAL SCIENCES, CHANDIGARH-160012

History 

The antiglobulin test was discovered by Coombs et al.(1945) for detecting incomplete antibodies in serum. He was traveling on an ill-lit wartime train from London to Cambridge, trying to read some papers by Ehrlich on the side-chain theory and speculating idly( Kekule on the tetravalent carbon atom) on the behaviour of red cells and antibodies, when he visualized the cells, already coated with molecules of incomplete antibody, which was of course a globulin, but still floating free, becoming linked together by molecules of another antibody, an antiglobulin antibody!

Robert Royston Amos Coombs 

Coombs¶

et al in 1947 showed that 'incomplete' antibody mainly responsible for positive anti-globulin test results is IgG antiwhile the 'complete' antibody is IgM. on,Dacie et al.(1957) showed that complement components attached to RBCs could also be detected by this test 1964,same workers performed DAT to detect in vivo RBCs sensitization in HDN. 

Later 

In

Introduction
For the detection of non-agglutinating Abs, especially nonIgG1 and IgG3 or complement components (C3d) affixed to RBCs in vivo or in vitro with the use of AHG reagent and Anti C3d or Anti-C3b. Anti-

Classification of Antiglobulin Tests a)Direct antiglobulin test (DCT) - detection of
sensitization of RBC s (coated with Abs and complement components) in vivo

b)Indirect antiglobulin test (ICT) - detection of
sensitization of RBCs in vitro

Principle of Anti-globulin Tests AntiWashed red cells which are coated with immune incomplete anti-bodies (IgG) and anticomplement components(C3d & C3b) will show agglutination with broad spectrum AHG reagents.The sensitization of red cells can occur in vivo or in vitro following incubation at 37°C with serum containing antibody.

Antibodies
Antibodies (also known as immunoimmuno-globulins) are gamma globulin proteins that are found in blood or other bodily fluids of humans and are used by the immune system to identify and neutralize antigens such as bacteria and viruses. They are typically made of basic structural units²each with units² two large heavy chains and two small light chains. Antibodies are produced by activated B-cell called as Bplasma cell.

Antigen and Antibody Reactions
Definition:Interactions between antigen and antibody involve non-covalent binding of an antigenic determinant (epitope) to the variable region (complementarity determining region, CDR) of both the heavy and light immunoglobulin chains. These interactions are analogous to those observed in enzymesubstrate interactions and they can be defined similarly.  Lock and Key Concept  Non-covalent Bonds ± Hydrogen bonds ± Electrostatic bonds ± Van der Waal forces ± Hydrophobic bonds  Reversible  Multiple Bonds

Mechanisms of Immune Destruction
Complete Antibodies (IgM Class)
ize ± Large. Occur in pentameric form. RBCs sensitized with IgM are not directly removed from circulation b¶coz macrophages don¶t have receptors for Fc portion of IgM. IgM antibodies cause intravascular hemolysis through complement activation. IgM antibodies may also cause extravascular hemolysis. Agglutination in saline at 37°C. ° Complete complement Activation

Incomplete Antibodies (IgG Class) 
ize - Small  Occur in monomeric form.  IgG antibodies involved in extravascular red cell 
  



destruction by: Antibody-mediated adhesion of Fc portion of IgG with Fc receptors on Macrophage and then, Phagocytosis of sensitized red cells occur or Macrophage ³pits´ the anitgen- antibody complex ± after continued pitting cell becomes rigid and is phagocytized No Agglutination in saline at 37°C,So use of HG is ° required,which causes their agglutination. Incomplete complement Activation.

Preparation of AHG Reagent 

Antihuman globulins (AHG) from immunized animals bind to human globulins either free in serum or attached to RBCs ± Polyclonal - Animals hyperimmunized with human globulins; bleed for antisera to obtain high titered, high avidity, specificity antibodies to human ± Monoclonal - Hybridoma cells from mice; collection of culture or ascites fluid yields antisera. 
mice hyperimmunized with human globulins  prepare cell suspension from spleens; fuse with immortalized myeloma cells  screen hybridoma clones for desirable specificities and affinities  maintain cultures of clones in vivo or in vitro.

Polyclonal Abs

Monoclonal Abs

Specificity of AHG Reagent 

Polyspecific AHG
± Abs to human IgG, and ± Abs to human C3d (C3b breaks down to C3c and C3d) ± Advantage is that polyspecific AHG may detect complement-dependent Abs complementon RBCs ± Disadvantage - more nuisance positives 

Monospecific AHG
± Abs to human IgG only or human C3d only ± Fewer nuisance positives ± may miss an important Ab

Anti Human globulin antibodies
AntiAnti-IgG antibodies react with the IgG isotype of human immunoglobulins. The Anti IgG recognizes the Fab region of IgG and reacts with all subclasses of IgG. IgG is the most abundant and the longest lived immunoglobulin in humans. The monomeric 150 kDa molecule is produced by B cells and is expressed in secreted and membrane-associated forms. membraneFunctions of IgGs are:are:1)Promotion of the phagocytosis of antibody-coated antibodyparticles 2)Feedback inhibition of B cell activation 3)Complement activation, 4)Antibody4)Antibody-dependent cell-mediated cytotoxicity cell-

Interaction b/w IgG and AHG serum 
IgG attached to the red cell memberane by Fab portion of the immunoglobulin.  IgG molecule attach to red cells unable to bridge the gap b/w sensitized red cells which are separated from each other by the ±ve charge on their surface.Thus, the senstized cells do not agglutinate. When AHG serum is added to the washed senstized the Fab portion of AHG molecule(Anti-IgG0 reacts with the Fc portion of two adjacent IgG molecules,thereby bridging gap b/w sensitized red cells causing agglutination.

Anticomplement Antibodies
The anticomplement antibodies are directed against the complement components such as C3b and C3d. The roles of anticomplement antibodies in AHG reagent are: 1)To detect complement binding antibodies of kidd,kell and Duffy systems(not picked by Anti-IgG). Anti2)To enhance of complement binding antibodies. 3)To detect IgM antibodies which invariably bind to the complement. 4)To detect IgM which elute off the red cells with increase in temperature. Most commonly used are anti-C3b and anti- C3d. antianti-

Complement Activation By Antibodies
Classical

IgG or IgM

Control Cells for Coombs Tests
Positive Control: sensitized O+ve cells Negative Control: sensitized O-ve cells Autocontrol: Patient s cells + Normal Saline

PREPARATION OF COOMBS CONTROL CELLS
(1) Prepare a 5% suspension of group 'O' RhoD positive cells in isotonic saline. (2) Mix equal volumes of diluted Anti-D reagent and 5% suspension of 'O' RhoD positive cells and incubate at 37°C for 15 minutes. (3) Decant and wash thoroughly with isotonic saline at least thrice. (4) Resuspend in isotonic saline to make a 5% suspension of coombs control cells.

Preparation of O+ve sensitized cells
Procedure:Procedure:1)Take O+ve red cells from CPDA/ACD containing blood bag in a labelled tube. 2) Wash the red cells three times with N/S at 2500-3000 2500rpm for 30 mins and make 5% cell suspension. 3) Add two drops 5%washed cell suspension of O+ve cells and one drop of Anti-D. Anti4)Wash the cell suspension three times in N/S and decant completely. 4) Mix,incubate at 37°C for 60-90 mins. 37° 605) Check for sensitization by adding 1 drop of AHG serum to 1 drop of the 5% washed sensitized cells 6) Check for agglutination in the tube.

Direct Antiglobulin Test(DAT/DCT) Test(DAT/DCT)
Principle:Principle:-The DCT is used to detect
incomplete antibodies or Anti-complement antibodies that are bound Antito the surface of red blood cells; a blood sample is taken and the RBCs are washed and then incubated with antihuman globulin and look for agglutination.

Sample Requirements:
1)EDTA sample(prevent the uptake of complement in vitro)Testing should be performed within 48 hours of collection. 2)Clotted should be as fresh as possible(not more than 24 old). 3)Cord blood sample

Requirements
Appartus
12 x 75 mm test tubes Pasteur pipettes Cenrifuge Marking pen Normal saline Microscope

Reagents
Polyspecific AHG AntiAnti-D AntiAnti-C3d Coombs Control Cells

Procedure:Procedure: 

Add ½ ml of patient blood from the EDTA to a tube labelled with the patient¶s initials. Wash this tube 3 times with N/S. 

  

  

Prepare a 2-5% suspension from the washed 2red cells. Label four tubes with the patient¶s test, C3, +ve & -ve control tubes. Add one drop of the 2-5% washed cells to each 2of the tubes labelled in step 4 above except +ve control tube . Add 2-5% suspension of O+ve sensitized cells in 2+ve control tube. Wash cell suspension in four tubes one more time, decant well and blot the last drop of saline. Add one drop of Anti-IgG to Patients¶ test tube & Anti+ve control tube, 1 drop Anti-C3d to the C3 tube, Antiand 1 drop saline to the ±ve control tube. Centrifuge all tubes at 1000 rpm for 1 min.

Incubate C3 tube for 5-10 mins. 5Resuspend the cell button gently and examine for agglutination in all tubes microscopically.  Read, grade and record results in the appropriate columns of the worksheet.  Confirm any negative Patient¶s test and C3 reactions with the Coombs Control cells. 


INTERPRETATION:INTERPRETATION:DCT is +ve when agglutation is seen in Patient¶s test and Anti C3 d tube. DCT is ±ve when no agglutination is seen in Patient¶s test and Anti C3d tube except using coated red cells. A ±ve DCT does not necessarily mean absence of coating antiglobulin antibody. +ve Control - agglutination in +ve Control tube -ve Control - No agglutination in ±ve µ¶ µ¶

Applications of Direct Coombs Tests
a)Diagnosis of haemolytic disease of newnewborn(HDN) b)Diagnosis of Acquired Immune Haemolytic Anaemia C)Investigation of Drug induced red cell sensitization d)Investigation of Haemolytic Transfusion reactions

Indirect Antiglobulin Test(IAT/ICT) Test(IAT/ICT)
Principle:Principle:- The ICT detects antibodies
against RBCs that are present unbound in the patient's serum. Serum is extracted from serum. the blooda nd the serum is incubated with antigenicity. RBCs of known antigenicity.

Procedure:Procedure:a)Place 2-4 drops of the test serum in a tube. 2b)Add 1 drop of 2-5% suspension of O+ve 2cells. c)Mix and incubate at 37 °C for 60-90 mins. 60-

g)Add 1-2 drops of AHG serum. 1h)Centrifuge at 1000 rpm for 1 min. i)Gently shake the tube to dislodge the button and examine for agglutination. j)Leave an ±ve test at RT for 5 min,centrifuge and read again. K)Confirm any negative Patient¶s test Anti-IgG coated AntiCoombs Control cells.

Interpretation: Interpretation:ICT is +ve when agglutation is observed either after immediate spin or after spin following RT incubation. ICT is ±ve when no agglutination is seen at either phase except using coated red cells. Controls are same as for DCT

Factors Affecting the Sensitivity of ICT
1)Temperature:1)Temperature:- Best temperature is 37 °C for most IgG ab and red cell reactions. <37° <37°C Decreases the rate of association b/w Ag and Ab. >37° >37°C Damaging of red cells or antibody molecule. 2)Incubation Time:Time:for saline or albumin techniques at 37°C for 30 mins 37° For LISS medium incubation time is 10-15 mins 103)Serum:Red Cells Ratio Increasing ratio increases the degree of antibody coating on red cells Common ratio is 2 drops of serum to 1 drop 2-5% cell 2suspension

Applications of Indirect Coombs Tests
a)Compatibility testing b)Screening and detection of unexpected antibodies in serum c)Maternal antibodies in pregnant women(Rh+ve) serum against fetusRh-ve fetusRhd)Detection of red cell antigens not detected by another techniques(Le,Fy,Jka)

Applications of Suspending Medium
The sensitivity of ICT can be increased with the addition of the following suspending media:media:22% Bovine Albumin Enzymes LISS

Bovine Albumin ( 22% )
Principle ::Albumin increases the dielectric constant of the medium and thus reduces the Zeta potential. Due to this effect the electrical repulsion b/w the red blood cells is less and the cells agglutinate so it increases the sensitivity of ICT.

Methods ::a) b)

One stage ( Additive ) method. Two stage ( Layering) method.

One Stage Method (Albumin- Additive) (AlbuminProcedure:Procedure:Add 2-3 drops of serum to a labelled tube. 2Add 1 drop of 2-5% O+ve red cells suspensions 2in the tube. Add 2 drops of 22% of Bovine Albumin. Mix and incubate 37*C for 15-20 min. 15Centrifuge at 1000rpm for 1-2 min. 1Add 1-2 drops of AHG serum. 1Gently resuspend the cell button and observe for agglutination. Confirm all negative results under microscope.

Two stage Method(Albumin Layering) Procedure:Procedure:1)Add 2-3 drops of serum to a labelled tube. 22)Add 1 drop of 2-4% red cell suspension in the tube. 23)Mix and incubate at 37°C for 30 mins. 37° 4)Centrifuge at 1000 rpm for 1-2 mins. 15)Add two drops of 22% albumin in the tube.Aibumin will form a layer on the top of the cells.Do not mix. 6)Incubate at 37°C for 10-20 mins. 37° 107)Add 1-2 drops of AHG serum. 18)Gently resuspend the cell button and observe for agglutination/haemolysis. 9)Confirm all negative results under microscope.

Enzymes Techniques
Principle:Principle:The action of enzymes on red cell potentiates agglutination in two ways: 1) Enzyme removes sialic acid from the red cell surface and thus,reduces the surface charge(Zeta potential) and allowing the cells to come closer to one another. 2)Enzyme may potentiate the agglutination by removing structures which sterically interfere with the access of antibody molecule.

Methods:Methods:1)Two Stage Enzyme Technique 2)One Stage Enzyme Technique

Two Stage Enzyme Technique
Reagents:Reagents:Preparation of enzyme stock solution a)Papain is dissolved in 0.85% N/S. b)Trypsin is dissolved in N/20 HCl. c)Ficin is dissolved in phosphate buffer saline PH 7.4 d)Bromoline is dissolved in phosphate buffer saline PH 5.5 All stock solutions are always prepared as 1% strength. Preparation of enzyme working solution 1 VOL of stock + 9 VOL of diluent

Procedure:Procedure:PrePre-treatment of RBCs
1)Take 0.1 ml of 3 times RBCs washed in N/S in a test tube. 2)Add 0.2 ml of Papin working solution. 3)Incubate at 37°C for 15-30 mins. 37° 154)Wash the red cells three times with N/S. 5)Make 2-4% cells suspension in N/S. 2-

PostPost-treatment of RBCs
a)Take 2 vol of serum in a test tube. b)Add 1 vol of papainised red cells. c)Add 1-2 drops of AHG serum. 1d)Incubate at 37°C for 30 mins. 37° e)See for agglutination.

Advantages of Two Stage Technique
1)Detection & identification of IgG antibodies and also for crossmatching. 2)More sensitive than one stage technique 3)In one stage technique proteolytic effect of enzymes is inhibited by the serum.

One Stage Enzyme Technique Reagents: Reagents:Preparation of Papain Cysteine Reagent Papain - 10gm Cystein HCl - 4.85 gm Phosphate buffer PH 6.2 - 1000 ml Store in small aliquots at -20°C. 20°

Method:Method:1)Take 1 VOL of serum in a test tube. 2)Add 1 VOL of 2% cell suspension. 3)Incubate at 37°C for 1hr. 37° 4)Add 1-2 drops of AHG serum. 15)See for agglutination.

LISS(Low Ionic Strength Salt Solution) Solution) Principle:Principle:In N/S Na+ and Cl- ions form ionic clouds around oppositely Clcharged sites on Ag and Ab molecules respectively and partially neutralizes them.When the ionic strength of the reaction mixture is reduced,the no. of ions available to form ionic clouds around cells or protein molecules is decreaesd and they unit more effectively and they unit more effectively.It results increased 2-4 folds in comparison with 2N/S.

Preparation of LISS Solution
0.17M saline - 180ml 0.15M phosphate buffer PH 6.7 - 20ml 0.3M sodium glycinate PH 6.7 - 800ml

Method:Method:1)Wash the RBCs twice in N/S. 2) Wash these cells once in LISS. 3)Make 2-3% cell suspension in LISS. 24)Take equal volume of serum and LISS suspened cells in a tube. 5)Incubate at 37°C for 10-15 mins. 37° 106)Add 1-2 drops of AHG serum. 17)See for agglutination.

Sources of Errors in Coombs Tests
Technical errors:errors:False ve Results 1)Adequate washing of sensitized cells b coz traces of globulin left causes neutralization of AHG serum. 2)If testing is delayed or interrupted,they may be elute of antibodies attached to red cells & neutralization of AHG serum. 3)Use of heavy red cell suspension prevent optimum coating of antibody or too light to make reading difficult 4)In appropriate serum to cell suspension. 5)Insufficient temperature and incubation time. 6)Failure to add AHG reagent

7)Low speed of centrifugation 8)Failure to check ±ve rxns microscopically 9)Improper storage of test cells,resulting loss of sensitivity. False +ve Results 1)Enzyme treated cells may react with residual non specific antibody in AHG serum. 2)Over centrifugation packs red cells so tightly that can not be dispersed completely. 3)Use of agglutinated RBCs before washing & so on their persistence throughout the washing.

Non Technical Errors False +ve Results 1)Inactivation of AHG reagent may be due to improper storage. 2)In bacterial contamination of test cells or septicemia in the patient, Anti-T in the AHG serum react with TAntiTactivated red cells. False -ve Results 1)Improper storage of test cells,resulting in the loss of sensitivity 2)Use of plasma sample 3)Inaccurate temperature of water bath or incubator 4) Inaccurate control of speed and temp. of centrifuge

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