Blood Group Types and Methods Dr.P.K.Bandi Dr.

Gaurav Pawar

Blood Group Typing and Methods

Study of blood Group Systems and their Antigens and corresponding Antibodies on the basis of red cell serological techniques. An introduction to antigens , antibodies , antigen - antibody reactions . Factors affecting antigen- antibody reactions Basic concept of genetics in relation to immunohaematology. Blood group systems , their Ag and Ab.

Antigen
An antigen is a substance either protein or non- protein, which

if introduced into and individual causes the production of an antibody .

In the practice of blood transfusion , red cell antigens are of

major clinical importance, though platelets, leucocytes and plasma proteins exhibit strong antigenic systems
The Four blood groups are determined by the presence or

absence of blood group Antigens ( agglutinogens) on the red cell and so individuals group is A,B, AB or O.

Antibody
Under normal circumstances, the new born has no ABO

antibodies.However after 10- 20 wks , later moderate amt of Ab are present which appear after specific Ag stimulus. So called as Naturally occurring Ab.
Ab are proteins,more specifically Igs which are recognized by

their interaction with Ag.

The Ab are mainly gamma - globulin in nature, Ab involved in

transfusion - related serological reactions are mainly IgG, IgM,IgA.

Blood Group Antibodies

Immunoglobulin Synonyms Properties NO. of subclass Mole. weight Sedimentation const. Transplacental transfer Serological activity

IgM
Naturally occurring Ab complete/cold 4deg/saline 1 900000 19 s no Heat labile

IgG
Immune(Acquired) incomplete/warm 4 150000 7s yes Unaffected

conti... Antibody( immune response)

On first exposure to an Ag the body initiates a Primary

response, slow and takes months for Ab to form

Once the individual has been sensitized to a particular Ag, a

subsequent exposure will cause faster and more pronounced response k/a Secondary response.
Primary response - cause production of IgM Ab. Secondary will predominantly produce IgG. Naturally occurring Ab - Anti- A and Anti- B are found in yhe

sera of individuals lacking the corresponding Ag.

 These naturally occurring Ab are believed to

occur on exposure to certain bacteria or plant Ag which share same structures with ABO red cell Ag, so crossreact with these.They are commonly IgM in nature.
Develop by the age of 6-8 mths and

maintained throughout life.

Antibody-Antigen Reaction
Agglutination- It is defined as clumping of particles or red

cells that have Ag corresponding to the Ab on their surface, most common type in blood group serology , occurs in two stages 1) Attachment (sensitization) 2) Agglutination - cross linking of sensitized cells occurs by bridges of Ab molecules leading to a lattice formation.
Sensitization- It is defined as coating or binding of Ab on red

cell surface without bringing about agglutination in saline .

Haemolysis- Red cell lysis indicates the presence of Ag-Ab

reaction and consumption of complement.

 Neutralization( Inhibition)- Soluble form of

Blood group subs. Ag can inhibit the reaction by neutralizing the corresponding Blood gp Ab.Soluble Ag are added to the serum containing the specific Ab , if the activity decreases or it disappears completely an AbAg reaction can be assumed to take place. Test used in testing saliva for A,B and H subs. to determine the secretor status.
Precipitation,Complement fixation,

Immunofluorescence,Radioimmunoassay,ELI

Reagents used in DetectingAg-Ab Reaction

Saline- Basic medium in which cells are suspended, pH of

which should be 6.8 to 7.2 for optimal reaction.

Bovine Albumin- Bovine albumin in reaction mix reduces the

zeta potential by dispensing sod. ions and thus raising the dielectric constant of medium,causes red cell to move closer together and allows IgG mole. to cause agglutination.
Enzymes- Proteolytic enzymes like papain,trypsin,ficin, have

been used to enhance agglutination.They act by removing sialic acid from red cell membrane,reducing the surface charge.

reduction in strength of suspension medium, decreases the ionic strength of Ag sites and increases the Ab uptake.Helps in sensitization phase of Agglutination.
Macromolecular potential medium- Synthetic

medium e.g polyvinyl pyrrolidone,dextran,gum acacia. Polybrene (hexadimethrine bromide) has been used in serology.
Anti-human globulin serum- Most commonly

Blood Group Systems Inheritance and Genetics

Experiments with blood transfusions have been carried out for hundreds of years. Many patients have died and it was not until 1901, when the Austrian Karl Landsteiner discovered human blood groups, that blood transfusions became safer. He found that mixing blood from two individuals can lead to blood clumping. The clumped RBCs can crack and cause toxic reactions. This can be fatal. There are more than 20 genetically determined blood group systems known today

The AB0 and Rhesus (Rh) systems are the most important ones used for blood transfusions.
Group of Allelic Ag encoded by a single gene locus or closely linked loci which do not crossover form a Blood gp System.Blood gp Ag are component of red cell membrane and are immunogenic,Chemically these are either Glycoprotein or Glycolipids, polysaccharide component determines the specificity and amino acid fraction for antigenicity.

Summary: 29 blood group systems, 40 genes, 707 alleles Also detailed: non-human counterparts for H/h, MN, Rh
System ABO Chido- Rodgers Colton Cromer Diego Dombrock Duffy Gerbich (Ge) GIL H/h I Indian (IN) JMH Kell (with Kx) Kidd Knops Locus Funcion Alleles System LandsteinerWeiner Lewis Locus Function Alleles 3 36 ABO enzyme 115 C4A, factor 7+ C4B AQP1 channel 7 DAF receptor 13 SLC4A1 exchanger 78 DO unknown 9 FY receptor 7 GYPC structure 9 AQP3 channel 2 FUT1, enzymes 57 FUT2 GCN2 enzyme 8 (IGnT) CD44 adhesion 2 SEMA7A signaling 0 KEL, enzyme 67 XK SLC14A1 transport 8 CR1 receptor 24+ ICAM4 adhesion (LW) FUT3, enzymes FUT6, FUT7 LU adhesion GYPA, unknown GYPB, GYPE BSG adhesion A4GALT, enzymes B3GALT3 CD151 RHCE, transport RHD, RHCG RHAG, RHBG ERMAP adhesion XG, adhesion CD99 (MIC2) ACHE enzyme

Lutheran MNS

16 43

OK P-related RAPH-MER2 Rh

5 27 3 126

Scianna Xg YT

4 0 4

antigen defining blood group system

ABO group system

ABO was first to be discovered , also is the most important in transfusion practice becoz of the invariable presence of naturally occurring anti-A and anti-B Ab.

ABO blood group genes consist of multiple alleles which are located on the long arm of Chromosome 9, the four alleles are A1,A2,B and O. O is a silent gene or amorph and no gene product is expressed, A and B are codominant and O is recessive.
What do co- dominant gene mean This meant that if a person inherited one A group gene and one B group gene their red cells would possess both the A and B blood group antigens. These alleles were termed A ( which produced the A antigen ), B (which produced the B antigen) and O (which was "non functional"and produced no A or B antigen)

ABO blood group genes do not code directly for the specific Ag instead they code for specific transferases namely N- acetyl galactosaminyl transferases and D - galactosyl trasferase. These Enzymes transferase the immunogenic sugars N- acetyl galactosamine and D- galactose resp. to H substance to confer A and B specificity. The H gene and its allele h are present at a different locus from ABO genes. The H gene produces an enzyme L - fucosyl transferase which will transfer sugar fucose to the precursor carbohydrate chains to form the H antigen.Now the H Ag will form substrate for A and B enzymes, while the homozygous hh is exceedingly rare and results in production of Bombay phenotype, with no H Ag. The ability to secrete A,B and H substance is determined by the presence of Se secretor gene in homozygous or heterozygous SeSe.80% of individuals are secretors and 20% are non- secretors sese of AB and H Ag. AB and H are widely distributed in the body tissues except the CNS.

The ABO blood groups

The most important in assuring a safe blood transfusion. The table shows the four ABO phenotypes ("blood groups") present in the human population and the genotypes that give rise to them.

Blood Antigens on Antibodies in Serum Group RBCs

A B AB O A B A and B Neither Anti-B Anti-A Neither Anti-A and anti-B

Genotypes

AA or AO BB or BO AB OO

Each person has two copies of genes coding for their ABO blood group (one maternal and one paternal in origin)

Bombay Blood group(Oh)

Was discovered in 1952 by Bhende et al, characterized by absence of A,B and H antigens on the red cell surface and in body and expressed as phenotype Oh. Frequency of Oh is about 1:7600 approx, Sera of individuals contain anti - A and anti-H and react with O group cells which express H Ag,these individuals have ABO & secretor genes , and ABand H are not found becoz of absence of H gene. The lectin of Ulex europeus has anti-H activity and can be used to detect H Ag

Rh blood group system blood produce The majority of Rh-negative individuals transfused with Rh-positive
immune Rh Ab. Importance in HDN, HTR. Of described 40 only 5 are important and common,they are protein products present on chromosome 1.Rh Ag ( D,C,c,e,E) are products of their genes . Concept of Fisher- race is adequate to study the clinical imp problems. Rh Antigen-Unlike ABO Ag only D Ag are present on red cell ,Rho (D) Ag is clinically imp in the Rh system beccause of immunogenicity which is greater than of other systems .95% of indians are Rh positive and 15% are Rh negetive Rh antigens are transmembrane proteins with loops exposed at the surface of red blood cells.

They appear to be used for the transport of carbon dioxide and/or ammonia across the plasma membrane.
They are named for the rhesus monkey in which they were first discovered.

RBCs that are "Rh positive" express the antigen designated D.

Du phenotype- A weaker variant of D Ag is termed as Du and detected only by AHG technique.

Its significance implies that red cells have fewer D Ag sites and therefore may be diagnosed as D -ve .By the techniques currently used and by many monoclonal reagents these will be grouped as Rh(D) +ve ( through previous sensitization ) is likely to develop haemolysis .

A sensitized Rh -ve mother having anti-D Ab can produce HDN in a Du- positive foetus.

Tranfusion of Du blood to an Rh -ve individual can theoretically produce Ab against D and Du.

Recently introduced monoclonal Anti-D reagents gives positive reaction in 95% Du individuals in the tube method and 99% in the microtitre plate.

Red Cell Serology

Red cell serological reactions form the basis of lab testings in blood transfusion.The Ag-Ab reaction is detected by the followingHaemagglutination Complement fixation Neutralization Absorption and Elution Precipitation

ABO Grouping

The principle of ABO grouping is based on a specific agglutination reaction b/w Ag on red cells and IgM Ab in the typing serum.
Cell grouping done by testing an individual red cell with known blood grouping reagents and corresponding serum group with Known A cell, B cell, O cell.

ABO grouping sera - It is essential to use reliable grouping reagents to correct results ,commercially prepared polyclonal, monoclonal,antisera are available.
Preparation of Red cells for ABO testing -Preparation of reagent red cells in addition to patients and donors red cells is required daily while performing the serum grouping and cell grouping. Reagent red cells When performing an ABO serum grouping, it is important to ensure that the cells prepared are fresh and well-selected A cells-

three times with normal (0.9%) saline, make a 2-5% suspension in saline for use. B cells Pool known fresh B group cells from 1-2 donors. Wash three times with normal saline, make a 2-5% suspension in saline for use. 0 cells Pool known fresh C group cells from 1-2 donors. Wash three times with saline and make a 2% suspension in saline for use. Cells are washed in isotonic saline to remove all traces of plasma or serum. They must then be accurately diluted to give a standard suspension in saline by either using a graduated tube (Wintrobes tube) or by counting drops to make a 2-5% cell suspension.

cont..... using a graduated tube (Wintrobes tube) or by counting drops to make a 2-5% cell suspension. When prepared, the cells should be checked against anti-A and anti-B. The cells should be kept at 4C in the refrigerator when not in use. If low-ionic strength saline solution (LISS) is being used in the laboratory as an enhancing medium, then the last wash should be in LISS and the cells are thereafter suspended in LISS to make a 2-3% cell suspension. Patient and donor cells The test cells also should be washed at least once with saline and suspended to make a 2-5% suspension in saline. When dealing with large number of samples, adequate precautions must be taken to avoid mislabelling of tubes and the cells must be dispensed in already labelled tube for washing.

Blood grouping - when performing blood grouping: Methods & Procedure Three manual methods can be used
- Glass slide or white porcelain tile - Glass test tube - Microwell plate or microplate Newer techniques - Column technique (sephadex gel) - Solid phase tests Slide or Tile Testing

This technique may be used for emergency ABO grouping tests or for preliminary grouping particularly in an outdoor camp, however it should always be supplemented with a cell and serum grouping using any one of the other

This technique may be used for emergency ABO grouping tests or for preliminary grouping particularly in an outdoor camp, however it should always be supplemented with a cell and serum grouping using any one of the other above mentioned techniques.

Slide or tile testing is not recommended for routine use because it is not reliable for

- weakly reactive antigens on cells

- serum grouping with low titre anti-A or anti-B

Disadvantage-- c

conti..

Less sensitive than the tube test

- Drying up of the reaction mixture can cause aggregation of cells, giving false positive results.

- Weaker reactions are difficult to interpret.

Procedure-1. Place 1 drop of anti-A and 1 drop of anti-B reagent separately on a labelled slide or tile.

2. Add 1 drop of 20% test red cell suspension to each drop of the typing antiserum (the suspension may be prepared by adding 20 parts of red cells to 80 part of normal saline).

3. Mix the cells and reagent using a clean stick. Spread each mixture evenly on the slide over an area of 10-15 mm diameter.

4. Tilt the slide and leave the test for 2 minutes at room temperature (22-24C). Then rock again and look for agglutination.

5. Record the results.

Tube Testing -

Test tubes either of glass or plastic may be used, of lOx75mm size. The tube technique is more sensitive than slide technique for ABO grouping.

Advantages of tube testing -

- It allows for fairly long incubation without drying up of the tubes contents.

conti..

- It allows for fairly long incubation without drying up of the tubes contents.

- Centrigugation involved enhances the reaction allowing weaker antigens and antibodies to be detected.

Simplicity of reading and grading of results.

- Clean and more hygienic.

- Requires smaller volume of reagents.

Procedure - conti.....

Saline agglutination test for cell and serum grouping by tube method

1. Cell grouping / forward grouping

2. Serum grouping / reverse grouping

Reagents required

1. Known antisera (anti-A, anti-B, anti-AB), monoclonal or polyclonal.

2. Red cell suspension - reagent cells (Ac, Bc and Cc); test red cells (patients or donor)

Cell and serum grouping

1. Spin test sample to separate serum.

2. Set up 6 tubes correctly labelled with donor/patient no. Anti A, Anti-B, Anti-AB, Ac, Bc and Cc.

3. Prepare once washed 2-5% suspension of the test cells.

4. Add I drop of anti-A in tube labelled A, anti-B in tube labelled B, and anti-AB in tube labelled AB.

5. Add 1 drop of 2-5% test cell suspension in the three tubes A,B and AB.

6. Add 2 drops each of the test serum in tubes labellaed Ac,Bc and Cc.

7. Add I drop each of reagent A cells in labelled tube Ac, B cells in labelled tube Bc and 0 cells in tube labelled Oc.

8. Mix all the 6 tubes and centrifuge at 1000 rpm for 1 minute.

9. Resuspend cell button by gently shaking the tubes and read against well-lit background. Record the result

9. Resuspend cell button by gently shaking the tubes and read against well-lit background.

defining the strength of reaction ( grading of agglutination)

 slide or tile testing

Microplate Technique - Microwell plate consists of a small tray with 96 small wells each of which can hold about 200-300u1 of reagent. Microplate technology is gaining widespread popularity due to increasing workload in blood transfusion laboratories and recent availability of packaged automated system.

Three types of microplates are available -a. U-type well ,b. V-type well ,c. Flat-bottom

The U-type well is generally used in red cell serological work as it is easier to read the results in U- bottom plates.

Flat bottom plates are useful in ELISA technology but are unsuitable for liquid-phase blood group serology.

U-well are preferred to V-well plates for resuspension technique because of the case of suspension prior to reading and their superior optical quality when using automated plate reader.

Advantages of Microplate ABO grouping-

Small volumes and low concentration of sera and red cells are used, making it cost-effective.

- Easy handling of a microplate, which can replace 96 test tubes.

- Batching of samples can be achieved with considerable economy in space and time.

- If larger laboratories acquire microplate hardware items e.g. reagent dispenser, sample handler and cell washer it may further reduce the operation time.

- Large batches of plates can be predispensed with antisera and reagent red cells before testing.

Equipment required for microplate grouping

Microplate

- Untreated rigid polystyrene microplates

-Pipettes

-Manual / automated single or multi-channel dispenser

-Variable volume dispenser (20 - 50 ul)

-Centrifuges

-Variable volume dispenser (20 - 50 ul)

-Centrifuges

-Bench-top centrifuge with head for carrying microplates

-Microplate shaker

-Two/four place shakers

Blood Grouping - Newer Techniques

Column technique-

conti.............

Recently interest has been generated in use of column technology for serological testing. The aims of this technology is to standardize red cell agglutination reactions and trap the agglutinates to permit simple and reliable reading.

The gel within the microtubes acts as a sieve, unagglutinated cells form a button at the bottom of the microtube and agglutinated red blood cells are trapped in the gel.

The Sephadex gel may be- Neutral,Specific reagent-------AHG, ANTI -A , ANTI -B, ANTI-B, ANTI-D, ANTI- KELL.

Advantages of gel system --- Requires small amount of red cells and serum for testing (1O-40m1), making it ideal for neonatal and paediatric use.

- Reactions are easily visible and can be graded

- Superior in sensitivity to conventional tube test without loss of specificity.

- Easy to use.

Reactions are easily visible and can be graded.Reactions using gel techniques are stable for 48 hrs and gel cards can be photocopied providing a permanent record for future use.

 READING AND

GRADING OF HAEMAGGLUTINATION IN GEL SYSYTEM

Naturally occurring anti-Al in A2 and A2B individuals

Human anti-Al(by adsorption of group B anti-A serum by A2 cells)

Dolichus biflorus lectin reacts specifically with Al antigen and causes agglutination. It is stored at 4-8C and may be frozen at -20C for prolonged storage.

Procedure - 1. Place 1 drop of anti-Al reagent into a clean, dry test tube. 2. Add 1 drop of 2-4% saline suspension of patients cells. 3. Mix and leave at RT for 30-60 minutes. 4. Gently agitate and examine for agglutination.

Reactions showing agglutination indicate Al blood group.

RH (D) GROUPING

Practical Aspects of Rh Grouping Rh grouping in routine use for donors and patients involves testing for Rh (D) antigen only, however tests for other important Rh antigens e.g. C,c,E and e may be done for Rh genotyping. Reagents for Rh (D) Grouping I) Polyclonal human anti-D serum (IgG) -Anti-D serum (IgG) for saline or rapid tube test (high protein medium) This contains macromolecular additives and give reliable results. Anti-D for saline tube test 2 types Anti-D IgM Anti-D IgG - Chemically modified II )Monoclonal antibodies---IgM anti-D monoclonal reagent,IgM and IgG anti-D monoclonal reagent,Blend of IgM monoclonal + IgG polyclonal reagent These antibodies are highly specific, react equally well at 20C as well as 37C and are reliable for slide and rapid test tube technique.

reagents from two different batches of different firms following the manufacturers recommended technique (antiDi, anti-D2)

III) Controls for Rh (D) grouping -Known 0 Rh (D) positive and 0 Rh (D) negative cells may be used as controls with monoclonal anti-D reagent.AB serum or diluent control provided with the anti-D reagent or 22% bovine serum albumin may be used as negative control with the test cells

Rh (D) Grouping-- 1. Slide technique (The slide test is not recommended for routine test as it may not pick up weak reactions, thus giving negative results) .2. Tube technique 3. Microplate technique.

Tube Technique-

a. Saline Agglutination test for Rh (D) Typing.

1. Prepare 2-5% washed red cell suspensionof test sample. 2. Place 1 drop of anti-D (Dl) in cleaned tube labelled Dl and place 1 drop .of anti-D (D2) from a different manufacturer in a clean tube labelled D2.

3. Place 1 drop of 22% bovine albumin/control reagent in another tube labelled C.

CONTI...........

4. Add 1 drop of 2-5% test cell suspension to each tube.

5. Mix well and centrifuge at 1000 rpm for 1 minute (in case of using IgG anti-D, incubate at 37C for 10mm. and centrifuge (spin tube method) or incubate at 37C for 60 minutes (sedimentation method).

6. Re-suspend the cell button and look for agglutination. All negative results must be confirmed under microscope.

Interpreation- - Positive test : Agglutination in anti-D (both tubes) and smooth suspension in control tube.

Negative test : Smooth suspension in all the tubes (test and control)

For all microscopically negative reactions in donor grouping, Du testing should be performed, hereas some workers suggest that if the two anti D reagents used are potent and specific, it is not necessary to perform Du testing.

b. Albumin technique for Rh (D) typing- Principle--- Albumin increases the dielectric constant of the medium and thus reduces the zeta potential. Due to this effect, the electrical repulsion between the red blood cells is less and the cells agglutinate. Mostly 22% bovine albumin is used, as higher concentrations can cause rouleaux formation.

Albumin can be used in ---- Albumin addition technique additive to serum & cell mixture & Albumin layering technique layered on cell button

Albumin layering technique -- Procedure--

1. Place 1 drop of anti-D in a labelled tube. 2. Add 1 drop of 2-5% test red cell saline suspension. 3. Incubate at 37C for 45-60 minutes.

4. Allow 1 drop of 22% albumin to run down the inside wall of the tube. Albumin will form a layer on top of the red cells. Do not mix.5. Incubate further at 37C for 15-20 minutes.

6. Examine for agglutination after gentle shaking and confirm all negative results under microscope.

Indirect antiglobulin test (Du Testing) - If using IgG anti-D in routine saline agglutination Rh (D) grouping proceed from step 6 in all negative reactions otherwise perform the following procedure :

1. Take 1 drop of anti-D (IgG) in a cleaned labelled test tube (T).

2. Take 1 drop of appropriate diluent control in another tube (C).

3. Add 2-5% washed test red cell suspension to both the tubes.

4. Mix and incubate at 37C for 45-60 minutes.5. Centrifuge at 1000 rpm for 1 minutAgglutination in test sample and negative reaction in control sample shows a positive test and the sample is labelled Rh (d) positive.e.

6. Gently suspend the cell button and look for agglutination, if positive test, no need to proceed as the sample is Rh (D) positive. 7. If negative, wash the cells 3-4 times with saline and decant the last washing.

8. Add 1-2 drop of anti-human globulin reagent (AI-IG-Coombs reagent). Mix gently and centrifuge at 1000 rpm for 1 minute. 9. Resuspended the cell button gently, examine for agglutination and record the results . Agglutination in test sample and negative reaction in control sample shows a positive test and the sample is labelled Rh (d) positive.

Microplate technique for Rh (D) grouping as described earlier in ABO grouping.

Rh(D) Grouping In Haemolytlc Disease of the Newborn---In haemolytic disease of the newborn, the babys red cells may be coated with immunoglobulin and a saline reactive Rh antiserum is usually necessary for testing.

When the cells are heavily coated with antibody, no free antigenic sites remain for reaction, resulting in a negative test. This is suspected when the infants cells show a positive direct antiglobulin test (DAT) and a negative test with anti-D reagent.In such instances it is recommended that the antibody should be eluted by gentle elution (heating at 45C for 30 minutes) to expose the antigenic sites before testing.

AntiGlobulin Test

Antiglobulin test is one of most important serological test done in routine. It utilizes the anti-human globulin {AHG}reagent to bring Agglutination of red cells coated with immunoglobulin or complement component , which do not show agglutination in saline . Principle - Red cells are coated with incomplete IgG Ab show agglutination on addition of AHG or Coombs reagent .The coating can occur either in vivo or vitro following incubation with serum containing AB.The majority of incomplete AB are IgG which attach to the red cell membrane by Fab portion.Addition of AHG reagents results in the Fab portion of the AHG molecule combining with the Fc portion of two IgG molecule,so causing Agglutination. There are two types of antiglobulin test- 1} Direct 2} Indirect

Direct antiglobulin test (DAT/DCT)

Direct antiglobulin test is used to detect in-vivo sensitization (coating) of red cells with immune antibody (IgO) or the complement component (C3d or C3c) in Diagnosis of haemolytic disease of the newborn (HDN) Diagnosis of autoimmune haemolytic anemia (AIHA) Investigation of haemolytic transfusion reaction Investigation of drug induced red cell sensitization

Indirect antiglobulin test (IAT/ICT)

This test is used to detect the presence of incomplete antibodies and complementbinding antibodies in the serum after coating on to the red cell in-vitro in. Screening and identification of unexpected (irregular) antibodies in serum. Compatibility testing Detection of red cell antigens using specific antibodies reacting only in antiglobulin test (K, Fya, Fyb, Jkb, etc.) Investigation of haemolytic disease of the newborn.

Anti-Human Globulin (AHG) Reagent

Anti-human globulin reagent is produced by immunizing rabbits, goats or sheep with human serum or purified add type antigen. Animals are bled after a specified period and the reagent is purified by absorbing unwanted antibodies. Anti-human globulin reagent can be polyspecific and monospecific. Polyspecific antiglobulin reagent contains antibodies to human IgG, C3 and C4 components of the complement. Monospecific antiglobulin reagent may be against any one of the human IgM, IgO, IgA or complement component C3 or C4. Monoclonal AHG is now available against IgG or complement components.

Direct Antiglobulin Test (DAT)

Sample collection-- Blood sample for DAT should be collected in EDTA (Na2 or K2) to prevent in-vitro uptake of complement. Reagents --Anti-human globulin reagent (AHG) Positive Control : Sensitized 0 Rh (D) positive cells Negative Control : Sensitized 0 Rh (D) negative cells Unsensitized 0 Rh (D) positive cells Preparation of 0 Rh (D) positive sensitized red cells -conti----

Take 0.5 ml of 5-6 times washed and packed 0 Rh (D) +ve red cells in a test tube.

Add 2-3 drops of IgG anti-D (select a dilution (titre 1:4) of anti-D which coats the red cells but does not agglutinate them at 37C).

Mix and incubate at 37C for 30 minutes. If there is agglutination, repeat the procedure using more diluted anti-D.

Wash 3-4 times and make 5% suspension in saline for use.

Perform a Direct antiglobulin test which should give a 2+ reaction. If no agglutination occurs, repeat the test by using less diluted anti-D serum.

0 Rh(D) negative sensitized red cells are also prepared by treating 0 Rh(D) negative cells in the same manner. The preparation should give a negative direct antiglobulin test (DAT).

Procedure (DAT) --

Place 1 drop of 2-5% suspension of red cells in a clean labeled test tube.

Wash the red cells 3-4 times with saline and decant the final wash completely.

Add 1-2 drops of Al-IG reagent.

Mix and centrifuge at 1000 rpm for 1 minute.

Shake the tube gently to dislodge the cell button and read the results using a concave mirror.

If result is negative, incubate the test for further 5 minutes at room temperature, centrifuge and look for agglutination and record the results. conti............

Interpretation-- Agglutination of red cells indicates a positive DAI Controls tubes should be read before final interpretation. A positive reaction after immediate spin indicates presence of IgG coating antibodies. Reactions due to IgG become weaker after incubation. A positive reaction after a 5 minute incubation indicates coating by complement component.

Indirect Antiglobulin Test (IAT)

Sample collection- Blood sample for IAT should be collected in plain labeled test tube. Reagents-Anti-human globulin reagent. Reagent 0 cells, commercially available or prepared in the laboratory (0I arid OIl) Obtain poled 0 Rh (D) positive cells in 2 test tubes 0 I and 0 II. In each tube, cells from 2 donors are taken. Wash 3 times with normal saline. Prepare a 5% suspension for use. Procedure (IAT)-- conti..........

Centrifuge the tubes at 3000 rpm for 5 minutes to separate the serum.

Add 2 drops of serum in each of the tubes labeled 0I and 0II (sample should be fresh for detecting complement - binding antibodies otherwise fresh AB serum should be added to it).

Add 1 drop of 5% suspension of 0Icells to tube labeled 0I and I drop of O IIcells to tube labeled 011.

Mix and incubate both tubes at 37C for 45-60 minutes.

Spin at 1000 rpm for 1 minute and examine for agglutination. Record the results. Agglutination will not occur if incomplete antibodies are present.

Wash the cells in each of the tube 3-4 times with warm saline. Decant the saline completely after the last wash over a filter paper. conti........

Add 1-2 drops of AHG reagent to each tube. Centrifuge immediately and look for Agglutination.

If negative, incubate at room temperature (22-24C) for further 5 minutes.

Re-centrifuge and look for agglutination.

Confirm negative test by adding a drop of IgG sensitized 0 Rh (D) positive cells. Agglutination should be seen.

Interpretation - Agglutination in one or both the tubes indicates presence of unexpected antibody in the test serum. If no agglutination occurs, use enhancing techniques. Results of control tubes should be considered before final interpretation.

Factors Affecting the Sensitivity of IAT---

1)Temperature- Optimal temperature : 37C. Incubation at higher or lower temperature may give false results.

2)Serum Cell ratio- Increasing the ratio of serum to cells increases the antibody coating. Commonly used ratio in saline suspension is 2:1 but in LISS suspending cells, use equal volume of serum and 2% cell suspension.

3) Incubation time- Saline, Albumin or enzyme technique : 45-60 minutes LISSsuspended cells - Routine 15 minutes Emergency : 5 minutes

Suspension medium--The sensitivity of IAT can be increased with addition of 22% bovine albumin, enzyme or by using LISS suspended cells.

The following enhancing media may be used to increase the sensitivity of the indirect antiglobulin test. 1. Albumin 2. Enzyme 3. LISS (Low ionic strength saline) solution 1) Albumin Albumin has no effect on sensitization of the antigen by the antibody. It enhances agglutination by reducing the repulsion charges between red cells. Procedure-First three steps are same as in IAT. 2. At step 4, incubate mixture for 15-20 minutes at 37C. 3. Add 1 drop of 22% bovine albumin along the side of the tube. 4. Incubate further for 15-20 minutes. Spin at 1000 rpm for 1 minute and examine for agglutination. 5. Spin at 1000 rpm for 1 minute and examine for agglutination.

Enhancing Media for IAT

Reagent 1% solution of papain-cystein ,

0I and OIl reagent cells

Procedure -

Add 1 drop to test serum in each of the tubes labelled 0I & OII

2. Add I drop of papain-cystein solution to each of the tubes.

3. Add 1 drop of 2-5% cell suspension of 01 cells to the tube labelled Ol and I drop of OII cells to the tube labelled 0II. Mix and incubate at 37C for 45 minutes. Spin at 1000 rpm for 1 minute, examine for agglutination and record the results. Proceed to the antiglobulin test from step 6 onwards in the IAT.

Two stage method for enzyme --

Reagent -- 0.1% solution of papain-cystein

Procedure --

Place 1 drop of washed 5% 01 and OII cells in labelled test tubes.

Add 2 drops of papain-cystein solution to each of the tubes.

Mix and incubate at 37C for the duration which has been determined by the enzyme standardization procedure. This is usually around 15 minutes

Wash 3 times with saline and decant last wash completely on filter paper. conti...

Add 1-2 drops of serum, mix and incubate at 37C for 15-30 minutes. Spin at 1000 rpm for 1 minute. Look for haemolysis or agglutination and record the results.

Proceed onwards as from step 6 in the IAT.

LISS (Low ionic strength saline) solution LISS reduced the ionic strength of the reaction medium and thereby enhances antibody uptake by the red cell antigens. This helps in increasing the sensitivity of the test

Preparation of 0 cells in LISS Pool known 0 Rh (D) positive cells from 2 donors each, in each of the 2 tubes labelled 01 & Oil. Wash twice in normal saline and once in LISS. Make 2-5% cell suspension in LISS.

Procedure is identical to IAT except that the incubation period is reduced to 15 minutes at step 5. In an emergency even 5 minute incubation will be sufficient.

Note: It is imperative to put positive and negative controls, as LISS may give few falsepositive reactions.

Washing of red cells-- While performing direct or indirect antiglobulin test it is mandatory to make sure that pipette, saline, test tubes, etc. are serum-free and absolutely clean.

Always wash the cells throughly after incubation (sensitization) to remove all traces of human serum except that coating the red cells. Presence of the smallest amount of human globulin can neutralize the antihuman globulin reagent and give a falsenegative result.

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