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 individual’s group is A,B, AB or O.

later moderate amt of Ab are present which appear after specific Ag stimulus.globulin in nature. So called as Naturally occurring Ab. • The Ab are mainly gamma . the new born has no ABO antibodies. • Ab are proteins.related serological reactions are mainly IgG.However after 10.Antibody • Under normal circumstances. Ab involved in transfusion .20 wks .IgA. IgM. .more specifically Ig’s which are recognized by their interaction with Ag.

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 .Blood Group Antibodies Immunoglobulin Synonyms Properties NO. of subclass Mole.

B are found in yhe sera of individuals lacking the corresponding Ag. a subsequent exposure will cause faster and more pronounced response k/a Secondary response..A and Anti. Antibody( immune response) • On first exposure to an Ag the body initiates a Primary response. • Secondary will predominantly produce IgG.cause production of IgM Ab. • Primary response . slow and takes months for Ab to form • Once the individual has been sensitized to a particular Ag.. .Anti. • Naturally occurring Ab .conti.

• Develop by the age of 6-8 mths and maintained throughout life. .• 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.

. most common type in blood group serology . • Sensitization. occurs in two stages 1) Attachment (sensitization) 2) Agglutination .Antibody-Antigen Reaction • Agglutination.cross linking of sensitized cells occurs by bridges of Ab molecules leading to a lattice formation. • Haemolysis.It is defined as clumping of particles or red cells that have Ag corresponding to the Ab on their surface.Red cell lysis indicates the presence of Ag-Ab reaction and consumption of complement.It is defined as coating or binding of Ab on red cell surface without bringing about agglutination in saline .

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

• Enzymes.2 for optimal reaction.Reagents used in DetectingAg-Ab Reaction • Saline. • Bovine Albumin. ions and thus raising the dielectric constant of medium.Basic medium in which cells are suspended.Bovine albumin in reaction mix reduces the zeta potential by dispensing sod.ficin.Proteolytic enzymes like papain. to cause agglutination. .They act by removing sialic acid from red cell membrane.trypsin.reducing the surface charge.causes red cell to move closer together and allows IgG mole. pH of which should be 6.8 to 7. have been used to enhance agglutination.

• Anti-human globulin serum.g polyvinyl pyrrolidone. decreases the ionic strength of Ag sites and increases the Ab uptake.gum acacia.dextran. • Macromolecular potential medium.Helps in sensitization phase of Agglutination.Most commonly .Synthetic medium e. Polybrene (hexadimethrine bromide) has been used in serology.reduction in strength of suspension medium.

The clumped RBCs can crack and cause toxic reactions.Blood gp Ag are component of red cell membrane and are immunogenic. that blood transfusions became safer.Chemically these are either Glycoprotein or Glycolipids.Blood Group Systems Inheritance and Genetics • Experiments with blood transfusions have been carried out for hundreds of years. 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. He found that mixing blood from two individuals can lead to blood clumping. polysaccharide component determines the specificity and amino acid fraction for antigenicity. • . This can be fatal. Many patients have died and it was not until 1901. when the Austrian Karl Landsteiner discovered human blood groups.

MN. Rh System ABO Chido. 707 alleles Also detailed: non-human counterparts for H/h.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. enzyme 67 XK SLC14A1 transport 8 CR1 receptor 24+ ICAM4 adhesion (LW) FUT3. enzymes B3GALT3 CD151 RHCE. RHBG ERMAP adhesion XG. 40 genes.Summary: 29 blood group systems. transport RHD. 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. unknown GYPB. RHCG RHAG. enzymes FUT6. FUT7 LU adhesion GYPA. GYPE BSG adhesion A4GALT. 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 . enzymes 57 FUT2 GCN2 enzyme 8 (IGnT) CD44 adhesion 2 SEMA7A signaling 0 KEL.

antigen defining blood group system .

O is a silent gene or amorph and no gene product is expressed.B and O. These alleles were termed A ( which produced the A antigen ). the four alleles are A1. • ABO blood group genes consist of multiple alleles which are located on the long arm of Chromosome 9.ABO group system • ABO was first to be discovered . What do co. A and B are codominant and O is recessive. also is the most important in transfusion practice becoz of the invariable presence of naturally occurring anti-A and anti-B Ab.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. B (which produced the B antigen) and O (which was "non functional"and produced no A or B antigen) • • • • .A2.

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

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

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

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

. • • • • • They appear to be used for the transport of carbon dioxide and/or ammonia across the plasma membrane.• Rh blood group system blood produce The majority of Rh-negative individuals transfused with Rh-positive immune Rh Ab.e.they are protein products present on chromosome 1.c.Rho (D) Ag is clinically imp in the Rh system beccause of immunogenicity which is greater than of other systems . They are named for the rhesus monkey in which they were first discovered. Importance in HDN. Of described 40 only 5 are important and common.race is adequate to study the clinical imp problems.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.C.Rh Ag ( D. Concept of Fisher.E) are products of their genes . • • RBCs that are "Rh positive" express the antigen designated D. HTR. Rh Antigen-Unlike ABO Ag only D Ag are present on red cell .

• A sensitized Rh -ve mother having anti-D Ab can produce HDN in a Du. • Its significance implies that red cells have fewer D Ag sites and therefore may be diagnosed as D -ve .A weaker variant of D Ag is termed as Du and detected only by AHG technique.• Du phenotype. • Recently introduced monoclonal Anti-D reagents gives positive reaction in 95% Du individuals in the tube method and 99% in the microtitre plate.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 . • Tranfusion of Du blood to an Rh -ve individual can theoretically produce Ab against D and Du. .positive foetus.

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 • • • • • .

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.antisera are available. Cell grouping done by testing an individual red cell with known blood grouping reagents and corresponding serum group with Known A cell. Reagent red cells When performing an ABO serum grouping.It is essential to use reliable grouping reagents to correct results .commercially prepared polyclonal. B cell. O cell. • • ABO grouping sera . monoclonal. it is important to ensure that the cells prepared are fresh and well-selected ‘A’ cells- • • • • .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.

‘0’ cells Pool known fresh C group cells from 1-2 donors. • • • • • .three times with normal (0.9%) saline. 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. 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. They must then be accurately diluted to give a standard suspension in saline by either using a graduated tube (Wintrobe’s tube) or by counting drops to make a 2-5% cell suspension.

If low-ionic strength saline solution (LISS) is being used in the laboratory as an enhancing medium. using a graduated tube (Wintrobe’s tube) or by counting drops to make a 2-5% cell suspension. The cells should be kept at 4°C in the refrigerator when not in use.• cont.. then the last wash should be in LISS and the cells are thereafter suspended in LISS to make a 2-3% cell suspension.. When prepared. • • • • • . 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.. the cells should be checked against anti-A and anti-B.. 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.

when performing blood grouping: Methods & Procedure Three manual methods can be used .Microwell plate or microplate Newer techniques .Glass test tube .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.Glass slide or white porcelain tile . however it should always be supplemented with a cell and serum grouping using any one of the other .Column technique (sephadex gel) .• • • • • • • • Blood grouping .

weakly reactive antigens on cells • . however it should always be supplemented with a cell and serum grouping using any one of the other above mentioned techniques.c .serum grouping with low titre anti-A or anti-B • Disadvantage-. • Slide or tile testing is not recommended for routine use because it is not reliable for • .• This technique may be used for emergency ABO grouping tests or for preliminary grouping particularly in an outdoor camp.

Mix the cells and reagent using a clean stick. 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). Spread each mixture evenly on the slide over an area of 10-15 mm diameter. giving false positive results.• conti. • .. • Procedure-1. .Weaker reactions are difficult to interpret. • Less sensitive than the tube test • .Drying up of the reaction mixture can cause aggregation of cells. • 3. • 2. Place 1 drop of anti-A and 1 drop of anti-B reagent separately on a labelled slide or tile.

Then rock again and look for agglutination. . of lOx75mm size.• 4. The tube technique is more sensitive than slide technique for ABO grouping. • Advantages of tube testing - • . Tilt the slide and leave the test for 2 minutes at room temperature (22°-24°C). • Tube Testing - • Test tubes either of glass or plastic may be used.It allows for fairly long incubation without drying up of the tubes’ contents. Record the results. • 5.

Centrigugation involved enhances the reaction allowing weaker antigens and antibodies to be detected..It allows for fairly long incubation without drying up of the tubes’ contents. . • Procedure . • Simplicity of reading and grading of results. • .• conti..Requires smaller volume of reagents...conti.. • .Clean and more hygienic. • . • .

Red cell suspension . • 2. monoclonal or polyclonal. Serum grouping / reverse grouping • Reagents required • 1. Spin test sample to separate serum.reagent cells (Ac.• Saline agglutination test for cell and serum grouping by tube method • 1. anti-AB). test red cells (patients or donor) • Cell and serum grouping • 1. . Known antisera (anti-A. anti-B. Bc and Cc). Cell grouping / forward grouping • 2.

anti-B in tube labelled B.B and AB. Prepare once washed 2-5% suspension of the test cells. • 5. Resuspend cell button by gently shaking the tubes and read against well-lit background. Add I drop each of reagent A cells in labelled tube Ac. Add I drop of anti-A in tube labelled A. Mix all the 6 tubes and centrifuge at 1000 rpm for 1 minute. Resuspend cell button by gently shaking the tubes and read against well-lit background. Bc and Cc. • 4. • 9. .Bc and Cc. Anti A. Anti-B. Set up 6 tubes correctly labelled with donor/patient no. Add 1 drop of 2-5% test cell suspension in the three tubes A. and anti-AB in tube labelled AB. • 8. Add 2 drops each of the test serum in tubes labellaed Ac.• 2. • 6. • 3. Ac. • 7. B cells in labelled tube Bc and 0 cells in tube labelled Oc. Record the result • 9. Anti-AB.

defining the strength of reaction ( grading of agglutination) .

• slide or tile testing .

Microplate technology is gaining widespread popularity due to increasing workload in blood transfusion laboratories and recent availability of packaged automated system. • . • Advantages of Microplate ABO grouping- • Small volumes and low concentration of sera and red cells are used.Microwell plate consists of a small tray with 96 small wells each of which can hold about 200-300u1 of reagent.Easy handling of a microplate. • Flat bottom plates are useful in ELISA technology but are unsuitable for liquid-phase blood group serology. Flat-bottom • The U-type well is generally used in red cell serological work as it is easier to read the results in U. . • 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.c. V-type well . U-type well . which can replace 96 test tubes.b. • Three types of microplates are available -a. making it cost-effective.• Microplate Technique .bottom plates.

Large batches of plates can be predispensed with antisera and reagent red cells before testing.Untreated rigid polystyrene microplates • -Pipettes • -Manual / automated single or multi-channel dispenser • -Variable volume dispenser (20 . • Equipment required for microplate grouping • Microplate • . • .Batching of samples can be achieved with considerable economy in space and time. reagent dispenser.If larger laboratories acquire microplate hardware items e. sample handler and cell washer it may further reduce the operation time. • .• .50 ul) • -Centrifuges .g.

.Newer Techniques • Column technique- • conti.50 ul) • -Centrifuges • -Bench-top centrifuge with head for carrying microplates • -Microplate shaker • -Two/four place shakers • Blood Grouping .. ...• -Variable volume dispenser (20 .........

ANTI-B. • .Neutral. ANTI-D. ANTI -B.KELL. ANTI. unagglutinated cells form a button at the bottom of the microtube and agglutinated red blood cells are trapped in the gel. ANTI -A .Reactions are easily visible and can be graded • . • The Sephadex gel may be. 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. • Advantages of gel system --.Reactions using gel techniques are stable for 48 hrs and gel cards can be photocopied providing a permanent record for future use. making it ideal for neonatal and paediatric use.Superior in sensitivity to conventional tube test without loss of specificity.Easy to use.Requires small amount of red cells and serum for testing (1O-40m1).• Recently interest has been generated in use of column technology for serological testing. • .Specific reagent-------AHG. • Reactions are easily visible and can be graded. .

• READING AND GRADING OF HAEMAGGLUTINATION IN GEL SYSYTEM .

3. dry test tube. Gently agitate and examine for agglutination. . 4. Place 1 drop of anti-Al reagent into a clean.• 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. • Reactions showing agglutination indicate Al blood group. It is stored at 4-8°C and may be frozen at -20°C for prolonged storage. • Procedure .1. 2. Add 1 drop of 2-4% saline suspension of patient’s cells. Mix and leave at RT for 30-60 minutes.

g. C. Anti-D for saline tube test 2 types Anti-D IgM Anti-D IgG . react equally well at 20°C as well as 37°C and are reliable for slide and rapid test tube technique.RH (D) GROUPING • • Practical Aspects of Rh Grouping Rh grouping in routine use for donors and patients involves testing for Rh (D) antigen only. 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.E and e may be done for Rh genotyping.Blend of IgM monoclonal + IgG polyclonal reagent These antibodies are highly specific. • • • • • • • .c. however tests for other important Rh antigens e.Chemically modified II )Monoclonal antibodies---IgM anti-D monoclonal reagent.IgM and IgG anti-D monoclonal reagent.

...reagents from two different batches of different firms following the manufacturers’ recommended technique (antiDi. • 1..2. Tube technique 3. 2. Saline Agglutination test for Rh (D) Typing. CONTI.of anti-D (D2) from a different manufacturer in a clean tube labelled D2. .1. 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. Slide technique (The slide test is not recommended for routine test as it may not pick up weak reactions..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-. • 3.. thus giving negative results) . Prepare 2-5% washed red cell suspensionof test sample. Microplate technique.... Place 1 drop of 22% bovine albumin/control reagent in another tube labelled C. Place 1 drop of anti-D (Dl) in cleaned tube labelled Dl and place 1 drop .. • Tube Technique- a.

All negative results must be confirmed under microscope.Positive test : Agglutination in anti-D (both tubes) and smooth suspension in control tube. . Mix well and centrifuge at 1000 rpm for 1 minute (in case of using IgG anti-D. hereas some workers suggest that if the two anti D reagents used are potent and specific. Re-suspend the cell button and look for agglutination.• 4. Du testing should be performed. • 5. • 6. it is not necessary to perform Du testing. Add 1 drop of 2-5% test cell suspension to each tube. • Negative test : Smooth suspension in all the tubes (test and control) • For all microscopically negative reactions in donor grouping.. • Interpreation. incubate at 37°C for 10mm. and centrifuge (spin tube method) or incubate at 37°C for 60 minutes (sedimentation method).

Add 1 drop of 2-5% test red cell saline suspension. Albumin will form a layer on top of the red cells.5. Place 1 drop of anti-D in a labelled tube. 3. Mostly 22% bovine albumin is used. Allow 1 drop of 22% albumin to run down the inside wall of the tube. Albumin technique for Rh (D) typing.• b.Procedure-- • 1. Due to this effect. • 4. Incubate at 37°C for 45-60 minutes. • 6. Do not mix. as higher concentrations can cause rouleaux formation. Examine for agglutination after gentle shaking and confirm all negative results under microscope.Principle--. 2. .Albumin increases the dielectric constant of the medium and thus reduces the zeta potential. Incubate further at 37°C for 15-20 minutes. • Albumin can be used in ---.Albumin addition technique additive to serum & cell mixture & Albumin layering technique layered on cell button • Albumin layering technique -. the electrical repulsion between the red blood cells is less and the cells agglutinate.

Agglutination in test sample and negative reaction in control sample shows a positive test and the sample is labelled Rh (d) positive. 7.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 appropriate diluent control in another tube (C). if positive test.e. examine for agglutination and record the results . 9. If negative. Mix gently and centrifuge at 1000 rpm for 1 minute. • 8. • 2. Gently suspend the cell button and look for agglutination.• Indirect antiglobulin test (Du Testing) . Add 2-5% washed test red cell suspension to both the tubes. Take 1 drop of anti-D (IgG) in a cleaned labelled test tube (T). Mix and incubate at 37°C for 45-60 minutes. • 6. no need to proceed as the sample is Rh (D) positive. wash the cells 3-4 times with saline and decant the last washing. • 4.5. Resuspended the cell button gently. . • 3. Add 1-2 drop of anti-human globulin reagent (AI-IG-Coombs’ reagent). 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.

.• Microplate technique for Rh (D) grouping as described earlier in ABO grouping. the baby’s 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. This is suspected when the infant’s cells show a positive direct antiglobulin test (DAT) and a negative test with anti-D reagent. resulting in a negative test. no free antigenic sites remain for reaction. • Rh(D) Grouping In Haemolytlc Disease of the Newborn---In haemolytic disease of the newborn.In such instances it is recommended that the antibody should be eluted by gentle elution (heating at 45°C for 30 minutes) to expose the antigenic sites before testing.

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

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----

• • •

• •

The preparation should give a negative direct antiglobulin test (DAT). • Mix and incubate at 37°C for 30 minutes. repeat the test by using less diluted anti-D serum.• Take 0. repeat the procedure using more diluted anti-D. If there is agglutination. .5 ml of 5-6 times washed and packed 0 Rh (D) +ve red cells in a test tube. • Perform a Direct antiglobulin test which should give a 2+ reaction. • 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 37°C). • 0 Rh(D) negative sensitized red cells are also prepared by treating 0 Rh(D) negative cells in the same manner. • Wash 3-4 times and make 5% suspension in saline for use. If no agglutination occurs.

..• Procedure (DAT) -- • Place 1 drop of 2-5% suspension of red cells in a clean labeled test tube.. centrifuge and look for agglutination and record the results. • Add 1-2 drops of Al-IG reagent.. • If result is negative. conti. • Shake the tube gently to dislodge the cell button and read the results using a concave mirror.. • Wash the red cells 3-4 times with saline and decant the final wash completely...... • Mix and centrifuge at 1000 rpm for 1 minute. . incubate the test for further 5 minutes at room temperature..

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

cells from 2 donors are taken.Blood sample for IAT should be collected in plain labeled test tube.Indirect Antiglobulin Test (IAT) • • Sample collection.. Prepare a 5% suspension for use..... Wash 3 times with normal saline.conti. Procedure (IAT)-.... 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. Reagents-Anti-human globulin reagent.. In each tube. • • • • .

• Add 1 drop of 5% suspension of 0Icells to tube labeled 0I and I drop of O IIcells to tube labeled 011.. • Wash the cells in each of the tube 3-4 times with warm saline.• Centrifuge the tubes at 3000 rpm for 5 minutes to separate the serum..binding antibodies otherwise fresh AB serum should be added to it). • Add 2 drops of serum in each of the tubes labeled 0I and 0II (sample should be fresh for detecting complement . • Spin at 1000 rpm for 1 minute and examine for agglutination. Agglutination will not occur if incomplete antibodies are present. Record the results.. conti.. ... • Mix and incubate both tubes at 37°C for 45-60 minutes. Decant the saline completely after the last wash over a filter paper..

use enhancing techniques. . Results of control tubes should be considered before final interpretation. • Re-centrifuge and look for agglutination. Agglutination should be seen. Centrifuge immediately and look for Agglutination. • If negative.Agglutination in one or both the tubes indicates presence of unexpected antibody in the test serum.• Add 1-2 drops of AHG reagent to each tube. • Confirm negative test by adding a drop of IgG sensitized 0 Rh (D) positive cells. • Interpretation . If no agglutination occurs. incubate at room temperature (22°-24°C) for further 5 minutes.

Optimal temperature : 37°C. use equal volume of serum and 2% cell suspension. enzyme or by using LISS suspended cells.• Factors Affecting the Sensitivity of IAT--- • 1)Temperature.Routine 15 minutes Emergency : 5 minutes • Suspension medium--The sensitivity of IAT can be increased with addition of 22% bovine albumin. • 3) Incubation time. .Increasing the ratio of serum to cells increases the antibody coating.Saline. Commonly used ratio in saline suspension is 2:1 but in LISS suspending cells. Incubation at higher or lower temperature may give false results. • 2)Serum Cell ratio. Albumin or enzyme technique : 45-60 minutes LISSsuspended cells .

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

Mix and incubate at 37°C for 45 minutes. Add I drop of papain-cystein solution to each of the tubes. 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. examine for agglutination and record the results. Proceed to the antiglobulin test from step 6 onwards in the IAT. 0I and OIl reagent cells • Procedure - • Add 1 drop to test serum in each of the tubes labelled 0I & OII • 2.• Reagent 1% solution of papain-cystein . . Spin at 1000 rpm for 1 minute. • 3.

. • Mix and incubate at 37°C 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.0. conti.. • Add 2 drops of papain-cystein solution to each of the tubes.• Two stage method for enzyme -- • Reagent -. .1% solution of papain-cystein • Procedure -- • Place 1 drop of washed 5% 01 and OII cells in labelled test tubes.

• Procedure is identical to IAT except that the incubation period is reduced to 15 minutes at step 5. mix and incubate at 37°C for 15-30 minutes. Make 2-5% cell suspension in LISS. • 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.• Add 1-2 drops of serum. 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. Spin at 1000 rpm for 1 minute. Wash twice in normal saline and once in LISS. in each of the 2 tubes labelled 01 & Oil. In an emergency even 5 minute incubation will be sufficient. Look for haemolysis or agglutination and record the results. • Proceed onwards as from step 6 in the IAT. .

etc. saline. test tubes. as LISS may give few falsepositive reactions. are serum-free and absolutely clean.While performing direct or indirect antiglobulin test it is mandatory to make sure that pipette. • Washing of red cells-.• Note: It is imperative to put positive and negative controls. • 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.

• Thank You .

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