INTRODUCTION

Karl Landsteiner in 1900 first described the existence of major human blood groups. At present more than 20 blood group systems having more than 400 red blood cell antigens have been identified, most of which are inherited in a Mendelian dominant fashion and only few are clinically important. The two most important blood group systems are ABO and Rh systems.

ABO BLOOD GROUP SYSTEM

ABO Antigens

According to the ABO blood group system, there are four different kinds of blood groups: A, B,AB or O. The antigen on red cells may be A/B/AB or no antigen at all. There are two subgroups in group A, namely A1 and A2. A and B antigens are inherited as per the Mendelian laws. Each individual inherits two ABO genes, one from each parent and these genes determine the ABO antigens on their red blood cells. Absence of both antigens, A and B on the red cells is seen in blood group O.

ABO Antibodies

Natural ABO Antibodies

The serum of individuals (who have not been previously transfused) who lack the corresponding red cell antigen contains naturally occurring antibodies. These antibodies are against red cell A (anti-A)/B (anti-B)/AB (anti-AB) antigens (Table 58.1). 

These are of IgM type and do not cross the placental barrier. Group A has two subgroups namely A1 and A2, and similarly AB has two subgroups namely A1B and A2B. But these are not of major significance.

The serological typing indicates the phenotype and family studies indicate the genotype. Genotype refers to the genes present and the phenotype is the red cell antigen on the cell surface.

Acquired ABO Antibodies

•• An individual lacking a particular antigen may develop an antibody after (acquired) exposure to red blood cells carrying the corresponding antigen.

•• Exposure occurs due to parenteral introduction of red cell antigens. This may develop during:

–– Transfusion of red cells: These acquired antibodies cause hemolytic transfusion reaction.

–– Pregnancy: Passage of fetal red cells (having paternal antigens foreign to the mother) into the maternal circulation during pregnancy may result in mainly IgG type of antibodies. They can cross the placental barrier resulting in hemolytic disease of the newborn. The most important acquired antibody is anti-D, which is a major cause of hemolytic disease of the newborn.

H Genes and H Antigens

The expression of A and B antigens are dependent on the presence of H gene. Most of the individuals are homozygous for the H gene. The sequence of events in the formation of A and B red cell antigens is:

•• A and B red cell antigens are glycoproteins and their formation starts with basic precursor substance (Fig. 58.1 above).

•• Basic precursor substance is first converted into H substance (by transferase) under the influence of H gene.

•• H substance is partially converted under the influence of A and B genes (and specific transferase) into A and B antigens. Some of the H substance remains unconverted.

•• Since O group individuals do not have A and B genes, neither A nor B antigen is formed and these have only H substance (H substance remains unchanged).

Note: A, B and H antigens may be detected in the saliva and other body fluids also. Such individuals are called secretors, while the remaining without it are non secretors.

Bombay Blood Group

In 1952, Bhende, Bhatia and Deshpande discovered a new blood group known as Bombay blood group (phenotype Oh). These individuals lack the H gene and therefore the basic precursor substance cannot be converted into H substance. This in turn, results in failure to form A or B antigen. When their blood sample is tested for routine ABO grouping, they will be labeled as blood group O. However, their serum contains anti-A, anti-B and anti-H antibodies.

These individuals, therefore, should be transfused with only Bombay blood group.

ABO System and Disease

•• Group O individuals have increased risk of developing peptic ulcer.

•• Group A individuals have increased risk of developing gastric carcinoma.

ABO Grouping Technique

ABO grouping is carried out by making a 2% saline suspension of red cells and adding anti-A, anti-B and anti-AB sera. The different methods available are:

•• Slide or tile technique

•• Tube technique

•• Microplate method

•• Microtyping system

•• Automated or semi-automated method.

The slide technique is easier to do and is described below.

Slide or Tile Technique

•• Take a slide/white tile and mark it anti-A, anti-B and anti-AB.

•• Put one drop each of anti- A, anti-B and anti-AB sera on the marked slide.

•• Add one drop of washed 2% red cell suspension to each anti-sera.

•• Mix each one separately with clean applicator stick and spread the mixture over an area of 2 cm.

•• Rock the slide gently and look for “agglutination” within 5 minutes.

Interpretation:

Blood group A, B, AB or O is interpreted depending on the agglutination in the anti-sera (Table 58.2 and Fig. 58.2).

Tube Technique

•• Take three test tubes (75 × 10 mm size) and label them as anti-A, anti-B and anti-AB respectively.

•• Add one drop each of the corresponding serum to each tube (i.e. anti-A, anti-B and anti-AB).

•• Add one drop of 2% red cells suspension to each one of the three test tubes and mix them gently.

•• Leave the tubes at room temperature for 90 minutes (or centrifuge for 15–20 minutes).

•• Look for agglutination.

•• Transfer one drop of mixture from each tube onto a glass slide, using a thin bore Pasteur pipette. Spread the cells over an area of 2 × 1 cm, using the pipette.

•• Examine the slide under microscope for “agglutination”.

Interpretation

Blood group is interpreted depending upon the agglutination in the anti-sera (Table 58.2).

Controls

Each blood group test should preferably have controls, both positive and negative.

•• Positive control: This is run by performing the above techniques by using cells of known groups A, B, AB and O. This is especially useful for testing the potency of anti-sera.

•• Negative control: Run the above tests by adding saline instead of anti-sera. If agglutination develops it indicates autoagglutination or pseudo agglutination.

Microplate Method

Microplate method is ideal for testing large number of blood samples and has replaced test tube method in many laboratories. Microplates are polystyrene plates having 96 small well. It saves time and cost.

Microtyping System (Gel Card)

Microtyping system (MTS) is a gel-based technology in which plastic cards contain microtubes filled with buffered dextran gel. The gel contains sodium azide (preservative) and group specific antisera (anti-A, anti-B, anti-AB, anti-D) or antiglobulin (for Coombs test). These constituents are incorporated into gel at the time of manufacture.

Technique

•• Red cells are poured over the microtube.

•• Incubate at 37°C and centrifuge.

•• The cells settle down and react with the specific antisera (antibody) incorporated into the gel resulting in hemagglutination.

Interpretation

•• Positive test: The gel matrix acts as a sieve and agglutinated red cells are too large to pass through the gel. Red cells are trapped at various places within the gel and form a line in the microtube indicating positivity of the test.

•• Negative test: If the specific antigen is not present on the red cells, hemagglutination does not occur and the red cells pass through the gel forming a pellet at the bottom of the microtube.

Advantages

•• Simple, safe, easy and cost-effective technique.

•• Greater sensitivity when compared to conventional techniques.

•• Technical errors are eliminated.

•• For Coombs test, red cells do not need saline washing.

Uses of microtyping system

•• Blood grouping and pre transfusion cross matching.

•• Coombs test.

•• For diagnosis of: 

–– Syphilis, measles, diphtheria, parvovirus infection

–– PNH, sickle cell disease.

Note:

•• A group has two subgroups namely A1 and A2, which can be differentiated by using anti-A1 lectin.

•• Presence of H substance can be detected by anti-H.

Rh BLOOD GROUP SYSTEM

Rh blood group system is the second system of clinical significance in transfusion medicine. This is a complete system of antigens which are labeled as D, d, C, c, E and e. The D antigen is highly immunogenic. Cc and Ee antigens are weak antigens and therefore risk of sensitization to these antigens is less than the risk of sensitization to D.

Rh (D) System

Rh Antigen

Rh positive (+ve)

Individuals who have D antigen on the red cell surface are Rh positive (+). This may be present as homozygous (DD) or heterozygous (Dd) state.

Rh negative (–ve)

Individuals who lack the D antigens are called as Rh-negative.

Rh Antibodies

•• In contrast to ABO system, there are no naturally occurring antibodies against Rh antigens in Rh negative individuals.

•• Acquired (immune) antibodies: Rh antibodies develop after exposure to Rh antigens either following transfusion (Rh +ve blood given to Rh –ve patients) or pregnancy (Rh +ve fetus in an Rh –ve mother). These antibodies are of IgG type and do not bind complement. They can be detected by antiglobulin test (Coombs test).

Clinical Significance

•• Rh incompatibility between donor and recipient results in hemolytic transfusion reactions.

•• Rh incompatibility between mother and fetus results in hemolytic disease of the newborn(HDN). 

Rh (D) Typing Techniques

The different methods available are:

•• Slide or tile technique

•• Tube technique

•• Microplate method

•• Microtyping system

•• Automated or semi-automated method.

Slide or Tile Techniques

•• Place one drop of anti-Rh (D) reagent (should be monoclonal IgM type) on a slide/white tile.

•• Add one drop of 2% red cell suspension.

•• Mix them using a clean applicator stick.

•• Observe for agglutination after 2 minutes.

Interpretation: Presence of agglutination indicates that the blood sample is Rh +ve.

Tube Techniques

•• Add one drop of anti-D to a small test tube (75 × 10 mm size).

•• Add one drop of 2% saline red cell suspension.

•• Mix them and incubate at 37°C for 30 minutes or centrifuge for 15–20 seconds.

•• Look for agglutination.

•• Transfer one drop of the mixture onto a glass slide, using a thin bore Pasteur pipette. Spread the cells over an area of 2 × 1 cm using the pipette.

•• Examine the slide under microscope for “agglutination”.

Interpretation: Presence of agglutination indicates Rh +ve blood group. Controls

Known Rh +ve and Rh –ve samples should be run as positive and negative controls.

OTHER BLOOD GROUP SYSTEMS

These are weak antigens and have no clinical significance(Box 58.1).


Points to note:

•• There are several blood group systems, important being ABO and Rh systems.

•• Individuals with Bombay blood group lack the H gene and there is failure to form A or B antigen.

•• Blood grouping can be performed by slide/tube/microplate/automated/semi-automated method or by microtyping system.

•• Blood grouping is important before blood transfusion and is also of medicolegal value.