Determination of Human blood groups: ABO and Rh system

 

Introduction: Agglutination Reaction

The interaction between antibody and its corresponding particulate antigen results in visible clumping called agglutination .Antibodies that produce such reactions are called agglutinins.  Agglutination reactions are routinely performed to determine the type of red blood cells Ag. In typing for the ABO antigens RBCs are mixed on a slide with antisera to the A or B blood group antigens. If the antigens are present on the cells they agglutinate forming a visible clumping on the slide. These agglutinations of RBCs are called hemagglutination.

BLOOD GROUP SYSTEM: 

Approximately 500 blood group antigens have been described, many of which fall within the 20-30 so-called blood group systems. Some examples of blood groups are :ABO, Rh, MNS, P, Lutheran, Kell, Lewis, Duffy, Diego, Yt, Li, XG, Dombrok and Colton blood group system. Fortunately, only ABO and Rh system are of major clinical importance than others. It is because of one or more of following reasons:

We now know these four classifications as the ABO blood group system.

International or Landsteiner Group	Antigen on RBCs	Antibodies in serum	May donate blood to	May receive blood from
A	A	Anti B	A & AB	O & A
B	B	Anti  A	B & AB	O & B
AB	A  & B	None	AB	Universal recipient*
O	None	AntiA + Anti B	Universal donor*	O

ABO AND Rh GROUPING TECHNIQUE

Establishing the ABO group of an individual usually involves so-called forward and reverse grouping. Forward grouping (cell grouping) involves testing of patient’s RBCs against known anti-An anti-B, and anti-A,B sera; while in reverse  grouping (serum grouping) patient's serum is tested against red cells obtained from group A, and B individuals. The results of forward and reverse typing must agree before a blood group is reported.

The sera for forward grouping is of humun origin, usually collected from individuals whose “natural” antibodies have been stimulated to high titers (1:256). However, present day sera are monoclonal in nature and are usually obtained by hybridism technology.

Blood grouping may be done either by side test or modified tube test.

 

Principle

Human red cells possessing A, B or D antigens will agglutinate in presence of antibody directed towards the respective antigens. Agglutination of red blood cells is a positive test result and indicates the presence of the corresponding antigen.

Requirements

Lancet .Normal saline solution (0.85%NaCl),.Cavity slides or plastic card, coverslips. Typing: anti-A, anti-B and anti-D Sera, 70%  alcohol.

RAPID SLIDE OR TILE METHOD

 

 

Procedure

·       Clean the fingertip with alcohol and wipe it dry.

·       Make a stab puncture deep enough to allow easy flow of blood without excessive squeezing.

·  Wipe off the first drop and collect 2-3 drops of blood in about 2 ml of normal saline solution(This makes about 10%suspension of red cells from the individual).

·       Mark 4 areas, A, B, D, C (control) on a clean glass slide.

·    Add one drop of anti-A, one drop of anti-B, and one drop of anti-D grouping reagent to the respective areas.

·       Put a drop of normal saline in the area labelled as C.

·      Add one drop of 10%red cell suspension (or oxalated whole blood) to each ofthe 4 areas  on  the  glass  slide.

·      Mix each separate cell –serum mixture thoroughly with different applicator sticks,and rock or rotate the tile for 1-2 minutes until clumping (agglutination ) is apparent.

Clinical significance:

·    Blood transfusions: The compatibility between donor red cell antigen & recipient’s plasma must be ensured by blood group matching cross matching.

·       Medicolegal Aspects: In paternity disputes the blood grouping of all parties concerned can no more than to exclude one of the parents. Usually a father is in dispute &is excluded if antigen that he genetically must pass on is not present in the child & if the child possess an antigen which both he & the mother lack the disputed father must be excluded.

·    Forensic Aspects: The determinations of the blood group antigens of an individual are nearly as exclusive as fingerprints. These facts are used frequently by police department throughout the world during the investigations of the criminal cases. The blood group substances are determined from the dried stains of blood, saliva, semen, &other body fluids.