Blood Group

Blood group System:

 ABO, MNS, P, Rh, Lutheran, Kell,Lewis, Duffy, Kidd,Diego, Yt, Xg,Scianna, Dombrack, Colton, Landseiner- weiner, Chido / Rogers,Hh, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph.

 

Principle: 

Landsteiner's Law

    If an agglutinogen (antigen) is present on the RBCs the corresponding agglutinin (antibody) must be absent in the plasma.

    If an agglutinogen (antigen) is absent on the RBCs the corresponding agglutinin (antibody) must be present in the plasma.

ABO Grouping.

    There are two methods for ABO grouping:

• Cell Grouping (Forward grouping): Red cells are tested for the presence of A and B antigens employing known specific anti-A and anti-B (and sometimes anti-A, B) sera.
• Serum Grouping (Reverse grouping): Serum is tested for the presence of anti-A and anti-B antibodies by employing known group A and group B reagent red cells.  

Both cells and serum grouping should be done since each test acts as a check on the other.

There are 3 methods for blood grouping: Tube, Microplate,and Slide.

Micro plate and Tube methods are employed better and are employed in blood bank.

Slide Test:

Principle:

    Red cells from the specimen are reacted with reagent antisera (anti-A and anti-B). Agglutination of red cells indicates presence of corresponding antigen (Agglutinogen) on red cells.

Specimen:

    Capillary blood from finger prick, or venous blood collected in EDTA anticoagulant.

Reagents:

    ABO antisera.

Procedure:

1. A clean glass slide is divided into two sections with a glass marking pencil. The sections are labeled as anti-A and anti-B to identify the antisera.
2. Place one drop of antisera-A at the one end of the slide and one drop of antisera-B at the another end of the slide.
3. Add one drop of blood sample to be tested to each drop of antiserum.
4. Mix antiserum and blood by using a separate stick or a separate corner of the slide for each section over an area about 1 inch in diameter.
5. By tilting the slide backwards and forwards, examine  for agglutination after exactly 2 minutes.

Positive: Little clumps of red cells are seen floating in a clear liquid.

Negative:Red cells are floating homogeneously in a uniform suspension.

• Slide test is quick and needs only simple equipment. It can be used in blood donation camps and in case of emergency.
• It is not recommended as a routine test in blood bank.
• Result of slide test should always be confirmed by cell and serum grouping by tube method.

Note:

• Three types: Anti-A,Anti-B,Anti AB.
• Anti-A blue colored;
• Anti-B Yellow colored.
• Anti-AB Colourless.
• Sodium azide is added to prevent the growth of bacteria.
• Anti sera are kept stored at 4/6 degree Celsius to preserve their potency.
• Routinely, Anti A and Anti B are used for blood grouping.
• Indication for using Anti AB :  For confirmation of cell grouping in new borns, and  To  resolve ABO discrepancies.
• Anti sera may be polycolonal or monoclonal. Monoclonal Antisera are Specific, avid and can detect weak antigens. Monoclonal Antisera are commonly used.

Tube Method:

Test tube method is more reliable than slide test, but takes longer time and more equipment.

Procedure: (Cell grouping/Direct grouping/Forward grouping)

• Mark two tubes with patient's number/ ID.
• Mark one tube Anti A or just A and other tube Anti B or B.
• Make 2% suspension in a saline of red cell
• Add 2 drops of Anti A to the tube marked A and 2 drops of Anti B to the tube marked B.
• Add 2 drops of cell suspension (2%) to each tube.
• Mix well and centrifuged both tubes at 1000 rpm (Revolution Per Minute) for 1 minute
• Remove the tube and hold the tube over concave mirror and inspect the deposited bottom of cells in the bottom.
• Look for agglutination and report .

Procedure: (Serum grouping/ Indirect grouping/Reverse grouping)

• Take 2 test tube and mark as 1 and 2.
• Place 2 drops of patient's sera /serum into each of the tubes.
• Add 2 drops of 2-5% suspension of group A cells in tube 1.
• Add 2 drops of 2-5% suspension of group B cells in tube 2.
• Tubes are shaken and centrifuged at 1000 rpm. for 1 minute.
• If clumping occurs in tube 1 and no clumping in tube 2, it belongs to blood group 'B'.
• If clumping occurs in tube 2 and no clumping in tube 1, it belongs to blood group 'A'.
• If there is clumping in tube 1 and 2' then it is blood group 'AB'.
• If no clumping occurs in tube 1 and 2,then it is blood group 'O'.

Microplate Method:

    It is a polysterene plate consisting of 96% micro wells of either U or V Shape.

    Grouping is carried out in micro wells. 

    This method is sensitive and ideal for large number of samples.

False Reaction In ABO blood grouping.

1. Autoagglutination.
2. Rouleaux formation.
3. False-negative result due to inactivated antisera.
4. Age.

Note: Infants start producing ABO antibodies by 3-6 months of age and serum grouping done before this age will yield false negative result.

Rh Blood Grouping:

The name Rh is taken from rhesus monkeys.

First of all antibodies against RBCs of rhesus monkey were obtained by injecting monkey RBCs into rabbits and guinea pigs.

Technique of Rh Grouping / Procedure:

Slide test

1. One drop of 40% cell suspension of patient on a clean slide is taken.
2. to,it 1 drop of anti-D sera is added.
3. Mix  and look for clumping , if blood sample in Rh-positive.

Tube Test:

1. Take 1 drop of 5% cell suspension of patient in a tube.
2. To it, add 1 drop of anti -D sera.
3. Centrifuge at 1000 rpm for 1 minute.
4. Look for clumping with naked eye and microscope.

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