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Blood grouping - Methods & Procedure

You are here : Home/ Blood Bank Zone/ Red Cell Serology/ 4. Blood grouping - Methods & Procedure

4. Blood grouping - Methods & Procedure

Three manual methods can be used when performing blood grouping:
- 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 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


- 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.


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°-24°C). 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.
- 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.


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.

10. Record results according to grades of agglutination.

Defining the Stregth of Reaction (Grading of Agglutination)

To record the difference in the strength of reaction, it is necessary to have a system of grading or scoring the reactions, as depicted in figure.

Grades Description
4+ 1 big clump
3+ 2 or 3 clumps
2+ many small clumps with clear supernatent
1+ many small clumps with turbid supernatent
w granular suspension
Zero or - smooth suspension
H partial or complete haemolysis
(positive reaction)


Cell grouping Serum grouping  
Anti-A Anti-B Anti-AB Ac Bc Oc Result
+ - + - + - A
- + + + - - B
- - - + + - O
+ + + - - - AB
- - - + + + Oh or any other irregular


1. Always use throughly cleaned test tubes for the test.

2. While reading the result, shake very gently to dislbdge the button.

3. Use correct speed and time centrifugation to avoid erroneous results.

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.
- The technique of microplate grouping may be automated by on-line data capture in larger laboratories, which may help in
---- reduction in reading and transcription errors
---- saving in staff time
---- use of bar codes for samples and microplate identification
---- integration into a comprehensive computer system for storage of data.

Equipment required for microplate grouping


- Untreated rigid polystyrene microplates
-Manual / automated single or multi-channel dispenser
-Variable volume dispenser (20 - 50 ul)
-Bench-top centrifuge with head for carrying microplates
-Microplate shaker
-Two/four place shakers
-Reading aid
-Automated plate readers
-Automated plate reader based on ELISA reading system

Blood bank zone Next Articles
  1. Red Cell Serology Introduction
  2. ABO Grouping
  3. Preparation of Red Cells for ABO Testing
  4. Blood grouping - Methods & Procedure
  5. Blood Grouping - Newer Techniques
  6. Reagents
  7. Rh Grouping
  8. Rh Genotyping
  9. Antiglobulin Test
  10. Enhancing Media for IAT
  11. Pretransfusion Testing (Compatibility Testing)
  12. Labelling and issue of Blood
You are here : Home/ Blood Bank Zone/ Red Cell Serology/ 4. Blood grouping - Methods & Procedure

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