What are the Procedures and Techniques of Blood Grouping and Cross Matching?–Explained!

The A blood type has only the A antigen and the B blood type has only the B antigen. The AB blood type has both A and B antigens, and the O blood type has neither A nor B antigen.

By the time a person is six months old, he naturally will have developed antibodies against the antigens his red blood cells lack.

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That is, a person with A blood type will have anti-B antibodies, and a person with B blood type will have anti-A antibodies.

A person with AB blood type will have neither antibody, but a person with O blood type will have both anti-A and anti-B antibodies.

Although the distribution of each of the four ABO blood types varies between racial groups, O is the most common and AB is the least common.

The ABO grouping is the first test done on blood when it is tested for transfusion.

Principle:

This ABO grouping test is based on the principle of haemagglutination reaction.

Haemagglutination of red cells occur when the red cell antigens (agglutinogens) react with corresponding antibody (a) positive agglutination is observed by the clumping of red cells as seen on slide or by button formation in the test tube, (b) negative reaction is observed by uniform distribution of red cells on slide and lack of button formation in test tube.

Procedure:

There are two basic methods to observe the haemagglutination reactions in ABO blood grouping (i) slide method and (ii) test tube method.

Former is easier to perform and the latter is more sensitive. Many laboratories of developing countries perform the tube test only in case the result of the slide test is doubtful.

However, tube method is recommended for reliable results as per FDA approved guidelines.

Slide Method

Requirement:

Glass slides, Pastuer pipettes, Applicator sticks and centrifuge. Reagents

1. Anti-A sera (blue color):

Human polyclonal or murine monoclonal.

2. Anti-B sera (yellow color):

Human polyclonal or murine monoclonal.

3. Normal saline:

0.9 g/dl sodium chloride in distilled water.

Specimen:

Clotted blood is generally used. Centrifuge the clotted blood at 1500 rpm for few min. to separate serum.

With the help of Pastuer pipette, separate the red cells from the clot and suspend them in saline.

Anti-coagulated blood with proper anticoagulant like EDTA can be used. Store the specimen at 2-8oC if there is any delay in examination.

Blood obtained by finger puncture may be tested directly by the slide method. To avoid clotting of the collected blood (on the slide), it should be mixed quickly with antisera.

Procedure:

1. Prepare a 10% suspension of red blood cells in normal saline as follows:

(i) Mix 0.05 ml (5 drops) of sedimented red cells with 2 ml of normal saline, (ii) Centrifuge at 1,500 rpm for 1 to 2 min.

Discard supernatant, (iii) Add 2 ml of normal saline to the sedimented red cells. Mix well. This gives a 10% suspension of red cells.

2. On one-half of a glass slide, place 1 drop of anti-A sera.

3. On the other-half slide, place 1 drop of anti-B sera.

4. Using Pastuer pipette add one drop of the red cell suspension to each half of the slide.

5. With separate applicator sticks, mix each cell- serum mixture well.

6. Tilt the slide back and forth and observe for agglutination.

Interpretation:

a. Tests that show no agglutination within two minutes are considered negative

b. Do not interpret peripheral drying or fibrin stands as agglutination.

Tube Method:

Requirement:

1. Test tubes (10 x 75 mm or 12 x 75 mm)

2. Microscope

Procedure:

1. Prepare a 5% suspension of red blood cells in normal saline as follows: (i) Mix 0.05 ml (5 drops) of sedimented red cells with 2 ml of normal saline, (ii) centrifuge at 1,500 rpm for 1 to 2 min.

Discard supernatant, (iii) Add 4 ml of normal saline to the sedimented red cells. Mix well. This gives a 5% suspension of red cells.

2. To a small test tube, add one drop of anti-A sera.

3. To a second test tube, add one drop of anti-B sera.

4. Using a Pastuer pipette, add one drop of 5% red cell suspension to each of the two test tubes.

5. Mix well and centrifuge both the tubes at 1,500 rpm for one min. or incubate at room temperature for one min.

6. Examine for agglutination: If the tube is centrifuged red cell sediment will be seen at the bottom of the tube, which is called a button.

Gently tap the button from the tube by a spring action of right index finger and dislodge the cell button.

If, red cells form one or more clumps with clear supernatant fluid, the agglutination is present.

If, red cells re-suspend easily, without any visible clumping, agglutination is absent.

7. In case of any doubt, take a drop of the suspension on a slide and observe under the 10X objective for agglutination.

Forward and Reverse Grouping:

To determine ABO blood group, there are two ways:

1. When the individuals red cells are tested with a known anti – A and anti- B sera, this procedure (as described above) is called forward grouping or front typing or cell typing.

2. When the individuals serum is tested with known group A cells and group B cells, the procedure is called reverse grouping or back typing or serum typing (Table 32.2).

Rh Blood Typing:

Rh typing (also called as Rh blood grouping) is next important to ABO blood grouping. It detects only the presence of Rh antigen (or D antigen) out of all Rh factors on the red cells.

Principle:

It is based on the principle of haemagglutination that, the red cells with Rh antigen (D antigen) will clump with anti-D antiserum at room temperature in presence of protein.

The technique is similar to ABO blood grouping and hence, Rh typing is done along with ABO blood grouping.

Procedure:

The Rh typing can also be done by two methods:

1. Slide method and

2. Tube method

Slide Method

Requirements:

Same as that of ABO slide method.

Reagents:

1. Anti-D sera (human polyclonal or human monoclonal).

2. Normal saline

Specimen:

Same as that of ABO method.

Procedure:

1. On a pre-warmed glass slide, place one drop of anti-D serum.

2. By using a Pastuer pipette add one drop of 10% suspension of red blood cells (in case of anaemic patients, use one drop of sedimented red cells)

3. With an applicator stick, mix cell-serum mixture well.

4. Tilt the slide back and forth and observe for agglutination.

5. Tests that show no agglutination within two min. are considered negative.

Tube Method

Requirements:

Same as that of ABO tube method.

Reagents:

1. Anti-D sera (human polyclonal or human monoclonal).

2. Normal saline.

Specimen:

Same as that of ABO method.

Procedure:

1. Prepare a 5% suspension of red blood cells in normal saline.

2. To a test tube add one drop of anti-D serum.

3. Add one drop of cell suspension with the help of Pasture pipette.

4. Mix well and centrifuge both the tubes at 1,500 rpm for one min. or incubate at room temperature for one min.

5. Examine the agglutination reaction in each tube by dislodging the button gently. If necessary, use a magnifying hand lens.

6. Interpretation: Agglutination will be recognised by the formation of small clumps in a clear liquid. As the bottom of the test tube is tapped, the clumps whirl up and then settle down.

This will be marked as positive reaction and the cells are identified as Rh- positive. If the red cells re-suspend homogeneously with no visible clumps, it should be marked as negative reaction and the cells are identified as Rh- negative.

7. All the Rh negative cells must be tested for Du.

Cross-matching:

Cross-matching is the final step in the pre-transfusion testing. It is commonly referred to as compatibility testing.

For the safe transfusion, blood group of donor and recipient must be same and match according to the antigen and antibody in blood (in vivo). If the blood groups are matched (in vitro), there are 99% chances of their compatibility. However, there are some occasions, in which though, blood groups are matched, but cross-matching is incompatible.

These are—a. The donor may have unexpected antibodies in his serum or b. The patient may have unexpected antibodies in his serum or c. There may have been a mistake in performing, reading or recording of the blood grouping and Rh typing results.

Considering all these possibilities, a compatibility test is essential before all transfusions. This procedure is performed in two parts:

Major cross match:

The donor’s cells are mixed with the patient’s serum. This brings reaction of donors red cells with patients serum or plasma to detect antibodies that could destroy donors red cells.

Minor cross match:

The patient’s cells are mixed with the donor’s serum/plasma. This brings reaction of patients’ red cells with donor serum or plasma to detect antibodies that would destroy patients’ red cells.

Requirements:

1. Small test tubes (10 x 75 mm)

2. Pasteur pipettes

3. Normal saline

4. Centrifuge.

Specimen:

1. Patient’s blood and serum

2. Donor’s blood and serum / plasma.

Procedure:

1. Prepare 5% cell suspensions of patient’s blood (P) and donor’s blood (D) in two separate tubes.

2. To the patient’s tube (P), add two drop of patient’s serum and one drop of donor cell suspension (Major cross match).

3. To the Donor’s tube (D), add two drops of donor’s serum and one drop of patient’s cell suspension (minor cross match).

4. Mix the contents of the tubes gently and keep the tubes at room temperature for 30 minutes. Centrifuge at 1500 rpm for one minute.

5. Examine for the agglutinations both macroscopically and also microscopically.

Interpretation:

1. Blood which shows a major incompatibility should never be transfused.

2. The minor cross-match results are also important. But in an emergency it is possible to use blood which is minor incompatible, (but not major incompatible) without leading to a transfusion reaction. This is because of the fact that, in patient’s body the ‘minor side” would be the donor plasma (about 200 to 300 ml).

This may contain the antibodies. But these antibodies will get mixed and diluted with about 2500 ml of patient’s serum so that there will not be a major reaction. Only following two conditions may show significant incompatibility.

(a) High titre of donor antibodies and

(b) Multiple transfusions of minor incompatible blood. The saline tube method requires longer incubation but weak reactions are detected better by this method.

Reasons for Blood Typing and Cross-matching:

i. Avoid a transfusion reaction in a previously sensitized patient.

ii. Prevent formation of isoantibodies which may cause a delayed transfusion reaction and subsequent haemolytic anaemia.

iii. Provide maximum survival of transfused cells.

iv. Prevent sensitization of recipient to future incompatible transfusions.

v. Avoid sensitization of future breeding stock and subsequent haemolytic disease of newborns through receiving colostral antibodies.

Limitations of Cross matching:

i. Haemolyzing antibody may not be detected in a cross-match looking for agglutination.

ii. Will not detect clinical errors.

iii. Does not guarantee the survival of donor cells.

Coombs’ cross-match:

For faster results, use the red cells in thermophase with protein. Where the red cells are suspended in the antibody (serum) with 22% albumin (protein) and incubated for 30 min. at 37°C. Now centrifuge at 1500 rpm for one min. and examine for agglutination. If negative, wash it three times with normal saline to remove unbound albumin. Add AHG, centrifuge at 1500 rpm for one minute and look for agglutination.

In an emergency, when there is not enough time for blood typing and crossmatching, O red blood cells may be given, preferably Rh-negative. O blood type is called the universal donor because it has no ABO antigens for a patient’s antibodies to attack. In contrast, AB blood type is called the universal recipient because it has no ABO antibodies to attack the antigens on transfused red blood cells.