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In this article we will discuss about the determination of blood group.
Blood type (also called blood group) is a classification of blood based on the presence or absence of inherited antigenic substances (antigens) on the surface of red blood cells. If the red blood cell had only “A” antigen on it, that blood was called type A.
If the red blood cell had only “B” antigen on it, that blood was called type B. If the red blood cell had a mixture of both antigens, that blood was called type AB. If the red blood cell had neither antigen, that blood was called type O.
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In biological terms, a foreign chemical causes the creation of antibodies against it. So in type A blood, there anti-B antibodies because the immune system recognizes type B red blood cells as foreign. In type B blood, there anti-A antibodies. In type AB there are no antibodies against either type A or B antigens. In type O blood, there are antibodies against both type A-and B antigens
Procedure:
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1. Prepare a 10% suspension of red blood cells in normal saline (preparation method as given below):
i. Mix 5 drops (0.05ml each) of sediment red cells with 2ml of normal saline.
ii. Centrifuge at 1,500 RPM for 1 to 2 minutes. Discard supernatant.
iii. Add 2 ml of normal saline to the sediment red cells. Mix well. This gives a 10% suspension of red cells.
2. On one half of a glass slide, place I drop of Anti-A blood groping serum.
3. On the other half of the slide, place I drop of Anti-B blood grouping serum.
4. Using a Pasteur pipette add 1 drop of the cell suspension to each half of the slid.
5. With separate applicator sticks, mix each cell-serum mixture well.
6. Tilt the slide back and forth and observe for agglutination.
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Note:
a. Test that show no agglutination within two minutes are considered negative.
b. Do not interpret peripheral drying or fibrin stands as agglutination.
Interpretation:
Note:
Blood obtained by finger puncture may be test directly by the slide method. To avoid clotting, the collected blood (on the slide) should be moved quickly with the antisera.
Qualitative Test for ABO Grouping by Tube Method:
Additional Requirements:
1. Test tunes (10 × 75mm or 12 × 75mm)
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2. Microscope
Procedure:
1. Prepare a 5% suspension of red blood cells in isotonic saline.
i. Mix 5 drops (0.05ml) of sedimentary cells with 2 ml of normal saline,
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ii. Centrifuge at 1, 500 RPM for 1 to 2 minutes. Discard supernatant.
iii. Add 4ml of normal saline to the sediment red cells, mix well.
2. To a small test tube, add one drop of Anti-A blood grouping serum.
3. To a second test tune add one drop of Anti-B blood grouping serum.
4. Using a Pasteur pipette, ad on drop of 5%, cell suspension to each of the two test tunes
5. Mix well and centrifuge both the tubes at 1,500 RPM for one minute.
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6. Responds the cells by gentle agitation.
7. Observe for agglutination. Use microscope if necessary.
Additional Information:
1. Anti AB serum can be used to confirm the result of slide and tube tests.
The expected observations are as follows:
Key:
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O – No agglutination; + Agglutination
Note:
Only group ‘O’ blood will not agglutinate with Anti-AB.
1. Suggested centrifuged speed and duration
a. 3000 RPM: 15 second
b. 1500 RPM: 1 minute
Note:
Over centrifugation, causes the cells to adhere to the bottom of the test tube. This requires vigorous agitation before the cells can be re-suspended. During such agitation weak agglutination may be dispersed leading to false negative reaction.
2. In the case of positive reaction for group A. use anti serum to determine the sub group A.
Key:
0 – No agglutination
+ – Agglutination
Rh Blood Grouping:
Rh typing is next important Blood grouping system. ABO blood grouping presence of Rh antigen (or D antigen) all the red cell is determined by the haem agglutination reaction of the RBC after reacting the letter with Anti-D Anti-serum at the body temperature. The technique is similar to ABO grouping and hence Rh tying is done with ABO blood grouping.
Principle:
Rh blood typing refers to the determination of the presence or absence of D- antigen all RBCs when reacted with anti-D serum. Rh positive cells shows agglutination with anti-D.
Specimen:
– Red cell
– Anti D
-Slide
Reagent:
Anti-D follows manufacture instruction in the use of anti-D Rh-negative blood group cell suspension.
Prepare a 4% red cell suspension for the tube method and cell suspension for slide method is 45%.
Procedure:
Slide Method:
I. Place a slide on a drying and allow it to warm up.
II. Add 2 drops 40-45% of cell suspension or whole blood on the slide.
III. Add 1 drop of anti-D serum.
IV. Mix the cell with the anti-serum
V. Continue mixing while tilting the slide back and forth
VI. Look for agglutination which is recognized by the clumping of red-cell do not observed longer than 2 minutes.
VII. A control must be run once a day in order to option reliable result.
Tube Method:
1. A 4% suspension in saline of washed cell or used diluted whole blood with saline. Take 2 applicator stick together and transfer sufficient amount of cells from the clotted or whole blood specimen to a test tube with saline.
Make concentration of cell suspension to about 4%. Place small test tube on a rack and label tham. S- specimen, +ve Rh positive control and -ve for Rh negative., S.A. for albumin control.
2. Add drop of anti-Rh in to the first 3 test tube. And the albumin in the tube mark- S.A.
3. Add 2 drops of cells suspension in S. tube and S.A. add 2 drops of control reagent cell in the tube marked as positive-and negative mix all the tube while gently shaking.
4. Incubate all the tube add 37°C for 30min
5. Centrifuge the tube 1500 rpm for 1min.
6. Examine the agglutination reaction in each test tube while dislodging the button gently.
7. Agglutination will be recognize by the formation of small clumps in pear liquid this will be marked as Rh-positive. If the RBC re-suspend homogenously with no visible clumps. It is negative reaction.
Source or Error:
Rh:
1. Always run Rh controls at least once a day.
2. Check the temp of the water-bath and slide box frequently.
3. Follow the manufactures instruction.
4. A fresh specimen is always desirable, specimen that are several days old may not yield desired and accurate result.
ABO Grouping:
1. False Positive Result:
Bring on the slide, use of infected sera, contaminated specimen, presence of unexpected antibody and roulex formation are some of the causes of false result.
2. False Negative Result:
The serum may be in active it may have been omitted from the tube. The red cells are not in good condition or the technique may have been wrongly performed. Reliability of antiserum is extremely important for optioning correct blood grouping. Avoid exposing the anti-sera to room temp when not in use. Check the anti-sera with occasionally with known cell. Discard out dated contaminated or weak anti-sera which may give false result.
3. Sometimes the red cells are not agglutinated but are piled upon each other like a stack of coins exhibiting rouleaux formation. This gives the appearance, of false agglutination. In that case add 2 drops of isotonic sodium chloride solution to the drop of the red cell-serum mixture and examine under microscope.
i. In false agglutination rouleaux disappears.
ii. In true agglutination the clumps remain.
50 Contaminated anti-sera should not be used. These sera appear cloudy whitish and give a foul smell.
4. After use, store the antisera at 2-8 °C in the refrigerator.
5. Source material used in the preparation of antisera is tested for hepatitis-B virus and HIV (AIDS) antibodies. But since no test method can offer complete assurance that HIV, hepatitis virus or other infectious agents are absent; the antisera should be handled carefully as recommended for any other specimen.
6. Every antiserum is carefully tested for specificity, potency, avidity and freedom from rouleaux formation properties Donor’s blood collected for the preparation of different antisera should be free from hepatitis-B surface antigen and HIV (AIDS) antibodies 0. If marine monoclonal Anti-A and Anti-B reagents are used, these are very safe and free from these viruses, besides being very, potent and specific.
7. Anti-D serum can be obtained from Rh negative mothers, who have recently delivered infants suffering from hemolytic disease of the new born or by immunization of Rh-negative volunteers with D-positive red blood cells. It is suitable blended with bovine serum albumin.
