Blood Grouping test is intended for knowing the type of blood group the person is having. This information becomes crucial when there is need to transfuse blood to the person. When the exact blood group and Rh type of the person is not known, there is a chance of transfusing different type of blood, which is fatal. Hence, knowing the blood group and Rh Type(1) is very much important. This test is also used for identifying maternal and infant ABO and Rh blood types for predicting potential hemolytic disease of the newborn.
- 1 How to Check Blood Group in Laboratory?
- 1.1 What is the Procedure of Blood Grouping & Rh Typing Tests?
- 1.2 How Results are Interpreted in Blood Grouping & Rh Typing Tests?
How to Check Blood Group in Laboratory?
Blood Grouping & Rh typing test is based on direct hemagglutination principle. Erythrocytes of a person have specific antigens on its surface, based on their blood group type. When these antigens are made to get in contact with corresponding antibodies, the antigen-antibody reaction will occur. This will lead to the clumping of erythrocytes. This will occur when corresponding antibodies react with specific antigens. That is, if the erythrocytes possess A / B / AB antigens, they will clump or agglutinate when mixed with Anti A / Anti B / Anti A and B antibodies respectively.
Agglutination will not occur when the antigens-carrying red cells have immune antibodies adsorbed on to their surface. Such blood group antigens can be identified through reaction with anti-0human gamma globulin reagent.
When there is a proper proportion of antigen and antibodies, visible agglutination occurs.
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How to Collect Sample for Blood Grouping & Rh Typing Tests?
- Whole blood should be used as a sample.
- 2 mL of blood should be collected in 3.6 mg K2 EDTA vacutainer.
- The sample should be processed within 1 hour of collection.
- Insufficient, contaminated and clotted samples should NOT be used.
In case there is going to be a delay in testing (after collecting sample), red cells from clotted samples, Heparinized / EDTA anticoagulant samples have to be separated from plasma, washed and stored in specific red preservative solution at 2 to 80 C for not more than 35 days.
What are the Chemicals / Reagents Required for Blood Grouping & Rh Typing Tests?
- 0.9% Isotonic Saline
- Applicator sticks
- Glass slides
- Anti A1 Lectin
- Anti D blood grouping reagent
- Anti AB blood grouping reagent
- Anti B blood grouping reagent
- Anti A blood grouping reagent
There is no equipment required for performing a blood grouping test. It is very simple, but one should be cautious to know which reagent they are adding and how to interpret the results.
What is the Procedure of Blood Grouping & Rh Typing Tests?
There are multiple methods of performing blood grouping & Rh typing tests. Following methods are the most common and most accurate ones.
Slide Agglutination Method
This is the most common and easiest method for determining the blood group & Rh type(2) of the person. Here is the step-by-step process for performing this test in this particular method:
- Clean, grease-free microscope slide should be taken
- Draw a line in the middle of the slide using wax glass marking the pencil
- Label the left portion as Anti-A and the right one as Anti-B
- Place a drop of Anti-A to the left side and add a drop of Anti-B to the right side
- Add 1 drop of well mixed 3 – 5 % cell suspension to both the sides of the slide
- Using a separate toothpick or match stick for each side of the slide, mix the contents and anti-sera well.
- Rotate the slide for mixing gently
- Position the slide against the white background
- Examine the slide after 2 minutes for agglutination (both macroscopic examination and microscopic examination).
Tube Agglutination Method
As the name suggests, this method uses a tube for finding out if agglutination occurs. Here is the step-by-step procedure for finding out blood grouping & Rh typing in tube agglutination method (3):
- Water bath should be turned on and its temperature should be set at 37o
- 2 tubes of size 12 x 100 mm should be taken
- One tube should be marked Anti A while others should be marked Anti B
- 2 drops of Anti A should be added to the tube which is marked Anti A
- In the same way, 2 drops of Anti B to be added to tube marked Anti B
- 2 drops of 3 – 5 % cell suspension should be added to each tube
- Both the tubes are to be centrifuged at 1500 rpm for at least 1 minute
- Now, remove the tubes from centrifuge for examining their bottom
- By gently shaking or tapping the tube, observe the dislocation of cell button
- In case there is agglutination, ‘Positive’ should be reported
- Keep the tubes (in which no agglutination observed) in the water bath for 30 minutes at 37o C
- Remove those tubes from the water bath and centrifuge them again to observe the cells carefully by shaking the tube gently
- In case agglutination is observed, report ‘Positive’
- Results are to be double-checked microscopically by observing 2 to 3 drops from each tube under the microscope. 3 – 5 % suspension of test red cells has to be prepared
- All these suspensions are to be prepared in autologous serum / saline / plasma
- Position 1 drop of appropriate A, B, AB blood grouping agents on a clean, dry glass slide at normal temperature
- Add 1 drop of a prepared suspension of red cells to each drop of reagent on a glass slide.
- Cells and reagents are to be mixed thoroughly over a wide circular area (of nearly 20 mm size), using fresh applicator stick for each reagent
- Rotate slides gently for examining macroscopic hemagglutination. Agglutination may commence within few seconds but the observation should not be continued for more than 2 minutes.
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How Results are Interpreted in Blood Grouping & Rh Typing Tests?
- If agglutination is found with Anti A blood group reagent, then the patient’s blood group is A.
- If agglutination is found with Anti A1 Lectin reagent, then the patient’s blood group is A1.
- If agglutination is found with Anti B blood group reagent, then the patient’s blood group is B.
- If agglutination is found with both Anti A and Anti B blood group reagent, then the patient’s blood group is AB.
- If agglutination is NOT found with both Anti A and Anti B blood group reagent, then the patient’s blood group is O.
- If agglutination is found with Anti D blood group reagent, then the patient’s Rh type is Positive. Those samples which got Rh negative are to be reconfirmed with Du testing.
What are the Precautions to be Taken during Blood Grouping & Rh Typing Test?
- Test slides are to be cleaned and dried thoroughly before testing.
- Reagents and samples should be at room temperature before use.
- Samples are to be managed carefully as they are potentially infectious
- Reagents are to be managed carefully and their contact with eyes, skin, and mouth should be avoided
- Used samples and reagents are to be discarded carefully using standard procedures
What are the Potential Sources of Variability in Blood Grouping & Rh Typing Tests?
- Sometimes, unexpectedly weak reactions may be seen with blood samples of weak A & B subgroups or with cord blood red cells from newborn infants.
- Inaccurate or unexpectedly weak reactions can occur when the samples are processed after prolonged storage or improper storage. (4)
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Health Counselor and Primary Care Physician by profession, I have 12 years experience in primary health care services. Years of practical experience in women’s health care & ANC services and study have gained me a lot of knowledge on several health issues and I wish to share these medical advice and suggestion through this blog.