Blood Grouping

Human bloods are different from each others  due to the presence or absence of certain antigen and antibodies. Different combinations of these genetically inherited protein molecules provide different types of blood in every individual.

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ABO & Rhesus Grouping

• Forward - Blood cell + anti-A, anti-B, anti-AB, anti-D
• To test the presence of antigen
  
  
• Reverse - Serum + ABO red blood reagent, (Blood cell + anti-D )
• To test the presence of antibodies
  
  
  

2 Methods:

Tile method (30% suspension)

1. Place 1 drop of EDTA blood + 1 drop of the reagent at the particular square on the tile.
2. Fully mix the solution using a toothpick and agitate for reaction.

O Blood sure no agglutination because there is no "Anti-O"

Add 1 drop of reagent and 1 drop of blood.

Result ( the photo shown is "AB" positive)

Tube method (5% suspension)

• Washing *3 times* : (Reverse ✗ need)

1. Fill up the test tube with 1 drop of blood and top up with normal saline
2. Centrifuge at 3000 rpm for 1 minute
3. Dispose the supernatant.

• Resuspend the pellet in 19 drops of normal saline (5%) (Reverse ✗ need)
• Prepare and label 4 clean tubes with Anti-A, Anti-B, Anti-AB and Anti-D. (Reverse use ABO reagents)
• Withdraw 50μl of the sample solution and release them in each of the 4 tubes.
• Centrifuge the 4 tubes in 3000 rpm for 15 seconds. 
• Gently swirl the 4 tubes under light for result observation.
   

Result Interpretation:

• Positive result: Agglutination of RBC
• Negative result: The button dissolved followed by no agglutination. *even under microscope.*

• The Forward and Reverse test should have inverted/opposite result.
  Except : Baby  (Baby might not yet produce antibodies therefore the reverse result will be negative) 

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Du test and anti-CDE are performed when agglutination is not shown in Anti-D test.


3%~5% of antigen is the maximum amount which will form reaction with the particular amount of antibodies. Thus, 5% of suspension is used in the test.

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Reference:
http://www.edu.pe.ca/threeoaks/teacherpages/higginbotham/Biology%20521%20Webpage/resources.htm

Du test.

D^u test 

• Confirmation for Rhesus-negative.

Procedure:

When the blood sample did not form agglutination with the Anti-D reagent, indicates that the sample is Rhesus-negative. 

1. Incubate the sample solution in water bath for 15 minutes.
2. Spin the test tube at 3000 rpm for 15 seconds.
3. If the result remain negative, wash three times at 3000 rpm for 1 minute each.
4. Dispose the supernatant and add 2 drops of AHG.
5. Spin the test tube at 3000 rpm for 15 seconds.
6. Look for any agglutination under microscope.

Cross Matching

Cross matching is conducted when there is a case for blood transfusion. The correct blood should be provided to the patient in order to avoid any transfuse reaction which may worsen the patient's health condition.

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Procedure:

• Blood Grouping (Both Forward and Reverse)
  *Tile method is used for rapid test during emergency*
  
• Centrifuge the plain tube.
  
• Take the requested unit of the relevant blood bag ( packed cell / whole blood )
  And cut 1 segment from each blood bag to release small amount of the blood to different labelled test tubes.
  Wash 3 times and make 5% suspension.
  
• Fill up the recipient card, place the card together with the blood bag and put them back to the fridge if the blood is not going to be collected immediately.

1. Add 100μl of patient's serum (from red-capped plain tube)  and 50μl of the Donor's red blood cells into a clean test tube.
2. Centrifuge for 15 seconds.
3. If no agglutination occurred, add 2 drops of LISS reagent and incubate at 37°C for 15 minutes.
4. Centrifuge for 15 seconds.
5. If no agglutination, wash 3 times  and add 2 drops of AHG reagent.
6. Centrifuge for 15 seconds.
7. View under microscope if agglutination is not observed visually.

Result interpretation: 

• No agglutination, add ccc ( Coomb's control cell/ checkcells ) to test the reliability of AHG
  *Should be have agglutination after centrifuge if the AHG reagent is valid *
• Agglutination indicating the donor's blood is not compatible with the patient's body. thus the blood must not be transfused.

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3 Phases

• RT (Room temperature) phase   -- to test when cold antibodies can function.  Eg. IgM ( Lewis....)
• LISS phase -- warm antibodies can function.  Eg:  1gG ( ABO )
• AHG phase

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Important Notes:

For newborn baby (less than 4 months), mother's blood/serum should be used for cross matching. Because, babies don't have antibodies yet.

Anti-CDE

Anti-CDE

• This test is performed due to the reason there are antigen and antibodies other than A,B,AB and D such as C, E, c, e.

Procedure:

1. Place 1 drop (50μl) of reagent and 1 drop of whole blood on a slide.
2. Mix well with the applicator stick.
3. Observe under microscope to see any agglutination occurred.

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* However, if Anti-D shows negative result while anti-CDE shows positive result, the patient will still be considered as Rhesus negative for safety purpose. *

Blood Bank

Not to lose out any knowledge I gained from the industrial training, I would like to share my experience during the training at the Blood Bank for the past 2 weeks before I went to biochemistry department.

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Blood Bank is the most important among all the departments as it involve blood collecting, processing, storing and providing.

• Blood Donation
• Blood Grouping
• Condition for Blood Storage
• Cross matching for Blood Transfusion

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My First Training Tray

Blood Bag.

Total, there are three types of blood bag, namely single blood bag, double blood bag and triple blood bag.

Needle on the blood bag that used to puncture into blood donor's vein.

• Single Blood Bag"
• It is used to collect whole blood only.

  

  
  

• Double Blood Bag"
• Can be used to collect whole blood and further separated into packed cells and plasma.

  

  
  

  
  
  
  
  
• Triple Blood Bag"
• The collected whole blood can be further divided into packed cells, platelets and plasma.

  

  
  





  








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The Potential of  Human Blood


• Whole Blood
  - From donation

  

  *Use plasma separator to divide the whole blood into packed cells,platelets and plasma.*

  
  
  
• Packed cell/ Red Blood cells
• Function: treat severe anemia by increase the amount of RBC in the patient.
• Storage: can be refrigerated for 42 days ( freeze for 10 years ) 

• Platelet concentrate. 
• Function: treat bleeding by increasing the platelet levels.
• Storage: 6 days in room temperature.

Placed on rotater to avoid agglutination takes place.

• Cryoprecipitate
• Function: treat fibrinogen deficiency
• Storage: 3 years in deep freezer.
  
  

• FFP ( Fresh Frozen Plasma )
• consists of all coagulation factors.
• Function: treat massive bleeding by correct the deficiency in coagulation factor.
• Storage: 3 years in deep freezer.

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Platelet Apheresis: Consists of 6 units of platelet concentrates from single donor.

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References:

1. http://ijtm.in/view_article.php?id=16
2. https://sites.google.com/site/vaaccs/home/vaaccs-blood-bank/blood-bags---what-do-i-need