Blood Grouping Slide & Tube Methods

M. Zaharna Practical Blood Bank 2009 ABO blood group antigens present on red blood cells and IgM antibodies present in the serum M. Zaharna Practical Blood Bank 2009

M. Zaharna Practical Blood Bank 2009 Blood Grouping tests Slide test for determination of ABO group of RBCs Tube test for determination of ABO group of RBCs and serum M. Zaharna Practical Blood Bank 2009

Slide test for determination of ABO group of RBCs M. Zaharna Practical Blood Bank 2009

Interpretation of ABO grouping Slide test Strong agglutination of RBCs in the presence of any ABO grouping reagent constitutes a positive result. A smooth suspension of RBCs at the end of 2 minutes is a negative result. Samples that give weak or doubtful reactions should be retested by Tube test ABO grouping M. Zaharna Practical Blood Bank 2009

Tube test for determination of ABO group of RBCs M. Zaharna Practical Blood Bank 2009

Forward and reverse typing ABO typing is the first thing to be done before transfusion A person must receive ABO matched blood because ABO incompatibilities are the major cause of fatal transfusion reactions To guard against these incompatibilities typing is done in two steps: 1- Forward typing 2- Reverse typing M. Zaharna Practical Blood Bank 2009

M. Zaharna Practical Blood Bank 2009 1- Forward typing Front or forward type using monoclonal anti-A and anti-B (commercial) Patient blood is mixed with serum that contains antibodies against type A blood, then with serum that contains antibodies against type B blood Determination of the blood type is based on the whether or not the blood agglutinate in the presence of these sera M. Zaharna Practical Blood Bank 2009

M. Zaharna Practical Blood Bank 2009 1- Forward typing Anti A Anti B Anti A Anti B A B Anti A Anti B Anti A Anti B AB O M. Zaharna Practical Blood Bank 2009

M. Zaharna Practical Blood Bank 2009 2- Reverse Typing Back or reverse type with A and B cells Commercially available A and B cells are added to two tubes of serum AB B A O A B A B A B A B M. Zaharna Practical Blood Bank 2009

Grading System for Reactions M. Zaharna Practical Blood Bank 2009

M. Zaharna Practical Blood Bank 2009

M. Zaharna Practical Blood Bank 2009 Ratio of serum to RBCs The ratio of serum to red cells may dramatically affect the sensitivity of agglutination tests. Consistent preparation of either 2 to 5% red cell suspension is critical to any agglutination test. M. Zaharna Practical Blood Bank 2009

Washed 3% Cell Suspension M. Zaharna Practical Blood Bank 2009

M. Zaharna Practical Blood Bank 2009 Principle Washing cells to be tested removes serum or plasma which may contain proteins that interfere with testing, causing non-specific agglutination or rouleaux formation. Washing also removes fibrinogen, which may cause small clots. The ratio of serum to cells markedly affects the sensitivity of agglutination tests. Preparation of a 2-5% cell suspension provides cells in an optimum concentration to detect weak antibodies. M. Zaharna Practical Blood Bank 2009

M. Zaharna Practical Blood Bank 2009 Procedure Label a 12 x 75 mm tube Transfer 2-4 drops of blood from the sample to the labeled tube Forcibly squirt saline from the wash bottle into the tube until it is about 3/4 full Centrifuge 45-60 seconds at high speed (3400 rpm) Decant supernatant and shake to resuspend completely If gross hemolysis is present, repeat steps 3 to 5 until supernatant is reasonably clear After the final wash, shake the tube to completely resuspend the cells and add saline to a final concentration of approximately 3% M. Zaharna Practical Blood Bank 2009

M. Zaharna Practical Blood Bank 2009 Notes and Precautions To prevent contamination, do not touch tubes with the tip of the saline bottle. Resuspend the cell button thoroughly between washes before adding more saline to ensure complete washing. Do not attempt to mix a tube full of saline. Do not mix cells by using your gloved finger as a stopper. To prevent cells from spraying out during centrifugation, fill tubes no more than 3/4 full. To ensure good resuspension of cells, add the saline in a forceful stream. M. Zaharna Practical Blood Bank 2009

Other methods for blood grouping Gel Cards The cards containing specific typing reagents (monoclonal antibodies to the various red cell antigens). Interpretation of Results A positive reaction is recorded when red cells are retained in or above the gel column after centrifugation A negative reaction is recorded when a distinct button of cells sediment to the bottom of the column after centrifugation. M. Zaharna Practical Blood Bank 2009

M. Zaharna Practical Blood Bank 2009

M. Zaharna Practical Blood Bank 2009 Microplate Test Microplate techniques can be used to test for antigens on red cells and for antibodies in serum. A microplate can be considered as a matrix of 96 “short” test tubes; the principles that apply to hemagglutination in tube tests also apply to tests in microplates. Add reagent and patient sample( red cells/ serum) Incubation, Centrifugation Red cell resuspension,   Reading of results   Interpretation of results M. Zaharna Practical Blood Bank 2009

M. Zaharna Practical Blood Bank 2009 Procedure M. Zaharna Practical Blood Bank 2009

M. Zaharna Practical Blood Bank 2009 Reagents Anti-A antibodies Anti-B antibodies Anti-AB antibodies (optional) Anti-D Group A & B RBCs M. Zaharna Practical Blood Bank 2009

RBCs testing (Forward) Place one drop of anti-A serum in a clean labeled test tube, anti-B serum in a second clean labeled test tube & anti-D in a third one Add to each tube one drop of a 2-5 % suspension of RBCs to be tested Mix the contents of the tubes gently and centrifuge for 15-30 seconds at approx. 900-1000 x g Gently resuspend the RBCs buttons and examine for agglutination If the Rh test is negative, add a second drop of anti-D and incubate 15 minutes at 37oC, then centrifuge and read again. M. Zaharna Practical Blood Bank 2009

Serum testing (Reverse) Label 2 clean test tubes (A, B ) Add 2-3 drops of serum to each tube Add one drop of (A) reagent RBCs to the tube labeled A Add one drop of (B) reagent RBCs to the tube labeled B Mix the contents of the tubes gently & then centrifuge for 15-30 seconds at 900-1000 x g Examine the tubes for evidence of hemolysis. Gently resuspend the RBCs buttons and examine them for agglutination M. Zaharna Practical Blood Bank 2009

Interpretation of results Agglutination in any tube of RBCs test or hemolysis or agglutination in serum tests constitutes positive test results A smooth suspension of RBCs after resuspension of an RBCs button is a negative result M. Zaharna Practical Blood Bank 2009

Interpretation of results Reaction of serum tested against Reaction of cells tested with ABO Group O cells B Cells A cells Anti-B Anti-A O - + A B AB M. Zaharna Practical Blood Bank 2009