Practical Blood Bank Lab 1 ABO Grouping

Introduction q ABO blood group antigens present on red blood cells (Natural Antigens) q Ig. M antibodies present in the serum

RBC Precursor Structure RBC Glucose Precursor Substance (stays the same) Galactose N-acetylglucosamine Galactose

Formation of the H antigen RBC Glucose Galactose H antigen N-acetylglucosamine Galactose Fucose

Formation of the A antigen RBC Glucose Galactose N-acetylglucosamine Galactose Fucose N-acetylgalactosamine

Formation of the B antigen RBC Glucose Galactose N-acetylglucosamine Galactose Fucose Galactose

Methods for determination of ABO group of RBCs

Reagents ◦ ◦ ◦ Anti-A antibodies Anti-B antibodies Anti-AB antibodies (optional) Anti-D Group A & B RBCs ◦ Slides, or Test tubes. ◦ Wooden applicator. ◦ …. .

Procedure q Verify that patient information on the sample matches information on the worksheet. q Centrifuge the sample and remove the serum to a labeled tube. q Add 2 -3 drops of the patient’s cells to the WC tube, and prepare a washed 3% suspension. q Use Patient cell suspension in forward typing q Use patient serum for confirmation ( reverse grouping or backtyping). q At the End, Discard all materials in the isolation trash containers.

Blood Grouping: Forward and reverse typing q q q 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 catch these incompatibilities, typing is done in two steps: q Forward typing q Reverse typing

Forward typing Front or forward type using monoclonal anti-A and anti-B (commercial) , Anti-D q Patient blood is mixed with serum that contains antibodies against type A blood, and type B blood. q Determination of the blood type is based on the whether or not the blood agglutinate in the presence of these sera q

Reverse Typing Back or reverse type with A and B cells • Commercially available A and B cells are used. • Patient serum added to the known cells. • Used as confirmatory for the forward method.

Grading System for Reactions

Washed 3% Cell Suspension q q q Used in forward typing , tube methods. 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.

Washed 3% Cell Suspension Principle q Washing cells to be tested removes serum or plasma which may contain q proteins that interfere with testing, causing non-specific agglutination or rouleaux formation. q Washing also removes fibrinogen, which fibrinogen may cause small clots. q 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.

Washed 3% Cell Suspension Procedure 1. Label a 12 x 75 mm tube 2. Transfer 2 -4 drops of blood from the sample to the labeled tube 3. Forcibly squirt saline from the wash bottle into the tube until it is about 3/4 full 4. Centrifuge 45 -60 seconds at high speed (3400 rpm) 5. Decant supernatant and shake to resuspend completely 6. If gross hemolysis is present, repeat steps 3 to 5 until supernatant is reasonably clear 7. After the final wash, shake the tube to completely resuspend the cells and add saline to a final concentration of approximately 3%

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

Slide Method – Forward Typing Principle: When red cells are mixed with various reagent antiseras (soluble antibody), agglutination will occur on the slides containing cells positive for (possessing the antigen) the corresponding antigen. No agglutination will occur when the red cells do not contain the corresponding antigen.

Procedure: 1. On the section of slide labeled anti-A place one drop of antibody A. 2. On the section of slide labeled anti-B place one drop of antibody B. 3. On the section of slide labeled anti-AB place one drop of antibody AB. 4. On new slide labeled anti-D place one drop of antibody D. 5. Place one drop of cells in each antibody containing circle. 6. Carefully mix each solution with a separate applicator stick. 7. Tilt slowly for one minute, then observe for the agglutination.

Interpretation of the results: q q q 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

Tube Methods - Forward Typing - Prepare 2 -5% cell suspension - Label Test tubes - Add 2 drops of Anti sera A, B , and D

- Add one drop of 2 -5% Patient Red Blood Cell suspension. - 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 37 o. C, then centrifuge and read again.

Read and record agglutination reaction Reaction of cells tested with Interpretation Anti-A Anti-B Cell Ag ABO Group - - No Ag O + - A A - + B B + + A, B AB

Serum testing (Reverse) 1. Label 2 clean test tubes (A, B ) 2. Add 2 -3 drops of serum to each tube 3. Add one drop of (A) reagent RBCs to the tube labeled A 4. Add one drop of (B) reagent RBCs to the tube labeled B 5. Mix the contents of the tubes gently & then centrifuge for 15 -30 seconds at 900 -1000 x g 6. Examine the tubes for evidence of hemolysis. Gently resuspend the RBCs buttons and examine them for agglutination

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

Interpretation of Both (Forward and Reversed Typing) Reaction of cells tested with Reaction of serum tested Interpretation against A Anti-B cells B Cells O cells ABO Group - - + + - O + - - + - A - + + - - B + + - - - AB

Other methods for blood grouping Gel Cards containing Anti-A, Anti-B, and Anti-A, B are used to test patient or donor red blood cells for the presence or absence of the A and/or B antigens. The results of red blood cell grouping should be confirmed by reverse (serum) grouping, i. e. testing the individual’s serum with known A 1 and B red blood cells. In the Gel Test™, the specific antibody (Anti-A, Anti-B, or Anti-D) is incorporated into the gel. This gel has been pre-filled into the microtubes of the plastic card. As the red blood cells pass through the gel, they come in contact with the antibody. Red blood cells with the specific antigen will agglutinate when combined with the corresponding antibody in the gel during the centrifugation step.

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. ◦ A positive reaction in the MTS Control microtube indicates a false positive reaction may have occurred in the corresponding blood grouping microtube, thus invalidating the blood grouping tests. ◦ Drying, discoloration, bubbles, crystals, other artifacts, opened or damaged seals may indicate product alteration A buffered gel suspension is contained in two (2) microtubes of the A/B/D Monoclonal and Reverse Grouping Card™. Sodium Azide (0. 1% final concentration) is added as a preservative.

ABO/D + Reverse group cards Procedure: 1. Suspend 50 µL WB or 25 µL RBCs in 0. 5 ml diluent. 2. Identify the card with patient's name. 3. To microtubes l, 2, 3 & 4 add l 0 µL of suspension. To microtube 5 add 50 µL Al cells + 50 µL plasma. To microtube 6 add 50 µL B cells + 50 µL plasma. 4. Centrifuge for l 0 minutes and read.

Microplate Technique 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 microplate. ◦ ◦ ◦ Add reagent and patient sample( red cells/ serum) Incubation, Centrifugation Red cell resuspension, Reading of results Interpretation of results

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