Practical Blood Bank Lab 1 ABO Grouping IMMUNOLOGIC

Practical Blood Bank Lab 1 ABO Grouping

IMMUNOLOGIC PRINCIPLES • primary immunological components: antigens & antibodies provides basis for blood bank testing and reactions CARDINAL RULE IN BLOOD BANK: The antigens are found on the surface of red blood cells and the antibodies are found in serum or plasma

IMMUNOLOGIC PRINCIPLES ANTIGENS • substances that have the capability to stimulate the production of an antibody Chemical composition of antigens: 1. Glycoproteins & lipoproteins – most potent 2. Glycolipids 3. Pure polysaccharides – not immunogenic except in humans and mice 4. Pure lipids & nucleic acids – not immunogenic but can be antigenic serve as haptens

IMMUNOLOGIC PRINCIPLES ANTIBODIES • also called immunoglobulins characteristics: • Protein • Produced in response to stimulation by an antigen • Specific for the stimulating antigen • Consists of 2 heavy chains & 2 light chains held together by disulfide bonds • Produce 3 fragments when cleaved by enzymes 2 Agbinding fragments (Fab) & 1 crystallizable fragment (Fc)

Complement activation ◦ Ig. G = will do it if conditions are optimal ◦ Ig. M = very good complement activator Placental transfer ◦ Ig. G is small enough to easily cross placenta and is the only immunoglobulin capable of doing so. ◦ Ig. M and the other classes do not cross placenta Optimum temperature of reactivity ◦ a. Ig. G = 37 o. C ◦ b. Ig. M = 4 o. C (may react at any temperature below 30 C)

Characteristics of Ig. G and Ig. M antibodies Clinical significance ◦ Clinical of red cell antibodies in blood bank depend on whether they can cause in vivo hemolysis, which in turn will cause transfusion reactions or hemolytic disease of the newborn. ◦ Ig. G will frequently cause in vivo hemolysis due to antibody coating the red blood cells. ◦ Ig. M, with a few important exceptions, usually does NOT cause in vivo hemolysis. The most important of these exceptions are ABO antibodies.

Important terms • Alloantibodies • • Reacts with foreign Ag not present on patient’s own RBC • Most produced as result of immune stimulation via transfusion or pregnancy (usually during delivery) Autoantibodies Reacts with an Ag on patient’s own cells & with that same Ag on the cells of other individuals • Cold antibody (cold agglutinin): Antibody whose optimal temperature of reactivity is less than 30 o. C • Warm antibody: Antibody whose optimal temperature of reactivity is greater than 35 o. C

Isoagglutinin: Name commonly given to blood group antibodies anti-A and anti-B Saline agglutinin: Antibody capable of causing direct agglutination of antigens suspended in a saline medium without requiring any enhancement techniques Hemolysin: Antibody capable of causing hemolysis when reacting with corresponding antigen Naturally occurring antibodies Formed without prior exposure to foreign antigen

Immunogenicity of Blood Group Antigens A, B and D (Rho) – most immunogenic Kell (K) Duffy: Fya Fyb Kidd: Jka Jkb

Importance of ABO Grouping The critical nature of ABO grouping stems from two characteristics of the system. First, unlike other blood group systems, antibodies of the ABO system are present in the serum of almost every person who does not have the corresponding antigen. Second, the all agglutinins of the ABO system fix complement and are capable of causing intra vascular hemolysis of incompatible red cells. For these reasons, an error in ABO grouping of a patient or donor could be fatal in a transfusion setting. While the crossmatch affords an additional measure of protection, this may not be done in every case.

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

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. ◦ …. .

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

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.

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

Grading System for Reactions

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

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 blood agglutinate the presence of these sera q

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

Washed 3% Cell Suspension Principle q Washing cells to be tested removes serum or plasma which may contain or plasma q proteins that interfere with testing, causing nonproteins specific agglutination or rouleaux formation. q Washing also removes fibrinogen, which may fibrinogen cause small clots. q The ratio of serum to cells markedly affects the sensitivity of agglutination tests. sensitivity of agglutination Preparation of a 2 -5% cell suspension provides cells in an optimum concentration to detect weak antibodies.

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 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 do not touch with the tip of the saline bottle q Resuspend the cell button thoroughly between Resuspend the cell button 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 gloved 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

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

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

Notes Samples for Blood Bank Testing ◦ Most samples for blood banking are drawn into a red top tube ◦ Serum is preferred on plasma in blood bank testing due to its content of calcium which is essential for complement activation and must be tested while fresh to ensure good complement activity ◦ No clot activation tube should be used since the patient's red cells may also need to used and no other chemicals should be present ◦ A few tests require an EDTA sample if complement is not to be activated. ◦ Antigens on cells are stable longer (months) in a clot tube.

Reverse grouping is performed on tube method only, because the titer of antibody in the patient serum may be low and give false negative result on slide method. A 1 cells must be used in reverse grouping to ensure its reaction of both anti A and anti A 1 that may present in the serum of B individuals, A 2 cell will react only with anti

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 prefilled 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 A positive reaction above the gel column after centrifugation ◦ A negative reaction is recorded when a distinct button of cells A negative reaction sediment to the bottom of the column after centrifugation. ◦ A positive reaction in the MTS Control microtube indicates a false the MTS Control 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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