Practical Blood Bank - Lab 1 - ABO Grouping Lab 1

Characteristics of IgG & IgM 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. IgG  will frequently cause in vivo hemolysis due to antibody coating the red blood cells. IgM, with a few important exceptions, usually does NOT cause in vivo hemolysis.  The most important of these exceptions are ABO antibodies. Complement activation IgG = will do it if conditions are optimal IgM = very good complement activator

Placental transfer IgG is small enough to easily cross placenta and is the only immunoglobulin capable of doing so. IgM and the other classes do not cross placenta Optimum temperature of reactivity IgG = 37oC IgM = 4 oC (may react at any temperature below 3 oC)

Important Terms Alloantibodies Autoantibodies 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 oC. Warm Antibody Antibody whose optimal temperature of reactivity is greater than 35 oC.

Naturally Occurring Antibodies  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 cross-match affords an additional measure of protection, this may not be done in every case.

Introduction ABO blood group antigens present on red blood cells (Natural Antigens) IgM antibodies present in the serum

Methods for determination of ABO group of RBCs A. Forward Method B. Reverse Method Microplate Method Slide Method Tube Method Gel Card Method

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 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: Forward Typing Reverse Typing

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

Forward Typing - Slide Method 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: On the section of slide labeled anti-A place one drop of antibody A. On the section of slide labeled anti-B place one drop of antibody B. On the section of slide labeled anti-AB place one drop of antibody AB. On new slide labeled anti-D place one drop of antibody D. Place one drop of cells in each antibody containing circle. Carefully mix each solution with a separate applicator stick. Tilt slowly for one minute, then observe for the agglutination.

Interpretation Of The Results: 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

Forward Typing -Tube Methods 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 37oC, then centrifuge and read again.

Washed 3% Cell Suspension 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.

Principle Washed 3% Cell Suspension 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.

Procedure Label a 12 x 75 mm tube. Washed 3% Cell Suspension 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 re-suspend 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 re-suspend the cells and add saline to a final concentration of approximately 3%.

Notes and Precautions Washed 3% Cell Suspension 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.

Grading System for Reactions

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

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

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 serum tested against Reaction of cells tested with ABO Group O cells B Cells A cells Anti-B Anti-A 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. A1 cells must be used in reverse grouping to ensure its reaction of both anti A and anti A1 that may present in the serum of B individuals, A2 cell will react only with anti A and give false negative result with anti A1

Other methods for blood grouping Gel Cards 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 A1 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 micro-tubes 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. Anti-A,B reagent agglutinates red blood cells possessing A and/or B blood group antigens. Group O red blood cells will not react with the reagent. In addition, Anti-A,B is particularly useful in detecting some weak subgroups of A and B which may not agglutinate with Anti-A or Anti-B reagents.

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. Micro Typing Systems, Inc.

Procedure: Suspend 50 µL WB or 25 µL RBCs in 0.5 ml diluent. ABO/D + Reverse group cards Procedure: Suspend 50 µL WB or 25 µL RBCs in 0.5 ml diluent. Identify the card with patient's name. To microtubes l, 2, 3 & 4 add l0 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. Centrifuge for l0 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 Shape of microplate (U, V, and strait bottom) - the U bottom is used for blood grouping.

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