Lecture #4 James K. Miyasaka [e-mail] February 13, 2008 Factors Affecting RBC Antigen-Antibody Reactions Speaker: Gregory Garraty, PhD, FRCPath Scienctific Director American Red Cross Blood Services Southern California Region Methods for Detecting Antigen-Antibody Reactions: (methods for blood transfusion) Precipitation: older ways look at it as lines of identity that merge into one product. Complement fixation: test for sifilys, uses sheep cells as antibody. Hemolysis: use routinely complement Agglutination: used every day. Inhibition: sample mixed in saline solution and A, B, and O will be in the saline. Labeled antibodies: routinely applied, binds to RBC and labels the antibody. o Flourochromes: (immunofluorescence) o Isotopes(RIA): use radio isotopes. o Enzymes(EIA): uses a particular one, it will change the blood color. o Ferritin/gold(EM): electron microscopy, look for black dots. Chemiluminescence: tagged antibodies give you use laminators. Solid phase RBC indicator: uses for platelets, sold in IGG bonded plate kits. Humagglutination has two stages. Stage 1: Red blood cells moves and causes collisions where antibodies will get on RBC. o Sensitization: antibodies goes onto RBC, bonds to antibody binding site. Stage 2: glutination o Aggulatination: is the clumping of cells together. Important Factors Influencing Binding of Antibody to RBC's Collision: RBC continuously moving, could collide. The negative charges causes a repel. Affinity(goodness of fit): add antibody to antigen it will reach equilibrium. You can graph it Concentration vs. Time. Law of Mass Action: Ab + Ag &lt;-==-&gt; Ag +Ab Equilibrium Constant of antibody (K0)=Ka/Kb=[AgAb]/([Ab]x[Ag]) Affinity Effect: antibody fits good when there are not spots attaching to the antigen, which causes high affinity. Low affinity is when the antigen does not fit to well in the antibody. Factors affecting the First stage of Agglutination (sensitization stage) Concentration of antigen and antibody pH Temperature <a href="/keyword/ionic-strength/" >ionic strength</a> . *make sure you do the test fast because you can wash off the antibody. 1 Lecture #4 James K. Miyasaka [e-mail] February 13, 2008 You can change the concentration by varying the number of antigen sites or varying the number of antibody molecules. Control of the Antigen-Antibody ratio in Antibody Detections compatibility test: Always use larger droplet for antibody Antibody to antigen ration should be 80:1 If you use more antibody than antigen you will pick up weaker antibodies. Optimal pH ranges from 7-7.4 and optimal temperature is 37oC. At this temperature it helps speed up the reaction. The reaction will still work at a lower temperature but it may take longer to finish or hit equilibrium. A test was done with different temperatures and proved this that at a variety of different temperatures 370C worked the best as the reaction hit equilibrium faster. <a href="/keyword/ionic-strength/" >ionic strength</a> o If you lower it down the <a href="/keyword/ionic-strength/" >ionic strength</a> of saline from 0.9% to 0.3% it will become hypotonic so you need osmotic pressure so you can use sucrose. o Lower the saline solution, will help anti-globulin bind faster. RBC has a &quot;-&quot; charge. A lot of ions are +/- charged so we need to reverse some of the ions. Zeta Potential (mV): Red blood cells have negative charge so they don't clump. Bovine albumin and enzymes are used. The distance between charges will determine the Zeta potential. Field potential causes cells to come closer together. Some potentiating antigen-antibody reactions o Albumin Albumin-suspended RBC's Albumin Replacement and addition o Enzymes Trypsion (animal) Papain (papaya) Facin (pig) Bromelin Methods: o Enzyme Method Two stages-Treat RBCs first with enzymes then was RBCs free of enzyme One-stage-mix serum+enzyme+RBCs o Disadvantage of Enzymes False positives, over treatments of RBCs Cold antibodies, rouleauz Enzyme panagglutinins. o Advantages of Enzymes: Increased sensitivity MUCH faster reaction 2 Lecture #4 James K. Miyasaka [e-mail] February 13, 2008 Denaturing of some antigens help identify mixtures of antibodies. o Low <a href="/keyword/ionic-strength/" >ionic strength</a> solution (LISS) you can make yourself. LISS suspended RBCs (older method) What we use now: LISS-additive (2 drops serum + 1 drop 3-4% NISSsuspended RBCs-add LISS-37C 10-15mins-centrifuge/read-wash-IAT) Final <a href="/keyword/ionic-strength/" >ionic strength</a> is the same. o <a href="/keyword/polyethylene-glycol/" >polyethylene glycol</a> o Polybrene Is useful when you can't use the other techniques but rarely used now days. Mostly used in Asia. History of Highlights of RBC Antigen-Antibody Detection Mehtods: 1945: Antiglobulin test and albumin 1945-59: enzymes 1960s: AutoAnalyzer 1964/74: Solid Phase 1989: Gel test 2000: Transfusion Service automation. Antiglobulin Test: Indirect Antiglobulin Test: o Detection and identification of antibodies o Compatibility testing Direct antiglobulin test o Diagnosis of immune hemolytic anemia o Hemolytic disease of fetus o Auto immune hemolytic anemia. Antiglobulin serum injected in rabbits: Help break down products. o Polyclonal: injected in rabbits o Monoclonal: made in labs Factors affection Sensitivity Ratio of RBCs(Ag) to serum (Ab) Incubation time/temperature and medium(<a href="/keyword/ionic-strength/" >ionic strength</a> , pH, etc) Factors affection Sensitivity of DAT and IAT Washing phase o Volume of wash solution o Number of washers o Temperature of washing process o Efficiency of cell washer Reading of test: o Centrifugation 3 Lecture #4 James K. Miyasaka [e-mail] February 13, 2008 o Resuspension of cell button o Macro vs. Micro Sources of Error o Washing is off, reading is off, serum contamination, elution, antiglobulin serum o False positive result, refreigerated clots, bacterial contamination, readings Gel test: 1988- commercial kit by DiaMed SA, Switzerland came out. Principles of Gel test: Serum+RBCs mixed in chamber above a column of dextran gel Gel may be neutral or contain antiserum Upon centrifugation, unagglutinatied RBCs pass through the gel to pellet at bottom Agglutinated RBCs remain in gel, at the top. Advantages of gel test: Less serum used, better serum ratio. Overcomes main disadvantages of test tube, resuspension of agglutinates by agitation No washing needed for AGT Reactions are stabe. Disadvantage of gel test: Special equipment Cost Increased number of positive DATs/ autocontrols There is no perfect test or technique for solving all problems. Dear Classmates, If there are any questions about this lecture or if I missed anything important that you may have in your notes, please feel free to email me at [e-mail] Thanks, ~Jimmy James K. Miyasaka 4