Immunology and Serology Lab - 01 Serial Dilution and Basic Serologic Techniques

IMMUNOLOGY & SEROLOGY LABORATORY 3. SERIAL DILUTION AND BASIC SEROLOGIC LABORATORY TECHNIQUES OUTLINE • Serial Dilution o Materials/Supplies o Procedure o Dilution Factor o Antibody Titer • 4. Basic Serologic Laboratory Techniques o Blood Specimen Preparation o Types of Specimen Tested o Pipettes 5. 6. SERIAL DILUTION • • Antibodies (Abs) are usually found in serum hence, it is the preferred specimen in different serologic assays. The quantitative estimation of antibodies present in serum formed during disease can be done by making serial dilutions. The titer or strength of antibody present in the sample is indicated by the highest serum dilution that will show a positive (agglutination) reaction. In this exercise, dilutions will be done in two ways by using serologic pipettes and doubling dilutions which can be made with micropipettes. MATERIALS/SUPPLIES • • • • • • • • • Serum/picric acid NSS/tap water Kahn test tubes and Test tube rack Serologic pipette (ml) & aspirator bulb Labeling material Micropipettes with disposable tips (ul) Microtiter plate Anti-A serum/Anti-B serum 4% Type A/B red cell suspension 7. o Add one drop of Anti-A antiserum to wells 1 and 2. Using 20-uL micropipette, mix well 2 by drawing up and down in the pipette several times. Wipe the outside of the pipette tip, and transfer 20 uL to well number 3. Repeat this process with successive wells through well number 5. After well 5 is mixed, discard 20 uL from it. Add one drop of 4-percent type A red blood cells to all wells. Rotate plate for 1 minute, making a concentric circular pattern. Stand for 30 minutes at room temperature. Observe for agglutination. Report the titer in the last well which agglutination can be seen Interpretation ▪ Positive result shows an irregular or crenulated pattern whereas negative result shows a smooth button on the bottom of a well. DILUTION FACTOR • • • A dilution factor is used to correct for having used a diluted sample in a determination rather than the undiluted sample. The result (answer) using the dilution must be multiplied by the reciprocal of the dilution made. For example, a dilution factor by which all determination answers are multiplied to give the concentration per 100 mL of sample (blood) may be calculated as follows: o First, determine the volume of blood that is actually analyzed in the procedure. Using a simple proportion, it is evident that 0.5 mL of blood diluted to 10 mL is equivalent to 1 mL of blood diluted to 20 mL: PROCEDURE • • Use of serologic pipettes 1. Prepare and label 5 test tubes in a test tube rack and mark the last tube as discard tube. 2. Dispense 2.0 milliliter (ml) of normal saline solution to all tubes except the discard tube. 3. Using a clean pipette, add 2.0ml.serum to the first tube and mix contents by tapping. 4. Using the same pipette, transfer 2.0 ml. of the contents of tube number 1 to tube number 2. 5. Mix content of tube no. 2. Following the same procedure, transfer 2.0 ml. of its contents to tube no. 3. Then mix contents of tube number 3 then transfer 2.0 ml. of the mixture to tube number 4. Do the same for the discard tube. o Interpretation ▪ The highest serum dilution that will show agglutination reaction is the titer. Use of micropipettes 1. Label one row of microtiter plate as follows: 10, 20, 40, 80 and 160. 2. Add one drop(10 ul) of saline to wells 2 through 5 on the microtiter plate. o The concentration of specimen (blood) in each milliliter of solution may be determined by the use of another simple proportion to be 0.05 mL of blood per milliliter of solution. EXAMPLE OF PREPARATION OF A SERIAL DILUTION • Use of Dilution Factors: o 1:5 dilution of a specimen is prepared and an aliquot (one of a number of equal parts) of the dilution is analyzed for a particular substance. o The concentration of the substance (C) in the aliquot is multiplied by 5 to determine its concentration in the original specimen. If the concentration of the dilution is 100 mg/dL, the concentration of the original specimen is: C =100 mg/dL × 5 (dilution factor) = 500 mg/dL in blood • Schematic of a twofold serial dilution\ • Contamination with alkali or acid must be avoided because these substances have a denaturing effect on serum proteins and make the specimens useless for serologic testing. PIPETTES • • Pipettes are used in the immunology-serology laboratory for the quantitative transfer of reagents and the preparations of serial dilutions of specimens such as serum. Semiautomated micropipettes have replaced traditional glass pipettes in the laboratory, traditional methods may still be needed at times. TYPES OF MANUAL PIPETTES (TO DELIVER) • Sample Problem: o Make serial dilution for 6 test tubes. Tube 1 contains 10 ml of solute. o Tubes 2-6 contains 5 ml of water each. Transfer 5ml of liquid from Tube # 1 (aliquot) • ANTIBODY TITER • • Defined as the reciprocal of the highest dilution of the patient’s serum in which the antibody is still detectable. That is, the titer is read at the highest dilution of serum that gives a positive reaction with the antigen. If a serum sample has been diluted 1:64 and reacts positively with the antigen suspension used in the testing process, and if the next highest dilution of 1:128 does not give a positive reaction, the titer is read as 64. BASIC SEROLOGIC LABORATORY TECHNIQUE BLOOD SPECIMEN PREPARATION • • • • After blood has been obtained from a patient, in a plain evacuated tube, without anticoagulant. it should be allowed to clot and the serum should be promptly removed for testing. Complete clot retraction normally takes about 1 hour. After clot retraction, the clot should be loosened from the sides of the test tube with an applicator stick and centrifuged for 10 minutes at a moderate speed. TYPES OF SPECIMENS TESTED • • • Most immunology tests are done on serum, although body fluids may also be tested. Lipemia, hemolysis, or any bacterial contamination can make the specimen unacceptable. Icteric or turbid serum may yield valid results for some tests but may interfere with others. Blood specimens should be collected before a meal to avoid the presence of chyle, an emulsion of fat globules that often appears in serum after eating, during digestion. • Graduated Pipette o Delivering a particular amount of liquid is to deliver the amount of liquid contained between two calibration marks on a cylindrical tube, or pipette. o Graduated pipettes are used primarily for measuring reagents but are not calibrated with sufficient tolerance for measuring standard or control solutions, unknown specimens, or filtrates. Serologic Pipettes o Recognized by a frosted ring at the noncalibrated end, with calibrations extending to the tip. o The letters TD (to deliver) appear on the pipette and, for quick recognition, each size of pipette has an imprinted, color-coded band that indicates the volume AUTOMATIC PIPETTE • • Steps in using piston-type automatic micropipette. 1. Attaching proper tip size for range of pipette volume, and twisting tip as it is pushed onto pipette to give an airtight, continuous seal. 2. Holding pipette before use. 3. Follow instructions for filling and emptying pipette tip.