Chapter Two Serologic techniques Acknowledgements Addis Ababa University
Chapter Two Serologic techniques
Acknowledgements Addis Ababa University Jimma University Hawassa University Haramaya University of Gondor American Society for clinical Pathology Center for Disease Control and Prevention Ethiopia
Learning Objective At the end of this chapter, the students should be able to: 1. List material and equipment for serological tests 2. Collect, preserve and prepare serological specimens 3. Run complement inactivation procedure and state its importance 4. Run serial dilution, determine end point and titer.
Outline 2. 1. Introduction 2. 2. Materials necessary for basic serologic tests 2. 3. Collection, preparation and preservation of serologic al tests 2. 4. Shipment of serological specimens 2. 5. Complement inactivation 2. 6. Dilution 2. 6. 1. Serial dilution
2. 1. Introduction Dilution is the act of making a weaker solution from a strong solution. Serial dilution The systematic re-dilution of a fluid number of times is called a Serial dilution Titer is the reciprocal of the highest dilution showing a positive reaction Complement is a group of non-immunoglobulin plasma proteins that are sequentially activated by Ag–Ab complexes
2. 2. Materials necessary for basic serologic tests Types of glassware include: § Test tubes § Glass slides § Serological pipette with a size of 10 ml, 5 ml, 2 ml and 1 ml.
2. 2. Materials necessary for basic serologic tests Glassware § Dirty glassware easily affects serological tests. § After using all the glassware (test tube, beaker, pipette, etc) they should be soaked in detergent for several hours and rinsed several times in tap water. § Finally, allow drying by placing in a dry oven or dust free place. Test tubes and pipettes should not be scratched or broken, which will interfere with the reading of a test.
Glassware's and plastic wares
2. 2. Materials necessary for basic serologic tests Constant Temperature Device Incubators and water baths are used in serological tests. These materials are electrically operated and have thermostat that hold the temperature within the required limits. These devices should be checked prior to use by a thermometer.
2. 2. Materials necessary for basic serologic tests Rotating Machine Rotating machines are required to facilitate antigen antibody reactions. Such machines have a flat plate, which rotate at a prescribed rate of speed. A knob located on the front of the machine controls the number of revolutions per minute.
2. 3. Collection, Preparation And Preservation Of Specimens For Serologic tests § Specimens that are used for serologic test include: serum, plasma and cerebrospinal fluid. § Serum or plasma samples could be obtained from venous blood, which can be collected by the laboratory personnel. § CSF should be collected by a physician or trained nurse.
2. 3. Collection, Preparation And Preservation Of Specimens For Serologic tests Serum or plasma sample collection Collect 2 -3 ml of venous blood from a patient using a sterile syringe and needle. If serum is required, allow the whole blood to clot at room temperature for at least one hour, Centrifuge the clotted blood for 10 minutes at 2000 rpm.
2. 3. Collection, Preparation And Preservation Of Specimens For Serologic tests Transfer the serum to a labeled tube with a paster pipette and rubber bulb. Plasma samples are obtained by treating fresh blood with anticoagulant, Centrifuge and separate the supernatant.
2. 3. Collection, Preparation And Preservation Of Specimens For Serologic tests § The specimen should be free from hemolyzed blood. § Finally, seal the specimen containing tube; the tube should be labeled with full patient's identification (age, sex, code number, etc). § The test should be performed within hours after sample collection, if this could not be done preserve it at -20 oc.
2. 4. Shipment of serological specimens § Most health center and clinic laboratories are limited in the diagnostic procedures that can be carried out and have to ship serologic specimens to other laboratories. § Before shipment, the following things should be considered. § Don't ship whole blood unless the tests to be performed require whole blood. § Don't inactivate serum or plasma.
2. 4. Shipment of serological specimens Serum, plasma, and CSF should be handled as follows: § Collect and process specimens under sterile conditions. § Ship specimens by the fastest route as soon after collection as possible. § Don't ship whole blood unless the test to be performed required whole blood. § Remove cells from plasma and clot from serum before shipment.
2. 4. Shipment of serological specimens § Don't inactivate serum or plasma before mailing. § Keep the specimen and packing container in the refrigerator until time of shipment. § Shipment is requires several days preserve by refrigeration in transit. First, freeze the specimen; then pack and ship in a well-insulated container with dry ice.
2. 5. Complement inactivation § Complement is a group of non-immunoglobulin plasma proteins that are sequentially activated by Ag–Ab complexes (or directly by microbial constituents) and cause irreversible damage to membrane of cellular target
2. 5. Complement inactivation § Complement molecules circulate in the blood in an inactive form but activation of the first complement component sets in motion a ripple effect. As each component is activated, it acts in turn on the next component in a precise sequence called complement cascade.
2. 5. Complement inactivation § Some tests need inactivated serum. Others do not. § Inactivation may be important since complement promotes lysis of erythrocytes and can contribute to false test results in tests using RBCs. § Some complement components may also cause false agglutination in some tests.
2. 5. Complement inactivation § Complement components can be inactivated by of three mechanism § Spontaneous decay § Enzymatic degradation of C 4, C 3 and C 5 rapidly decay § Stoichiometric inhibition
2. 5. Complement inactivation § The complement in serum must be inactivated usually by stoichiometric inhibition for most serological testing. § To inactivate complement, place tubes of serum in hot water bath (56 c) for 30 min § If the protein complement is not inactivated it will promote lysis of the red cells and other types of cells and can therefore produce invalid results
2. 5. Complement inactivation § Complement is also known to interfere with certain tests for syphilis. § Serum samples to be tested more than 4 hours after inactivation should be reheated at 560 c for 10 minutes temperature and allowed to cool to room
2. 6. Dilution § Dilution is the act of making a weaker solution from a strong solution. § Adding a diluent such as water or saline, which contains none of the material being diluted, is used to do this. § Dilutions are usually expressed as 1 unit of the final solution.
2. 6. Dilution techniques § Dilutions can be used in the laboratory to change the concentration of the body fluids, such as serum so that it is consistent with the range of an assay. § Making dilutions can also be necessary to prepare reagents and standards. § Dilution has two parts: diluents and solute.
2. 6. Dilution § A dilution involves adding of a substance, the diluent to other substances, the solute. § Dilutions show the relative amount of the solute in the dilute solution. § It is an indicator of concentration, not volume.
2. 6. Dilution § Expression of dilution Dilution is usually expressed as: a to b a: b a/b Whereas; a, is the volume of the original materials that was diluted e. g. serum (solute) b, is the total volume to which it was diluted. It contains solute a and diluent b.
2. 6. Dilution § The dilution factor is the inverse of the dilution statement. For a 1: 10 dilution, the dilution factor is 10. For a : b dilution the dilution factor is b.
2. 6. Dilution Technique § Two liquids of very different compositions (density, or surface tension) is required § An exact volume of concentrated solute is added to a calibrated flask or container, and then diluent is added to the required volume. § Adequate mixing must take place to ensure homogeneity
2. 6. Dilution E. g. , if you want to prepare 1: 10 dilution § Take 1 ml solute 1 st § Take 9 ml solvent 2 nd § Then mix
2. 6. Dilution Method § Add 1 -ml solute into 10 ml graduated volumetric flask and then add water up to the 10 -ml mark or graduation of the flask.
2. 6. Dilution § When a solution is diluted with water, its concentration is decreased and its volume is increased. But the total amount of solute remains constant. § Mathematical expressions of the dilutions are; C i V i = C f. V f Where, Ci is initial concentration Vi is initial volume. Cf final concentration Vf is final volume.
2. 6. Dilution 2. 6. 1. Serial dilutions § The systematic re-dilution of a fluid number of times is called a "serial dilution". § Serial dilutions are most commonly employed in serological procedure to obtain quantitative estimations of antigen or antibody content.
2. 6. Dilution § Serial dilutions are a unique type of dilution techniques. § In serial dilution, all dilutions, except the 1 st are prepared from the previous dilution and all dilutions made after the initial dilution are the same.
2. 6. Dilution § Serial dilutions are used to prepare sets of standard solutions and are also used to prepare patient's samples to analyze components that can exist over a wide concentration range, such as antibody titers. § Serial dilutions must be prepared with care as errors can be compounded during the serial technique.
2. 6. Dilution 0. 5 0. 1 0. 5 0. 9 initial Tube Dilution 0. 5 1 2 1: 10 1: 20 1: 40 1: 80 20 40 80 factor 10 3 4 0. 5 5 0. 5 6 0. 5 7 8 1: 160 1: 320 1: 640 160 320 640 0. 5 1: 1280 0. 5 9 10 1: 2560 1: 5120 2560 5120
2. 6. Dilution An example of the serial dilution is as follows: - § Into each of ten test tubes is measured 0. 5 ml of saline 1/2 ml of serum is placed in the 1 st tube and mixed. § Since there is 0. 5 ml of serum in a total volume of 1. 0 ml; a 0. 5: 1 or a 1: 2 dilution exists in the first tube.
2. 6. Dilution § Now, 0. 5 ml of this solution is removed and mixed with the 0. 5 ml of saline in the 2 nd tube; this gives another 1: 2 dilution, but since the 0. 5 ml of solution put into the 2 nd tube is already a 1: 2 dilution of the serum, the dilution of serum in the 2 nd tube is one half that of the 1 st tube or 1/2 of ½ =1/4 or 1: 4. § This and, by applying the above reasoning, the dilutions of serum are found to be (1/2)10 = 1/1024 or 1: 1024 in the 10 th tube.
2. 6. Dilution Class work Q. Calculate the volume of serum in 2 nd tube and next respective tubes?
2. 6. Dilution The titer § The titer (French; Titer = standard) may be defined as the quantity of a substance required to produce a reaction with a given volume of another substances or the amount of one substances required to correspond with a given amount of another substances. § It is also defined as the reciprocal of the highest dilution showing a positive reaction (agglutination, hemolysis, etc, ).
2. 6. Dilution § In clinical serology titer is usually referred to as a measure of the number of antibody molecules per unit volume of the original serum and gives and indication of the antibody concentration in the patient’s serum.
2. 6. Dilution § An antibody titer of serum is the highest dilution of serum that will give a reaction with antigen. § § For example, if the last tube showing a ratio contains 1 ml. Volume, and the serum in this tube is 1 part in a total of 640 parts, the titer is 640 units/ml of serum, or 1: 640. Generally a maximum dilution of a specific antibody that gives a measurable reaction with a specific antigen; usually expressed as the respect of that dilution is called titer
Review questions Try the following problems § For ASO titer, tube 1 contains 0. 8 ml 0 f saline, tubes 2 to 5 contain 0. 5 ml of saline; 0. 2 ml of serum is added to tube 1, and serial dilutions using 0. 5 ml are carried out in the remaining tubes. What is the dilution in each tube? § Explain the shipment complement inactivation. of specimen and
Reference 1. Tizard. Immunology an introduction, 4 th edition , Saunders publishing, 1994 2. Naville J. Bryant Laboratory Immunology and Serology 3 rd edition. Serological services Ltd. Toronto, Ontario, Canada, 1992 3. Mary Louise . Immunology and Laboratory medicine 3 rd edition Serology in
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