Chapter 2 Serologic Techniques PDF

Chapter Two Serologic techniques Learning Objective At the end of this chapter, the students should be able to: 1. List material and equipment for serological tests 2. C o l l e c t , p re s e r v e a n d p re p a re s e ro l o g i c a l specimens 3. Run complement inactivation proc...

Chapter Two Serologic techniques Learning Objective At the end of this chapter, the students should be able to: 1. List material and equipment for serological tests 2. C o l l e c t , p re s e r v e a n d p re p a re s e ro l o g i c a l 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 Introduction Antibodies that have been produced in response to a specific stimulus can be identified easily in the serum. Serological reaction produces an observable change in the mixture. The reaction takes different forms, because of variations in the condition of the antigen, the Introduction… Dilution is the act of making a weaker solution from a strong solution. Serial dilution The systematic re-dilution of a f luid 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  Materials necessary for basic serologic tests Types of glassware include:  Test tubes  Glass slides  Serological pipette with a size of 10ml, 5ml, 2ml and 1ml. 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 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. Materials necessary for basic serologic tests… Water Bath A water bath is an instrument where water is heated and the set temperature is maintained at a constant level. It can provide temperature ranging from room temperature to 100Oc. It is used to incubate liquid substances. Chemical tests react best at a specific temperature. Many tests react at room temperature (18 to 22 oC) and others require a specific temperature as body temperature (35 to 37o C). Materials necessary for basic serologic tests… Rotating Machine Rotating machines are required to facilitate antigen antibody reactions. Such machines have a f lat 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. 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.  Blood specimens should be collected before meal Collection, Preparation And Preservation Of Specimens For Serologic tests… Serum or plasma sample collection Collect 2-3ml 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. Transfer the serum to a labeled tube with a paster pipette and rubber bulb. Collection, Preparation And Preservation Of Specimens For Serologic tests… Plasma samples are obtained by treating fresh blood with anticoagulant. Centrifuge and separate the supernatant. The specimen should be free from hemolyzed blood. Finally, seal the specimen containing tube; the tube should be labeled with full patient's identif ication (age, sex, code number, etc). The test should be performed within hours after sample collection, if this could not be done preserve it at -20oc. Shipment of serological specimens  Most health center and clinical 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. 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. 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.  If 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. Shipment of serological specimens… Some times it is necessary to ship a specimen to another laboratory, a large reference laboratory, for testing. Specimens are shipped if: Tests are infrequently performed in the laboratory If tests need specialized technology at a central or reference laboratory setting If tests require special tests Shipment of serological specimens… Since biological specimens are potentially infectious care must be taken to ship them safely. All specimen containers to be shipped must be labeled with the necessary patient identification, and other important information. The mailing package must include properly completed request form for the test to be done. Complement inactivation  Complement is a group of non-immunoglobulin plasma proteins that are sequentially activated by Ag –Ab c o m p l e x e s ( o r d i re c t l y b y m i c ro b i al constituents).  They can cause irreversible damage to membrane of cellular target.  Complement molecules circulate in the blood in an inactive form but activation of the f irst complement component sets in motion a ripple effect. Complement inactivation …  As each component is activated, it acts in turn on the next component in a precise sequence called complement cascade.  Some tests need inactivated serum, while 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 Complement inactivation …  Complement components can be inactivated by of three mechanism  Spontaneous decay  Enzymatic degradation of C4, C3 and C5 rapidly decay  Stoichiometric inhibition Complement inactivation …  The complement in serum must be i n a c ti v a te d u s u a l l y b y s to i c h i o m e tr i c inhibition for most serological testing.  To inactivate complement, place tubes of serum in hot water bath (56c) for 30min.  If the protein complement is not inactivated it will promote lysis of the red cells and other types of cells and can therefore produce 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 56 0 c for 10 minutes and allowed to cool to room temperature 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. Dilution… Dilution techniques  Dilutions can be used in the laboratory to change the concentration of the body f luids, 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. 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. 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. 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. 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 f lask or container, and then diluent is added to the required volume.  Adequate mixing must take place to ensure homogeneity. Dilution… E.g., if you want to prepare 1:10 dilution  Take 1 ml solute 1st  Take 9 ml solvent 2nd  Then mix Dilution… Method  Add 1-ml solute into10 ml graduated volumetric flask and then add water up to the 10-ml mark or graduation of the flask. Dilution…  When a solution is diluted with water, its c onc entration is dec reased and its volum e is increased.  But the total amount of solute remains constant.  Mathematical expressions of the dilutions are; CiVi = CfVf Where, Ci is initial concentration Vi is initial volume. Cf final concentration Vf is final volume. Dilution… Serial dilutions  The systematic re-dilution of a f lu id for a 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.  Serial dilutions are a unique type of dilution techniques. Dilution…  In serial dilution,  All dilutions, except the 1 st are prepared from the previous dilution.  All dilutions made after the initial dilution are the same.  Serial dilution is decreasing the volume of serum progressively by maintaining a constant volume of fluid.  Most common serial dilutions are two fold that is each dilution is half as concentrated as the preceding one, while the total volume in each tube is the same. Dilution…  Occasionally, the f irst dilution may be different from the rest.  The f ir st dilution determines the starting point and the remaining dilutions determine the magnitude of the increase.  Serial dilutions are used:  to prepare sets of standard solutions  to prepare patient's samples to analyze components that can exist over a wide concentration range, such as antibody titers. Dilution… 0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.9 0.5 0.5 0.5 0.5 0.5 0.5 0.5 initial Tube 1 2 3 4 5 6 7 8 9 10 Dilution 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120 Dilution factor 10 20 40 80 160 320 640 1280 2560 5120 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 1st 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. Dilution…  Now, 0.5 ml of this solution is removed and mixed with the 0.5 ml of saline in the 2nd tube; this gives another 1:2 dilution, but since the 0.5 ml of solution put into the 2nd tube is already a 1:2 dilution of the serum, the dilution of serum in the 2nd tube is one half that of the 1st 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 10th tube. Dilution… Class work Q. Calculate the volume of serum in 4th tube and next respective tubes? Dilution… The titer  The titer (French; Titer = standard) may be def ined 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 def in ed as the reciprocal of the highest dilution showing a positive reaction (agglutination, hemolysis, etc,). Dilution…  In clinical serology titer is usually referred to as a measure of the number of antibody molecules per unit volume of the original s er u m an d gi v es an d i n di c ati on of th e antibody concentration in the patient’s serum. 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 1ml. 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 specif ic antigen; usually expressed as the respect of that dilution is called titer Dilution… Determination of end point and titer After serially diluting the patient serum, equal amount of an antigen is added to each dilution to observe the immunologic reaction. The last tube that shows a visible immunologic reaction is known as the end point of the test, the dilution of antiserum (antibody) at the end point is known as the titer. The reciprocal of the greatest reacting dilution of the serum is considered as the measure of titer or the concentration of antibody. Review questions Try the following problems  For ASO titer, tube 1 contains 0.8ml 0f saline, tubes 2 to 5 contain 0.5ml of saline; 0.2ml of serum is added to tube 1, and serial dilutions using 0.5ml are carried out in the remaining tubes. What is the dilution in each tube?  Explain the shipment of specimen and complement inactivation. Reference 1. Tizard. Immunology an introduction,4 th edition , Saunders publishing,1994 2. Naville J. Bryant Laboratory Immunology and S e ro l o g y 3 r d e d i t i o n. S e ro l o g i c a l s e r v i c e s Ltd.Toronto,Ontario,Canada,1992 3. Ma r y L o ui se.I m m uno l o g y a nd S e ro l o g y i n Laboratory medicine 3rd edition