Serological Testing Principles PDF

Section II: Basic immunological procedures Part 1: Principles of Serological Testing LM335 Dr. Hassan Kofahi Serum The most commonly used specimen in the immunology lab is serum. What is serum? Serum is the liquid component of blood that...

Section II: Basic immunological procedures Part 1: Principles of Serological Testing LM335 Dr. Hassan Kofahi Serum The most commonly used specimen in the immunology lab is serum. What is serum? Serum is the liquid component of blood that does not contain blood cells or clotting factors (fibrinogen). What is the difference between serum and plasma? Clotting factors are present in the plasma but are not in the serum. Serum=plasma-clotting factors How do you obtain serum? Serum is obtained by first allowing the blood sample to clot in a clean plain (no additives) tube followed by centrifuging the blood sample and separating the clear supernatant. How do you obtain plasma? Plasma is obtained by collecting the blood sample in a tube containing an anticoagulant (such as heparin or EDTA) followed by centrifugation and collecting the supernatant. Serology: the study of serum. Serology usually refers to the diagnostic identification of certain antigens or antibodies in the serum. Specimen collection, preparation and storage Blood is collected aseptically by venipuncture into a clean, dry, sterile tube. Hemolysis must be avoided because it may affect the test results. Fresh serum is usually recommended for testing. If testing cannot be performed immediately, serum may be stored between 2°C and 8°C for up to 72 hours. If there is any additional delay in testing, the serum should be frozen at –20°C or below. Inactivation of the complement Inactivation of the complement: is the process that destroys complement activity in the serum. Complement is known to interfere with the reactions of certain tests. In these tests, complement must be inactivated in the sample prior to testing. Complement can be inactivated in the sample by heating to 56° C for 30 minutes. Dilution Dilution is required for most of the serological tests. Reagents are often provided in a concentrated forms that need to be diluted prior to performing the test. Many serology tests require dilution of the serum before performing the test Dilution involves two elements: The solute: the material to be diluted (serum or a reagent). The diluent: the medium that makes the rest of the solution. Dilution is expressed as a ratio or fraction 1:10 or 1/10 dilution means that the solute was diluted 10 times (10X) by mixing 1 volume of the solute with 9 volumes of the diluent. In most of the serologic tests, serum samples are diluted in normal saline (NS), phosphate buffered saline (PBS) or a diluent provided by the manufacturer. Dilution = Total volume (Vt) = Volume of solute (Vs) + Volume of diluent (Vd) Example 1: 2 mL of a 1/20 dilution is needed to run a specific serological test. How much serum and how much diluent are needed to make this dilution?  Vt = 2 dilution=1/20  Dilution= Vs/ Vt 1/20 = Vs /2 Vs = 2/20 = 0.1 ml Total volume (Vt) = Volume of solute (Vs) + Volume of diluent (Vd) 2=0.1+ Vd Vd = 1.9 ml This solution can be prepared by mixing 0.1 ml of serum and 1.9 ml of diluent.  Example 2: What is the dilution if you add 0.1 mL of a serum sample to 9.9 mL of diluent? Example 3: In a serological test, you need 50 ml of a working reagent to run the test. However, the manufacturer provides the reagent as a 10 fold concentrated stock (10X) that needs to be diluted in distilled water to the working concentration (1X) before use. How much reagent and how much water are needed to prepare the working reagent? Example 4: A 1:5 dilution of patient serum is necessary to run a serological test. There is 0.1 mL of serum that can be used. What amount of diluent is necessary to make this dilution using all of the serum? Serial dilution Serial dilution is a set of dilutions in which the dilution factor is exactly the same at each step. The concentration decreases by the same quantity in each successive step. A first dilution is prepared, then this dilution is used to prepare a second dilution and so on. Serial dilution techniques: Serial two-fold dilution (Also known as doubling dilution, used frequently in immunology/serology lab) Serial ten-fold dilution (rarely used in immunology/serology lab) Two-fold serial dilution The serum is diluted by the factor of ½ in each step. Dilution Ten-fold serial dilution The serum is diluted by the factor of 1/10 in each step Dilution 1:10 1:102 1:103 1:104 1:105 Titer The reciprocal of the highest dilution in which positive reaction occurs. Used as an indicator for the strength of an antibody. To determine the titer, two-fold serial dilution is done first. Then each dilution is tested. The titer corresponds to the reciprocal of highest dilution that still yields a positive result. Example A serial 2-fold dilution was prepared as shown in the figure below. If the first three tubes gave positive results when tested for antinuclear antibodies (ANA) and the remaining tubes gave a negative result. What is the titer of ANA? Answer: The highest dilution in which + + + - - positive result occurs equals ½ X ½ X ½ = 1/8  The titer is 8 Test Parameters Many clinical tests are used to confirm or refute the presence of a certain disease. In an ideal test: All of the patients with the disease test positive. 1. True positive: the patient has the disease and the test is positive. All of the individuals without the disease test negative. 2. False positive: the individual does not have the disease but the test is positive. Unfortunately, most of the tests are not 3. True negative: the individual does not have the ideal: disease and the test is negative Some of the patients with the disease test 4. False negative: the patient has the disease but negative (False negative). the test is negative. Some of the individuals without the disease test positive (False positive). Sensitivity Vs Specificity Sensitivity Proportion of people who have a disease or condition and who have a positive test Sensitivity (%) = × 100 Sensitivity indicates how small an amount can be measured and still produce a positive test result Highly sensitive test can give positive results even when the specimen contains small amounts of the substance to be measured. Specificity Proportion of people who do not have a disease or condition who have a negative test Specificity (%) = × 100 Highly specific test measures the substance that it is designed to measure, not interfering substances. Example 1: A certain new laboratory test was used with a particular population. The results were as follows: 200 patients were tested and there were 160 true positives, 20 true negatives, 8 false positives, and 12 false negatives. What is the sensitivity and the specificity of the test? Sensitivity (%)= TP/(TP+FN) = 160/(160+12) = 93% Specificity (%)= TN/(TN+FP) =20/(20+8) =71.4% Example 2: A new laboratory assay gave the following results: number of patients tested = 100; number of true positives = 54, number of true negatives = 42; number of false positives = 2; number of false negatives = 2. What is the sensitivity and the specificity of this assay?  The sensitivity and specificity of a quantitative test are dependent on the cut-off value. Increasing the cut-off value increases the specificity but decreases the sensitivity. Decreasing the cut-off value increases the sensitivity but decreases the specificity. Cut-off value: the test value that acts as a dividing point between positive and negative. In most of the cases, test result is considered positive if it is greater than the cut-off value. Frequency Test Result