Serology lab manual

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Train Better. Think Better. Be Better. Joe De Sena. Jessamyn Stanley. Related Audiobooks Free with a 30 day trial from Scribd. Pamela Peeke, M. Ethics of Laboratory 2. Collection of blood sample by vein puncture, separation and preservation of serum 3.

Widal Test 4. C-Reactive Protein Latex agglutination 7. Rheumatoid factor RF Latex agglutination 8. Perform Demonstrate various serological tests in identification of different diseases 2. Medical laboratory Technology Vol. Introduction about Phlebotomy: Phlebotomy is the process of making an incision in a vein with a needle. Introduction Venipuncture: Venipuncture is the process of obtaining blood samples from veins for laboratory testing, It is probably the most common procedure in the medical field, usually performed for the following reasons.

It occurs because the wall of a blood vessel wall, artery, vein, or capillary, has been damaged and blood has leaked into tissues If drawn here, could cause incorrect test results. Reference: 1. Result: …………………………………………………………………………………………………………… ……………………………………………………………………………………… Clinical Significance of Serum: …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………………………………………………………………….. Typhi———-O antigen 2 S. Tyhhi———- H antigen 3 S.

Paratyphi ——AH antigen 4 S. In acute typhoid fever, O agglutinins can usually be detected 6—8 days after the onset of fever and H agglutinins after 10— 12 days. Antigen- 9. Method: Rapid slide test method. Clean the glass slide or test card supplied in the kit well and make it dry. Place a drop of undiluted test serum in each of the four labelled circle 1, 2, 3 and 4 ie O, H, AH and BH and place a drop of Negative control serum in circle 5 and Positive control in circle 6.

Mix the content of each circle with a separate wooden applicator stick and spread to fill the whole area of the individual circle. Rock the test card for a minute and observe for agglutination. Observation: ………………………………………………………………………………………………… ………………………………………………………………………………………………… ………………………… Result: ………………………………………………………………………………………………… ………………………………………………………………………………………………… ………………………………………………………………………………………………… ……………………………………..

Clinical significance: ………………………………………………………………………………………………… ………………………………………………………………………………………………… ………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………………………………… Quantitative tube test: 1. Take a set of 8 clean dry test tubes Kahn tubes and label as 1, 2,3, 4, 5, 6, 7 and 8 for O antibody detection.

Similarly, take 3 sets of 8 test tubes and label then as 1, 2…8. Continue this serial dilution till tube no. Now the dilution of the serum sample achieved in each set is as follows: Tube No. Add a drop of appropriate widal test antigen to all the test tubes 5. Antibody titre is the highest dilution of serum showing clear agglutination. Observation: ………………………………………………………………………………………………… ………………………………………………………………………………………………… ………………………… Result: ………………………………………………………………………………………………… ………………………………………………………………………………………………… ………………………………………………………………………………………………… ……………………………….

False negative Widal tests may be due to antibody responses being blocked by early antimicrobial treatment or following a typhoid relapse. Variations also exist between laboratories in the performance and reading of Widal tests which compromise further the reliability of the test. Cardiolipin reacts non-specifically to cardiolipin auto-antibodies IgM and IgG to form flocculation floccular agglutination. For th test, at first a drop of antigen is placed on a slide and then a drop of serum is added to it.

The slide is rotated to mix the content. In case of positive test, flocculation occurs. The cardiolipin antigen is an alcoholic solution composed of 0. The heat inactivated to destroy complement serum is mixed with VDRL antigen.

If the specimen contains reagin, flocculation occurs which can be observed using microscope. Non- reactive specimens appear as homogeneous suspension. Reagents should be mixed gently to avoid possible deterioration of the antigen-carrier complex.

Reagents are stable until the expiration date printed on the label. Stabilized combination of 0. The antigen suspension is packaged in ampules. An unopened ampule of antigen is stable up to the expiration date. Liquid reactive R , minimally reactive Rm , and nonreactive N control serum specimens of graded reactivity. If quantitative tests are to be performed, a control serum that can be titered to at least a dilution should be used.

Bring the VDRL antigen suspension, controls and samples to room temperature. Add one drop of well-mixed VDRL antigen next to the test specimen, positive control and negative control. Using a mixing stick mix the test specimen and the VDRL reagent thoroughly spreading uniformly over the entire reaction circle. Rotate the slide gently and continuously either manually or on a mechanical rotor at r. Observe for flocculation microscopically at 8 minutes.

Observation ………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………. Result: ………………………………………………………………………………………………… ………………………………………………………………………………………………… ……………………………………………………………………… II. Using isotonic saline prepare serial dilutions of the test sample positive in the qualitative method , , , ,, , and so on as follows : 1. Mix the mixture. Avoid formation of bubbles. Repeat this serial dilution procedure in tube 3 to 4, and then 5.

Tubes 1 to 5 now represent a dilution series as follows: Tube Number 1 2 3 4 5 Dilution Conversely, a false positive VDRL can be encountered in infectious mononucleosis, lupus, antiphospholipid antibody syndrome, hepatitis A, leprosy, malaria and, occasionally, pregnancy. Limitations 1. Biological false-positive reactions can occur with cardiolipin antigens, mainly in specimens from persons who abuse drugs; who have diseases such as lupus erythematosus, mononucleosis, malaria, leprosy or viral pneumonia; or who have recently been immunized.

Reactive specimens should be subjected to further serologic study, including quantitation, whenever feasible. A prozone reaction may occur.

In a prozone reaction, reactivity with undiluted serum is inhibited. The prozone phenomenon may be suspected when a specimen produces only a weakly reactive or a rough nonreactive result in a qualitative test.

The VDRL may be reactive in persons from areas where yaws is endemic. There will not be a detectable immune response for days after exposure. The SLO toxin has direct toxic effects on heart tissue and joints. Principle: ASO latex agglutination is the rapid and simple test for the qualitative and semi-quantitative measurement of antibodies to Antistreptolysin-O in human serum.

This method is based on an immunological reaction between streptococcal exoenzymes bound to biologically inert latex particles and streptococcal antibodies in the test sample. Sample Collection and Handling: Only fresh serum specimens should be used.

Plasma must not be used since fibrinogen may cause non-specific agglutination of the latex. It is preferable to test samples on the same day as collected. Materials used in the ASO Test Shake well prior to use. Sufficient disposable pipettes. Glass test slide. Patient serum sample Procedure: 1. Bring all reagents and specimens to room temperature. Mix well using disposable stirrer spreading the mixture over the whole test area and tilt the slide gently.

Agitate for about 2 minutes with rotator or by hand and observe for the presence or abscence of agglutination. Measurement of ASO antibody titre is important in the investigation of post- streptococcal diseases, particularly acute poststreptococcal glomerulonephritis and rheumatic fever.

Limitations of the Test 1. Highly haemolyzed and lipemic serum as well as plasma interfere with the test. Since streptolysin is only one of several Streptococcus A exoenzymes, the ASO test will not detect the other antibodies to exoenzymes of Streptococcus A.

CRP is classified as an acute phase reactant, which means that its levels will rise within a few hours after tissue injury, the start of an infection, or other cause of inflammation. High CRP levels may put patients at increased risk for coronary artery disease, which can cause a heart attack. This is a slide agglutination test for the qualitative and semiquantitative detection of C- Reactive Protein CRP in human serum.

Or The C-Reactive Protein test is based on the principle of the latex agglutination. Materials Required: 1. Negative Control: Human serum containing 0.

Procedure Qualitative Test 1. Bring all reagents and serum sample to Room Temperature and mix latex reagent gently prior to use. Do not dilute the controls and serum. Place 1 drop of Serum, Positive control and Negative control on separate reaction circle on glass slide. Then add 1 drop of CRP latex reagent to each of the circles. Mix with separate mixing sticks and spread the fluid over the entire area of the cell. Tilt the slide back and forth slowly for 2 minutes observing preferably under artificial light.

Observe for visible agglutination. Observation ………………………………………………………………………………………… ………………………………………………………………………………………….. Result ………………………………………………………………………………………… ………………………………………………………………………………………… Result Interpretation of CRP Test: Agglutination of latex particles is considered a positive reaction, indicating the presence of C-reactive protein at a significant and detectable level.

Specimens which do not contain human CRP will not cause agglutination. If controls do not give expected reactions the test is invalid and must be repeated.

Limitations of CRP Test 1. The strength of the agglutination reaction is not indicative of the CRP concentration. Weak reactions may occur with slightly elevated or markedly elevated concentrations. A prozone phenomena antigen excess may cause false negatives. It is advisable, therefore, to check all negative sera by retesting at a dilution.

Reaction times longer than specified may produce apparent false reactions due to a drying effect. Strongly lipemic or contaminated sera can cause false positive reactions. Only serum should be used in this test. A quantitative titration procedure on positive specimens is required to observe increasing or decreasing levels. Patients with high titers of rheumatoid factors may give positive results.

Of these IgM and IgG are the most common. However, it is also detectable sometimes in the serum of patients with Systemic Lupus Erythematosus SLE and in certain non-rheumatic conditions. Reagent used is a suspension of polystyrene latex particles in glycine-saline buffer with pH: 8. Materials required: 1. Positive Control: Human serum containing known RF Place one drop of the positive control and 40ul of the patient serum into separate circles on the slide. Gently and add one drop of RF latex reagent on each circle of sample to be tested and control.

Tilt the slide back and forth for 2 minutes in a rotary shaker so that the mixture rotates slowly.