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False negative interpretation of HBsAg ELISA by interference of very high rheumatoid factor level
India (MSAI) - Parul Institute of Medical Sciences & Research, Vadodara, Gujrat
Dr NisargSavjiani, Dr K K Shiromani
Type of Research Project
- Clinical Project with Laboratory work
What is the background of the project?
Rheumatoid factor (RF), a kind of autoantibody against the fragment c portion of IgG, can be of any isotype of immunoglobulins i.e. IgA, IgG, IgM, IgE, IgD. High levels of serum RF occur in about 80% of patients with rheumatoid arthritis. RF is sometimes mentioned as an important factor causing positive interference in immunoassay. In two-site immunoassays, RF can bridge the capture antibody and HRP(Horseradish peroxidase)-labeled antibody together and falsely increase the patient’s value. Immunoassays using either polyclonal or monoclonal antibody can be affected. The hepatitis B surface antigen (HBsAg) is the first marker that appears in the blood following infection with hepatitis B virus (HBV). The presence of HBsAg in human serum indicates an ongoing HBV infection, either acute or chronic. 3 rd generation ELISA for detection of HBsAg is a very reliable test with sensitivity and specificity of 99% and above. Immunomodulated patients may have chances of revealing discrepancies in ELISA results. It may prove to be a more or less false negative in patients having very high rheumatoid factor levels. This discrepancy becomes a big challenge in getting to the right result in this patients. An extra precaution and caution has to be taken into the mind with proper history taking of the patient with clinico pathological correlation.
What is the aim of the project?
To assess the relevance of HBsAg ELISA in patients with very high level of rheumatoid factor
What techniques and methods are used?
The serum samples of patients with moderate to high concentration of rheumatoid factor are collected. Rheumatoid factor quantification is done by turbidimetric immunoassay method on semi automated biochemistry analyser. Serum samples of HBsAg positive patients is also collected by 3 rd generation ELISA. The serum samples are stored at -20 degree Celsius. Blend of 25 micro litre of HBsAg positive serum with 225 micro litre of moderate or high concentration of rheumatoid factor sera is made. Control serum is made by mixing the same HBsAg positive serum with normal serum at the ratio of 1:9 respectively.3 rd generation ELISA in Qualisamicrowells provided by Qualpro Diagnostics is performed. The OD value by ELISA reader is taken and statistical analysis is done by comparing results of diluted and undiluted sera with controls.
What is the role of the student?
- The student will mainly observe
- The student will observe the practical experiments but will be highly involved in the analysis of the results
What are the tasks expected to be accomplished by the student?
The student is supposed to do rheumatoid factor estimation on semi automated biochemistry analyser. Would be doing ELISA under guidance once they are prepared to do after observing the technique on first few days. Students are explanation) supposed to analyse statistical data and make the inference out of it. Once the data is compiled the student is supposed to make poster/paper presentation. Simultaneously student must learn about clinical implication of rheumatoid factor and HbsAg.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Yes, There will be daily 1 hr of teaching of theoretical aspect of the procedures as well as about the disease by any of the above mentioned tutors.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a presentation - The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
☐ Subjects passed: Biochemistry, Pathology, Microbiology ✓ Previous experience with: Biochemistry, Pathology, Microbiology ☐ Certificate of: ☐ None
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system - Dental medicine students (IADS members)
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