TPHA examination Lecture Notes

TPHA examination Basic theory : Treponema Pallidum Hemagglutination Assay(TPHA) is a serological examination for syphilis and is less sensitive when used as a screening (early or primary stage) of syphilis. The benefits of TPHA examination as a confirmatory examination for syphilis and detecting specific serological responses forTreponema pallidum in the advanced or late stages of syphilis. For screening for syphilis, VDRL or RPR examination is usually used if the results are reactive, then followed by TPHA examination as confirmation. TPHA is a very specific test to see whether antibodies to treponema are present. If there are these bacteria in the body, the test result is positive. This test will become negative after 6 – 24 months after treatment. Bacteria other than the treponema family cannot make this test result positive. TPHA examination is carried out based on the presence of antibodiesTreponema Pallidumwhich will react with treponemal antigens attached to erythrocytes so that agglutination of the erythrocytes is formed. The advantages of the TPHA method for syphilis examination compared to other methods: 1. The technique and reading of the results are easy, quite specific and sensitive (can detect very low titers) 2. Bacteria other than those from the familyTreponemacannot give positive resultsHowever, the TPHA method has several disadvantages, including: 1. It's expensive 2. The process requires a long incubation time, almost 1 hour. Things that need to be considered in the TPHA examination include: 1. Do not use hemolyzed serum because it can affect the examination results. 2. Serum or plasma must be free from blood cells and microbiological contamination 3. If there is a delay in the examination, the serum is stored at 2-80C which can last for 7 days and when stored at -200C, the serum can last longer. 4. Serum or plasma that is frozen before the examination must be thawed and homogenized properly before the examination. 5. Reagents should be stored at 2-80C if not in use and do not store in the freezer. 6. The TPHA test shows reactive results after 1-4 weeks after the formation of the chancre. 7. When carrying out the examination, positive controls and negative controls must be included Principle : Specific antibodies forT. pallidumcontained in the patient's serum will agglutinate with preserved bird erythrocytes contained in the Plasmatec TPHA reagenthas been coated with pathogenic antigenic componentsT. pallidum(Nichol Strain) shows an agglutination pattern in the microtitration wells. Tool : And Micropipette 190 µl, 10 µl, 25 µl, and 75 µl Microplate Yellow tip Reagents: Plasmatec TPHA Test Kit contains: R1 : cell test R2 : control cells R3 : diluent R4 : positive control R5 : negatife control Material : serum Ways of working Qualitative Test 1. Tools and materials are prepared 2. Each kit component and sample is conditioned at room temperature. 3. All reagents are homogenized slowly 4. 190 µl of diluents was added and 10 µl of the sample was added to well 1 then homogenized 5. 25 µl of the mixture in well 1 was pipetted and added to wells 2 and 3 6. Control cells of 75 µl were added to well 2 and then homogenized 7. 75 µl of test cells were added to well 3 and then homogenized 8. Wells are incubated at room temperature for 45 – 60 minutes. 9. The agglutination that occurs is observed 10. Samples that show positive agglutination results are continued to semiquantitative testing. Note:Positive and negative controls are always included in each test without needing to be diluted. Semi Quantitative Test 1. Tools and materials are prepared 2. Each kit component and sample is conditioned at room temperature 3. All reagents are homogenized slowly 4. Microtitration wells are prepared and labeled no. 1 to 8 5. Sample dilution is made in a different well from the microtitration well by mixing 190 µl of diluents and 10 µl of sample 6. Microtitration well no. 1 is left blank 7. Microtitration well no. 2 – 8 added 25µl of diluent 8. In microtitration well no. 1 and 2 added 25 µl of diluted sample. 9. 25 µl of the mixture in well 2 was pipetted and added to well 3, then homogenized. And so on until well 8 10. 25 µl of the mixture in well 8 was pipetted and discarded 11. Control cells of 75 µl were added to microtitration well no. 1 then homogenized 12. 75 µl of test cells were added to microtitration well no. 2-8 then homogenized 13. The wells are incubated at room temperature for 45 – 60 minutes 14. The agglutination that occurs is read, and the titer is determined Interpretation of Results Qualitative Test Positive hemagglutination is indicated by the presence of red dots on the surface of the well, negative results appear as a red dot in the middle of the bottom of the well. Degree of agglutination: + 4 : : red dots are evenly distributed over the entire surface of the well + 3 : red dits are found on most the surface of the well + 2 : the red circle that forms is not large and looks like a ring + 1 : the red circle isndmall and looks like a bright ring +/- : a ring appears with faint red cirlce – : a red dot appears at the bottom of the well