The document discusses tests for HIV and hepatitis B virus (HBV). It describes several tests used to detect HIV and HBV, including chemiluminescence immunoassay, CD4 receptor counting, ELISA, reverse transcriptase PCR, and rapid tests. For HBV detection, it outlines HBsAg ELISA, PCR, and rapid tests. The tests are important to screen blood donations and prevent transmission of HIV/AIDS and hepatitis.
3. Tests of virology in blood bank
• In blood bank, it is mandatory to test for Hepatitis B surface antigen
and anti-HIV
• These tests are done to prevent transmission of AIDS and hepatitis
• Only those samples, which are found to be HBsAg and anti HIV
negative are released for donation purpose
• This information is handled with complete confidentiality
4. Human Immunodeficiency Virus (HIV)
• HIV is an RNA retrovirus that attacks mainly to the CD4+ T-cells
causing immune suppression AIDS
• It is transmitted by sex, blood transfusion, infected blood sharp
instruments and from infected mother to child
• Its window period is typically 3–6 weeks
• Four stages can be recognized in the progression of HIV disease:
• Seroconversion
• Asymptomatic stage
• Symptomatic stage
• Late stage HIV
5.
6. Hepatitis B Virus
• Double stranded DNA enveloped hepadnavirus.
• It is spread by blood, body fluids, and close personal contact
• Causes most serious form of viral hepatitis, 60–80% of all primary
liver cancer, a major cause of cancer death in East and Southeast asia
• The virus carries
• hepatitis B core antigen (HBcAg)
• surface antigen (HBsAg)
• enveloped antigen (HBeAg)
• viral DNA
7.
8. HIV Tests
1. Chemiluminescence immuno-assay (CLIA)
2. Receptor CD4 test
3. ELISA
4. Rt-PCR
5. Rapid test
HBV Tests
1. ELISA
2. PCR
3. Rapid test
9. HIV Tests
HIV window period
• The time between
infection and when
the test can detect
that infection
10. Chemiluminescence immunoassay (CLIA)
• It is a alternative of colorimetric assay
in which emission of light occur by
chemical reaction
• Some enzyme reaction produce light
and this can be measured to detect
product formation
• This assay can be extremely sensitive
11. • When complimentary antigen antibodies are present they
form immune complex
• Estimating the levels of such immune complex by use of
labeled antibodies and addition of substrates.
• The intensity of emitted light is directly proportional to the
amount of labeled complexes present .
Principle:
12. Antigen or antibody coated well
Test Specimen
Washing
HRP labelled AbAg conjugate
14. CD4 Test
• CD4 T helper -lymphocytes are naturally
occurring defense cells that regulate cellular
and humoral immunity in body
• HIV infect cells bearing the CD4 antigen
receptor depleting CD4 cells
• Flow cytometry is the technique used to
count CD4 T-cells
• The normal CD4 T cell count for an adult is
just over 1000 cells/µl
15. Principle:
• Cell components are fluorescently labelled and then excited by the
laser to emit light at varying wavelengths.
• The fluorescence can then be measured by detectors to determine
the amount and type of cells present in a sample.
• This data is then analyzed by a computer that is attached to the flow
cytometer
17. Result
• The normal CD4 T cell count for an adult is just over 1000 cells/µl
• Positive: CD4 T <200 cells/µl OR
CD4 T < 20%
18. ELISA Test
Principle
• Indirect ELISA principle
• The microtiter wells coated with an
antigen react with antibody from
sample
• Upon adding enzyme labelled
secondary antibody and specific
substrates, color given measured by
ELISA reader.
19. Procedure
• Coat the micro titer plate wells with antigen.
• Block all unbound sites
• Add sample containing antibody to the wells
• Incubate the plate at 37°C
• Wash the plate
• Add secondary antibody conjugated to an enzyme
• Wash the plate
• Add substrate
• Record color
20.
21. Results
• Reactive test result: Value of test is equal or greater than Cut off
Value
• Non-reactive test result: Value of test is less than Cut off Value
• Negative control: Non-reactive test result
• Positive control: Reactive test result
22. Reverse transcriptase- PCR
Principle:
• RNA is released from cellular material through extraction
• The extracted sample is added to a reaction mixture which
contains reverse transcriptase enzyme, primers specific for the
target of interest and nucleotides
• If the target is present, primers anneal to the RNA strand
• Reverse transcriptase enzyme synthesizes a complementary
DNA strand, extending from the primer
23. • The temperature is raised to 95o C, and the DNA strands are
denatured
• The temperatures are lowered, allowing primers to anneal to the
newly formed cDNA
• Polymerase enzyme synthesizes a new DNA strand, extending
from the primer
• Multiple cycles geometrically increase the number of copies of
DNA
24. HIV Rapid Test Kit
Principle
• Rapid tests are a simplified
version of antibody ELISA tests.
They look for HIV antibodies in
the blood.
• The antigens for HIV are fixed on
one particular strip along the
rapid test stick.
• Towards the end of the testing
stick are control antigens to
show that the test worked
26. Hepatitis B Tests
1. HBsAg ELISA
2. Polymerase chain reaction (PCR)
3. Rapid kit test
27.
28. HBsAg ELISA
Principle:
• HBsAg ELISA is a solid-phase
enzyme-linked immunosorbent
assay based on the principle of
the antibody sandwich
technique.
29. Procedure
• Coat the micro titer plate wells with antibody
• Add the antigen-containing sample
• Incubate the plate at 37°C
• Wash the plate
• Add the enzyme-linked antibodies specific to also antigen
• Incubate at 37°C
• Wash the plate
• Add substrate
• Color recording at 450nm
30.
31. Polymerase chain reaction (PCR)
• For the detection of HBV DNA, following are the basic steps for PCR;
• DNA extraction from the sample
• Amplification
• Detection
• The first step is the extraction of DNA from the test samples that may be
blood
• The amplification is done on automated thermocycler in repeated cycles
containing;
• Extracted DNA
• Complementary primers
• Nucleotides
• DNA polymerase
• Reaction buffer
32. • The amplification process involves ;
• De-naturation of the double stranded DNA at 94°C
• Annealing of the primers with the complementary DNA sequence at 54-60°C
• Extension of the primer and formation of double stranded DNA molecule from
single stranded at 72°C
• After 25-30 cycles, one molecule of the target DNA can be
amplified to produce over 100 million DNA molecules
• The DNA can then directly be visualised after electrophoresis in
conventional PCR or quantified in real time- PCR
33. HBsAg Rapid Test
Principle:
• Based on flow immunoassay.
• HBsAg from sample forms a complex with anti-HBsAg conjugate dried
onto the test strip.
• The liquid migrates through the nitrocellulose membrane.
• If HBsAg is present, this binds to a second anti-HBsAg antibody
immobilized on the membrane, forming a visible red line.
34. Procedure
• Bring kit and sample to room temperature prior to testing. Once the
device pouch is opened, it must be used within one hour.
• Place 3 - 4 drops (~ 100 μL) of serum or plasma into the sample
window of the device.
• After 10- 15 minutes all the pink color from the conjugate will clear
from the membrane except for test and controls lines that form.
35. Results
• A reactive test shows as two colored lines, one in the test area (P =
Positive), one in the control area (C = Control).
• A non-reactive test shows only one colored line in the control area. If
the control line is not visible, the test is invalid.
36.
37. References
• Dacie and Lewis Practical Haematology, 12th edition, 2017.
• District Laboratory Practice in Tropical Countries, Part 2, 2nd edition, Monica Cheesbrough.
• Human Cytology, flow cytometry by Dr. Mubasher Rauf.
• https://m.youtube.com/watch?v=jB8RlreiN10
• https://www.labce.com/spg605456_reverse_transcriptase_pcr_rt_pcr.aspx#:~:text=RT%2DPCR%2
0is%20one%20of,on%20the%20Taq%20Polymerase%20enzyme.
• Manual of medicine laboratory, 3rd edition, 2005
38. • Kumar M, Khumar R, Aa M, Tn D. Role of CD4 Receptor in Diagnosis of AIDS. J Fam Med.
2017;4(1):4–7.
• China%20Testsealabs%20HIV%201_2%20Rapid%20Test%20Kit
• Indus hospital and health network training manual processing
• https://vawnet.org/sc/transmission-hiv
• preventing transmission of infectious agents in healthcare settings 2007.
http://www.cdc.gov/ncidod/dhqp/gl_isolation.html
• http://www.tulipgroup.com/Common_New/Tech_Pubs_PDF/CHEMILUMINESCENCE.pdf
