Basic Principle of Serodiagnosis Flashcards
Why are infections diagnosed serologically rather than by isolation or antigen detection?
- when difficult to isolate or identify
- when specimen for direct testing is difficult to obtain
- when specimen is called too late
- when antibiotic therapy was started for specimen collection (for bacteria or fungi)
Traditional serological assays detect primarily what?
IgG
4 facts about IgG
- “memory” immunoglobulin
- persists lifelong after most viral infections
- produced in highest quantities during infections
- it is the predominant immunoglobulin (76%)
What do we use when the quantity of the antibody is not important - we just want to know if it is there or not?
Qualitative determinations
What are specific reasons to use qualitative determinations?
- Immune status (herpes, “vaccine viruses”- HIV, hepatitis)
- unusual antibodies: HIV and hepatitis
What do we preform when the quantity of antibody, not simply presence or absence, is important?
Quantitative Determinations
Quantitative determinations are used in determining current or acquired infection requiring comparison of antibody level in an “____” sample collected early in the disease and in a “_____” sample called 2-3 weeks later
- acute
- convalescent
Antibody Profile in Acquired Infection:
- when do you test
- what does large change mean?
- what does no change mean?
This involves testing two samples collected 2 weeks apart (acute and convalescent)
- large increase expected in new infections
- no change if antibodies are from “old” infection and not involved in the current infection
Antibody Profile in Congenital Infection:
- when do you test?
- what does levels staying the same or increasing mean?
- what does levels decreasing mean?
Testing sequential samples, with one at birth an the others during the first 4-6 months of life
- antibody levels stay the same or increase, it means the baby is infected (and is producing own antibodies)
- if antibody levels decrease to very low or undetectable by 4-6 months, the antibodies present at birth were strictly maternal (baby was not infected)
In a serial dilution, a _____ fold or greater difference is a significant difference
4
If you have no reactivity detected in any of your dilutions, how do you report out?
report as “less than” the lowest dilution
!! DO NOT report as NEGATIVE!!!
If you have reactivity detected in all of the dilutions, how do you report it out?
Do not report; dilute further until an endpoint is achieved
Other quantitative comparisons can be made with assays such as EIA’s ect…
Comparing the index values is the most popular approach, but what must the manufacturer do in order for us to compare?
EIA manufacturer must give criteria that define a significant difference
Technical Hints: Heat inactivation: pretreatment of samples required for certain assays.
- What temp and how long?
- What does it destroy?
- 56C waterbath for 30 minutes
- destroys complement, enzyems, and lipoproteins that may interfere with reaction
Technical Hints: Serum quality
- what samples may be unacceptable (3)
Hemolyzed samples
Lipemic samples
Contaminated (bacterial, fungi) samples
Technical Hints:
- For comparing results (Quant Assays), test in _______
parallel
Technical Hints: CSF testing issues
- most serological assays are designed for testing what?
- testing other samples (i.e. CSF) cannot be done unless…
- serum sample should always be tested in _____ with any CSF being tested
- serum
- assay is FDA cleared
- parallel
Specimen Collection/Storage of Serum:
- Collect in what kind of tube
- tube without anticoagulant in it
Specimen Collection/Storage of Serum:
- what do you do after collection
- spin and remove serum
Specimen Collection/Storage of Serum:
- how can you store serum
- refrigerate, freeze (-20C)
What 5 factors can effect immunoserologic assays in vitro?
- Electrolyte dependency
- Time and Temp
- pH
- Reagent Quality
- Immunological events (primary vs. secondary)
Prozone
too much antibody for the amount of antigen (antibody excess)
Zone of Equivalence
equal amounts of antibody and antigen; where you get the highest amount of Ag/Ab complexes
Postzone
not enough antibody for the amount of antigen (antigen excess)
Particles coated with reactants are said to be “___”
sensitized
How many pipettes are used for preparing serial dilution?
How many pipettes are used for transferring (dilute to concentrated?
- single pipette
- single pipette
T or F: we vortex all mixtures?
F: not for all mixtures (RBC’s would hemolize)
4 things you should always remember to do in the serology laboratory
- use controls
- Label EVERYTHING
- Safety
- Confidentiality
