Diagnostic Tests - Overview + Serology Flashcards
Screening vs. Diagnosis clinical question
Screening: test to ID early disease states or risk factors in apparently healthy/asymptomatic subjects
Diagnosis: test to determine the presence or absence of a specific entitiy in a symptomatic patient
formula for sensitivity
[true +] / [(true +) + (false -)]
proportion of patients w/ dz and + test result
formula for specificity
[true -] / [(true -) + (false +)]
proportion of patients w/out dz and a negative test result
PPV vs. NPV
PPV: proportion of pts w/ (+) test that actually have disease
NPV: proportion of pts w/ (-) test that do NOT have disease
Requires knowledge on incidence & prevalence of disease in a population.
A negative test rules out disease:
SnNout: test w/ high sensitivity (produces very few false negatives)
- prone to false positives
A positive test rules in disease:
SpPIN: test w/ high specificity (produces very few false positives)
- prone to false negatives
What dx methods directly visualize viruses, parasites, and bacteria?
Light or electron microscopy
What dx methods detect antigen surface proteins?
Antibody-Antigen Based Tests
1. Enzyme-Linked Immunoabsorbent Assay (ELISA-Sandwich)
2. Immunofluorescence (IFA)
3. Immunohistochemistry (IHC)
detect ANTIGENS b/c of their surface proteins
What dx methods (3) detect DNA or RNA genomes?
- PCR & RT-PCR
- NGS
- In-situ hybridization
How does ELISA work?
Antigen immobilized in well -> detection antibody w/ enzyme attached, forming Ag-Ab complex -> enzyme-specific substrate generates signal => color reaction
Capture-Antigen
How do Direct IFA tests work?
Cells with high viral load fixed on slide -> antibody with fluorescent tag applied and links with antigen (conjugated) -> fluorescence microscope used to visualize complex
How does IHC work?
tissues fixed to slide -> antibody with enzyme applied -> enzyme-specific substrate generates color reaction if antigen present in the lesion or specific cells
How can IHC be used in neoplasia dx?
Tumor markers can have antibodies -> IHC detects antigens revealing info on cell of origin, proliferation stage, and prognostic indicators
In RT-PCR, how do you know if the sample has a low viral load?
A high Cycle Threshold (Ct)
More amplification cycles required to detect DNA/RNA
How can PCR be used to dx neoplasia?
ID clonal lymphocyte populations; detect chromosomal translocations, deletions, duplications; detect mutations in MCTs
What is NGS used for?
Unknown viruses are detected by sequencing the whole genome
What is in-situ hybridization used for?
To visualize pathologic virus in the tissue or at the site of infection
What is an indirect way to detect presence of a virus that you can’t do with other pathogens?
ID Cytopathic effect in cell culture
Cytopathic effect refers to structural changes in host cells that are caused by viral invasion. The infecting virus causes lysis of the host cell or when the cell dies without lysis due to an inability to replicate.
When are tests detecting organisms vs detecting antibodies most useful?
Detecting…
- Organisms: ID cause of acute disease, or determine cell of origin of a neoplasm for prognosis of tx
- Antibodies: ID organisms circulating within a population
Methods to detect antibodies using serology (7)
- Detect host response (IgM and IgG)
- ELISA (Direct and Indirect)
- Agar Gel Immunodiffusion (AGID)
- Virus Neutralization
- IFA
- IHC
- Agglutination
What is detected in the FIV/FeLV Snap Test?
- FIV Ab
- FeLV Ag
- (+) and (-) Controls
FIV antigen can go into latency -> preference is detecting antigen over antibody to determine clinical infection (Snap test only detects FeLV antigen).
SNAP tests = type of ELISA
How does AGID work?
Center well = antigen, test serum loaded in periphery -> a (+) charge is added to the gel and Abs move toward Ag, precipitating a band
How does Virus Neutralization work?
Used to determine viral antibody titer:
- serum from infected patient is cultured & loaded in wells -> diluted w/ Ab until virus neutralized
- highest dilution of serum that prevents cytopathic effect = Ab titer reported (e.g., canine distemper, BVDV)
Molecular diagnostics are direct detectors of what?
DNA or RNA genomes
What are 3 scenarios that can produce antibodies?
- Infection
- Exposure but no clinical disease
- Vaccination
Serological diagnostics are indirect detectors of what?
IgG or IgM antibodies (and antigens)
Purpose of PCR
to synthesize and amplify specific target genome sequence to determine pathogen load
IgG and IgM
IgG = most abundant; smallest; sticks to/neutralizes antigens
IgM = appears 1st in circulation; largest; sticks to/neutralizes antigens
IgG is the only Ab that can penetrate placenta & blood vessels
When are IgG levels highest?
During secondary response/after secondary antigen exposure (e.g., after booster shot)
Serostatus vs Serocoversion
Serostatus: specifies whether sample contains Ab/Ag (sero+) or no Ab (sero-)
Seroconversion: indicates a change from sero- to sero+ (or 4-fold increase in titer b/w acute & a convalescent sample taken 2-4 weeks later)
Sero- does NOT mean patient is NOT infected!! Only that no Ab present
Can ELISA tests differentiate b/w Ab produced from infection vs. vaccination?
No (i.e., FIV)
Direct vs. Indirect IFA
Direct: detects antigen; poor sensitivity
- Giardia & Cryptosporidium (feces)
- Rabies (brain tissue)
Indirect: tests antibody and helps establish serum antibody titer; more sensitive
- Anaplasma & Ehrlichia
How does IHC differ from IFA?
IHC: antibody is conjugated with horseradish peroxidase; visualized with light microscope
IFA: antibody is conjugated with a fluorescent tag; visualized with fluorescent microscope
How does Agglutination work?
- Detects Ab or Ag in serum
- relies on Ag-Ab complex bivalence property to produce assessable clumps
- protocol = series of dilutions of pt’s blood serum mixed w/ live organisms of pathogen -> highest titer = where ≥ 50% of Ag-Ab still cross-linked
- brucellosis, lepto, crypto, blood typing
How are antibody titers interpreted?
Highest dilution of Ab that will detectably interact w/ Ag
- aka lowest [Ab]
1 part serum, 160 parts saline -> it would take 160 parts of viral load for there to be no detectable antibodies left (aka, animal would need to be severely, severely infected to be clinically affected).
