Fundamentals Intro Flashcards
What are all the diagnostic tests you could run?
Radiology, clinical pathology, biopsy, necropsy, histopathology, virology, bacteriology, toxicology, parasitology, serology and molecular diagnostics
How do you choose a diagnostic test?
Think about…
-Type of Sample
-Intended Use
-Is this a screening test or diagnostic
-Performance characteristics of said test
What is the difference between screening and diagnosis (antibody vs antigen)?
Screening - detect early disease or risk factors for disease in apparently healthy subjects (antibody)
Diagnosis - determine presence/absence of specific entity in symptomatic patient
What is sensitivity?
Measure’s ability of a test to correctly identify individuals with a condition (true positive rate)
Tp/ (TP + FP)
SnNout - high sensitivity = Negative test rules out disease
What is specificity?
Evaluates test capacity to accurately ID those without the condition (true negative)
TN / (TN + FP)
SpPIN - high specificity a positive rule in disease
What is positive predictive value?
It is precision, probability that a positive test result accurately indicates the presence of a specific condition or disease
If my test is very sensitive but not very specific what does that mean in relation to the results?
It means that if you get a positive you can assume it is Truely positive. If you get a negative result it could be a false negative.
What is reliability?
does the test give similar results under consistent testing conditions
-Over time
-When interpreted by different observers
What are some diagnostic testing methods?
Direct Exam: Electron microscopy, bacterial culture, antigen detection, molecular diagnostics (DNA/RNA and protien)
Indirect Exam: Virus Isolation
Serology: Antibody detection IgM and IgG
How do you visualize virus, parasites or bacteria?
Light Microscopy
Electron Microscopy
How do you detect proteins? (Antibody-antigen)
Elisa, IFA (immunofluorescence) and IHC(Immunohistochemistry)
How do you detect DNA or RNA?
PCR and NGS
What are some examples of pathogens that can be detected by electron microscopy?
PEDV, Poxvirus, E. Coli
How does Enzyme-linked immunosorbent Assay (ELIZA) work?
Antigen based (Antigen Capture)
-Detects pathogen itself
-Conjugated primary or secondary antibody
-Substrate gives a color reaction
Ex. Felv antigen test
How does Immunofluorescent antibody test (IFA) work?
-Cell culture or tissues
-Antibody has a fluorescent tag and is red with fluorescent microscope
-Need high viral titer
How does immunohistochemistry work?
-paraffin embedded tissues mounted on glass slides
-Sections are incubated with specific antibodies tagged with an enzyme
-Add substrate - if antigen of interest is in the tissues then color reacts
-Allows detection of the antigen in the lesion or specific cells
How can you use immunohistochemistry (IHC) in neoplasia cases?
Antibodies against tumor markers to detect cells of origin, stage differentiation and prognostic indicators
How do molecular diagnostics work?
Qualitative or quantitative measure of:
-Genomic detection DNA/RNA - real time PCR, In situ hybridization, microarray, sequencing
-Proteins - flow cytometry, MALDI-TOF
What components does a Realt time Polymerase Chain reaction (PCR) test need to be successful?
DNA primers and probes specific to target DNA, enough good quailty target DNA or RNA to amplify, validated test method, well-maintained equipment, limiting cross contamination
What are the general steps to PCR?
- Increase Temp - denatures dna
- Annealing- attach primers and probes
3.Polymerization and signal generaration - make new stands DNA, separate
What is the ct value?
Cycle threshold - Cycle number at which the signal passes a fixed threshold
Signal is proportional to the amount of amplified product. The higher the starting copy number of the target DNA in a sample the soon the signal increases in fluorescence. What does that mean in reference to CT value?
Low CT value (High viral load)
What is key to remember about CT value?
Its not transferable, different among tests
How can PCR be used in neoplasia cases?
-ID of clonal lymphocyte population
-Detect chromosonal translocation, deletions and duplications
-Detect c-kit mutation in mast cell tumor
How does in-situ hybridization work?
- In-situ = place of origin
-Allows for visualization of a virus in the tissue or at the site of infection
-Allows for determination of which virus is responsible for pathologic lesion
What is next generation sequencing?
-Whole genome sequencing
-Can use to detect unknown viruses
There are portable sequencing options - MinION
How does virus isolation work?
look for the effects of the virus (cytopathic effect) - see syncytial cells and inclusion bodies
When are these tests most useful?
-ID cause of acute disease
-Determine cell origin of a neoplasm for prognosis or treatment
How do you know you have found a pathogen on serology?
-Detection of antibodies
-4x increase in antibody titers between acute or convalescent stages of infection, detection IgM in primary infection
Which antibody develops 1st? When does IgG get to the scene?
IgM (peak in 1 week then decrease)
IgG increase in 10-14 days
How can you detect antibodies?
Serology, Elisa, Immunodiffusion and Virus Neutralization
What is critical to think about when testing serology?
Think about the timing of your test relative to exposure
IgM without IgG may mean early exposure
So Elisa can detect antigen, but can it detect antibodies too?
Yes!
How does Elisa work?
-antibody based elisa
-dilutions allow for detecting antibody titers
-2nd antibody can detect IgM, IgA or IgG
Ex. Snap - FIV (antibodies) FelV (antigen)
How does immunodiffusion work?
Antigen is loaded at center of gel, test serum is loaded on the periphery, a charge is added and antibodies move toward the antigen and precipitate making a band (positive)
How does virus neutralization work?
-Antibodies in serum to inhibit virus replication
-titer inverse of highest dilution needed to neutralize
-highly specific
-slow or expensive b/c relies on inhibition of virus growth in cell culture
What is the best way to determine what organisms are circulating in a population?
Serology
What can produce antibodies?
Infection, exposure or vaccination
What do antibody levels do after continuous exposure?
Decrease
Why might a test not give you the results you expect?
Technical problem, early infection or recovery, false positive (low prevalence more false positives) , sequestering in tissues
