17. Referral Tests

Question 1

What is a direct coombs test?

Answer 1

detects the presence of Ab’s that are BOUND to the surface of the body’s RBCs
used to diagnose IMHA
a pos = visible agglutination of RBCs
canine bench top test kits are available, usually performed in ref lab

Question 2

What is an indirect coombs test?

Answer 2

detects FREE abs in patients serum
a serum sample is taken from patient and incubated w/ RBCs from a normal animal of the same species
if autoab’s are present, will bind to RBCs
species specific anti-gamma globulin is added to the sample causing agglutination of the RBCs

Question 3

What is immunodiffusion?

Answer 3

patient serum is placed in a well in agar and a known Ag is present in a neighbouring well
a pos = band of precipitation when the patients abs combine with test ag

Question 4

What is the fluorescent antibody testing?

Answer 4

performed in ref lab
a fluoroscent dye tag is used to indicate a pos result
sample is visualized under fluorescent microscope
very specific
can be used to detect specific antibodies or antigens in a sample

Question 5

What is direct fluorescent antibody testing?

Answer 5

Ag is tagged w/ fluorescent dye
labelled Ab is added to test slide containing sample
if patient has Ag, the labelled ab will bind to Ag and will be visible using fluorescent microscopy

Question 6

What is indirect fluorescent ab testing?

Answer 6

patients sample is added to a slide containing a specific ag
if sample has abs to the ag, ag-ab complexes are formed
fluorescent labelled antiab’s are added and bind to any patient ab’s that are present

Question 7

What is histology? what about histopathology?

Answer 7

histology - science of examining tissues
histopathology - ^^ to obtain a dx

Question 8

What is immunohistochemistry?

Answer 8

uses direct and indirect fluorescent Ab methods to identify certain ags on surface of cells
Ags may ID type of cell
Ags may belong to viruses that have infected the cell (ex. distemper)
Ags may indicate certain diseases (ex. cancer proteins)

Question 9

What are serum antibody titers?

Answer 9

measurement of ab lvls in serum
can be total ab in the serum OR abs specific to a certain ag (ex. rabies or EIA titers)
quantitative
most commonly used to determine if there has been prior exposure to a disease, or if a booster vaccination is required

Question 10

What happens with serum antibody titers once the sample is collected?

Answer 10

serial dilutions of serum are made
each dilution is then examined for the presence of the ab
each dilution is then examined for the presence of the antibody
a high titer indicates an active infection or immunity
a low titer indicates a previous exposure or not enough ab’s to prevent disease
a very low titer can indicate no prior immunity

Question 11

What are molecular diagnostic tests

Answer 11

analyze DNA ro RNA
includes: testing for bact, viral or fungal pathogens
tests for genetic defects
genetic tests for pedigree
tests for DNA and proteins associated w/ certain cancers and other disease
Very complex, only performed in ref labs

Question 12

What are the benefits of molecular diagnostic tests?

Answer 12

Increased sensitivity and specifity
req a very small amount of sample
fast turnaround time (hours as opposed to days w/ bact cultures
less interference from enviro variables
disadvantages - cost, equipment required, risk of contamination, and requires specific technical skills

Question 13

What is the polymerase chain reaction (PCR)

Answer 13

common molecular dx test
takes a sm amount of DNA and amplifies it until it can be detected
advantages - very specific, tiny sample required (a few cells)
disadvantages - easily contaminated, DNA sequence of target gene must be known

Question 14

What are the 5 steps of a PCR?

Answer 14

1. denaturation
2. annealing
3. extension
4. amplification
5. visualization

Question 15

What is denaturation?

Answer 15

sample containing DNA is heated to separate the double-stranded DNA molecule into 2 strands
each strand becomes a template to form new strands of dna

Question 16

What is annealing

Answer 16

sample is cooled and primers are added
primers are short pieces of DNA that bind (anneal) to the beginning and end of the sections of DNA that are to be copied

Question 17

What is extension/elongation

Answer 17

temp is raised
DNA polymerase and free nucleotides are added to sample
this enzyme reads the original strand of DNA and builds onto the primer piece to create a new double-stranded piece of DNA
now have two complete DNA molecules

Question 18

What is amplification?

Answer 18

steps 1-3 are repeated 25-30 times until there is enough DNA strand to work with

Question 19

What is visualization?

Answer 19

Visualize the DNA that has been amplified
there are many rapid and automated processes for reading the DNA sequence and determining how much DNA was present in the original sample
Sample may be cut into small pieces and run thru an agarose gel

Question 20

What is the modified PCR tests?

Answer 20

reverse transcriptase polymerase chain reaction - identifies RNA instead of DNA in a sample, RNA needs to be converted to double strange DNA before PCR can be performed, RNA samples degrade very easily - storage and handling are critical to ensure test is accurate
REAL TIME PCR - faster, more easily automated, easier to run, less contam risk, fluorescent probe added to sample mix, probe attaches to the DNA segments if they are present, if sample has enough fluorescence the sample is considered positive