Referral tests

Question 1

What is a direct coombs test

Answer 1

Detects the presence of antibodies that are bound to the surface of the body’s RBCs
Used to diagnose immune-mediated hemolytic anemia
A positive test result results in visible agglutination of the RBCs
Canine bench top test kits are available, though usually performed in a reference lab

Question 2

What is an indirect coombs test

Answer 2

Detects free autoantibodies in the patient’s serum
A serum sample is taken from the patient and incubated with RBCs from a normal animal of the same species
If autoantibodies are present, they will bind to the RBCs
Species specific anti-gamma globulin is added to the sample causing agglutination of the RBCs

Question 3

Immunodiffusions is done how

Answer 3

An older technique that is sometimes still used
Patient serum is placed in a well in agar and a known antigen is present in a neighbouring well
A positive test results in a band of precipitation when the patient’s antibodies combine with the test antigen

Question 4

What is the Fluorescent antibody testing

Answer 4

Performed in referral labs
A fluorescent dye tag is used to indicate a positive result
The sample is visualized under a fluorescent microscope
Very specific
Can be used to detect specific antibodies or antigens in a sample

Question 5

Direct fluorescent antibody testing is

Answer 5

An antibody is tagged with a fluorescent dye
The labelled antibody is added to the test slide containing the patient sample
If the patient sample has the antigen, the labelled antibody will bind to the antigen and will be visible using fluorescent microscopy

Question 6

Indirect fluorescent antibody test is

Answer 6

The patient’s sample is added to a slide containing a specific antigen
If the sample has antibodies to the antigen, antigen-antibody complexes are formed
Fluorescent labelled antibodies are added and bind to any patient antibodies that are present

Question 7

immunohistochemistry is and used for

Answer 7

Histology is the science of examining tissues
Histopathology is the science of examining tissues to obtain a diagnosis
Immunohistochemistry uses direct and indirect fluorescent antibody methods to identify certain antigens on the surface of cells
Antigens may identify the type of cell
Antigens may belong to viruses that have infected the cell (ex. distemper)
Antigens may indicate certain diseases (ex. Cancer proteins)

Question 8

Serum antibody titer are and are testing for

Answer 8

Measurement of antibody levels in serum
Can be total antibodies in the serum OR antibodies specific to a certain antigen (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
A serum sample is collected
Serial dilutions of the serum are made
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 antibodies to prevent disease
A very low titer can indicate no prior immunity

Question 9

Molecular diagnostic tests are wht types of test

Answer 9

Tests that analyze DNA or RNA
Types of tests include
Testing for bacterial, viral or fungal pathogens
Tests for genetic defects
Genetic tests for pedigree
Tests for DNA and proteins associated with certain cancers and other diseases
Very complex, only performed in reference labs

Question 10

What are the benefits of a molecular diagnostic test

Answer 10

Increased sensitivity
Increased specificity
Requires a very small amount of sample
Fast turnaround time (hours as opposed to days with bacterial cultures)
Less interference from environmental variables

Question 11

What are the disadvatanges of a molecular diagnostic test

Answer 11

Cost, equipment required, risk of contamination, and requires specific technical skills

Question 12

Polymerase chain reaction (PCR) is

Answer 12

Common molecular diagnostic test
Takes a small amount of DNA and amplifies it until it can be detected

Question 13

What are the advantages of a PCR

Answer 13

Very specific
Tiny sample requirement (a few cells)

Question 14

What are the disadvantages of a PCR

Answer 14

Easily contaminated
The DNA sequence of the target gene must be known

Question 15

What are the steps of a PCR

Answer 15

Denaturation
Annealing
Extension
Amplification
Visualization

Question 16

What happens during the denaturation of a PCR test

Answer 16

The sample containing DNA is heated to separate the double- stranded DNA molecule into two strands
Each strand becomes a template to form new strands of DNA

Question 17

What is the annealing part of a PCR

Answer 17

The 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 18

What is the extension/elongation of a PCR test

Answer 18

The temperature is raised
DNA polymerase and free nucleotides are added to the 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 19

What is the amplification part of the PCR test

Answer 19

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

Question 20

What is the visualization part of the PCR test

Answer 20

The last step of the PCR process is to 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
The sample may be cut into small pieces and run through an agarose gel

Question 21

What are the types of modified PCR test

Answer 21

Reverse transcriptase polymerase chain reaction
Real time PCR

Question 22

What does a Reverse transcriptase polymerase chain reaction identify

Answer 22

Identifies RNA instead of DNA in a sample
The RNA needs to be converted to double stranded DNA before the PCR can performed
RNA samples degrade very easily–storage and handling are critical to ensure test is accurate

Question 23

What is a realtime PCR test

Answer 23

Faster, more easily automated, easier to run, less contamination risk
Fluorescent probe is added to the sample mix
The probe attaches to the DNA segments if they are present
If the sample has enough fluorescence, the sample is considered positive