Referral tests Flashcards
What is a direct coombs test
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
What is an indirect coombs test
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
Immunodiffusions is done how
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
What is the Fluorescent antibody testing
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
Direct fluorescent antibody testing is
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
Indirect fluorescent antibody test is
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
immunohistochemistry is and used for
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)
Serum antibody titer are and are testing for
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
Molecular diagnostic tests are wht types of test
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
What are the benefits of a molecular diagnostic test
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
What are the disadvatanges of a molecular diagnostic test
Cost, equipment required, risk of contamination, and requires specific technical skills
Polymerase chain reaction (PCR) is
Common molecular diagnostic test
Takes a small amount of DNA and amplifies it until it can be detected
What are the advantages of a PCR
Very specific
Tiny sample requirement (a few cells)
What are the disadvantages of a PCR
Easily contaminated
The DNA sequence of the target gene must be known
What are the steps of a PCR
Denaturation
Annealing
Extension
Amplification
Visualization
What happens during the denaturation of a PCR test
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
What is the annealing part of a PCR
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
What is the extension/elongation of a PCR test
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
What is the amplification part of the PCR test
Steps 1-3 are repeated 25-30 times until there is enough DNA to work with
What is the visualization part of the PCR test
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
What are the types of modified PCR test
Reverse transcriptase polymerase chain reaction
Real time PCR
What does a Reverse transcriptase polymerase chain reaction identify
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
What is a realtime PCR test
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
