topic 5: Diagnostic Techniques Flashcards
(114 cards)
list of diagnostic techniques
- blood and haematology
- blood serum biochemistry
- urine samples/urinalysis
- parasite detection
- culture samples/biological samples on agar plates
- cytology: evaluating changes in blood cell appearance
- ELISA
- monitoring disinfection/sanitation effectiveness (RODAC plate VS bioluminescence detection)
- necropsy + sending out of samples
- radiography/radiation
blood and haematology (plasma/serum)
- whole blood (EDTA tube - CBC)
- plasma: liquid portion of unclotted blood (Lit. Hep tube) > mix blood with anticoagulant gently to avoid damaging cells. centrifuge and collect the plasma
- serum: liquid portion of clotted blood (normal, plain tube) > antibodies/blood biochemistry. leave blood to clot with no anticoagulants. centrifuge.
- serum can identify lipedema
- serum may be jaundiced (icteric in appearance) if liver issues are present
handling blood for haematology
- after blood has been drawn, remove the needle before transferring the blood into the tube to minimise the lysis of blood cells
- tilt blood tubes when transferring the blood to drip the blood into the tube slowly
- use the correct size of needles (27G for rats and mice & 23G for dogs and cats)
- use proper syringes (3mL)
- proper blood tubes (EDTA? Lit. Hep? Plain? each tube has a different purpose based on what you want to collect & the test to be done)
- volume of blood (total volume is 6% of animal’s body weight. only 1% can be taken for a one time sampling. if repeated samplings must be done, limit it to 0.1%)
what is haemolysis? what causes it?
- rupture of RBC
- due to improper collection, wrong needle size, incorrect mixing or wrong tubes used
complete blood count (CBC)
- whole blood in EDTA tube
- RBC count: platelets, haematocrit, haemoglobin, packed cell volume (PCV) & RBC indices
- WBC count: neutrophils, lymphocytes, eosinophils, monocytes & basophils
diagnostic machines require __ maintenance before samples can be run
- DAILY!
- boot up the machine every morning before samples are to be run
blood serum biochemistry
- serum (Plain tube)
- studies the changes of biochemistry in the blood
- enzymes (e.g. alanine transferase)
- metabolic waste (BUN/blood urea nitrogen - indicator of kidneys)
- electrolytes (sodium & free ions)
- blood serum biochemistry is broad. Once an issue has been identified, more specific tests can be used to determine/confirm the severity
urine samples/urinalysis
- collected by manual expression cystocentesis (under ultrasound/catheterisation)
- expression is recommended as free catch can be difficult
- metabolic cages can be used for rodents (type of device that catches urine/faeces)
- fresh samples are required!
- check physical properties of the urine!
- dipstick to compare colour coding
- refractometer measurement (urine specific gravity/USG)
- prepare urine for centrifugation
- after centrifugation, prepare urine for microscopic evaluation (check for crystals)
- urinary sediment (sign of urinary tract disorder)
what does USG indicate?
- kidney function
- hydration
physical properties of urine
- colour
- clarity
- odour
- volume
- presence of foam
- USG
what does foam in urine indicate?
- protein in the urine (proteinuria)
parasite detection
- ectoparasites & endoparasites
- mites, pinworms, protozoa, lice, fleas, roundworms, tape worms, etc.
- skin scrape
- cellophane tape test
- gross microscopy/wet mount/direct smear
- faecal flotation test/faecal sedimentation
faecal flotation test STEPS
- place a teaspoon of faecal sample into the WHITE outer Fecalyzer container
- add Fecasol Solution to the halfway mark on the container
- stir the faecal sample and mix well with the Fecasol Solution using the GREEN INNER SHEATH
- after mixing, top up the Fecasol Solution to the top, forming a meniscus
- place a cover slip at the opening of the Fecalyzer container without touching the Fecasol Solution with your hands
- after 20 MINUTES, lift the cover slip DIRECTLY UPWARDS and place it (wet side down) on a microscope slide
- examine the slide at 100X magnification (check for presence of helminth eggs) and 400X magnification (for identification of helminth eggs)
how does faecal flotation work?
- it determines if eggs and larvae of metazoan parasites were present in the faecal sample
- helminth eggs will float to the surface of the Fecasol due to lower density!!
culture samples/biological samples on agar plates
- growth of swabs/plate streaking
- gram positive/negative
- detects microorganisms
- antibiotics resistance testing
- sensitivity testing
- fungus/virus medium
cytology
- vaginal cytology
- aspiration of lumps/pustules
- skin scraping
ELISA (enzyme linked immunosorbent assay): what does it do? what are ANTIGENS and ANTIBODIES?
- detects and counts certain antibodies, antigens, proteins & hormones in a sample (may be blood/plasma/urine/saliva/CSF)
- antibodies = substances made by the body that binds to unwanted substances in order to remove them
- antigens = markers that antibodies recognise (usually sugars/proteins found on cell surfaces of viruses)
ELISA data
- qualitative: Positive or Negative. results are compared to a blank well containing no ag or an unrelated ag as a control.
- false positive: shows substance is present when it is absent in reality
- false negative: shows substance is absent when it is present in reality
- quantitative: results are interpreted in comparison to a standard curve (dilution of a known, purified ag) to PRECISELY calculate the concentration of ag in a sample
- semi-quantitative: compares relative levels of ag in assay samples. signals vary according to the concentration
4 main steps of ELISA
- coating with ag/ab
- blocking (usually with BSA)
- detection (by adding a substrate like HRP (blue) or AP (yellow) that produces a colour)
- final read
- wash between each step
4 types of ELISA
- direct (ag-coated plate to screen for ab)
- indirect (ag-coated plate to screen for ab/ag)
- sandwich (ab-coated plate to screen for ag)
- competitive/inhibition (screening for ab. the other types can also be adapted into competitive formats)
potential interfering factors of ELISA
- PLATE ASSAY (shape/quality of the wells, material of the plates, uneven coating)
- BUFFER (pH, any contamination)
- ANTIBODY USED for detection (incubation time, temperature, specificity, titre (concentration/strength), affinity)
- BLOCKING BUFFER (cross-reactivity, concentration, contamination)
- TARGET AG (conformation, stability, epitopes (AAs))
- ENZYME CONJUGATE (type, concentration, function, cross-reactivity)
- WASHES (contamination, frequency, volume, duration, composition)
- SUBSTRATE (quality/manufacturer)
- DETECTION (instrument-dependent factors
- READER/HUMAN ERROR
advantages of ELISA
- high sensitive and specificity
- high throughput (can process a lot of samples at once, commercially available in 96 & 394 well plates)
- easy to perform (easy to follow, minimal hands-on time)
- quantitative (can determine the conc of ag in a sample)
- able to test a variety of samples (serum, plasma, cellular, tissue extract, urine, saliva, etc.)
disadvantages of ELISA
- temporary readouts (detection is based on enzyme/substrate reactions so readouts must be obtained within a short time span)
- limited information about the ag (results obtained are limited to the amount or presence of ag in the sample)
epitopes
- group of AA/other chemical group exposed on the surface of a molecule
- can generate antigenic responses
- binds antibodies