topic 5: Diagnostic Techniques Flashcards
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
direct ELISA Steps
- COATING: buffered solution of ag is added to each well of a microtitre plate
- incubate for 1h at 37°C or overnight at 4°C to allow the ag to adhere to the plastic through charge interactions (immobilises them to a solid surface)
- BLOCKING: a solution containing non-reactive proteins (Bovine Serum Albumin) is added to each well to cover any uncoated plastic surface in the well
- wash with a suitable buffer solution
- primary ab with an a conjugated (attached) enzyme (amplifier) is added
- incubate for 2h to allow the primary ab to bind to the ag that has coated the well
- wash with a suitable buffer solution
- DETECTION: add a SUBSTRATE for the enzyme. usually HRP (Horse Radish Peroxidase).
- the substrate chosen should change colours upon reaction with enzymes
- if ab is binded to the ag, the enzyme should react with the substrate and change colours
- FINAL READ: high concentration of ag = stronger colour change (measure with spectrometer)
indirect ELISA Steps
- similar to direct but with an additional step of adding a secondary enzyme-linked ab that is complementary to the primary detection ab
- COATING: buffered solution of ag is added to each well of a microtitre plate
- incubate for 1h at 37°C or overnight at 4°C to allow the ag to adhere to the plastic through charge interactions (immobilises them to a solid surface)
- BLOCKING: a solution containing non-reactive proteins (Bovine Serum Albumin) is added to each well to cover any uncoated plastic surface in the well
- wash with a suitable buffer solution
- primary ab is added (WITH NO CONJUGATED ENZYME)
- incubate for 2h to allow the primary ab to bind to the ag that has coated the well
- wash with a suitable buffer solution to remove any excess
- EXTRA STEP DIFFERENT FROM DIRECT ELISA: ENZYME-CONJUGATED SECONDARY AB IS ADDED & incubated for it to bind to the primary ab
- wash with a suitable buffer solution to remove excess
- DETECTION: add a SUBSTRATE for the enzyme. usually HRP (Horse Radish Peroxidase).
- the substrate chosen should change colours upon reaction with enzymes
- if ab is binded to the ag, the enzyme should react with the substrate and change colours
- FINAL READ: high concentration of ag = stronger colour change (measure with spectrometer)
direct VS indirect ELISA
- indirect has higher sensitivity
- indirect is more flexible & less expensive as there are more possible primary ab that can be used
- indirect has a higher risk of cross-reactivity between secondary detection antibodies (direct does not use any secondary antibodies)
- direct is faster (less steps & less incubation time)
sandwich ELISA
- requires 2 antibodies specific for different epitopes of ag (matched antibody pairs must be highly specific)
- 1 of the antibodies is capture ab (facilitates immobilisation of the ag)
- the other antibody is secondary ab (conjugated to an enzyme, facilitates the detection of the antigen)
- suitable for complex samples
- high flexibility: can use both indirect and direct methods
- demanding design: it is difficult to find 2 abs that work well together against the same target while recognising different epitopes
sandwich ELISA Steps
- capture ab is used to coat the well of the plate
- wash off excess capture ab with appropriate rinsing buffer
- Ag from the sample is added to the plate
- wash off excess Ag with appropriate rinsing buffer
- Enzyme-conjugated secondary ab is added and washed with suitable buffer to remove excess
- Incubate
- Substrate for the enzyme (usually HRP: Horse Radish Peroxidase) is added
- the substrate changes colour upon reaction with the enzyme
- observe for colour change
competitive/inhibition ELISA
- measures ag concentration by detecting signal interference
- low specificity (cannot use for diluted samples)
- sample ag competes with reference ag to bind to a specific amount of labelled ab (or vice versa)
- NEGATIVE = colour change (enzyme-conjugated ab binds to reference ag and not sample ag)
- POSITIVE = no colour change (presence of sample ag in test serum)
competitive/inhibition ELISA Steps
- Sample ag is incubated with labelled ag
- Reference ag is pre-coated onto the wells
- wash off excess with appropriate rinsing buffer
- Sample ag & labelled ab complex is added to the coated wells
- Incubate
- wash off excess with appropriate rinsing buffer
- substrate for enzymes (usually HRP: Horse Radish Peroxidase) is added.
- substrate changes colour upon reaction with enzymes
RODAC plate (monitoring disinfection/sanitation effectiveness)
- Replicate Organism Direct Agar Contact plates
- Touch the plates to the surfaces to be inspected
- areas to be inspected should be clean, dry & disinfected before touching the RODAC plate
- For irregular surfaces: sterile swab (with sterile water) is used to swab before rubbing on the RODAC plate
- Send for incubation and observe it after 24-48 hours.
- None = excellent!
- Slight = acceptable!
- Moderate = poor
- Heavy = not acceptable.
- used to measure cleanliness of the lab animal facility
Bioluminescence detector (monitoring disinfection/sanitation effectiveness)
- uses ATP detection as a rapid assessment of sanitation
- uses bioluminescence to detect ATP from any organic matter (microorganisms) that may be on the surface
- If there are microorganisms = lights up = sanitation was not effective
- Makes use of Firefly enzymes (luciferin-luciferase) which gives off light in the presence of ATP
- More intense glow = more microorganisms
- used to measure cleanliness of the lab animal facility
Necropsy & sending of samples
- When an animal dies, the cause of death is determined
- find out of the animal was normal during the experiment/colony health surveillance by checking case history
- Store in fridge not freezer
- Consistent, thorough, systemic approach (from external surfaces to the different body parts)
- Lesions, abnormalities etc. before making incisions and collecting specimens of organs
- Specimens are collected, placed in formalin and sent for histopathology
Imaging equipment (radiography & radiation)
- X-ray, fluoroscopy
- computed tomography CT
- ultrasound
- magnetic resonance imaging (MRI)
- positron emission therapy (PET)
Lab rodents that harbour diseases impact…
- research results
- animal health
We should purchase _____ animals from commercial vendors with ______________.
- disease-free animals
- strict health surveillance
- strict quarantine
- testing
conduct ______ within the lab animal facility periodically.
- colony surveillance
Diseases can be detected through ____ or through ____________. Some animals may not show clinical signs.
- PCR
- health surveillance serology tests
Mouse hepatitis virus (MHV): Causative agent & transmission
- Causative agent: Enveloped RNA coronavirus (digestive tract, suppresses immune system)
- Transmission: faecal-oral, direct contact, aerosol, fomite.
Mouse hepatitis virus (MHV): Clinical signs
- Diarrhoea
- weight loss
- low fertility
- focal hepatic necrosis
- Potential death in infant mice
Mouse hepatitis virus (MHV): Prevention
- cease breeding for 2-4 months
- rederivation of animals via caesarean/embryo transfer
- regular health surveillance testing of sentinel animals
Rederivation of animals via caesarean/embryo transfer
removing embryos from sick mothers and transferring them to a healthy one to be carried to full term
Mouse hepatitis virus (MHV): Diagnosis and treatment
- No treatment
- diagnosed via serology and clinical signs
Sialodacryoadenitis virus (SDAV): Causative agents & transmission
- Causative agent: Enveloped RNA coronavirus
- Transmission: direct contact, aerosol
- Animals that are infected are unfit to be used for experiments
- Contagious
- related to MHV