Diagnostic Techniques

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Diagnostic Techniques

• LAT Chapter 10

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To Make A Diagnosis

• Diagnosis responsibility of veterinarian.
• Diagnostic tests may be performed by a
  technician.
• Diagnostic techniques, history, clinical
  examination, and other information considered.
  
• Also used to define or establish health status of
  a clinically normal animal.
• Diagnostic techniques radiography, anatomical
  pathology, necropsy, microscopic examination of
  tissue sections, clinical pathology,
  microbiology, hematology, blood chemistry,
  immunoserology, parasitology and urinalysis

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Anatomical Pathology

• Study of structure of diseased tissues
• Gross pathology performed at necropsy.
• Perform necropsies as soon after death as
  possible.
• Animals that cannot be examined immediately
  should be refrigerated, but not frozen, to slow
  the process of decomposition.
• Proper protective clothing must always be worn
  while conducting a necropsy.
• Good necropsy has consistent, thorough, routine
  method for examining the entire carcass.
• Examine exterior of animal. Open abdomen and each
  internal organ is examined in a specific order.
  Next the thorax is opened, and heart and lungs
  are observed.
• Make a precise record of each tissue collected
  and label each container accurately with the
  types of tissues, date of collection and name of
  investigator.

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Necropsy

• Collected tissues are preserved by
  placing them in a solution of
  10-percent buffered formalin.
• Stops all tissue decomposition and fixes the
  tissue to preserve its anatomical structure.
• Place remains in leak proof bag and barrel.
• If radioactive or infectious, store and dispose
  of separately.
• Mark containers with hazardous material involved.
• Once fixed, slices of tissue are cut and placed
  in trays.
• Next they are treated with a number of chemicals,
  and then embedded in paraffin.
• Slices mounted on slides and stained with dyes.
• Procedure is referred to as histopathology (histo
  tissue).

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Clinical Pathology

• Analysis of blood, urine, feces,
  tissues, and exudates
• Diagnostic microbiology to isolate and identify
  microorganisms, interpret culture results, and
  perform antibiotic sensitivity tests
• Sterile swabs of various types are used for
  collection of cultures from the area that is
  thought to be infected.
• Throat swabs - dont touch lips, tongue, or other
  oral surfaces.
• When culturing wounds or abscesses, obtain sample
  from the edge, or wall, of the lesion.
• Fresh feces for microbiological examination
  should be collected in a clean container.
• If feces not available, a rectal swab is
  acceptable but less ideal.

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Bacterial Culture

• Prevent drying of the sample or swab.
• Ampule at bottom of culture container is crushed
  to release fluid transport medium to keep
  bacteria alive for 24 hrs.
• Sample is inoculated for culture and
  identification.
• Individual colonies are picked and grown as a
  pure culture.
• Tentative ID made based on colony shape and
  staining.
• Definitive ID requires biochemical, serological,
  and various tests.
• Antibiotic sensitivity
• Organisms inoculated over the surface of petri
  dish then several small disks containing various
  antibiotics placed on surface.
• If sensitive to a particular antibiotic, growth
  will be inhibited around that particular
  sensitivity disk.
• Supplies information for prescribing proper
  antibacterial therapy.

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Hematology

• Blood erythrocytes (red blood cells),
  leukocytes (white blood cells), platelets,
  and plasma
• Total blood volume 68 of body weight.
• Blood cell structures and normal ratios provides
  information to evaluate health status.
• Reptiles, amphibians, and birds all have
  nucleated erythrocytes.
• Rabbits and guinea pigs have neutrophils with red
  staining granules in the cytoplasm that make
  these cells appear more like the eosinophils of
  dogs.
• Some guinea pig lymphocytes also contain large,
  red-staining granules called Kurloff bodies.

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Blood Collection

• Red blood cells ruptured (hemolysis)
  during collection by pulling too hard
  during aspiration, or by forcing blood in a
  syringe through a needle.
• Can result in inaccurate values for clinical
  chemistry tests.
• Insert the needle bevel up to ensure smooth entry
  into the vein.
• Syringe plunger pressure just sufficient to pull
  blood in.
• Needle held firmly while attaching vacuum vial to
  prevent the needle from pulling out or being
  pushed through vein.
• Large animals - collect from veins on legs or
  from jugular.
• Mice / rats collect small amount of blood from
  the tail vein, heart or retroorbital.
• Rabbits - collect from the vessels in the ear.

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Blood Collection
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Serum Plasma

• EDTA, sodium citrate or heparin prevent clotting,
  which allows whole blood to be separated into
  plasma and the red cells, white cells and
  platelets.
• Whole blood will separate into clotted and a
  liquid fraction.
• Some tests require whole blood, others require
  serum or plasma.
• Red (orange) stopper indicates no anticoagulant.
• Used to collect blood that is going to be allowed
  to clot.
• Purple (lavender) stopper indicates EDTA.
• Prevents clotting and allow the collection of
  plasma.
• Green stopper indicates heparin.
• Centrifuge spins tube very rapidly and forces the
  cells or clot to bottom, separating liquid from
  cellular fractions.
• The liquid (serum or plasma) can then be removed.

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Blood Diagnostic Tests

• 1.Packed Cell Volume (PCV) hematocrit, is a
  measure of the of cells vs. liquid in the
  sample.
• 2. Differential Leukocyte Count is a count of
  different types of leukocytes in a drop of whole
  blood.
• 3. Total Red and White Cell Counts
• When a diagnostic laboratory receives a request
  to perform a Complete Blood Count (CBC) on a
  blood sample, the lab will do all of the tests
  listed above, plus several more not mentioned
  here.
• Clinical chemistry tests help to determine that
  the kidney, liver and other organs are
  functioning normally.

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Blood Chemistry

• Accuracy of tests depends on method of collecting
  and transporting specimen.
• If whole blood or plasma is required, sample is
  immediately mixed gently with a anticoagulant.
• If serum is required, the sample is allowed to
  clot.
• Proper withdrawal and submission procedures gt
  uniform, representative specimens.
• Tests for renal (kidney) function include
  analysis for blood urea nitrogen (BUN) and
  creatinine.
• Waste products produced during normal body
  metabolism.
• These waste products are eliminated mainly by the
  kidneys, and elevated blood levels of either one
  usually indicates an abnormality in the urinary
  system.

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Blood Chemistry (continued)

• Chemical tests of liver function include
  metabolic tests, excretion tests, and serum
  enzyme tests.
• Some substances formed in the liver are bile
  pigments, albumin, fibrinogen, prothrombin, and
  cholesterol.
• Excretion tests involve intravenous injection of
  dyes.
• At a standard time after injection, blood level
  of dye is measured and excretion rate calculated.
• Enzymes in high concentration within the cells of
  the liver.
• When damaged, enzymes are released into the blood
  stream.
• Serum ion levels important in disease diagnosis
  and postsurgical treatment.
• If uncorrected, severe illness or death may
  result.
• sodium (Na), potassium (K), magnesium (Mg),
  calcium (Ca), phosphorus (HPO4), and chloride (Cl)

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Immunoserology

• Disease agents act as antigens (immune system
  stimulators) gt protein molecules called
  antibodies.
• Antibodies in the serum indicate either an active
  infection, or exposure to disease.
• Serum antibody levels antibody titer.
• Techniques to measure antigen-antibody reactions
• complement fixation, fluorescent antibody
  precipitation, hemagglutination, and ELISA
• Consecutive samples determine if antibody level
  is rising, falling, or remaining constant.
• An indication of the immune competence of the
  host, as well as approximate stage of the disease.

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Immunoserology (continued)

• Viruses differ from bacteria mainly by being
  intracellular and requiring living cells in which
  to grow.
• Solutions containing cells known to support viral
  growth are used.
• Once inoculated cells in the culture become
  infected, damaged or killed in characteristic
  patterns.
• Testing tissues for the presence of DNA from
  specific organisms is a means of identifying a
  disease.
• A procedure called the polymerase chain reaction
  (PCR) has made it possible to detect very small
  numbers of organisms by artificially increasing
  (amplifying) the DNA they contain.

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Parasitology

• Therapy and prevention depend on accurate ID of a
  parasite or parasite ova.
• To detect helminth (worm) infections, fecal
  examination for ova is performed.
• Pinworms (Syphacia spp.), lay their eggs on the
  exterior of animal around the anus.
• Heartworm infestation of dogs and cats is
  diagnosed by microscopic examination of the blood
  for microfilaria (immature larval forms).
• External parasites, especially mites, may be
  identified by microscopic examination of the fur
  or by skin scrapings.

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University of Illinois Department of Entomology
http//www.life.uiuc.edu/entomology
Flea Lifecycle
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Roundworm Ova
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Pinworm Egg Syphacia species
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Pinworm Eggs
In The Uterus of Mature Adult ?
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Male Adult Pinworm
Syphacia mesocriceti
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Grain Beetle
Found in infested rodent feed bags
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Hamster Skin Mite (lateral view)
Demodex aurate.
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Hamster Skin Mite (ventral view)
Demodex aurate.
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Psoregates Mange Mite (ventral view)
Isolated from Non-Human Primate
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Urine Examination

• Physical, chemical tests microscopic exam of
  sediment
• culture and antibiotic sensitivity testing of
  bacteria isolated from urine for diagnosing and
  treating bacterial infections
• pH (acid or alkaline), specific gravity, color,
  and volume
• Chemical tests detect mainly proteins,
  carbohydrates, electrolytes, pigments, and
  hormones.
• Urine sediment examination identifies cells,
  renal tubular casts, and crystals.
• Location of a disease in the urinary tract
  determined through examination of the urinary
  sediment.
• Important collection consideration
• Specimen must be collected in a clean, dry
  container.

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Diagnostic Imaging

• Bone absorbs X-rays, air absorbs very few X-rays.
• The film directly beneath the bone appears white.
• Film placed under the lungs appears dark or
  black.
• Movement will cause blurring of the image and
  make interpretation of the image more difficult.
• Fluoroscope - X-rays of moving objects
• X-rays are ionizing radiation.
• Proper shielding worn by
  personnel at all
  times.
• Computerized Axial Tomography
• Positron Emission Tomography
• Magnetic Resonance Imaging
• Ultrasound scanning

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Summary

• Effective treatment can be initiated sooner if
  diagnostic results can be made quickly available
  to the clinician treating a disease outbreak.
• The diagnostic techniques discussed above are
  equally important in determining the true health
  status of normal-appearing animals, since
  subclinical infections can have devastating
  effects on research results.
• It is through the combined use of these
  techniques, coupled with clinical examination and
  daily observations by laboratory animal
  technicians, that the health status of both
  individual animals and entire colonies can be
  accurately defined.

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Summary (continued)
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Additional Reading

• 1. McCurnin, D.M. Clinical Textbook for
  Veterinary Technicians. W.B. Sanders,
  Philadelphia, PA, 1994.
• 2. Pratt, P.W. Laboratory Procedures for
  Veterinary Technicians. Mosby, St. Louis, MO,
  1996.
• 3. Sharp, P.E. and La Regina, M.C. The Laboratory
  Rat. CRC Press, New York, NY, 1998.