Diagnostic Techniques

1. Diagnostic Techniques LAT Chapter 10

2. LAT Presentations Study Tips • If viewing this in PowerPoint, use the icon to run the show. • Mac users go to “Slide Show > View Show” in menu bar • Click on the Audio icon: when it appears on the left of the slide to hear the narration. • From “File > Print” in the menu bar, choose “notes pages”, “slides 3 per page” or “outline view” for taking notes as you listen and watch the presentation. • Start your own notebook with a 3 ring binder, for later study!

3. 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

4. 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.

5. 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).

6. 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 - don’t 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.

7. 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.

8. Hematology • Blood = erythrocytes (red blood cells), leukocytes (white blood cells), platelets, and plasma • Total blood volume = ~ 6–8% 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.

9. 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.

10. Blood Collection

11. 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.

12. 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.

14. 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 => 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.

16. 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)

18. Immunoserology • Disease agents act as antigens (immune system stimulators) => 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.

20. 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.

21. 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.

22. University of Illinois Department of Entomology: http://www.life.uiuc.edu/entomology Flea Lifecycle

23. Roundworm Ova

24. Pinworm Egg Syphacia species

25. Pinworm Eggs In The Uterus of Mature Adult 

26. Male Adult Pinworm Syphacia mesocriceti

27. “Grain Beetle” Found in infested rodent feed bags

28. Hamster Skin Mite(lateral view) Demodex aurate.

29. Hamster Skin Mite(ventral view) Demodex aurate.

30. Psoregates Mange Mite(ventral view) Isolated from Non-Human Primate

31. 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.

32. 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

33. 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.

34. Summary (continued)

35. 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.