Transcript Laboratory Techniques

Laboratory
Techniques
Small Animal Technology
Laboratory Tests
 Used to diagnose & treat health problems
 Tests are performed in:
 Veterinary hospitals
 Zoos
 Research facilities
 Commercial labs
Who performs lab work?
 Vet. technician is typically responsible for collecting
samples & performing the tests.
 Vet. tech must know:
 Proper collection techniques
 Specimen handling
 Knowledge of complex equipment (using & maintaining)
 Accurately perform the tests (vet will decide treatment based
on results)
Different Types of Tests
 Hematology
 Urinalysis
 Susceptibility testing
 Circulatory System
 Excretory System
Circulatory System Functions
 Consists of:
 Heart
 Blood vessels
 Lymphatics
Circulatory System Functions
 Respiratory- O2 & CO2 exchange
 Excretory-removes waste from body cells
 Protection-clotting, & transporting white blood cells to
infections
 Nutrition-carries energy & food throughout the body
 Regulatory-helps to maintain pH & temperature
 Hormonal-transfers hormones to organs
Circulatory System Components
 Heart-muscular, four-chambered pump that drives the
circulatory system
 Pericardium-fibrous sac that encloses the heart
 Artery-an elastic vessel with thick walls to maintain high
pressure while carrying blood away from the heart
 Vein-a thin-walled vessel that carries deoxygenated blood
to the heart
Circulatory System Components
 Capillary-a microscopic vessel that forms a network
between arteries, veins, & body tissues
 Lymph system-consists of lymphatic vessels & tissues
(tonsils, thymus, spleen, lymph nodes) that play an
important part in immunity & disease prevention
 Lymph node-bean-shaped structures located throughout
the body that produce lymphocytes & monocytes, & filters
bacteria, foreign bodies, & malignant cells
Circulatory System Components
 Spleen-largest lymph organ, produces lymphocytes &
monocytes, stores red blood cells & iron, & destroys old
red blood cells
Blood Circulation
Through the Heart
 Important when evaluating a sick animal
 Problems in the right side of the heart will cause the
abdomen to fill with fluid (ascites).
 Problems in the left side of the heart will cause lung
congestion.
Blood Circulation
Through the Heart
 Blood flows through the heart in this order:
 Right atrium>right ventricle>pulmonary
arteries>lungs>pulmonary veins>left atrium>left
ventricle>aorta
Major Arteries & Veins
 Aorta-the largest artery that sends blood from the heart to
the body
 Brachiocephalic-branches from the aorta to send blood to
the head & right side of the body
 Common carotid arteries-(left & right) run up both side of
the neck & supply blood to the head
 Coronary arteries-wraps around the exterior of the heart &
supply blood to the heart muscle
Major Arteries & Veins
 Facial artery-wraps under lower jaw & is used to take the
pulse on a horse or cow
 Femoral artery-runs down the inside hind leg; used to take
the pulse on a cat or dog
 Mesenteric-supplies blood to the intestines
 Renal artery-supplies blood to the kidney
 Pulmonary arteries-carry deoxygenated blood to the lungs
Major Arteries & Veins
 Pulmonary veins-carry oxygenated blood to the left
atrium
 Cranial vena cava-returns blood to the heart from the
head, neck, & forelegs
 Caudal vena cava-returns blood to the heart from the
thorax, abdomen, & hind legs
 Cephalic vein-runs along the front of the foreleg
Major Arteries & Veins
 Jugular vein-runs down the neck & returns blood from the
head
 Renal vein-returns blood from the kidney to the caudal
vena cava
 Femoral vein-runs along the inside of the hind leg
 Saphenous vein-an extension of the femoral vein
Major Arteries & Veins
 Arteries generally are located deeper in the body than
veins.
The Structure of Blood
 Blood is composed of cells & plasma (the liquid portion of
blood).
 Cells make up 40% of blood.
 The cellular portion consists of erythrocytes (red blood
cells), leukocytes (white blood cells), and platelets.
 The other 60% is plasma.
The Structure of Blood
 The amount of blood contained in an animal varies by
species.
 Usually blood volume comprises 6%-8% of the animal’s
total body weight.
 Health & age will cause variations in blood volume within a
species.
Blood Cells
 The formation & development of blood cells is called
hematopoiesis: hem=blood, poiesis=formation &
development
 All blood cells develop in the bone marrow from one type
of cell called a hemocytoblast.
 In young animals, blood is produced in all the bones.
 Adults produce blood in the pelvis, ribs, vertebrae, femur,
& humerus.
Erythrocytes
 The red blood cell is the most abundant blood cell.
 Its function is to transport O2 throughout the body.
 Erythrocytes contain hemoglobin, a pigment that contains
iron & gives blood its red color when combined with O2.
 In mammals the red cell is non-nucleated, while reptiles &
birds have nucleated red cells.
 A red cell’s life span in dogs & humans is 120 days, but this
varies among different species.
Leukocytes
 Are colorless, nucleated cells that are capable of moving
throughout the body.
 Their function is mainly body defense.
 Divided into 2 categories: Granulocytes & Agranulocytes
Granulocytes
 Produced in the bone marrow, have lobed nuclei, &
granules in their cytoplasm.
 Neutrophils, basophils, & eosinophils are granulocytes.
Neutrophil
 Has red & blue granules in cytoplasm.
 Function is to stop or slow down foreign organisms.
 How they work 1) Phagocytosis-to eat bacteria & dead cells
 2) Bactericidal-kill bacteria
 How they get to infection site 1) sticky & can migrate through vessel walls
 2) release chemials to attract other neutropils to the infection
site
Basophil
 Has dark purple granules in cytoplasm.
 Functions are phagocytosis, to mediate allergic reactions,
& to produce heparin & histamine
Eusinophil
 Has organish-red granules in the cytoplasm.
 Function is to moderate the inflammatory response &
phagocytosis.
Agranulocytes
 Produced in lymphatic organs, have rounded nuclei, & no
granules in cytoplasm.
 Lymphocytes & monocytes are agranulocytes.
Lymphocyte
 Has a round nucleus & plays a vital role in immunity
 Divided into 2 categories:
 1) T-cells, also called memory cells, are long-lived & once
they are sensitized to an antigen remember it so that the
next time they can fight off that antigen.
 2) B-cells encounter an antigen & divide to form many cells
that all produce the same antibodies to fight the antigen.
Monocyte
 Has an irregular shaped nucleus.
 The largest cell in the blood, its function is phagocytosis.
Thrombocytes (platelets)
 Main function is hemostasis (clotting)
 Platelets are 1/3 the size of a red blood cell
 Stop bleeding by adhering to damaged vessels & clumping
together & releasing proteins that help form a clot
 Average life span of a platelet is 10 days
Hematology
 Study of the structure of blood & the tissues that produce
blood.
 Clinical hematology is a division of medicine that uses lab
tests performed on blood to determine the cause of an
illness.
 To correctly evaluate lab tests, it is necessary to have a
working knowledge of the circulatory system.
Hematology
 The lab tests are most commonly performed in veterinary
medicine are:
 PCV or hematocrit
 White cell count
 TPP (total plasma protein)
 Blood film evaluation
PCV (Packed Cell Volume)
 Measures the % of red blood cells in the total blood
volume.
 A capillary tube is filled with fresh anticoagulated blood,
sealed with clay, & centrifuged for 5 minutes.
 The results are read using a special scale.
 An animal with a PCV that is below normal is said to have
anemia.
Normal PCV Values
 Dogs: 37-55
 Cat: 30-45
 Horse: 32-48
 Bovine: 24-46
TPP (Total Plasma Protein)
 Measurement of proteins produced mainly by the liver.
 Measured using a capillary tube of blood.
 The tube is scored with the edge of a microscope slide,
broken at the plasma layer, and the plasma is placed on a
refractometer.
 The refractometer takes a measurement in g/dl.
 An elevated TPP is a sign of inflammation, infection, or
dehydration.
 A decreased TPP occurs normally in newborns, pregnant
animals.
Blood Film Evaluation
 Used to determine the size, color, & shape of cells &
abnormalities in their formation.
 Blood films are also used to look for blood parasites such
as the microfilaria of the heartworm.
 Blood films are prepared by spreading a drop of blood on a
slide, drying the slide, & then staining with Wright’s stain.
Blood Film Evaluation
 The film is evaluated by scanning under high power for
abnormalities.
 Abnormalities appear in the RBC as color changes in the
cell, abnormal cell shape & size, & foreign bodies within
the cell.
 WBC numbers are tallied & provide information about
infection.
 Platelet numbers are also evaluated.
The Urinary System
 Consists of the:
 Kidneys
 Ureters
 Urinary bladder
 Urethra
The Urinary System
 Main function is to extract & remove waste from the
blood.
 The kidneys are responsible for extracting & collecting
waste.
 They are paired organs located on both sides of the spinal
column & are bean-shaped in most species of animals.
 The kidneys of cattle have several lobes instead of the
bean shape.
The Urinary System
The Urinary System
Kidneys
 Consist of a cortex, medulla, & renal pelvis.
 Throughout the cortex & medulla are located nephrons;
nephrons are the functioning units of the kidneys & are
directly responsible for the filtering & collection of wastes
from the blood.
 Collecting ducts run through the medulla & drain into the
renal pelvis.
Kidneys
Nephrons
The Urinary System
 Urine then passes into the ureters & proceeds to the
bladder.
 The ureters are smooth muscled tubes that extend from
each kidney.
 They use peristalsis to move urine to the bladder.
 Urine is pushed into the bladder every 10-30 seconds
depending on the species.
 It flows in spurts rather than continuously.
The Urinary System
 The bladder, consisting of smooth muscle, is an extremely
elastic sac that is capable of holding large volumes of
urine.
 The bladder has 3 openings: 2 that receive urine from the
ureters, & 1 used to excrete urine to the urethra.
The Urinary System
 The urethra carries urine from the bladder to the exterior.
 The urethra varies in length & circumference in males &
females.
 The female urethra is shorter in length & runs directly from
the bladder to the exterior.
 Males have a narrower urethra that is longer to extend
through the accessory sex glands.
3 Phases of Urine Production
 1) filtration
 2) reabsorption
 3) secretion
 These phases occur in the nephrons, the functioning unit
of the kidneys.
1) Filtration
 Blood enters the glomerulus through the afferent arteriole
 Under various pressures, water, salt, & small molecules
move out of the glomerulus & into Bowman’s capsule
2) Reabsorption
 Occurs in the proximal convoluted tubule & the loop of
Henle
 Substances needed by the body such as water &
electrolytes will be reabsorbed by the body from the loop
of Henle
3) Secretion
 Substances are secreted into the collecting tubules &
transported to the renal pelvis
Urinalysis
 Provides information about how the kidneys are
functioning & if wastes are being properly filtered from the
body.
Specimen Collection
 1) Free Catch-simplest method of collecting urine.
 Samples from dogs can be caught with a pan or soup ladle.
 Use a metal pie plate for females.
 To collect from a cat, replace the cat litter with a shredded
plastic bag or plastic pellets.
Specimen Collection
 2) Manual Expression-involves palpating the bladder
through the abdomen then applying pressure to it to
encourage urination.
 Manual expression is mainly used for animals that are
unable to urinate on their own due to an injury or illness.
 Animals with obstructions should never be manually
expressed.
Specimen Collection
 3) Catheterization-performed by inserting a plastic, or
rubber catheter through the urethra into the bladder.
 The size & type of catheter used depends on the sex &
species of animal.
 Catheterization is performed aseptically to prevent
infection & is used in emergencies & for immobile animals
that need long-term care.
Specimen Collection
 4) Cystocentesis-performed by inserting a needle through
the abdomen into the bladder.
 Aseptic technique is used to prevent infection.
 Is performed to obtain a pure urine sample or to relieve
bladder pressure on an obstructed animal.
Urinalysis
 Evaluation-ideally evaluation of a urine sample should
occur within 30 minutes of collection, however samples
can be refrigerated overnight if necessary.
 Refrigerated samples should be brought to room
temperature before they are evaluated.
Urinalysis
 Samples are evaluated on the following:
 Color
 Transparency
 Specific Gravity
 Chemistry
 Sediment
Color
 In most species urine is a pale yellow to amber color.
 The color of the urine correlates to specific gravity.
 Lighter colored urine=lower specific gravity
 Darker colored urine=higher specific gravity
 Red urine=hematuria (red blood cells in urine)
 Yellowish-brown foamy urine=presences of bile pigments
 Some species, like the rabbit, have urine that is normally a
darker orange to reddish-brown.
Transparency
 Terms used to describe urine transparency are clear,
cloudy, or flocculent.
 Clear, fresh urine is normal for most species.
 Cloudy urine indicates the presence of cells, bacteria,
crystals, or fats, but in the horse, rabbit & hamster cloudy
urine is normal.
 Flocculent describes urine that has pieces of floating debris
in it caused by the presence of cells, fats, or mucus.
Specific Gravity
 Measures the concentration or density of urine compared
to distilled water.
 There are 3 ways to measure sg.
Specific Gravity
 1) Refractometer-a tool that refracts light through urine &
measures density by comparing it to the amount of light
that will pass through distilled water.
 The refractometer is also used to measure total plasma
protein.
Specific Gravity
 2) Urinometer-a bulb is floated in a cylinder filled with
urine.
 Specific gravity is read off a scale attached to the bulb.
 This method requires a larger sample than the other
methods.
Specific Gravity
 Reagent strips-contain a chemical pad that changes color
when dipped into urine.
 The color change is read using a scale on the reagent
container.
Specific Gravity
 Average Specific Gravities:
 Dog: 1.025
 Cat: 1.030
 Horse: 1.035
 Cattle & swine: 1.015
 Sheep: 1.030
Specific Gravity
 An increased sg could indicate dehydration, decreased
water intake, acute renal disease, or shock.
 A decreased sg could indicate increased water intake,
chronic renal disease, or other diseases.
Specific Gravity
 4) Chemistry
 The chemical components evaluated in urine are:
 pH
 Protein
 Glucose
 Ketones
 Bile
 Blood
 Yeast
 sperm
Specific Gravity
 Chemistry tests are performed using reagent strips.
 Several companies produce strips that will evaluate all the
chemical components on one strip.
 The chemical components provide information used to
diagnose problems such as diabetes, renal failure, liver
infections, muscle disease, inflammation of the urinary
tract, & ketosis.
Specific Gravity
 5) Sediment
 An examination of urine sediment provides information on
the types & numbers of cells present.
 Cells commonly seen are:
 RBC’s
 WBC’s
 Epithelial cells
Specific Gravity
 All of these cells are normal in small amounts; large amounts
indicate disease or infection.
 Excess RBC’s indicate hemorrhaging of the urinary tract.
 Excess WBC’s indicate inflammation of the urinary tract.
 Epithelial cells are sloughed from the urinary tract as they wear
out, but trauma to the urinary tract will also cause sloughing.
 Other components found in sediment are bacteria, crystals, &
casts.
Bacteria
 Indicate infection or contamination of the sample by
improper handling.
 If bacteria are present with an increased number of WBC’s
then infection is likely.
Crystals
 Form due to influences from pH, urine concentration, &
diet.
 Crystals do not necessarily indicate a disease, but they do
cause problems in large amounts by irritating the urinary
tract, causing blood in the urine (hematuria) & pain.
 Crystals bond together creating stones that can block
urine low & may eventually cause death.
 Stones & crystals are especially serious in males due to the
size & shape of the urethra.
Casts
 Tubular clumps of cells or other materials that form in the
collecting tubules of the kidney.
 Large numbers of casts indicate a problem in the collecting
tubules.
 The types of casts are:
 Hyaline
 Fine granular
 WBC/RBC
Susceptibility Testing
 Performed to determine how bacteria will respond to an
antibiotic since some types of bacteria do not respond in a
predictable manner.
 Testing is important so that an effective antibiotic can be
found.
 The main methods used to test antibiotic sensitivity are
broth dilution & agar diffusion.
Broth Dilution
 Uses a series of test tubes that contain varying
concentrations of the same antibiotic.
 The test tubes are inoculated with bacteria & incubated.
 The test tube that has the lowest antibiotic concentration
with no bacteria growth indicates the minimum amount of
antibiotic that is effective.
Agar Diffusion
 Uses petri dishes coated with bacteria.
 Disks containing antibiotics are placed on the petri dishes &
incubated.
 After incubation, the “zone of inhibition” is measured to
determine which antibiotic is most effective.
 The “zone of inhibition” is an area of no growth around an
antibiotic disk.
 The larger the “zone of inhibition”, the more effective the
antibiotic.