Urinalysis Laboratory Procedures Ch. 5

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UrinalysisLaboratory Procedures Ch. 5
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Urinalysis

• This test is often part of an initial data base
  for case work up of a clinically ill patient. 
• It is a very useful indicator of renal function,
  and should be performed on any animal suspected
  to have renal disease or urinary tract pathology

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Urinalysis

• A urinalysis consists of gross examination of
  urine, a specific gravity (SG), chemical
  analysis, and sediment evaluation.
• Equipment required clean glass or plastic
  collection container, a centrifuge and conical
  centrifuge tubes, chemical reagent strips, clean
  glass slides and coverslips, a refractometer and
  microscope. A sediment stain is recommended.

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• The best samples for urinalysis are morning
  samples. These are the most concentrated.
• Urine samples should be analyzed within 30
  minutes to 1 hour of collection.
• Physical properties of urine include volume ,
  color, odor, transparency, and specific gravity.

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Collection for analysis

• There are several different methods of collection
  for urinalysis and each has its benefits and draw
  backs.
• Collection methods will often be dictated by the
  information that you are looking to gather. 
• Analysis of urine samples should be performed
  only in samples taken before administration of
  therapeutic agents.

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Midstream, voided or Free Catch

• This collection method is often easiest for the
  animal but can be quite difficult for the
  collector. 
• Collection is made into a container directly from
  the patient. 
• This collection method will obviously contain
  contamination

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Manual Expression

• This collection method is most often performed on
  small dogs and cats.  It is sometimes difficult,
  and can result in trauma in the form of red blood
  cells in the urine.  This method will also
  contain contamination from the lower urinary
  tract

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Catheterization 

• Catheterization is the insertion of a
  polypropylene or rubber catheter into the bladder
  by way of the urethra
• This test can be used on male dogs for the
  assessment of urethral patentcy and upper urinary
  tract infection.  This method often results in
  iatrogenic presence of red blood cells in the
  urine.
• Lubricate the catheter

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Cystocentesis 

• This method requires penetration of the bladder
  through the body wall and can be accompanied by
  minimal bleeding. This is the best way to analyze
  the upper urinary tract for infection.
• Use a 22 or 20 gauge needle by 1 inch or 1½
  inches and a 10 ml syringe
• For male dogs, insert the needle caudal to the
  umbilicus and to the side of the sheath
• For female dogs and cats insert the needle on the
  ventral midline caudal to the umbilicus

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Volume

• Normal 24 urine production for dogs and cats is
  20-40 ml/kg.  An average sized saddle horse may
  produce between 5 and 15 L of urine in 24 hours. 
  
• An increase in this volume is termed polyuria and
  may be due to physiological, pharmacological or
  pathological causes. 
• Decreased urine volume is called oliguria, and
  occurs in dehydration, renal failure, or urinary
  blockages. 
• No urine is called anuria, and is an emergency
  condition that may be due to renal failure,
  urinary blockage or ruptured bladder.

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Color

• Urine color will vary between species, but it is
  normally some shade of yellow depending on the
  concentration. 
• Abnormal color changes in the urine could be due
  to drugs, increased urinary pigments or red blood
  cells.  Red to reddish-brown could be due to
  either hematuria, hemoglobinuria, or
  myoglobinuria. 
• Occasionally, unusual colors may be caused by
  dyes associated with food or drugs.

urine sample exhibiting hematuria
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Turbidity

• Urine is normally transparent in most animals,
  except for the horse.  The horse has a thick
  viscous urine that is cloudy on examination. 
• In small animals, turbidity suggests the presence
  of cells, casts, or crystals.  Often
  refrigeration will propagate the sedimentation of
  crystals in the urine, producing a cloudy
  appearance.  This is usually of no significance

The sample on the left is exhibiting turbidity. 
The sample on the right is a normal color and
clarity for canine urine
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Odor 

• Urine has a characteristic smell that varies
  slightly by species and concentration of the
  sample. 
• A particularly foul odor may occur in the
  presence of bacteria. Thus, strong smelling urine
  is common in cases of cystitis. 
• Ketonuria produces a very sweet smell as does
  glucosuria. 
• Sweet smelling urine is commonly associated with
  diabetes mellitus.

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Specific Gravity

• Specific gravity measures the concentrating
  ability of the kidney tubules.  It is the ratio
  of the weight of urine to the weight of an equal
  volume of water. 
• Normal values range from 1.001-1.060 in most of
  our domestic animals.  If the kidneys are unable
  to concentrate urine the specific gravity will
  approach that of the glomerular filtrate, at
  1.010.
• A small drop of the urine sample is placed under
  the slide on the top of the scope, and the
  measurement is made by looking through the eye
  piece to read the value indicated.

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The Chemical Analysis 

• A chemical analysis of urine is performed using a
  commercial dip stick reagent strip.
• If the sample is turbid, the chemistry tests are
  done on the supernatant.

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What is evaluated on chemical analysis?

• Protein
• Glucose
• Ketones
• Blood
• Bilirubin
• Urinary pH

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pH

• Urine pH will be affected by many things
  including the diet, handling of the sample, and
  acid-base balance of the animal.  An alkaline pH
  is most indicative of an infectious process. 
  Normal pH is between 6 and 8 for most animals
  depending on their diet.

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Glucose

• In the normal animal there should not be glucose
  in the urine. 
• If glucose is present, it is a classic response
  to hyperglycemia and should instigate an
  investigation into the possibility of diabetes in
  the patient. 

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Ketones 

• In the normal animal there will be no ketones in
  the urine.  An animal that is undergoing fat
  metabolism or is deficient in carbohydrates will
  have ketones in the urine.  Slight ketonuria
  should be expected in malnourished animals.  A
  ketonuria also frequently accompanies diabetes
  mellitus. 

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Bilirubin 

• Bilirubinuria increased concentrations of
  bilirubin due to biliary obstruction,
  cholestasis, or increased bilirubin production
  secondary to hemolysis.

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Blood

• There should not be any blood in the urine of a
  normal animal.  Most test strips cannot
  differentiate between red blood cells,
  hemoglobin, or myoglobin, thus some care should
  be taken in interpretation. 
• Hematuria is also evaluated in urine
  sedimentation microscopically and is reported as
  cells per high power field (or HPF).  Remember
  that collection methods may also cause blood to
  appear in the urine. Other causes of hematuria
  include infection, neoplasia, or trauma.

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Protein 

•  Urine normally contains a small amount of
  protein, which is due to normal leakage and
  secretion from the urinary tract lining. This
  normal amount of protein will not show on the
  dipstick.
• Proteinuria is an excess of serum proteins in the
  urine. This is an important factor of renal
  disease.
• Proteinuria can also be found in
  glomerulonephritis, congestive heart failure, and
  renal ischemia of all kinds.

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Sediment

•  Urine sedimentation may contain cells, casts and
  crystals and is examined microscopically after
  centrifugation of a urine sample. 
• A very small amount of all of the above sediments
  is normal.  Concern begins when any of these
  components is significantly elevated. 
• There are many different crystals, cell types,
  and casts that may be found in the urine of
  animals, and it varies from species to species.

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Urinary Crystals
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Crystals contd

• pH dependant
• May indicate a urolith (bladder stone)
• May lead to urethral obstruction

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Calcium Oxalate crystalsAcidic urine (6.5 and
below)
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Calcium Oxalate crystals in a slightly different
configuration
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Struvite Crystals / Triple PhosphateNeutral to
Acidic (7.0 and above)
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Struvite Urolith
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Casts

• Casts are elongated structures composed of
  protein from plasma and mucoprotein from the
  renal tubules.
• In general, they form in the distal tubules, in
  which the urine is more concentrated and acidic.
• Any structures that happen to be in the tubules
  at the time the casts form (RBCs, WBCs or
  epithelial cells), become embedded in the casts.

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Casts

• The presence of increased numbers of casts helps
  to localize the renal disease to the tubules, but
  the numbers to not necessarily correlate with the
  severity of the disease.
• The five main types of casts are
• Hyaline, Cellular, Granular, Waxy and Fatty

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A Hyaline cast B Fatty cast C Hyaline to
finely granular D Cellular cast E Cellular to
coarsely granular F Coarsely granular cast G
Finely granular cast H Granular to waxy cast I
Waxy cast.  
Cast Compilation The image below represents different casts seen in urine at the same magnification and lighting. Shown are hyaline, fatty, granular and waxy casts.Hyaline casts can be quite difficult to see in wet preparations of urine sediments with light microscopy, even with the condenser of the microscope racked down. They are much easier to visualize using phase contrast, however phase is usually not available on most microscopes. They become more visible with regular light microscopy if fat sticks to the protein matrix (Tamm-Horsfall mucoprotein) that makes up the hyaline cast (image B) or particulate material from degenerating cells is present within the cast matrix (image C). Cellular casts have distinct cells within the protein matrix - if the cells are of epithelial origin (i.e., not WBCs or RBCs), they are called epithelial casts (images D and E). As cells within the protein cast matrix break down, the cast becomes coarsely (image E and F) then finely granular (image G). Waxy casts are the final stage of cast degeneration (usually originating from cellular and granular casts). Compared to hyaline casts, they are readily observable because they have a smooth appearance, no internal texture, and are more refractile than the surrounding urine.

Legend A Hyaline cast B Fatty cast C Hyaline to finely granular cast D Cellular cast E Cellular to coarsely granular cast F Coarsely granular cast G Finely granular cast H Granular to waxy cast, I Waxy cast.
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Hyaline Casts

• Colorless, homogenous, and semitransparent. May
  be difficult to see unless the light is reduced.
  These casts occur in health and also in
  association with mild glomerular leakage.

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Cellular Casts

• Contain recognizable cells embedded in the
  protein matrix.
• Cellular casts may be epithelial cell casts that
  contained sloughed tubular epithelial cells, RBC
  casts that indicate renal hemorrhage, or WBC
  casts that indicate renal inflammation of
  pyelonephritis.

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RBC Cast
WBC Cast
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Granular Casts

• Casts derived from degenerating cells or cellular
  casts.
• Characterized by a nonspecific granular matrix
  and are designated as either coarsely or finely
  granular.
• This is the most common type of cast found in
  animals.

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Granular casts
Coarse
Fine
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Waxy Casts

• Homogenous and wide.
• Usually has distinct blunt or squared ends.
• Indicate a more chronic renal lesion.

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Fatty Casts

• Contain fat globules from degenerating tubular
  epithelial cells.
• Most common in cats because of high lipid content
  of feline tubular epithelium.

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Bacteria / Microorganisms

• Bacteria in a voided sample are usually not
  clinically significant contamination from
  distal urinary tract.
• Bacteria in a catheterized or cystocentesis
  sample clinical significance.
• Bacteria in a sample is most often correlated
  with the presence of WBC.

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Rod bacteria may appear singly or in chains.
Cocci bacteria may also be present.
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Bacteria Video
YouTube - Bacteria In Urine
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How to Microscopically Examine Urinary Sediment

• A sample of well-mixed urine (usually 10-15 ml)
  is centrifuged in a test tube at relatively low
  speed (about 2-3,000 rpm) for 5-10 minutes. The
  supernatant is decanted and a volume of 0.2 to
  0.5 ml is left inside the tube. The sediment is
  re-suspended in the remaining supernatant by
  flicking the bottom of the tube several times. A
  drop of re-suspended sediment is poured onto a
  glass slide and coverslipped. Another drop is
  stained using urinary sediment stain.

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• The sediment is first examined under low (10X)
  power to identify most crystals, casts, squamous
  cells, and other large objects. The numbers of
  casts seen are usually reported as number of each
  type found per low power field (LPF). Since the
  number of elements found in each field may vary
  considerably from one field to another, several
  fields are averaged.

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• Next, examination is carried out at high (40X
  -100X) power to identify crystals, cells, and
  bacteria. The various types of cells are usually
  described as the number of each type found per
  average high power field (HPF). Example 1-5
  WBC/HPF.

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RBCs

• Red Blood Cells
• Red cells may also contaminate the urine from the
  vagina in menstruating dogs or from trauma
  produced by bladder catherization. Theoretically,
  no red cells should be found, but some find their
  way into the urine even in very healthy animals.
  However, if one or more red cells can be found in
  every high power field, and if contamination can
  be ruled out, the specimen is probably abnormal.

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• RBC's may appear normally shaped, swollen by
  dilute urine (in fact, only cell ghosts and free
  hemoglobin may remain), or crenated by
  concentrated urine. Both swollen, partly
  hemolyzed RBC's and crenated RBC's are sometimes
  difficult to distinguish from WBC's in the urine.
  In addition, red cell ghosts may simulate yeast.

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RBC Cast
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• White Blood Cells
• Pyuria refers to the presence of abnormal numbers
  of leukocytes that may appear with infection in
  either the upper or lower urinary tract or with
  acute glomerulonephritis. Usually, the WBC's are
  granulocytes.
• If two or more leukocytes per each high power
  field appear in non-contaminated urine, the
  specimen is probably abnormal. Leukocytes have
  lobed nuclei and granular cytoplasm.

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WBC Cast
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Epitheleal Cells
Represent possible contamination of the specimen
with skin flora. Large polygonal with small
nuclei.
Renal tubular epithelial cells, usually larger
than granulocytes, contain a large round or oval
nucleus and normally slough into the urine in
small numbers. However, with nephrotic syndrome
and in conditions leading to tubular
degeneration, the number sloughed is increased.
Transitional epithelial cells from the renal
pelvis, ureter, or bladder have more regular cell
borders, larger nuclei, and smaller overall size
than squamous epithelium.
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• Squamos Epitheleal

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• Transitional Epithelial (A)

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• Renal Tubule Epithelial (B)