VET 131
Urinalysis

Required reading: 

Hendrix, “Laboratory Procedures for Veterinary Technicians”, Chapter 5, pages 151 - 180

Recomended reading:

Sink and Feldman: Laboratory Urinalysis and Hematology for the Small Animal Practitioner.  Sections 1 and 2, pages 1-46.

Required website:

http://diaglab.vet.cornell.edu/clinpath/modules/

Objectives

 After completing this unit, you should be able to: 

1.                   Describe four methods for collecting a urine sample, and know the pros and cons of each.

2.                   Explain why fresh samples of urine should be examined.

3.                   Describe methods of preserving urine, and know when to use each method.

4.                   Identify the characteristics which describe the appearance of urine.  Practice using accepted terminology -- "gunky" is not acceptable, but "flocculent" is.

5.                   Describe the procedures which can be used to measure the specific gravity of urine.  Know why specific gravity is important.

6.                   Describe the chemistries which can be tested with reagent strips.

7.                   Describe the various cells which can be found in a urine sediment.  Know normal or acceptable levels for cats, dogs and horses.

8.                   Describe the non-cellular constituents which can be found in a urine sediment.  Know normal and acceptable numbers.

 

Sites on the Web:  Material from the following websites MAY appear on exams...

http://www.vetmed.auburn.edu/distance/clinpath/newurine/

An on-line tutorial for veterinary practitioners from the Auburn University College of Veterinary Medicine's Clinical Pathology Section of the Department of Pathobiology.  Contains some excellent pictures and text of urinalysis procedures both dipstick and microscopic examinationation.  You may have to copy/paste this into your computer's browser, if the link doesn't work from this website.

Introduction

 Urinalysis is routinely performed by veterinary technicians in veterinary clinics.  It consists of a number of relatively simple and inexpensive tests which provide a great deal of information to the veterinarian, such as:  hydration status of the patient, kidney function, evidence of liver disease, urinary tract disease, endocrine disease, and electrolyte abnormalities.

 

Specimen Collection

Specimens can be collected through natural voiding (urination), catheterization, cystocentesis or by manually compressing the bladder.  Morning samples are preferable since they tend to be more concentrated and will more likely contain clinically significant components, if they are in the urinary tract.  The sample should be refrigerated if it is not examined within 15 - 30 minutes of collection.  Sunlight affects bilirubin and urobilinogen, therefore samples should be kept out of direct sunlight.  Any refrigerated sample needs to be warmed to room temperature (5 minutes on the counter) before analysis.

Voided sample

A voided sample is the easiest one to collect but is not satisfactory for a bacteriological study since the sample is often contaminated during urination.  If possible, the vulva or prepuce of the animal should be washed prior to sample collection in order to minimize contamination.

Voided samples should be collected mid-stream into a clean container.  Mid-stream samples tend to contain fewer bacteria than a first-stream sample.  Since some animals may stop urinating when you try to get a sample, you may want to collect the first stream of urine in one container, and then replace it with a second container to collect a mid-stream sample.

Technical tip:  Owners can use a dixie cup on the end of a coat hanger, or a flat plate, lid to a refrigerator dish, or a shallow dish to collect from small dogs.  Other tips???

Catheterization

During catheterization a sterile catheter is inserted into the bladder by way of the urethra.  The external genitalia should be cleaned using a sterile prep as for surgical scrub, and a sterile, water-soluble lubricant (e.g. K-Y Jelly) placed on the tip of the catheter.  Care should be taken in inserting the catheter since you do not want to traumatize the tissues.  Female dogs can be challenging to catheterize.  You should practice this technique on a cadaver either using a speculum, or blindly before attempting it on a live patient.

Once the dog is catheterized, the urine is collected by GENTLY aspirating into a syringe attached to the catheter.  The first portion of the sample is discarded since it may contain contaminated tissue collected as the catheter passed through the urethra.  Since there is some degree of trauma in catheterization, some red blood cells may be present in the urine sample.  More than 5- 10 RBCs/high power field (40X) is ABNORMAL.

Cystocentesis

Cystocentesis involves the insertion of a 1 -1 ˝ inch, 23 gauge needle into a distended bladder and gently aspirating the urine into a syringe.  As much urine as possible is removed from the bladder in order to minimize the possibility of pressure in the bladder causing urine to leak out of the bladder when the needle is removed.  Cystocentesis is the best method of urine collection for the culturing of bacteria, if the skin is aseptically prepped.  It may be performed blindly, by manually palpating the bladder, GENTLY immobilizing it against the body wall, and inserting the needle at an angle.  Cystocentesis may also be performed using ultrasound to guide your needle (preferred and safer method).  This method takes some practice -- you can practice by making a jello mold around a grape or orange or olive, and placing the ultrasound probe on the jello to visualize your needle as you attempt to "aspirate" the fruit object.

Manual expression of bladder

A small amount of urine can be collected by manually compressing the bladder.  This method is not recommended in awake animals since the animal naturally resists urination and the pressure buildup in the bladder can cause injury to the organ.  Also, in some cases, the urine can back up into the ureters and kidneys.  This is the method of choice in SEDATED or ANESTHETIZED cats.  DO NOT ATTEMPT THIS in animals that may be obstructed, as the bladder is easily ruptured.

 

Specimen preservation

 Ideally, samples should be examined within 30 minutes of collection.  It is important that fresh samples be used because of the following factors: 

1.

Bacteria proliferate

2.

Cells autolyse and cytological features deteriorate rapidly

3.

Casts may dissolve if the urine is alkaline

4.
5.

pH may increase because of bacteria action breakdown of urea
Crystal formation or dissolving due to temperature changes and pH changes when urine sits

If the sample is immediately refrigerated, it can be preserved for 6 to 12 hours without too many alterations in chemistry or cellular components.  Additional crystals may form when the urine cools.  Refrigerated samples should be warmed to room temperature before determining specific gravity and examining.

Samples can be held longer than six hours or preserved for shipment to an outside lab by one of the following methods:

1.

1 drop of 40% formalin in 1 oz. of urine

2.

Layer toluene on top of sample

3.

Addition of a single thymol crystal to sample

4.

1 part 5% phenol to 9 parts urine

Be sure to check with the outside lab to find out the preservation method they require.  Formalin is the best preservative for the cells in urine.  If formalin is used, chemical tests should be performed before the formalin is added to the sample, since formalin interferes with the chemical test.  Samples preserved with the above methods cannot be used for bacterial culture.

Samples can be frozen in order to preserve them.  Freezing retards the growth of bacteria making this preservation method acceptable for future bacteria culturing.  However, the cellular components of the urine are altered during the freezing process.

 

Gross Examination of Urine

The physical and chemical characteristics of the urine should be examined and the results recorded.  A typical report form is depicted below.  Always note the method of collection, the time of collection, and the animal's information:  reproductive status, age, breed, etc...

Report.jpg (361631 bytes)

Appearance

The volume of urine collected should be recorded.  This volume can be used to estimate the amount of urine an animal is producing, although a 24 hour sample is more accurate but less practical.  A normal 25 dog produces from 300 to 750 ml of urine per day, whereas a normal 10 lb cat produces from 45 to 90 ml of urine per day.  The following terms can be used to describe abnormal quantities of urine production:

Polyuria:

production of excess amount of urine

Oliguria:

decrease in production of urine

Anuria:

no production of urine

The color of the normal urine is pale yellow, yellow or amber.  The intensity of the color usually correlates with the concentration of substances within the urine.  A very dilute urine sample may be colorless, whereas a highly concentrated sample may be deep yellow or dark amber.  A reddish-brown urine is caused by the presence of red blood cells or hemoglobin, whereas a greenish urine indicates the presence of bile pigments.  Brown to black urine in horses may be present if the horse was recently exercised strenuously.  The color is due to the lysis of muscle cells and the subsequent release of myoglobin.  Brown urine may also result from various drug therapies and a milky white color may indicate pus, an abundance of phosphate crystals or the presence of milky chyle from the lymphatics. 

The transparency of a urine sample can be described as clear, cloudy or flocculent.  Normal, freshly voided samples are clear, except for horses which contain a high concentration of calcium carbonate crystals which can cause the urine to appear cloudy.  Cloudy samples usually contain large particles and sediment, whereas flocculent samples contain particles which are large enough to be seen with the naked eye.  

The odor of urine is not diagnostic but may be a useful piece information for a veterinarian in making a diagnosis.  Improperly stored samples typically has a strong odor, as does urine from male cats, goats and pigs.  A fruity odor is due to the presence of ketones in the urine.   The excretion of some drugs may affect the odor of the urine. 

A foam may result when a tube of urine is shaken.  A white foam indicates the presence of protein, whereas a green foam indicates the presence of bile.  Horses NORMALLY have protein and mucus in their urine.  This results in urine with a syrup consistency that foams easily when mixed.

Tech Tip:  Urine that is dark red can be centrifuged for 5 minutes to determine if the color is due to RBCs or hemoglobin.  If RBCs are present in the urine, they will settle to the bottom of the centrifuge tube and the sample color clears, while the pigment hemoglobin will remain in the urine, causing the color to remain.

Specific gravity  

The specific gravity of  urine is its weight (density) compared to an equal amount of distilled water.

                           Specific gravity =  Density of urine/Density of water

Since urine contains various solutes it always has a specific gravity greater than 1.  The normal range of specific gravity for dogs is 1.018 to 1.045, whereas the specific gravity for cats is 1.020 to 1.040.  An increase in specific gravity (Hypersthenuric) indicates a more concentrated urine, whereas a decrease in specific gravity (Hyposthenuric) indicates a more dilute urine.  There are three ways to measure specific gravity: refractometer, urinometer, and reagent strips.  ALWAYS record the method used to determine the specific gravity, and if both a strip and refractometer are used, record both values as an internal quality control check.  These values typically do not agree well, and if they consistently agree, you may want to check your refractometer to be sure it is calibrated and reading appropriately.

The refractometer is the most frequently used procedure to measure specific gravity.  A drop of  urine is placed on the glass plate of the refractometer, the plastic cover closed and the specific gravity determined by observing the internal scale.  Since specific gravity is influenced by temperature, the refractometer should be temperature corrected to ensure consistent and accurate readings.  A refractometer should be calibrated using distilled water before using.  Additionally, be sure the refractometer is clean and dry before using.

                          Refract1.jpg (390480 bytes)       Refract 2.jpg (409594 bytes)

                                            Refractometer                                     Plastic cover of refractometer

The urinometer is a less frequently used procedure because it requires a larger volume of urine and is not as consistent as the refractometer.  A urinometer is floated in a cylinder of urine and the specific gravity read on a scale printed on the instrument.

                          Urinometer.jpg (409505 bytes)    Urinometer

Reagent strips are available to measure specific gravity, but they are the least reliable method for doing so. 

 

Reagent strips for chemical constituents

Various chemical components of the blood can be tested with paper reagent strips containing chemicals that react with the chemicals in urine.  A color change on the strip is compared to a color chart and the value recorded.  Be careful to not allow the samples to mix between spots on the strip, as this can alter the results.

Strips.jpg (373573 bytes)

pH is a measure of the acidity or alkalinity of the urine.   The pH of an animal’s urine is affected by diet with herbivores producing more alkaline (higher pH) urine and carnivores producing more acidic (lower pH) urine.  The normal pH range for dogs is 5.2 to 6.8, whereas the normal pH range for cats is 6 to 7.

Protein is usually absent or present in trace amounts in normal urine.  Reagent strips detect primarily albumin and are less sensitive to globulin.   With increased muscle exertion or in convulsions there may be a transitory increase in the level of proteins in the urine.  If the urine is very dilute, a false-negative reading may occur.  Therefore, even if a trace amount of protein is found in a very dilute urine sample, this information should be noted.  When using test strips, an alkaline urine occasionally produces a false-positive reading. 

If the protein level in the urine is excessive, the levels should be confirmed by a sulfosalicylic acid turbidity test which is sensitive to both albumin and globulin.  This test is described on page 231 in Hendrix.

Glucose is normally present in very small amounts in the urine, but the small quantity is not detected by reagent strips.  The renal threshold for glucose is approximately 170 to 180 mg/dl for dogs.  If the blood level of glucose exceeds the renal threshold, the excess glucose will end up in the urine.  A positive reading of glucose in the urine may indicate diabetes mellitus.  Pharmacological agents such as morphine, epinephrine, progesterone and ACTH may produce a positive glucose reading.  If an animal is in fear, is excited or overly stressed, the resulting high epinephrine levels will lead to a transitory, positive glucose reading.  This is a common occurrence in cats.  If you are in doubt, allow the cat to settle down and resample in a couple of hours.

Ketones may be present in small amounts in normal urine.  Conditions associated with increased fat metabolism will result in a positive ketone reading.  Increased fat metabolism occurs in starvation, high fat diets, fasting and anorexia.  Ketosis in cows and pregnancy toxemia in ewes occurs when the cow or ewe is not able to ingest enough food to maintain the energy requirements for milk production.  The urine ketone test is a common "cow-side" screening test for subclinical ketosis.

Bile pigments, urobilinogen  and bilirubin, are commonly found in the urine.  Urobilinogen is normally found in all domestic animals.  Bilirubin is normally found is small amounts in cows and dogs.  An obstruction in bile flow, as well as some diseases of the liver may cause an increase in bile pigments in the urine.  Hemolytic anemia, especially in dogs, may also cause an increase in bilirubin in the urine.   Sunlight breaks down bilirubin resulting in a false-negative readings of bilirubin for urine samples exposed to sunlight.

Blood tests in urine test for intact RBC’s (hematuria), free hemoglobin (hemoglobinuria) and myoglobin (myoglobinuria).  Hematuria usually indicates bleeding in the urogenital tract.  Hemoglobinuria is usually due to hemolysis of RBC’s in blood vessels.  Myoglobinuria usually indicates severe muscle damage.

Assignment:

1.    Practice doing  a gross examination of a urine sample under the guidance of your mentor.
2.    Once your are confident with your technique, perform the tests on two samples and report your results on Lab Report #2.
3.     Be sure to answer the study questions in the lab.
4.     Email Dr. Durham a copy of your lab report #2

Microscopic Examination of Urine Sediment

 Microscopic examination of urine sediment must be a part of every urinalysis.  To obtain a sediment sample the following procedures should be employed: 

1.

Allow refrigerated samples to rewarm to room temperature.  Mix sample thoroughly.

2.

Place 5 ml of urine in a centrifuge tube.  If you do not have 5 ml. Indicate that QNS (quantity not sufficient)

3.

Centrifuge at 1000 – 1500 rpm for 5 minutes

4.

Drain off 4.5 ml of supernate.

5.

Resuspend sediment in the remaining supernate

6.

Place 1 drop of sediment on a slide

7.

Subdue light by lowering condenser and diaphragm

8.

Use low power (10X) to scan slide.  Excessive RBC’s can be lysed with 1:1 dilution with 5% acetic acid.  If sediment is too dense, it can be diluted with isotonic saline.  Note if you need to perform these operations.

9.

RBC’s are standard of measure.  Report as average number of RBC’s per high power field (40X) or average number of RBC’s per low power field (10X)

10.
 

11. 

Mix one drop of resuspended sediment with one drop of new methylene blue stain on a microscope slide, place a coverslip over this, and look at it using low power (10X) to scan the slide, and high power (40X) to look at the cellular components.
Bacteria are only visible using 100X and oil.  Remember that they will all stain purple using this technique, but you can make a judgement on their morphology -- i.e...rods, cocci, mixed population, few, many, etc...Beware of stain precipitate that can look like bacteria under 40X -- if this occurs, or you are in doubt, use fresh stain.
You should always look at BOTH an unstained and a stained urine sample!

Centrifuge tube.jpg (373255 bytes)  Centrifuge tube (a large red-top tube works in a pinch!)

 A microscopic examination of urine sediment can reveal the following components: Sink and Feldman has a good color atlas for Urine sediment examination, as well as the Cornell Diagnostic Lab website (required).

Leukocytes (WBC’s) are not usually found in urine.  If more than 2 or 3 are found per HPF (high power field) there is probably an inflammation in the urinary or genital tract.  Urine with a large number of leukocytes should be cultured for bacteria, even if they are not evident in the slide.

            White blood cell UA.JPG (23461 bytes)  White blood cells and sperm         urineWBC.JPG (7988 bytes)  White blood cells (from Auburn University)

Erythrocytes (RBC’s) may be found in the urine.  Normal urine contains 2 to 3 RBC’s per HPF. If there is bleeding in the urogenital tract the number will increase.  The trauma that occurs in catheterization, cystocentesis and manual expression usually increases the numbers of RBC’s in the urine sample. 

            urineRBC.JPG (4546 bytes)  Red blood cells (from Auburn University)

There are three types of epithelial cells that can be found in urinary sediment.  

Squamous:

Flat cells which are derived from the urethra, vagina, vulva or prepuce

Transitional:

Round or pear shaped cells derived from the bladder, ureters, pelvis or urethra. 

Renal:

Round, small cells that are derived from the renal tubules.

 Squamous epithelial cells are frequently observed, whereas transitional and renal epithelial cells are rarely found (0 to 1/HPF)

              See figure 5-9 thru 5-13 on pages 241 to 243 of Hendrix

Microorganisms may be found in urinary sediment.  They are reported as being few, moderate, or many.  Since bacteria proliferate rapidly, urine must be examined within 30 minutes or the sample must be refrigerated.  Bacteria can only be identified with high power magnification (100X).  The presence of a large number of bacteria and WBC’s usually indicates an infection and inflammation of the urinary tract.

              See figure 5-14 on page 244 of Hendrix

Casts are a mucoprotein matrix formed in the distal and collecting tubules of the kidney.  Casts take on the shape of the tubule and incorporate material present at the time of their formation.   They are large, and often found at the periphery of the coverslip when looking at a wet mount of urine on a microscope slide.

Hyaline casts are clear, colorless, refractile, cylindrical structures which are believed to be the basic structural unit of all other casts.  The ends of the casts have a rounded appearance.  Hyaline casts dissolve in alkaline urine and therefore are not observed in herbivores.  A few hyaline casts can be seen in normal urine,  but the number increases with fever, strenuous exercise and poor renal circulation.

            See figure 5-16 on page 246 of Hendrix

Granular casts are hyaline casts with granules which are derived from epithelial cells and WBC’s which are trapped in the cast and then degenerate.  Granular casts are the most frequently seen cast in urine sediment.   An abundance of granular casts occurs in acute nephritis.

              granularcast.JPG (7679 bytes)  Granular cast (from Washington State University - image database)

Epithelial casts contain epithelial cells.  Granular casts usually are present when there are epithelial casts.  The presence of epithelial casts indicates degeneration of the renal tubular epithelium.

Leukocyte casts contain white blood cells.  Free WBC’s are usually also present when there are leukocyte casts.   An inflammation in the renal tubules is indicated when leukocyte casts are observed.

Erythrocyte casts contain red blood cells.  They appear an orangish yellow color because of the hemoglobin.  Erythrocyte casts indicate renal bleeding.      

Waxy casts look like hyaline casts but have a “waxy” appearance and are wider with broken off ends,  They are present in chronic, severe degeneration of the renal tubules.

Fatty casts contain many small refractile droplets of fat.   Since cats have fat droplets in their urine, cats with renal disease frequently have fatty casts in their urine.  Dogs with diabetes mellitus may have fatty casts in their urine.

 

Crystals in the urine may or may not be of clinical significance and their presence is usually recorded as occasional, moderate or many.  The kidney normally secretes crystal forming substances and when the urine becomes oversaturated with these substances, crystals “grow” and appear in the urine.  The oversaturation can occur in vivo or vitro.  Factor such as diet, drugs, pH and temperature of the sample affect the occurrence of crystals.  Samples which have been refrigerated tend to have more crystals than fresh, warm samples.  Samples that sit for a long period of time will develop crystals.  Following are brief descriptions of some crystals found in urine. 

Triple phosphate

Eight-sided prism generally seen in alkaline urine.  Resembles a “coffin lid”

Amorphous phosphate

Granular precipitate seen in alkaline urine

Calcium carbonate

Round with lines radiating from center.  Can also be “dumbbell shaped”.  Commonly seen in horses

Amorphous urate

Granular precipitate seen in acid urine

Ammonium biurate

Yellow-brown spheres with long spicules.  Look like thornapples.  Common with severe liver disease

Calcium oxalate

Small square with an X across the crystal.  May also be dumbbell shaped.  Seen in ethylene glycol (antifreeze) poisoning.  May indicate predisposition to uroliths (stones)

Bilirubin

Delicate amber or reddish brown needles.  More common in acidic urine

Leucine

Circular with a spoke wheel appearance.  Found with liver disease

Tyrosine

Looks like stick or haystack bundles.  Found with liver disease

Cystine

Six sided flat plates.  Associated with renal dysfunction or presence of uroliths.

 Triple phosphate, calcium oxalate and bilirubin cyrstals:

            triple phosphate.JPG (24404 bytes)  Triple phosphate  calcium oxalate.JPG (18485 bytes)  Calcium oxalte  Bilirubin crystal.JPG (16097 bytes) Bilirubin (sperm in field)

Other objects are frequently observed in urine sediment and their presence should be noted.  These include:

Sperm are frequently seen in voided samples of intact male animals.  There is no clinical significance to their presence.

Sperm.JPG (14523 bytes)    Sperm

Fat droplets are refractile spheres that vary in size.   If a slide sits for a few minutes, the droplets rise to the top of the sample and are located just under the coverslip, in a different plane of view than the other components on the slide

 lipid droplet.JPG (10951 bytes)     Fat droplet

Mucus threads look like a twisted ribbon.  They should not be confused with casts.  Mucus threads are common in horses.

 

 

Assignment: Due September 14, 2007 midnight

1.    Practice doing  a complete urinalysis under the guidance of your mentor.
2.    Once your are confident with your technique in doing a urinalysis, perform the tests on two samples and report your results on Lab Report #3
3     Email Dr. Durham a copy of your lab report #3