EXAMINATION OF URINE

Urine Formation
 Ultrafiltrate of plasma
 Approx. 170,000 mL of plasma = average urine output of 1200 mL / day
Urine Composition
 95% water and 5% solutes (organic and inorganic chemicals)
 Factors that affect concentration of solutes: dietary intake, physical activity, body metabolism, and endocrine
functions
 Organic substances: urea, creatinine and uric acid.
 Inorganic substances: Cl-, Na+, K+, trace amounts of phosphate, ammonium and Ca 2+
 Other substances: hormones, vitamins, and medications
 Formed elements: cells, casts, crystals, mucus, and bacteria
Urine Volume
 Amount excreted usually depends on body’s hydration state.
Factors that influence urine volume:
1. Fluid intake
2. Fluid loss from nonrenal sources
3. Variations in the secretion of antidiuretic hormone
4. Need to excrete increased amounts of dissolved solids (glucose or salts)
Urine Volume
 Normal output: usually 1200-1500 mL (600 to 2000 mL is normal)
Oliguria
 Decrease in urine output (<400mL/day in adults, <0.5 mL/kg/hr in children or <1 mL/kg/hr in infants)
 Commonly seen when the body becomes dehydrated as a result of vomiting, diarrhea, perspiration, or severe
burns.
Anuria
 Cessation of urine flow; kidney damage or decreased renal blood flow
Nocturia
 Increased excretion of urine during the night
Polyuria
 Increase in daily urine volume (>2.5 L /day in adults, >3mL/kg/day in children)
 Often associated with DM (↑sg) and DI (↓sg)
 Can be induced by diuretics, caffeine, or alcohol
Specimen Collection, Transport and Handling
 Pre-examination variables: test requests, patient preparation, timing, specimen collection, handling, and
storage
 Requisition forms should include the ff:
1. Actual date and time of collection
2. whether the specimen was refrigerated before transporting
3. time the specimen was received in the laboratory and the time the test was performed
4. tests requested
5. area for specific instructions
6. patient identification information (patient’s sex, age or date of birth, source of specimen, time of
collection)
 Patient preparation (fasting or elimination of interfering medications), type and volume of specimen,
containers (opaque, sterile)
 Examined within 2 hours

Specimen Collection
Containers:
 Clean, dry, leak-prrof containers
 Disposable containers
 Routine analysis: wide mouth and a wide flat bottom to prevent overturning
 Made of clear material
 50 mL capacity
 Microbiologic urine study: individually packed sterile containers with secured closures
 Sterile containers: if > 2 hours elapse time between collection and analysis
Labels
 Patient’s name and ID number
 Date and time of collection
  Labels must be attached to the container
Requisitions
 Request form must accompany the specimen when delivered in the laboratory
 Matched information
 Additional information: method of collection or type of specimen, possible interfering medications, and patient’s
clinical information

Specimen Rejection
1. Specimen in unlabeled containers
2. Nonmatching labels and requisition forms
3. Specimens contaminated with feces or toilet paper
4. Containers with contaminated exteriors
5. Specimens with insufficient quantity
6. Specimens that are improperly transported

Specimen Handling
Specimen Integrity
 Delivered to the lab and tested within 2 hours
Specimen Preservation
 Refrigeration (2-8°C) - routinely used method of preservation
 Urine culture – refrigerated during transit and kept refrigerated until cultured up to 24 hours
 Ideal preservative: bactericidal, inhibit urease, preserve formed elements in the sediment and should not
interfere with chemical tests (if samples are to be transported over a long distance)
 The specimen must return to room temperature before chemical testing by reagent strips.

Types of Specimens
Random specimen
 Most received specimen
 Easy to collect and convenient
 Collected any time
 useful for routine screening tests
 may also show erroneous results resulting from dietary intake or physical activity

First Morning Specimen

 Ideal screening specimen; concentrated
 Prevents false-negative pregnancy tests and evaluates orthostatic proteinuria
 Collected immediately after waking up and deliver within 2 hours

24-hour (Timed) Specimen

 used to produce accurate quantitative results
 24-hour collection: used to measure substances that changes with diurnal variations and with daily
activities
 Shorter period: concentration of substance remains constant
 begin and end the collection period with an empty bladder
 24-hour sample must be mixed thoroughly and refrigerated during collection period

Catheterized specimen
 collected under sterile conditions by catheter through the urethra into the bladder
 Most requested test is bacterial culture

Midstream Clean-Catch Specimen

 Alternative to catheterized specimen
 safer, less traumatic alternative to catheterized specimen
 Bacterial culture and routine urinalysis
 Less contaminated by epithelial cells and bacteria
 Appropriate cleansing materials (mild antiseptic towelettes), sterile container and instructions

Suprapubic Aspiration
 A needle is inserted through the abdomen and into the bladder
 Bacterial culture and cytologic examination
Prostatitis Specimen
Three-glass Collection
1. Area is cleansed using male midstream clean-catch procedure
2. 1st container – first urine passed
3. 2nd container – midstream
4. Prostate is massaged
5. 3rd container – prostate fluid and remaining urine
 Quantitative culture – 3 specimens
 Microscopic examination – 1st and 3rd specimens
 Prostatic infection – 3rd specimen >10x WBC/hpf and bacterial count than 1st specimen
 2nd specimen – control for bladder and kidney infection

Pre- and Post-massage Test (PPMT)

1. 1st specimen - clean-catch midstream urine specimen
2. Prostate is massaged
3. 2nd urine sample
 >10x bacteriuria in the post-massage sample

Pediatric Specimen
 Soft, clear plastic bags with hypoallergenic skin adhesive for routine urinalysis
 catheterization or suprapubic aspiration – sterile specimen
Physical Examination
Color
 Normal Urine Color: Yellow, pale yellow, or dark yellow
 Yellow color: Urochrome
 Intensity of the yellow color in the urine could give rough estimates of the urine concentration
 Pink: uroerythrin
 Orange brown: urobilin in urine that is not fresh
 Dark Yellow/Amber/Orange: bilirubin (suspected when yellow foam appears when shaken)
 White foam: indicated the presence of protein
 Yellow Green: Biliverdin
 Yellow Orange: urobilinogen, phenazopyridine or azo-gantrisin (UTIs)
 Red/Pink/Brown: presence of blood
 Brown: Hemogblobin  methemoglobin, glomerular bleeding
 Red: Hemoglubinuria, Hematuria, and Myoglobinuria,
 Port wine: prophyrins
 RBC: Red and Cloudy
 Hemogblobin/Myoglobin: Red and Clear
 Brown/Black melanin (melanoma) or homogentisic acid (alkaltonuria)
 Blue/Green: bacterial infections (UTI: Pseudomonas)
 Green: Clorets
 Purple: Klebsiella or Providencia species

Clarity
 Transparency or turbidity
 Normal Clarity
 Freshly voided urine: clear
 White cloudiness: Precipitation of amorphous phosphates and carbonates
 Non pathologic Turbidity
 Epithelial cells and mucus (female)
 Specimens allowed to stand or refrigerated
 Semen, fecal contamination, radiographic contrast media, talcum powder and vaginal creams
 Amorphous phosphates: White ppt
 Amorphous urates: pink brick dust (uroerythrin)
 Pathologic Turbidity
 RBCs, WBCs, and Bacteria (infection of systemic organ disorder)
 Non squamous epithelial cells, yeast, abnormal crystals, lymph fluid, and lipids
Specific Gravity
 SG of plasma filtrate: 1.010
 Isothenuric: SG=1.010
 Hyposthenuric: SG<1.010
 Hypersthenuric: SG>1.010
 Normal random Spx: ranges from 1.002 to 1`.035
 SG < 1.002: not urine

Specific Gravity: Urinometer

• Consists of a weighted float attached to a scale that has been calibrated in terms of urine specific gravity
• The weighted float has been designed to sink to 1.000 in distilled water.
• Dissolved substances cause the float to displace a volume of urine smaller than that of distilled water.
• Disadvantages:
1. Less accurate than other methods
2. Requires a large volume of specimen (10-15 mL)
3. Requires correction for temperature
• 20°C – calibration temperature
• Cold specimen: - 0.001 for every 3°C below calibration temperature
• +0.001 for every 3°C above calibration temperature
• Corrections must be made for glucose and protein
a. Glucose: - 0.004 per gram
b. Protein: - 0.003 per gram

Specific Gravity: Refractometer

• Used to determine the concentration of dissolved particles in a specimen by measuring refractive index
• Refractive index - comparison of the velocity of light in air with the velocity of light in a solution
• The concentration of dissolved particles present in the solution determines the velocity and angle at which light
passes through a solution.
• Advantage:
1. uses a small volume of specimen (1-2drops)
2. temperature corrections are not necessary (15-38°C)
• Corrections must be made for glucose and protein
o Glucose: - 0.004 per gram
o Protein: - 0.003 per gram
• Amount of glucose and protein is based on chemical reagent strip
• Calibrated using distilled water (1.000)
• Further checked with 5% NaCl (1.022±0.001) or 9% sucrose (1.034 ± 0.001)

Specific Gravity: Harmonic Oscillation Densitometry

• Frequency of a soundwave entering the solution changes in proportion to the density of the solution.
• Relative density is calculated as shifts in harmonic oscillation are measured.
• was used in early automated urinalysis instruments
1. Urine sample enters a U-shaped glass tube with electromagnetic coil at one end and motion
detector at the other end.
2. Electric current is applied to the coil; sound waves pass through the urine sample.
3. Microprocessor measures change in sound frequency, compensates for
temperature and converts reading to specific gravity.

Specific Gravity: Reagent Strip

• change in pKa (dissociation constant) of a polyelectrolyte in an alkaline medium
• Polyelectrolyte ionizes and releases H+ in proportion to the number of ions in the solution
• ↑ Concentration of urine = ↑ H+ released = ↓ pH
• Indicator: bromthymol blue
• Blue 1.000 (alkaline) – shades of green – yellow 1.030 (acid)

Odor
 Aromatic Normal
 Foul; Ammonia like Bacterial decomposition, UTI
 Fruity, sweet Ketones
 Maple syrup Maple Syrup Urine Disease
 Mousy Phenylketonuria
  Rancid Tyrosinemia
 Sweaty Feet Isovaleric acidemia
 Cabbage Methionine malabsorption
 Bleach Contamination
Chemical Examinations

pH
Reagent Strip Reactions
• Measures urine pH between pH 5 to 9
• double-indicator system:
1. methyl red – red to yellow (pH 4 to 6)
2. bromthymol blue – yellow to blue (pH 6 to 9)
• Methyl red + H+→ bromthymol blue – H+
(Red-orange → yellow) (green → blue)
• No known interfering substances

Protein
Reagent Strip Reactions
• Protein error of indicators
• Protein (albumin) accepts hydrogen ions from the indicator.
• Indicator: tetrabromophenol blue (Multistix) or 3',3",5',5"-tetrachlorophenol, 3,4,5,6-tetrabromosulfonphthalein
(Chemstrip)
• Yellow →green →blue
• Reaction interference: highly buffered alkaline urine, technical error
• False positive: rxn does not take place in acidic conditions, highly pigmented urine, contamination with
quaternary ammonium compounds, detergents, and antiseptics

Sulfosalicylic Acid Precipitation Test

• cold precipitation test that reacts with protein
• Procedure:
1. Add 3 mL of 3% SSA reagent to 3 mL of centrifuged urine.
2. Mix by inversion and observe for cloudiness.
3. Grade the degree of turbidity.

Testing for Microalbuminuria

• based on immunochemical assays for albumin or albumin-specific reagent strips that also measure creatinine
(albumin:creatinine ratio)
• Micral-test (Roche Diagnostics, Indianapolis, IN) and ImmunoDip (Sakisui Diagnostics, Framingham, MA)
Micral-Test
• contain a gold-labeled antihuman albumin antibody-enzyme conjugate
• The amount of color produced represents the amount of albumin present in the urine.

ImmunoDip
• Immunochromographic technique
• blue latex particles coated with antihuman albumin antibody
• Unbound and bound particles continue to migrate up the strip
• Two bands:
o 1st band – unbound particles;
 o 2nd band – urine albumin
• color intensity of both bands is compared against the manufacturer’s chart
• Darker bottom band: <1.2 mg/dL (negative)
• Equal band colors: 1.2 to 1.8 mg/dL (borderline)
• Darker top band: 2.0 to 8.0 mg/dL (positive)

Albumin: Creatinine Ratio

• permits an estimation of the 24-hour microalbumin excretion
• Albumin reading can be corrected for overhydration and dehydration in a random sample
• Reagent strip:
o Albumin – DIDNTB dye has higher sensitivity and specificity for albumin
o Creatine - pseudoperoxidase activity of copper-creatinine complexes CuSO4 + CRE → Cu (CRE)
peroxidase
o -10, 50, 100, 200, 300 mg/dL, or 0.9, 4.4, 8.8, 17.7, or 26.5 mmol/L of creatinine
Glucose
Glucose Oxidase Reaction
• Testing area: glucose oxidase, peroxidase, chromogen, and buffer
• Chromogen:
o potassium iodide (Multistix) – green to brown
o tetramethylbenzidine (Chemstrip) - yellow to green
• Interferences:
o False-positive: peroxide or strong oxidizing detergents
o False-negative: strong reducing agents (ascorbic acid), unpreserved sample

Copper Reduction Test (Clinitest)

• one of the earliest chemical tests performed on urine
• Glucose reduces copper sulfate to cuprous oxide in the presence of alkali and heat
• copper sulfate, sodium carbonate, sodium citrate, and sodium hydroxide
• Does not provide a confirmatory test for glucose
• Interferences: reducing sugars, including galactose, lactose, fructose, maltose,
pentoses, ascorbic acid, certain drug metabolites, and antibiotics
Ketones
• Sodium nitroprusside reaction
o Acetoacetate (and acetone) + sodium nitroprusside + (glycine) →purple color
• Interference:
• False-positive: Large amounts of levodopa and medications containing sulfhydryl groups
• False-negative: improperly preserved specimen
• Acetest tablet - confirmatory test
Blood
• pseudoperoxidase activity of hemoglobin

• Hemoglobin/myoglobin: yellow → green →green-blue

• Intact RBCs: speckled pattern
• Interference:
o False-positive – menstrual contamination, strong oxidizing agents, vegetable peroxidase, bacterial
enzymes
o False-negative – ascorbic acid, crenated RBCs, formalin preservative, captopril or high
concentrations of nitrite

Bilirubin
• Diazo reaction
Bilirubin glucuronide + diazonium salt → azodye
• Interference:
 o False-positive - urine pigments (phenazopyridine compounds), indican and metabolites of Lodine
• False-negative – specimen is not fresh (most frequent), hydrolysis of bilirubin diglucuronide produces
free bilirubin, ↑ ascorbic acid and nitrite
• Ictotest – confirmatory test
blue-to-purple color
Urobilinogen
Ehrlich’s aldehyde reaction (Multistix)
• light to dark pink
• Erhlich units (mg/dL)
• Normal readings: 0.2 – 1; abnormal readings: 2, 4 and 8
• Interference:
o False-positive - Ehrlich-reactive compounds (porphobilinogen, indican, p-aminosalicylic acid,
sulfonamides, methyldopa, procaine, and chlorpromazine compounds
o False-negative – improperly preserved specimen, formalin preservative

Azo-coupling (diazo) reaction (Chemstrip)

• white to pink
• more specific for urobilinogen
• Interference:
o False-negative - High concentrations of nitrite, improperly preserved specimen, formalin preservative

Nitrite
• Griess reaction
Para-arsanilic acid or sulfanilamide + NO2 → diazonium salt (nitrite)
Diazonium salt + tetrahydrobenzoquinolin → pink azodye
• Result: Negative or positive
• Interference:
o False-positive: improperly preserved specimens, highly pigmented urine
o False-negative: Nonreductase-containing bacteria, insufficient contact time between bacteria and
urinary nitrate, lack of urinary nitrate, large quantities of bacteria converting nitrite to nitrogen, presence
of antibiotics, high concentrations of ascorbic acid, high specific gravity

Leukocyte Esterase
• hydrolysis of an acid ester
• trace, small, moderate, and large or trace, 1+, 2+, and 3+
• Interference:
o False-positive: strong oxidizing agents or formalin
o False-negative: high concentrations of protein (>500 mg/dL), glucose (>3g/dL), oxalic acid and
ascorbic acid, high specific gravity