URINALYSIS

[Publish Date]

PHYSICAL EXAMINATION OF URINE

URINE COLOR

How to examine urine?

 Under a good light source, looking down through the container against a white background

 Pale yellow - Dilute urine

 Dark yellow - Concentrated

Urine Pigments:
 Urochrome
o Causes yellow color of urine
o Named by Thudichum in 1864. Urochrome
o Produced at a constant rate
o Dependent on the body’s metabolic state
 Increased amounts produced in patients with thyroid conditions
 Or those in fasting states.
o Also increases in urine that stands at room temperature

 Uroerythrin
o Pink pigment
o Most evident in refrigerated specimens
o Resulting in the precipitation of amorphous urates in an acid urine
o Uroerythrin attaches to the urates, giving a pink color to the
sediment

 Urobilin
o Imparts an orange-brown color to urine that is not fresh
o An oxidation product of urobilinogen

COLOR CAUSE CLINICAL/LABORATORY CORRELATIONS

Colorles Recent fluid Commonly observed with random specimens
s consumption
Polyuria or diabetes Increased 24-hour volume and low specific gravity
Pale insipidus
yellow Diabetes mellitus Elevated specific gravity and positive glucose test result
Dilute random specimen Recent fluid consumption

Concentrated specimen May be normal after strenuous exercise or in first morning specimen
Dehydration Fever or burns
Dark Bilirubin Yellow foam when shaken and positive chemical test results for bilirubin
yellow White Foam -Increase protein amount
Photo-oxidation of large amounts of excreted urobilinogen to urobilin also produces a
yellow-orange urine; however, yellow foam does not appear when the specimen is shaken
Acriflavine Negative bile test results and possible green fluorescence
Nitrofurantoin Antibiotic administered for urinary tract infections
B complex vitamins

Phenazopyridine Azo-gantrisin compounds

Orange- (Pyridium) Drug commonly administered for urinary tract infections
yellow Produce a yellow foam when shaken, which could be mistaken for bilirubin
Phenindione Anticoagulant, orange in alkaline urine, colorless in acid urine
Sulfasalazine Anti-inflammatory drug
(Azulfidine)
Yellow- Bilirubin oxidized to Colored foam in acidic urine and false-negative chemical test results for bilirubin
green biliverdin
Green Pseudomonas infection Positive urine culture
Asparagus. Vitamin B

Amitriptyline Antidepressant
Methocarbamol Muscle relaxant, may be green-brown
(Robaxin)
Blue- Breath deodorizers None
green (Clorets)
Indican Bacterial infections, intestinal disorders
Klebsiella or Providencia species
Methylene blue Fistulas
Phenol derivatives When oxidized
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Propofol Anesthetic
Familial hypercalcemia “Blue diaper syndrome”
Indomethacin (Indocin, Indo- Nonsteroidal anti-inflammatory drug
Tivorbex)
Amitriptyline (Elavil)
RBCs Cloudy urine with positive chemical test results for blood and RBCs visible
microscopically
Hemoglobin Clear urine with positive chemical test results for blood; intravascular hemolysis + RED
Pink PLASMA
Red Myoglobin Clear urine with positive chemical test results for blood; muscle damage + No change in
plasma color
Beets Alkaline urine of people who are genetically susceptible
Rifampin/ Rifampin - Tuberculosis medication
phenolphthalein,
phenindione, and
phenothiazines
Menstrual Cloudy specimen with RBCs, mucus, and clots
contamination
Blackberries Red color in acidic urine

Port Porphyrins Negative test for blood, may require additional testing
wine Oxidation of porphobilinogen to porphyrins
Red- RBCs oxidized to Seen in acidic urine after standing; positive chemical test result for blood
brown methemoglobin If seen in fresh urine - Glomerular bleeding
Myoglobin

Homogentisic acid Seen in alkaline urine after standing; specific tests are
(alkaptonuria) Available
Homogentisic acid, a metabolite of phenylalanine
Brown Melanin or melanogen Urine darkens on standing and reacts with nitroprusside and ferric chloride
Black Melanin is an oxidation product of the colorless pigment melanogen, which is produced in
excess when a malignant melanoma is present
Phenol derivatives Interfere with copper reduction tests
Argyrol (antiseptic) Color disappears with ferric chloride
Methyldopa or levodopa Antihypertensive
Metronidazole (Flagyl) Darkens on standing, intestinal and vaginal infections
Chloroquine and Antimalarial drugs
primaquine
Methocarbamol Muscle relaxant
Fava beans, rhubarb, or None
aloe
Nitrofurantoin [Furadantin]); laxatives - cascara or senna;
Liver and kidney disorders and muscle injury from extreme exercise

CLARITY
 Refers to the transparency or turbidity of a urine specimen.
 Reporting clarity:
Urine Clarity
Clear No visible particulates, transparent
Hazy Few particulates, print easily seen
through urine
Cloud Many particulates, print blurred through
y urine
Turbid Print cannot be seen through urine
Milky May precipitate or be clotted

o Clear
o Hazy
o Cloudy
o Turbid
o Milky
 Normal urine - usually clear, particularly if it is a clean-catch midstream specimen.
 White cloudiness in an alkaline urine
o Precipitation of amorphous phosphates and carbonates

Nonpathological Causes of Urine Turbidity Pathological Causes of Urine Turbidity

1. Squamous epithelial cells 1. RBCs
2. Mucus 2. WBCs
3. Bacterial growth in improperly preserved 3. Bacteria
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urine 4. Yeast
4. Amorphous phosphates, carbonates 5. Trichomonads
 White precipitate in Alkaline urine 6. Nonsquamous epithelial cells
5. Amorphous Urates 7. Abnormal crystals
 Acidic Urine 8. Lymph fluid
 Resembles pink brick dust due to the  Chyluria
presence of uroerythrin. o Urine contains lymph fluid
6. Semen, spermatozoa o Associated with lymphatic
7. Fecal contamination obstruction: Filariasis
8. Radiographic contrast media - Acidic (W.broncrofti infections)
urine Tumors
9. Talcum powder 9. Lipids
10. Vaginal creams  Lipiduria - fat globules in urine
o Nephrotic syndrome
o Skeletral trauma, with
fractures to major long
bones and pelvis
 Contaminants
o Paraffin wax

SPECIFIC GRAVITY
 Defined as the density of a solution compared with the density of a similar volume of distilled water (SG 1.000) at a similar temperature
 Indicator of concentration of dissolved material in the urine
 Influenced by the number of particles present and by their size
 Terms
o Isosthenuric : specific gravity of 1.010
o Hyposthenuric : < 1.010
o Hypersthenuric : > 1.010

 Normal random values:

o Specific gravity of the plasma filtrate entering the glomerulus: 1.010
o Random: ~ 1.002 to 1.035
 Most random specimens fall between 1.015 and 1.030.
o Not urine: < 1.002
o Most random specimens fall between 1.015 and 1.030.

Current Urine Specific GravityMeasurements

Method Principle
Refractometry Refractive index Indirect
Reagent strip pKa changes of a polyelectrolyte by ions Indirect
present
Osmolality Changes in colligative properties by
particle number
Urinometry Based on the density of the solution Direct
Harmonic Frequency of a sound wave entering a Direct
Oscillation solution changes in proportion to the
Densitometry density of the solution

1. REFRACTOMETER
 Refractive index is a comparison of the velocity of light in the 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.
 Uses a prism to direct a specific (monochromatic) wavelength of daylight against a manufacturer-calibrated scale of specific gravity.
o The concentration of the specimen determines the angle at which the light beam enters the prism.
o Specific gravity scale is calibrated in terms of the angles at which light passes through the specimen

 Advantage
o Volume of specimen (one or two drops)
o Temperature corrections are not necessary because the light beam passes through a temperature-compensating liquid before
being directed at the specific gravity scale.
o Temperature is compensated between 15°C and 38°C.

 Corrections for glucose and protein:

o Subtracting 0.003 for each gram of protein
o Subtracting 0.004 for each gram of glucose
o The amount of protein or glucose present can be determined from the
chemical reagent strip tests.
 Calibration
o Distilled : reading of 1.000.
o 5% NaCl : 1.022 ± 0.001
o 9% sucrose : 1.034 ± 0.001.
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 Abnormally high—above 1.040:

o Who have recently undergone an IV pyelogram caused by the excretion of the injected radiographic contrast media.
o Who are receiving dextran or other high- molecular-weight IV fluids (plasma expanders)
o Can be measured using the reagent strip chemical test or osmometry because they are not affected by these high-molecular-
weight substances

2. REAGENT STRIP SPECIFIC GRAVITY

 The polyelectrolyte ionizes, releasing hydrogen ions in proportion to the number of ions in the solution.
 The higher the concentration of urine, the more hydrogen ions are released, thereby lowering the pH.
 Because ions, such as Na+, Cl-, and NH-4+ are important in evaluating renal concentrating ability, the reagent strip method provides
additional information and is not affected by nonionizing substances, including urea, glucose, protein, and contaminating substances such
as radiographic dye
 concentrating ability, the reagent strip method provides additional information and is not affected by nonionizing substances, including
urea, glucose, protein, and contaminating substances such as radiographic dye.
 Indicator: Bromothymol blue on the reagent pad measures the change in pH.
o As the specific gravity increases, the indicator changes from blue (1.000 [alkaline]), through shades of green, to yellow (1.030
[acid]).
o Readings can be made in 0.005 intervals by careful comparison with the color chart.

3. OSMOLALITY
 Principles : Freezing- point and vapor pressure osmometers
 Osmolality is affected only by the number of particles present.
 Values:
o Sodium (molecular weight 23)
o Chloride (molecular weight 35.5)
o Urea (molecular weight 60)
 An osmole is defined as 1 g molecular weight of a substance divided by the number of
particles into which it dissociates.
o Glucose (molecular weight, 180), contains 180 g per osmole
o Sodium chloride (NaCl) (molecular weight 58.5)
o If completely dissociated, contains 29.25 g per osmole.

4. URINOMETERY
 Urinometer consists of a weighted float attached to a scale that has been calibrated in terms of urine specific gravity.
 Weighted float displaces a volume of liquid equal to its weight and has been designed to sink to a level of 1.000 in distilled water.
 The additional mass provided by the dissolved substances in urine causes the float to displace a volume of urine smaller than that of
distilled water.
 The level to which the urinometer sinks, as shown in the figure, represents the specimen’s mass or specific gravity.
 Disadavantage:
o Less accurate than the other methods
o Not recommended by the Clinical and Laboratory Standards Institute (CLSI).
o Requires large volume of specimen ( 10 to 15 ml)

ODOR
 Seldom of clinical significance and is not a part of the routine urinalysis
 Freshly voided urine has a faint aromatic odor.
 As the specimen stands, the odor of ammonia becomes more prominent.
 The breakdown of urea is responsible for the characteristic ammonia odor

POSSIBLE CAUSES OF URINE ODOR

Aromatic Normal
Foul, ammonia- Bacterial decomposition,
like urinary tract infection
Fruity, sweet Ketones (diabetes
mellitus, starvation,
vomiting)
Maple syrup Maple syrup urine
disease
Mousy Phenylketonuria
Rancid Tyrosinemia
Sweaty feet Isovaleric academia
Cabbage Methionine
malabsorption
Bleach Contamination
Unusual or Ingestion of onions,garlic
Pungent and asparagus
Rotting Fish Trimethylaminuria
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CHEMICAL EXAMINATION OF URINE

 Two major types of reagent strips:
o Multistix (Siemens Healthcare Diagnostics, Inc., Tarrytown, NY)
o Chemstrip (Roche Diagnostics, Indianapolis, IN)

 PRINCIPLE OF THE REAGENT STRIP

o Consist of chemical-impregnated absorbent pads attached to a plastic strip
o A color - producing chemical reaction takes place when the absorbent pad comes in contact with urine
o Reactions are interpreted by comparing the color produced on the pad with a chart supplied by the manufacturer
o By careful comparison, results are described as TRACE, 1+, 2+, 3+ or 4+

 CARE OF THE REAGENT STRIP

o Store with desiccant in an opaque, tightly closed container
o Stored at room temperature below 30°C (but never refrigerated)
o Do not expose to volatile fumes
o Do not use past the expiration date
o Do not use if chemical pads become discolored
o Remove strips immediately prior to use

 QUALITY CONTROL
o Test open bottles of reagent strips with known positive and negative control every 24 hr
o Resolve control results that are out of range by further testing
o Test reagents used in backup tests with positive and negative controls
o Perform positive and negative controls on new reagents and newly opened bottles of reagent strips
o Record al control results and reagent lot numbers

pH
 DETERMINED BY THE CONCENTRATION OF THE FREE H+
o As H+ increases, pH decreases (becomes more acidic)
o As H+ decreases, pH increases (becomes more alkaline)
Normal Urine pH:
 First morning specimen: 5.0 to 6.0;
 Random specimens: 4.5 to 8.0
 With normal protein diet: 5.0 - 6.0
 After meals (alkaline tide): More alkaline pH
 Unpreserved: >8.5
 Contaminated: <4.0

Clinical Significance
 Systemic acid–base disorders of metabolic or respiratory origin
 Management of urinary conditions
 Respiratory or metabolic acidosis not related to renal function disorders: Acidic
 Respiratory or metabolic alkalosis is present: Alkaline
 Renal calculi formation
 Precipitation/ identification of crystals
 Determination of unsatisfactory specimen
 Treatment of UTI

DIAGNOSTIC METHODS
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Reagent Strip Method - Principle: Double indicator system of methyl red and bromthymol blue
- Methyl red at pH range 4.4 to 6.2 >>> red to yellow
- Bromthymol blue at pH range 6.0 to 7.8 >>> yellow to blue
- Nitrazine paper or Litmus paper - measures pH only
Strips Reagents Sensitivity
Multistix Methyl Red pH 5 - 9
Bromthymol Blue
Chemstrip Methyl Red pH 5 - 9
Bromthymol Blue
Phenolphthalein

pH Electrode - pH meter with glass electrode

- Used in patients with disturbance of acid - base balance
Titratable Acidity of - Measured by titrating an aliquot of 24 - hour urine with 0.1N NaOH with pH7.4 as endpoint
Urine - Normal titratable acidity:
o 200 to 500 mL 0.1 N NaOH
o 20 to 40 mEq/24h or 6 mL 0.1NaOh/kg BW

Note:
 Collecting specimens in containers other than the single-use laboratory-supplied containers can produce a pH above 8.5 if alkaline detergent
remains in the container.
 Care must be taken to prevent runover between the pH testing area and the adjacent, highly acidic protein testing area on Multistix, as this may
produce a reading that is falsely acidic in an alkaline urine.

PROTEIN
 Most indicative of renal disease
 Normal urine protein :
o < 10 mg/dL or 100 mg per 24 hours
o 150mg/24hrs (Henrys)
 Consists primarily of Low molecular weight proteins:
o Albumin - major serum protein found in normal urine.
o Small amounts of serum and tubular microglobulins
o Tamm-Horsfall protein (THP):
 A.k.a uromodulin
 Produced by the renal tubular epithelial cells
 A glycoprotein, is produced routinely in the ascending loop of Henle
 Forms the matrix of casts.
o Proteins from prostatic, seminal, and vaginal secretions.
 Quantification of proteinuria:
o Heavy proteinuria: > 4 g/day
o Moderate proteinuria: 1.0 to 4.0 g/day
o Minimal proteinuria: < 1.0 g/day

CLINICAL SIGNIFICANCE
 Clinical proteinuria: 30 mg/dL or greater (300 mg/L)
 Three major categories:
o PRERENAL or OVERFLOW
o RENAL
o POSTRENAL

I. PRERENAL PROTEINURIA
o Caused by conditions affecting the plasma before it reaches the kidney
o NOT indicative of actual renal disease
o NOT detected by reagent strip
o Transient
o As soon as the level of plasma proteins returns to normal, the proteinuria resolves
o Caused by increased levels of low-molecular- weight plasma proteins:
 Hemoglobinuria - after a hemolytic episode
 Myoglobinuria - follows muscle injury
 Acute-phase reactants - infections and inflammations
 Bence Jones Protein
 Seen in multiple myeloma (Proliferative disorder of the immunoglobulin-producing plasma cells)
 Contains levels of monoclonal immunoglobulin light chains that are markedly elevated.
 Suspected cases: perform serum electrophoresis and immunoelec- trophoresis.
 Screening test for Bence Jones protein is not performed routinely, as cases of multiple myeloma are easily
detected by chemical methods
 Screening Test for Bence Jones Protein:
o Coagulates between 40°C and 60°C
o Dissolves at 100°C
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oInterference due to other precipitated proteins can be removed by filtering the specimen at 100°C and
then observing the specimen for turbidity as it cools to between 40°C and 60°C.
 NOT all patients with multiple myeloma will excrete detectable levels of BJP and suspected cases should be
diagnosed by performing serum ELECTROPHORESIS & IMMUNOELECTROPHORESIS

II. RENAL PROTEINURIA

 Associated with true renal disease
 Results to increase excretion of albumin

Glomerular  Occurs in primary glomerular diseases or disorders that cause glomerular damage
Proteinuria  Major Causes:
o Amyloid material
o Toxic substances
o Immune complexes (Lupus erythematosus and streptococcal glomerulonephritis)

 Reversible causes:
o Strenuous exercise
o Dehydration
o Hypertension/ Preeclamptic state

 Benign proteinuria: (Not pathological, Transient)

o Strenuous exercise
o High fever
o Dehydration
o Exposure to cold

Microalbuminur  Indicator of early glomerular damage

ia  Proteinuria NOT detected by the routine reagent strip
 Signifies onset of renal complications of Diabetic nephropathy (reduced glomerular filtration)
 Albumin levels in the urine are 20 to 200 mg/L
 Increased risk of cardiovascular disease
 Historical testing:
o 24-hour urine for quantitative procedures
o 30 to 300 mg/24 hours
o AER 20 to 200 µg/min (Albumin excretion rate)
Orthostatic  Occurs frequently in young adults
(Postural)  Occurs after periods spent in a vertical posture and disappears when a horizontal position is
Proteinuria assumed.
 Usually caused by Increased pressure on the renal vein when in the vertical position
 Suspected patients are requested to empty the bladder before going to bed, collect a specimen
immediately upon arising in the morning, and collect a second specimen after remaining in a
vertical position for several hours.
o First morning specimen - Negative
o Second specimen: positive result

Tubular  Albumin that is normally filtered can no longer be reabsorbed.

Proteinuria  Causes:
o Toxic substances
o Heavy metals
o Severe viral infections
o Fanconi syndrome.
 Protein that appears in the urine after glomerular damage ranges from slightly above normal to
4 g/day
 Protein levels that are markedly elevated are seldom seen in tubular disorders.

III. Postrenal Proteinuria

 Protein can be added to a urine specimen as it passes through the structures of the lower urinary tract (ureters, bladder, urethra, prostate,
and vagina)
 Seen in:
o Lower UTI (ureters, bladder, urethra, prostate and vagina)
o Injury/trauma
o Menstrual contamination
o Prostatic fluid/ spermatozoa
o Vaginal secretions
 Inflammations produce exudates containing protein from the interstitial fluid.
 Presence of blood:
o Injury
o Menstrual contamination contributes protein
 Presence of prostatic fluid
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 Large amounts of spermatozoa.

DIAGNOSTIC METHODS

Reagent Strip Principle: Protein error of indicators

Reactions Test is more sensitive to albumin

Strips Multistix Chemstrip

Sensitivity 15 to 30 mg/dL Albumin 6 mg/dL Albumin
Reagents Tetrabromphenol 3′,3″,5′,5″-tetrachlorophenol, 3,4,5,6-
tetrabromosulfonphthalein
False Positive - Highly buffered alkaline urine
- Highly pigmented urine (phenazopyridine)
- Contamination of the container with quaternary ammonium
compounds, detergents, and antiseptics
- Urine that is visibly bloody
- High specific gravity.
False Negative - Proteins other than albumin

Sulfosalicylic Acid - A cold precipitation test that reacts equally with all forms of protein.
Precipitation Test - May be performed as a confirmatory test in certain situations.
(SSA) - Principle: Precipitation test
- Reagents: 3% SSA
- Sensitivity: Detects 5 to 10 mg/dL of proteins (Albumin, globulins, glycoproteins and Bence Jones protein)
- Procedure: Add 3 ml of 3% SSA reagent to 3 ml of centrifuged urine; Mix by inversion and observe for
cloudiness
- False positive:
o Phosphates and urates
o Drug metabolites: Tobutamide, sulfonamide, high dose penicillin, cephalothin and
chlorpromazine
o Abundant red blood cells or white blood cells
- False negative:
o Highly buffered alkaline urine

Grade Turbidity Protein Range

Negative No increase in turbidity Less than 6
Trace Noticeable turbidity 6–30
1+ Distinct turbidity, no granulation 30–100
2+ Turbidity, granulation, no 100–200
flocculation
3+ Turbidity, granulation, flocculation 200–400
4+ Clumps of protein

NOTE:
 The specific gravity of the urine specimen should be considered in evaluating urine protein, because a
trace protein in a dilute specimen is more significant than in a concentrated specimen.
 SSA (+) & RGT (+) Strip = Presence of ALBUMIN
 SSA (+) & RGT (-) Strip = Presence of proteins OTHER THAN ALBUMIN
Testing for o Usually detects small amounts of albumin and β2 macroglobulinemia
microalbuminuria
IMMUNOLOGIC METHOD - use antibodies against the protein
o Micral-Test (Roche Diagnostics, Indianapolis, IN)
o Semiquantitative method
o Strips contain a gold-labeled antihuman albumin antibody–enzyme conjugate
o Principle: Enzyme Immunoassay
o Sensitivity: 0 -10 mg/dL
o Reagents: Gold -labeled Ab galactosidase; Cholorphenol galactosidase
o Interference: False (-) in dilute urine
o Procedure: Strip dipped into urine and held for 5 seconds

o ImmunoDip (Sekisui Diagnostics, Framingham, MA)

o Principle; Immunochromographics
o Sensitivity: 1.2 to 8.0 mg/dL
o Reagent: ab - coated blue latex particles
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o Interference: False (-) in dilute urine

o Top band represents the bound particles (urine album
o Bottom band represents unbound particles.
o Darker bottom band;
 Represents less than 1.2 mg/dL
 Negative
o Equal band colors:
 Represent 1.2 to 1.8 mg/dL
 Borderline
o Darker top band:
 Represents 2.0 to 8.0 mg/dL of albumin
 Positive results.

o Albumin:Creatinine Ratio
Clinitest Microalbumin Strips/Multistix - Pro
(Clinitek microalbumin (Bayer Diagnostics,
Tarrytown, NY)
- Highly sensitive dye - binding
method
- Principle: Sensitive albumin tests
related to creatinine concentration to
correct for patient hydration
- Sensitivity:
 Albumin: 10 - 150 mg/dL
 Creatinine: 10 - 3000
mg/dL, 0.9 - 26.5 mmol/L
o Significant if Albumin: Creatinine
is >3.4 mg/mmol

o Advantage; With additional pad for

simultaneous measurement of creatinine
o Disadvantage: Not specific for albumin, dye
can bind to Tamm - Horsfall Mucoprotein

NEPHELOMETRIC METHOD

RADIOIMMUNOASSAY

Quantitative Protein o Uses 24-hour specimen

Determination o 30 - 300 mg/24 hrs
o Albumin excretion Rate (AER) f 20 to 200 ug/min

Precipitation:
o Precipitants;
 Sulfosalicylic acid (SSA) - poor precision
 Trichloroacteic acid (TCA) - use Biuret Reaction
o Resultant precipitate measured by: Photometer or nephelometer

Colorimetric;
o Coomasie blue method
o Ponceau S method
o Benzethonium chloride turbidity method (Dupont - ACA)
o Pyrogallol Red - Molybadate (Dade - Dimension)
Bence Jonce Protein  Heat and Acetic Acid Test
Determination o Bence Jones protein precipitate at temperature between 40 to 600C, and redisolves at 1000C
(Precipitation in the cold with salts, ammonium sulfate, and acid)
o Principle: Urine is coagulated by heat and precipitated by acetic acid (5-10%) and the degree of
turbidity produced is proportional to the amount of protein present
o Positive results:
 1+ = Diffuse cloud
 2+ = Granular cloud
 3+ = Distinct floccule
 4+ = Large Floccule, dense, something solid
 Toluene Sulfonic Acid (TSA) Test
 SSA Test/Exton Test (+) result
 Osgood Haskin (+) result
 Other Tests:
o Bradshaw - HCL; precipitation
o Putnam et al - 2M acetate buffer; precipitation
o Jacobson - Milner - Concentrated HNO3 and 25% Acetic Acid: precipitation
o Electrophoresis
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 Best method for detection

 Presence of either κ or γ light chains
 Methods:
 Amido Black Stain
 Coomasie brilliant blue stain
 Other tests for Albumin:
1. Esbach’s Test (Tsuchya’s Modification - Use Picric Acid and Ascorbic Acid)
2. Shevky and Staffords
3. Kingsbury’s Test
4. Kingsbury - Clark
5. Purdy’s

GLUCOSE

 Most frequent chemical analysis performed on urine

 Clinical significance:
o Detection of DIABETES MELLITUS
o Mellituria - presence of ant sugar (reducing or non-reducing) in urine
o Glycosuria - presence of any reducing sugar in the urine specimen
 Glucose, Lactose, Galactose, Fructose, Pentose
 RENAL THRESHOLD FOR GLUCOSE: 160 to 180 mg/dL
 Normal Urine Glucose: 15 mg/dL
 Fasting: 2 to 20 mg /dL per 100 mL urine

Glucosuria with Hyperglycemia Glucosuria without Hyperglycemia/Renal-

Associated
**Generally due to “overflow disorders” ** Generally results from accumulation of the renal
INCREASE BLOOD GLUCOSE type - caused by malfunctions in the tubular
INCREASE URINE GLUCOSE reabsorption mechanism
NORMAL BLOOD GLUCOSE
- Diabetes mellitus INCREASE URINE GLUCOSE
- Pancreatitis
- Pancreatic cancer - Fanconi syndrome
- Acromegaly - Nephrotic Syndrome
- Cushing syndrome - After nephorotic toxicity (Carbon monoxide,
- Hyperthyroidism lead, mercuric chloride)
- Pheochromocytoma - Advanced renal disease
- Central nervous system damage - Osteomalacia
- Stress - Pregnancy
- Gestational diabetes
- After certain types of anesthesia such as ether, and drugs
(thiazides, corticosteroids and adrenocorticotropic hormone)
- After myocardial infarction (occasionally and transient)
- Alimentary glucosuria
- Increase intracranial pressure (tumors, intracerebral hemorrhage,
skull fractures)

DIAGNOSTIC METHODS

Reagent Strip - GLUCOSE OXIDASE METHOD

- Test is based on a specific glucose oxidase and peroxidase method , a double sequential enzyme reaction

REAGENTS STRIPS DIFFER IN THE CHROMOGEN USED

Strips Chromogen Color Change/Comments
CLINISTEX O - toluidine Pink to purple
Detects 100 mg/dl of glucose/more sensitive to
interfering substance such as ascorbic acid
MULTISTIX Potassium iodine Green to brown at 30 seconds
CHEMSTRIP Aminopropyl - Blue to brown at 60 seconds
carbazol
CHEMSTRIP Tetramethylbenzidine Reagent pads have different sensitivities to urine
uGc glucose, ranging from 60 mg/dl to 5 g/dl.
The strips are read at 2 to 3 minutes
Yellow to green
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- Advantage;
o The glucose oxidase method is specific for glucose
o It does not react with lactose, galactose, fructose, or reducing metabolites of drugs
- Sources of errors;
o False positive
 Strongly oxidizing cleaning agents in the container (Hydrogen peroxide and
hypochlorites)
 Reducing sugars
o False negative
 Use of sodium fluoride as preservative
 Ascorbic acid
 Technical error of allowing specimens to remain unpreserved at room temperature for
extended periods, subjecting the glucose to bacterial degradation.
 High specific gravity and low temperature

Benedict’s Test - General test for glucose and other reducing sugars
- Reagent: Benedict’s Solution
- Principle: Relies on the ability of the glucose and other reducing substances to reduce copper sulfate to
cuprous oxide in the presence of alkali and heat
- RESULTS:
o Negative - clear blue, blue precipitate may form
o Trace - bluish - green color
o 1+ - green color, green or yellow precipitate
o 2+ - yellow to green color, yellow precipitate
o 3+ - yellow - orange color, yellow - orange precipitate
o 4+ - reddish - yellow color, brick red or red precipitate

Copper reduction - The test relies on the ability of glucose and other substances to reduce copper sulfate cuprous oxide in the
Test presence of alkali and heat

CLINITEST (REAGENT STRIP)

- Non - specific for glucose
- Based on the qualitative BENEDICT TEST (1909) that contained copper sulfate, sodium carbonate, and
sodium citrate buffer.
- Sensitivity: Minimum level of 200 mg/dl
- Regents:
o COPPER SULFATE - main reacting agent
o SOSIUM CARBONATE AND CITRIC ACID - effervescent
o SODIUM HYDROXIDE - provides alkaline medium
o Sodium Hydroxide with water and citric acid - provides heat
- 5 gtts urine + 10 gtts of H2O + Clinitest tablet then wait for 15 seconds
- False Positive:
o Non - glucose reducing sugars - Galactose, lactose, fructose, maltose and pentose
o Strong reducing substances - Ascorbic and gentisic acid or homogentisic acid
o Drugs give false positive results or unusual colors with clinitest, especially cephalosporins and
radiographic media

COPPER REDUCTION TABLET TEST

- Detects 150 to 250 mg glucose per deciliter of urine
- Copper sulfate, sodium hydroxide, sodium carbonate, and citric acid are incorporated into the tablet

**PASS THROUGH PHENOMENON:

- Occurs at very high glucose levels
- May cocur if > 2 g/dL sugar is present in urine
- Prevented by changing 5gtts to 2 gtts of urine
- Color produced passes through the orange stage and returns to a blue or blue green color. Final color
doesn’t compare with section of the color chart.; However, it corresponds most closely to a siginificantly
lower result.
- Five drop method
o Place five drops of urine in a dry test tubes and add 10 drops of water.
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o Add one clinitest tablet and immediately compare color change to the color scale
o Results correspond to the following approximately concentrations:
 Negative: 0.25 g/dl, 0.5 g/dl, 0.75 g/dl, 1.0 g/dl, 2.0 g/dl
o Pass through - it indicates that more than 2 g/dl sugar is present and this should be reported as
more than 2 g/dl.
- Two - drop method
o Report results as 1 g/dl, 2 g/dl, 3 g/dl, 5 g/dl, and more than 5 g/dl - if “pass through” reaction
occurs

CLINITEST RGT STRIP RESULT

+ + Presence of GLUCOSE
+ -- Presence of NON - GLUCOSE
REDUCING SUGAR
-- 1+ Presence of SMALL AMOUNT OF
GLUCOSE
-- 4+ FALSE - POSITIVE REACTION

SUMMARY OF GLUCOSE OXIDASE AND CLINITEST REACTIONS

Glucose Oxidase Clinitest Interpretation
Positive Positive Glucose present
1+ Negative Small amount of glucose present
4+ Negative Possible oxidizing agent interference
Negative Positive Possible oxidizing agent interference

OTHER TESTS FOR URINE GLUCOSE

Fehling’s Test Reagent A:

- Cupric Sulfate
- Distilled water
Reagent B:
- Rochelle Salt (Sodium - Potassium Tartrate, KOH, Distilled water)

(+) YELLOW PRECIPITATE

Phenyldydrazine/ Osazone Reagent:

Test - Phynylhydrazine
- Hydrochloride
- Sodium acetate

(+) CRYSTAL FORMATION (PHENYLGLUCOSAZONE)

Nylander’s Test Reagent:

- KOH or NaOH
- CUSO4

(+) YELLOW PRECIPITATE (CUPROUS HYDROZIDE)

Moore - Hellers Reagent: KOH

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NOTE: Keep in mind that table sugar is sucrose, a nonreducing sugar, which does not react with Clinitest or glucose oxidase strips. Therefore, it
cannot be used as a control or in preparation of a laboratory exercise for glucose testing

OTHER SUGARS IN URINE

Sugars Disorder Screening Test Confirmatory Test

Galactose Deficiency of galactose - 1 - phosphate - Glucose oxidase - Thin - layer chromatography
(most uridyl - transferase or galactokinase - Copper reduction test (urine)
significant) - Erythrocyte enzyme assay
Lactose Normal pregnancy or during lactation. - Lactose test: Add 3 g Lead Thin - layer chromatography
Deficient enzyme lactase acetate to 15 ml urine. Shake
and filter. Boil filtrate, add 2
ml concentrated NH4OH, and
boil.
- Lactose: brick - red solution,
red precipitate with clear
supernatant
- Glucose: Yellow solution and
precipitate
Fructose Parenteral Feedings - Glucose oxidase Thin - layer chromatography
Enzyme deficiency - Copper reduction test
- Resorcinal Tests
Pentose Large amount of fruit; exretion of L - - L - xylulose, at concentration Thin - layer chromatography
xylose and L - arabinose in amounts up of 250 to 300 grams reduces
0.1g/day Benedict’s reagent at 500C
(water bath) within 10 minutes
or at RT for several hours
Sucrose Sucrase deficiency a - dextrinase - Glucose oxidase Thin - layer chromatography
(isomaltase) - Copper reduction test

KETONES
 Normal Urine Ketone: NORMALLY NOT IN URINE
 Metabolized fats are completely broken down into CO2 and H2O in normal individuals
 Represents three intermediate products of increased fat metabolism due to abnormal utilization:
o Acetone (2%)
o Acetoacetic acid (20%)
o β-hydroxybutyrate (78%)
 Both acetone and β-hydroxybutyrate are produced from acetoacetic acid
 Most valuable in monitoring insulin - dependent (type 1) diabetes mellitus

CLINICAL SIGNIFICANCE OF URINE KETONES

 Diabetic acidosis
 INSULIN DOSAGE MONITORING
 Starvation
 Malabsorption/pancreatic disorders
 Cold exposure
 Strenuous exercise
 Vomiting
 Inborn errors of amino acid metabolism (see Chapter 9) Alcoholism
 Febrile state in children
 Diabetic Ketonuria
o Implies presence of ketoacidosis
o Provide a warning for impending coma in Type I DM
 Nondiabetic Ketonuria
o Usually seen in children and infants with:
 Acute febrile illness
 Toxic states accompanied by vomiting or diarrhea
 Cachexia
 Following anesthesia
 Hyperemesis of pregnancy
 Lactic acidosis
o Coexists with other conditions; Shock, DM, renal failure, liver disease, and response to drugs (phenformin and salicylates)
 Complications:
o Electrolyte imbalance
o Dehydration
o Acidosis
o Diabetic coma
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DIAGNOSTIC METHODS

DIACETIC ACID/ ACETOACETIC ACETONE β - HYDROXYBUTYRIC ACID

ACID
- Gerhard’s Test - Rothera’s Test - Hart’s Test
- Rothera’s Test - Legal’s Test - Orberg
- Acetest - Frommer’s Test
- Lange’s Test
- Acetest

*** Not routinely performed nowadays

Reagent Strip - Principle: Based on sodium nitroprusside reaction (Nitroferricyanide) (Legal’s Test) for
Reactions ketones
- Does not react with 3 - hydroxybutyrate acid
- Reagent strips without alkali reacts to acetoacetic acid and not to acetone

MULTISTIX
- Detects 5 to 10 mg/dL acetoacetic acid only
- Reagents:
o Sodium nitroprusside and buffers
o Acetoacetic acid + sodium
nitroprusside  Pink maroon

CHEMSTRIP
- Detects about 10 mg/dL of acetoacetic
acid and 70 mg/dL of acetone
- Reagents:
o Sodium nitroprusside, glycine,
disodium phosphate and
lactose
o Acetoacetic acid + sodium
nitroprusside + glycine +
Acetone  Purple color

REPORT SEMIQUANTITA QUALITATIVE

TIVE
Negative
Trace 5 mg/dL
Small 15 mg/dL 1+
Moderate 40 mg/dL 2+
Large 80 to 160 mg/dL 3+

- Advantage over reagent strips:

o It can be used to assay whole blood, plasma, and urine
- Sources of errors:
o False positive:
 Improperly timed reading of reagent strip
 Large of phenylketones
 Medications containing sulfhydryl groups:
 Mercaptoethane sulfonate sodium (MESNA)
 Captopril
 Levodopa
 Phthalein dyes: Phenolsulfonphthlein (PSP) and Bromsulphthalein (BSP)
o False negative:
 Volatilization of acetone
 Improperly preserved specimens

Nitroprusside Tablet - Confirmatory for questionable reagent strip results

Test - Not relevant to current laboratory practice
- Does not react with 3 - hydroxybutyrate acid

ACETEST (Bayer diagnostics. Elkhart, IN)

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- Can be used to test urine, serum, plasma or whole blood

- About 10x more sensitive to DIACETIC ACID than ACETONE
- Should be read within 30 seconds
- Detects:
o 5 to 10 mg/dL of acetoacetic acid
o 20 to 25 mg/dL of acetone
- Reagents:
o Sodium nitroprusside, glycine, disodium phosphate and lactose
o Lactose - for better color differentiation
o Acetoacetic acid + sodium nitroprusside + glycine + acetone  Purple color

Tube Tests (Other  Rothera’s test

tests) o Detects acetoacetic acid and acetone
o Modified sodium nitroprusside
o Detects acetoacetic acid and acetone
 Gerhardt’s test
o Detects acetoacetic acid
o Based on the reaction of FERRIC CHLORIDE with DIACETIC ACID to form a PORT
WINE or BORDEAUX RED COLOR
o Acetoacetic acid + Ferric chloride  Red color
 Hart’s test
o INDIRECT METHOD for Detecion of beta - hydroxybutyric acid
o Β - hydroxybutyric acid is converted to acetone through the addition of H2O2 then
acetone is tested by sodium nitroprusside reaction
o Ketones ---BOILING---> Acetoacetic acid and acetone
o Ketones ---BOILING---> Beta - hydroxybutyric acid + Hydrogen peroxide

BLOOD
 Normally, NO BLOOD IN THE FORM OF HEMATURIA, HEMOGLOBINURIA OR MYOGLOBINURIA SHOLD BE
DETECTED IN THE URINE
 Presence of > 5 rbcs/uL is CLINICALLY SIGNIFICANT (Microscopic Hematuria)

Hematuria Hemoglobinuria Myoglobinuria Hemosidenuria

INTACT RED CELLS NO RED CELLS NO RED CELLS MYOGLOBIN IN URINE
CLOUDY RED URINE CLEAR RED URINE CLEAR RED - BROWN
URINE
Bleeding is renal or Lysis of RBC produced in Characterized by “ cola drink” Associated with muscle
genitourinary urinary tract particularly in or “ black coffee” urine destruction
dilute, alkaline urine Reabsorption of filtered
May result from intravascular Associated with hemoglobin results in the
hemolysis “Rhabdomyolysis” appearance of large yellow-
brown granules of denatured
Heme portion of myoglobin is ferritin called hemosiderin in the
toxic to the renal tubules, and renal tubular epithelial cells and
high concentrations can cause in the urine sediment.
acute renal failure
Renal calculi Transfusion reactions Muscular trauma Hemochromatosis
Glomerulonephritis Hemolytic anemias Crush injuries Usually 3 days following an
Pyelonephritis Severe burns Prolonged coma acute hemolytic episode
Tumors Infections/malaria Convulsions Free Hemoglobin  Ferritin +
Trauma Strenuous exercise/RBC trauma Muscle-wasting diseases Hemosiderin
Exposure to toxic chemicals Brown recluse spider bites Alcoholism/overdose
Anticoagulants Drug abuse
Strenuous exercise Extensive exertion
During menstruation Cholesterol-lowering statin
Schistosoma haematobium medication
infection

DIAGNOSTIC METHODS

Plasma Examination - Hemoglobin - RED/PINK (with decreased haptoglobin)

- Myoglobin - PALE YELLOW (with increased CK & LD enzymes)
Bloodheim’s Test (2.8 - Hemoglobin - Clear supernatant with NEGATIVE REACTION in BLOD REAGENT STRIP
Ammonium Sulfate Test) - Myoglobin - Red supernatant with POSITIVE REACTION in BLOOD REAGENT SRIP
o Myoglobin levels must be 25 mg/dL before red pigmentation
** Ammonium sulfate precipitates hemoglobin
Reagent Strip - For heme compounds (Hemoglobin and intact red cell; myoglobin)
- Principle: Based on the peroxidase - like activity of heme from free hemoglobin, lyzed RBC or
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myoglobin. Erythrocyte are lyzed by the reagent strip this releasing hemoglobin.
- Reagents:
o Hydrogen peroxide (H2O2)
o Tetramethylbenzidine - chromogen
- Chemstrip and Multistix -= detects 0.05 to 0.3
mg hemoglobin
- Result:
o Green - blue color
o Spotted pattern - indicates intact red
cells
o Uniform pattern - indicates free
hemoglobin
- False positive:
o Oxidizing agents:
 Peroxide
 Sodium Hypochlorite
(Bleach)
o Menstruation
- False negative
o Ascorbic acid
o Formaldehyde
o Reduced sensitivity
 Very high Specific Gravity
 High nitrite (>10mg/dL)

Ammonium Sulfate - Historical method

Preipitation - Principle: Hemoglobin precipitates at 80% saturation with ammonium sulfate; myoglobin does
after 100 % saturation
- 5 ml urine + 2.8 g (NH4)
- Result: Red precipitate - Myoglobin
- Source error:
o This method would give false negative results for urine results for urine samples
containing <300 mg/L hemoglobin
Quantitative Test for - Radioimmunoassay - too laborious, time consuming
Myoglobin - High Performance Liquid Chromatography
- Electrophoresis
- Rapi - tex - latex agglutination (semi - quantitative)
- Immunoassay
Prussian Blue Reaction - Used to demonstrate iron present in hemosiderin
- Uses potassium ferrocyanide solution
- If positive, hemosiderin appears as blue granules singly or in groups, in renal tubular epithelial
cells, as amorphous sediment, or as blue granules in cast

BILIRUBIN
 Urine Bilirubin is excreted in very small amounts and normally should not be detectable in urine
 Only the conjugated form of bilirubin can appear in the urine
 Is a degradation product of hemoglobin
 BILIRUBIN METABOLISM:
o Bilirubin comes from the breakdown of hemoglobin released from red blood cells
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o Bilirubin released into the bloodstream from the peripheral tissues is WATER - INSOLUBLE and becomes reversibly
bound to ALBUMIN making it UNCONJUGATED BILIRUBIN (unable to pass the glomerular filtration barriers and
hence it cannot be excreted via the urine)
o When the blood passes through the liver sinusoids, the hepatocytes rapidly remove bilirubin from albumin and then
conjugates it with gluronic acid to produce WATER - SOLUBLE CONJUGATED BILIRUBIN.
o Normally, all conjugated bilirubin is transported into the bile duct and ultimately into the small intestines
o In the intestinal tract, it will be deconjugated and reduced by anaerobic intestinal bacteria to form the colorless tetrapyrrole
UROBILINOGEN
o About 20% of urobilinogen is reabsorbed and reenter liver via hepatic portal circulation (and then re - excreted again) while
some portion is reduced to STERCOBILINOGEN (cannot be reabsorbed)
o Urobilinogen and stercobilinogen are oxidized in the large intestine forming UROBILIN and STERCOBILIN which gives
the stool its characteristic color
o 2 to 5 % of urobilinogen normally remains in the bloodstream and can be excreted in the urine when it passes through the
kidneys
 Normal Conditions:
o Excreted in the feces in the form of urobilin
o Adult urine contains only 0.02 mg/deciliter
 Clinical significance:
o SCREENING OF ABNORMAL HEPATOBILIARY FUNCTION
o Appear in urine when the normal degradation cycle is disrupted by obstruction of the bile or when the integrity of the liver is
damaged
 Pre - hepatic jaundice (hemolytic anemia)
 Hepatic jaundice (hepatitis, cirrhosis)
 Post - hepatic jaundice (biliary obstructions, gallstones, carcinoma)
 Hepatotoxic drugs or toxins

Urine Bilirubin and Urobilinogen in Jaundice

Urine Bilirubin Urine Urobilinogen
Bile duct obstruction +++ Normal
Liver damage + or – ++
Hemolytic disease Negative +++

DIAGNOSTIC METHODS

Reagent Strip - Principle: Based on Diazo reaction

Tan or pink to violet

STRIP MULTISTIX CHEMSTRIP

Reagent 2,4-dichloroaniline di-azonium salt 2,6-dichlorobenzene-diazonium-
tetrafluoroborate
Sensitivity 0.4 - 0.8 mg/dL 0.5 mg/dL
Interference False positive:
- Highly pigmented urines, phenazopyridine
- Indican (intestinal disorders)
- Metabolites of Lodine
False negative:
- Specimen exposure to light
- Ascorbic acid greater than 25 mg/dL
- High concentrations of nitrite
- Testing of specimens that are not fresh - are the most frequent
errors associated with bilirubin testing.
Correlation to Urobilinogen
others

Diazo Tablet/Mat - Confirmatory

Method - 4X more sensitive than the reagent strip
- Sensitive to 0.05 to 0.10 mg/dL of bilirubin
- Less subject to interference
- Ictotest (Siemens Healthcare Diagnostics, Inc., Tarrytown, NY)
- Reagents:
o p-nitrobenzene- diazonium-p-toluenesulfonate
o Sulfosalysylic acid (SSA)
o Sodium carbonate
o Boric acid
- Result: Bluish - Purple color of the mat around the tablet after 30 seconds
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- Sources of error: Same as reagent strips

Fouchet’s Test (Acidified - Principle: Ability of ferric chloride dissolved in trichloroacetic acid to oxidize bilirubin to
Ferric Chloride) biliverdin producing a green color
- Source of error:
o False - positive: Aspirin metabolites, Urobilin or indicant, Urobilinogen
o False - negative: Oxidation of bilirubin, if examination is delayed

OTHER TESTS
Smith’s (Iodine Test) - (+) Emerald green color
Gmelins: Concentrated Nitric acid - Concentrated Nitric acid: (+) Play of colors (green - blue, yellow & red)
Ultzman’s Test - KOH, HCL: (+) Green color
Foam Test - Yellow foam when shaken
Huppert’s Test - Mild of lime, HCl, Ethyl
Harrison spot Test - Ferric chloride in the presence of TCA will oxidize yellow bilirubin to green
biliverdin
- Barium chloride: Fouchets reagent
o Biliverdin (green)
o Bilicyanin (blue)
o Choleitilen (yellow)

UROBILINOGEN
 NORMAL URINE UROBILINOGEN:
o < 1 mg/dL or 1 ERLICH UNIT
o 0.5 to 25 mg/units/24 hours
o This is never reported as negative in urinalysis report
 A colorless pigment formed from the breakdown of bilirubin in the intestines
 Appear in urine because as it circulates in the blood en route to the liver, it may pass through the kidney and be filtered by the
glomerulus
 Urobilinogen excretion reaches peak levels between 2 PM and 4 PM
 Same clinical significance as bilirubin
 Clinical Significance:
o Occur whenever the liver is unable to efficiently remove the reabsorbed urobilinogen from the portal circulation and more
urobilinogen than normal is routed to the kidney.
o Hepatocellular damage:
 Viral hepatitis
 Drug toxicity
o Hemolytic disorders

TYPE OF JAUNDICE URINE BILIRUBIN URINE UROBILINOGEN

Pre - hepatic Jaundice Negative +++
Hepatic Jaundice + or Negative ++
Post - hepatic Jaundice +++ Normal

DIAGNOSTIC METHODS

Reagent Strip - Based on Diazo or Erlich’s reaction

- Principle:
o Based on Erlich’s reaction (Multistix)
 Urobilinogen utilizes Erlich’s reagent ( p- diaminobenzaldehyde) in acid medium
producing colors ranging from tan to orange
o Based on Diazo reaction (Chemstrip)

STRIPS ERLICH’s Reaction Azo Dye Reaction

(Multistix) (Chemstrip)
Reagents p-dimethylaminobenzaldehyde 4-methoxybenzene- diazonium-
tetrafluoroborate
Sensitivity 0.2 mg/dL urobilinogen 0.4 mg/dL urobilinogen
Source of error False positive: False positive:
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- Porphobilinogen - Highly pigmented urine

- Indican False negative:
- p-aminosalicylic acid - Old specimens
- Sulfonamides - Preservation in formalin
- Methyldopa - High concentrations of
- Procaine nitrite
- Chlorpromazine
- Highly pigmented urine
False negative:
- Old specimens
- Preservation in formalin
Remarks Unreliable if urine bilirubin is Specific for urobilinogen
high

Note:
The urobilinogen test pad on the Multistix Pro11 and Clinitek Microalbumin strips has been replaced by
the protein - low test pad.
Tube Test - Not specific, may also employed for testing indicant, sulfonamides, p-aminosalicylic.
porphobilinogen
- Principle: Based on Erlich’s reaction
- Reagent:
o p-dimethylaminobenzaldehyde
o Sodium acetate (added to enhance reaction)
- Source of error: Same as reagent strips tests (Multistix)
- Semi quantitative method:
o Test serial dilutions of urine
o Observe for the presence of pink color (enhanced by adding sodium acetate)
- Results:
o Reported in Erlich unit (= to 1 mg/ml of urobilinogen)
o CHERRY RED COLOR
- Normal value:
o Males = 0.3 to 2.1 Erlich units
o Females = 0.1 to 1.1 Erlich units
- Recommended specimen collection: After the noon meal (2 to 4 pm)
- Corresponds to the time of greatest

Watson - Schwartz - Differentiates between urobilinogen and porphobilinogen

Differentiation Test - Note:
o Urobilinogen is soluble in both chloroform and butanol
o Porphobilinogen is insoluble in both chloroform and butanol
o Other Erlich reactive compounds are soluble in butanol and insoluble in chloroform
- Procedure:

Extraction with UROBILINOGEN PORPHOBILINOG OTHER ERHLICH

EN REACTIVE
COMPOUNDS
BUTANOL
Top - Butanol RED colorless RED
Bottom - Urine colorless RED colorless
Chloroform
Top - Urine colorless RED RED
Bottom - Chloroform RED colorless colorless
Hoesch Test - Rapid screening test and monitoring for urine pophobilinogen (> 2 mg/dL)
(Inverse Ehrlich) - Principle: Based on Erlich reaction; Addition of an acid inhibits urobilinogen
- Reagent: HOESCH REAGENT (EHRLICH REAGENT DISSOLVED IN 6M HCL)
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- Result: Presence of phorphobilinogen = RED upon addition of reagent

- Procedure:
1. 2 drops of urine + Hoesch reagent (Erlich’s reagent dissolved in 6 M HCL)
2. Observe top of the solution for appearance of a red color - pophbilinogen
3. Shaking of solution - red solution seen all throughout
- Source of error:
o False - positive reaction;
 Methyldopa
 Indican
 Highly pigmented specimens
- Watson - Schwartz test: detects > 6 mg/dL of pophobilinogen (more sensitive0
- Hoesch test: detects > 11 mg/dL of porphobilinogen

OTHER TESTS

TESTS FOR UROBILIN

TEST REAGENTS (+) RESULTS
Schlesinger’s Tests Lugol’s Iodine Greenish Fluorescence
Zinc acetate
Schmidts Tests 10% Hg2Cl2 Green color

TESTS FOR INDICAN

TEST REAGENTS (+) RESULTS
Obermeyer HCL Indigo Blue
FeCl3
Chloroform
Schmidts Test HCL Blue color
Chloroform
Calcium Hypochlorite
Jolles Test Lead acetate Violet color
Thymol
Chloroform
Obemayer’s reagent

NITRITE
 Clinical Significance:
o Cystitis
o Pyelopnephritis
o Evaluation of antibiotic therapy
o Monitoring of patients at high risk for urinary tract infection
o Screening for urine culture specimens
o Periodically screen people with recurrent infections
o Patients with diabetes
o Pregnant women
 Most common organisms that infect the urinary tract;
o Proteus spp.
o E.coli
o Klebsiella pneumonia
o Pseudomonas aeruginosa
o Enterobacter spp.

DIAGNOSTIC METHODS

Reagent Strip - Rapid screening test for the presence of UTI

- Indirect Method
- Principle: Based on the ability of certain bacteria to reduce nitrate to nitrite
o Nitrite at an acidic pH reacts with an aromatic amine (para - arsanilic acid or sulfanilamide)
(Greiss reaction) to form diazonium compound that reacts with tetrahydrobenzoquinolin to
form a pink - colored azo dye

Multistix Chemstrip
Reagents p-arsanilic acid Sulfanilamide
Tetrahydrobenzo(h)-quinolin-3-ol Hydroxytetrahydrobenzoquinoline
Sensitivity 0.06–0.1 mg/dL nitrite ion 0.05 mg/dL nitrite ion
Interference False negative:
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- 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

False positive:
- Improperly preserved specimens
- Highly pigmented urine

Correlations with other tests:

- Protein
- Leukocytes
- Microscopic

Note:
 Many laboratories use the nitrite test in combination with the LE test to determine the necessity of performing urine cultures.
 Nitrite tests should be performed on first morning specimens or specimens collected after urine has remained in the bladder for at least
4 hours

LEUKOCYTE ESTERASE
 Indicates pyuria & that an inflammatory process is occurring in the kidney or urinary tract
 Principle: Based on the action of Leukocyte esterase to catalyze the hydrolysis of an acid ester to produce aromatic compound
and acid
 Extracts of human neutrophilic azurophilic granules
 Detects the presence of esterase in the:
o Granulocytic white blood cells (neutrophils (most frequent), eosinophils, basophils)
o Monocyte
o Trichomonas
o Histiocytes
 Clinical Significance:
o Bacterial and nonbacterial urinary tract infection
o Inflammatory of the urinary tract
o Screening of urine culture specimens
 Do not contain leukocyte esterase:
o Lymphocytes
o Erythrocytes Bacteria
o Renal tissues
 Contain leukocyte esterase:
o Trichomonas
o Histiocytes
 UTI caused by bacterial infection: (+) for nitrite and leukocyte test

DIAGNOSTIC METHODS

Multistix Chemstrip
Reagents Derivatized pyrrole amino acid ester Indoxylcarbonic acid ester
Diazonium salt Diazonium salt
Sensitivity 5–15 WBC/hpf 10–25 WBC/hpf
Interference False positive:
- Strong oxidizing agents
- Formalin
- Highly pigmented urine,
- nitrofurantoin
False negative:
- High concentrations of protein (> 500 mg/dL)
- Glucose (> 3 g/dL)
- Oxalic acid
- Ascorbic acid
- Gentamicin, cephalosporins, tetracyclines
- Inaccurate timing
Correlations with other tests Protein
Nitrite
Microscopic
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ASCORBIC ACID or VITAMIN C

 Source of interference due to its strong reducing property leading to FALSE - NEGATIVE results
 As hydrogen donator, ascorbic acid readily oxidizes to dehydroascorbic acid, a colorless compound.
 Reagent strip tests that use hydrogen peroxide or a diazonium salt are subject to ascorbic acid interference
 Whether these compounds are impregnated in the reaction pad or are produced by a first reaction, they are removed by ascorbic acid,
which prevents the intended reaction
 As a result, colorless dehydroascorbic acid is produced, no positive color change is observed, and a false - negative or a falsely low
result is obtained
 Affects blood, bilirubin, leukocyte esterase, nitrite and glucose

Chemstrip Stix and Multistix

- Impregnated with phosphomolybdates buffered in an acid - Impregnated with methyl green
medium - Methyl green is reduced by ascorbic acid to its colorless
- Detects 5 mg/dL of ascorbic acid in urine after 10 form
seconds - Detects 25 mg/dL of ascorbic acid in urine at 60 seconds
- False (+) results may be due to gentisic acid and L - dopa - Neutral red provides a background color and the overall
color changes from blue to purple at level of 150 mg/dL

SPECIFIC GRAVITY
 Part of physical examination
 Principle: Based on the change in pKa (dissociation constant) of a polyelectrolyte in an alkaline medium
 The higher the concentration of urine, the more hydrogen ions are released, thereby lowering the pH.
 Incorporation of the indicator bromothymol blue on the reagent pad measures the change in pH.
o As the specific gravity increases, the indicator changes from blue (1.000 [alkaline]), through shades of green, to yellow
1.030 [acid]).

Clinical Significance;
 Monitoring patient hydration and dehydration
 Loss of renal tubular concentrating ability
 Diabetes insipidus
 Determination of unsatisfactory specimens due to low concentration

DIAGNOSTIC METHODS

Multistix Chemstrip
Reagents Poly (methyl vinyl ether/maleic anhydride) Ethylene glycol diaminoethyl ether tetra-acetic acid,
bromothymol blue bromothymol blue
Sensitivity 1.000–1.030
Interference False positive: High concentrations of protein
False negative: Highly alkaline urines (greater than 6.5)
 URINALYSIS
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