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Urinalysis

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I. Color
• Normal urine color
– The color of urine is caused by a pigment named urochrome-chief pigment.
– Urochrome is a product of endogenous metabolism and under normal
conditions it is produced at a constant rate.

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The first three tubes show different shades of yellow: straw, light yellow, dark yellow. The fourth
tube illustrates cloudy urine.

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Causes of Urine Color

* Pathologic Condition

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* Pathologic Condition

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* Pathologic Condition

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*Pathologic Condition

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Blood in the urine

Tube 1 (from the left) is pinkish- small amount of red blood cells/blood. Tube 2 is red showing increased RBCs. Tube 3 is brown/black
indicating glomerular bleeding.
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Urine Clarity/Appearance
• Can range from clear to milky
• Typically clear in a normal patient
• Hazy
• Cloudy
• Milky

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Causes of Urine Turbidity

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Specific Gravity
• The density of a substance compared with the density of a similar volume of
distilled water at a similar temperature; influenced by the size and number of
particles.
– Measure of dissolved substances present in a solution.

Density of Urine
Specific Gravity = (5 - 1)
Density of an equal volume of water

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• Urinometer
– Consists of a weighted float attached to a long narrow calibrated stem-a scale
that has been calibrated in terms of urine-specific gravity (1.000-1.040)
– The weighted glass 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.

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• Refractometer
– Reading depends on:
– Wavelength of light used
– Size and number of particles
– Concentration of the solution (higher concentration)
decrease angle of refraction, decrease velocity)
– Temperature of the solution

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Advantages of Refractometry

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• Refractometer
– Correction of specific gravity
▪ A gram of protein/d L of urine will raise the urine-specific gravity by 0.003.
eci iter

▪ A gram of glucose/d L will raise the S.G. by 0.004.

eci iter

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• Reagent strip
– Polyelectrolyte, a pH indicator and an alkaline buffer
– Ionic solutes cause hydrogen ions to be released by the polyelectrolyte,
decreasing the pH of the test pad
(more acid).
– As pH changes, the pH indicator changes color.

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• Clinical correlations of specific gravity

– Specific gravity of plasma filtrate entering glomerulus is 1.010
– Isosthenuric
▪ Neither concentration or dilution may point to renal disease
▪ Sthenos = strength

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• Clinical correlations of specific gravity

– Urines with specific gravity below 1.010 are hyposthenuric.
– Lowest specific gravity possible: 1.002

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• Clinical correlations of specific gravity

– Hypersthenuric = specific gravity > 1.010
– Normal range: 1.015-1.025, which is the majority of specimens.
– Normal random specimens can range from 1.003 to 1.035.

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Causes of increased Specific Gravity

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Chemical Examination of the Urine: The Reagent Strip

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• Reagent strips
– Plastic strips with chemical-impregnated absorbent pads for each test
– When the pad comes in contact with a urine sample that contains the
substance, a color is produced depending on the concentration of the analyte.

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Reagent Strip Procedure

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• Handling and storage of reagent strips

• Reagent strip technique
• Quality control

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pH

• Measure of the hydrogen ion concentration of a solution,

in this case urine
• A solution that is neither acid nor alkaline has a pH of 7
– Acidity indicated by a number less than 7
– Alkalinity by a number greater than 7
• Clinical significance

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Factors affecting pH of the urine

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pH

• Reagent strip reactions

– Reagent strips usually measure pH in one-unit
increments between 5 and 9.
– Methyl red covers the acid range from 4 to 6,
changing from red-orange to yellow.
– Bromthymol blue, the alkaline indicator, changes from
green to blue and covers the pH from 6 to 9.

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Conditions Associated with Acidic Urine

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Conditions associated with Alkaline urine

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Protein

• Protein in the urine can be an important indicator of renal

disease.
• Proteinuria
– Increase in the loss of protein in the urine
– Often associated with early renal disease

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Protein

• Clinical significance
– Prerenal proteinuria
– Renal proteinuria
– Postrenal proteinuria
– Benign proteinuria

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Causes of proteinuria

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Causes of Benign Proteinuria

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Protein
• Clinical significance
– Orthostatic or postural proteinuria
▪ Diagnosed in young adults following periods spent in a vertical position
(standing up) and disappears when a horizontal position is resumed

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Bence Jones protein

• Bence-Jones (B-J) protein

– Associated with multiple myeloma, a proliferative
disorder of immunoglobulin-producing plasma cells
• Serum contains marked increase of immunoglobulin light
chains, either kappa or lambda, called Bence-Jones
protein, which are LMW proteins filtered and excreted in
the urine.

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Protein Testing
• Screening tests
• Reagent strip reactions
– Utilize the “protein error of indicators” to produce a visible colorimetric reaction

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Protein Testing

• Reagent strip reactions

– Based on the property of some pH indicators to change color in the presence
of protein.
▪ At a constant pH, they are one color when protein is absent and another
color when protein is present.

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Protein Testing
• Interfering substances
– False positive
– False negative
• Precipitation test
– Confirmatory test
– Cold precipitation test using sulfosalicylic acid (SSA)

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Protein Testing
• Discrepant results
– When the results of one test for an analyte do not agree with the results of
another

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Discrepant Results of Reagent Strip and SSA

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Glucose
• Glucose testing is used to detect and monitor diabetes mellitus.
• Clinical significance
– Glucosuria
▪ Prerenal
▪ Renal

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Causes of Pre renal and Renal Glucusoria

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Glucose

Glucose testing
– Reagent strip: glucose oxidase reaction
Glucose
Glucose  O 2  gluconic acid  H2 O 2
oxidase

peroxidase
H2O2  chromogen  oxidized colored compound
 H2O

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Glucose

Glucose testing
– Interfering substances
▪ False positive
▪ False negative
– Clinitest
heat
CuSo4 reducing substance Cu2O
   oxidized substance
 cupric ions   e.g., glucose  cuprous oxide
alkali

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Clinitest Procedure

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Glucose
• Glucose testing
– Clinitest
▪ “Pass-through” phenomenon
– When large amounts of glucose or other reducing substances are
present in the urine
– Reaction goes throughout the entire color range very quickly and back to
dark greenish-brown.

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Glucose
• Glucose testing
– Clinitest
▪ “Pass-through” phenomenon
– During the pass-through phenomenon the cuprous oxide is reoxidized to
cupric oxide.
• Discrepant results

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Reducing substances other than glucose resulting in a positive clinitest

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Discrepant Results Glucose Oxidase and Clinitest

Reactions

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Ketones
• Intermediate products of fat metabolism formed during the catabolism or
breakdown of fatty acids
• The three ketones are:
– acetone
– acetoacetic acid (diacetic acid)
– β-hydroxybutyric acid.

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Ketones

• Clinical significance
– Most valuable in the management and monitoring of diabetes mellitus
– Positive ketones unrelated to diabetes can be caused by increased loss of
carbohydrates from vomiting and diarrhea and digestive disturbances.

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Ketones
• Ketone Testing
– Reagent strip test
▪ Based on Legal’s test, the reaction of acetoacetic acid with
nitroprusside/nitroferricyanide.
▪ In an alkaline medium, acetoacetic acid reacts with sodium nitroprusside to
form a purple color.
▪ Glycine is added to allow acetone to react.

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Ketones
• Ketone Testing
– Acetest tablets
▪ Confirmatory test in tablet form
– The tablet contains sodium nitroprusside, glycine, disodium phosphate,
and lactose (for better color differentiation).

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Ketones
• Ketone Testing
– Interfering substances
▪ False positive
▪ False negative
• Correlation of glucose and ketones
– Conditions are found when one is present and the other is negative.

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Correlation between ketones and Glucose

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Bilirubin
• Degradation product of hemoglobin with 85% of serum bilirubin derived from the
breakdown of old red blood cells (RBCs)

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Bilirubin
• Clinical significance
– Bilirubinuria
▪ Early indicator of liver disease that may be present before jaundice
– Jaundice
▪ Accumulation of bilirubin in the body, leading to yellow pigmentation of the
skin, sclera (whites of the eyes), and mucous membranes

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Bilirubin
• Clinical significance
– Jaundice
▪ Prehepatic jaundice
– Caused by increased RBC destruction or hemolysis (e.g., hemolytic
anemia).
▪ Hepatic jaundice
– Usually seen in liver damage and is an early warning sign of liver
disease.

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Bilirubin
• Clinical significance
– Jaundice
▪ Posthepatic jaundice
– Caused by bile duct obstruction (e.g., gallstones or cancer).

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Prehepatic, hepatic, and post hepatic

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Bilirubin

• Bilirubin Testing
– Screening test
– Reagent strip
▪ Diazo reaction
– Bilirubin is coupled with a diazonium salt in an acid medium to form
azobilirubin, a colored compound.
– Ictotest
▪ A tablet test that yields a more sharply colored diazo reaction.

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Ictotest procedure

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Urobilinogen
• A bile pigment
• Formed from the degradation of hemoglobin
• Produced in the intestine from the reduction of bilirubin by the intestinal bacteria

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Urobilinogen
• Clinical Significance
– Increased urine urobilinogen seen in liver disease (e.g., hepatitis and cirrhosis)
– Results in the impairment of liver function and the liver’s ability to process
urobilinogen recirculated from the intestine

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Urobilinogen

• Urobilinogen Testing
– Reagent strips
▪ Modification of Ehrlich’s reaction
▪ ρ-dimethylaminobenzaldehyde turns a cherry red color in the presence of
urobilinogen
▪ Multistix reagent strip reaction

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Urobilinogen

• Urobilinogen Testing
– Reagent strips
▪ Chemstrip reaction
– More specific than Multistix;
uses an azocoupling reaction
– 4-methoxybenzene-diazonium-
tetrafluoroborate in an acid
medium resulting in a red azo
dye
• Interfering substances

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Urobilinogen

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Urobilinogen
• Correlation of bilirubin and urobilinogen
– Bilirubin and urobilinogen results can point toward prehepatic, hepatic, or
posthepatic jaundice.

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Prehepatic, hepatic and posthepatic

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Blood

• Hematuria
– Presence of blood in the form of intact RBCs in the urine
• Hemoglobinuria
– Presence of hemoglobin from hemolyzed RBCs in the urine

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Blood
• Clinical significance
– Myoglobin
▪ A muscle hemoglobin
▪ Should be suspected in conditions associated with muscle destruction,
rhabdomyolysis

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Blood

• Clinical significance
– Myoglobin
▪ A muscle hemoglobin
▪ Should be suspected in conditions associated with muscle destruction,
rhabdomyolysis

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Conditions Associated with Hematuria and

Hemoglobinuria

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Conditions Associated with Hematuria and

Hemoglobinuria

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Conditions Associated with Myoglobinuria

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Blood

Blood testing
– Reagent strip reactions
▪ Chemical tests for blood utilize the peroxidase
activity of hemoglobin to catalyze a reaction
between hydrogen peroxide and a chromogen,
tetramethylbenzidine, to produce an oxidized
chromogen, which is blue-green.
heme
H2O2  Chromogen
 Oxidized chromogen  H2O
 tetramethylbenzidine 
 peroxidase activity 
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Blood and Hemoglobin

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Leukocytes

• Leukocyturia (pyuria)
– Presence of white blood cells in the urine
– Often indicates the presence of an infection in the
upper or lower urinary tract

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Leukocytes

• Clinical significance
– Leukocyte esterase reaction should be interpreted in
conjunction with nitrite, which is present in UTIs or
bacteruria.

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Leukocytes

Leukocyte testing
– Reagent strip
▪ Leukocyte esterase reaction is based on an acid
ester and a diazonium salt

Leukocyte Esterase
Indoxylcarbonic acid ester  indoxyl + diazonium salt  purple azo dye

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Leukocyte Esterase and sediment

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Nitrite

• Produced by the reduction of nitrate by the enzyme

nitrate reductase by gram negative bacteria
• Not normally present in urine
– Can be used to screen for UTIs

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Nitrite
• Clinical significance
– Can detect UTIs
– Can be used to monitor or evaluate the effectiveness of antibiotic therapy

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Nitrite

Nitrite testing
– Reagent strip
– Nitrite test pad based on the Greiss reaction
▪ Involves diazotization of nitrite with an aromatic
amine to form a diazonium salt

Acid pH
Nitrite + para-arsanilic acid  or sulfanilamide   diazonium compound  pink 

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Causes of false negative nitrite

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Microscopic
• Microscopic examination
– Low power (10×)
▪ Scan for epithelial cells, casts, and crystals and count them.
– High power (40×)
▪ Count red blood cells, white blood cells, yeast, and other smaller elements.

– In each case
▪ Count 10 fields and the average number of cells per milliliter is calculated
and reported

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Acid or neutral Urine

Precipitate as pink brick

dust after settling or
centrifugation. Large
Yellow –Brown numbers can obscure
Amorphous urates important elements. Balls or spheres
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Birefringent. Seen in
Calcium oxalate patients post ethanol dumbbell shapes, ovoid,
monohydrate Colorless ingestion. longitudinal

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Calcium oxalate Birefringent; cause of renal

dihydrate Colorless calculi. Octahedral or envelope

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Uric acid Crystals (50*, Brightfield); (B) Uric Acid Crystal, Barrel Form (50×, Brightfield); (C) Uric Acid Crystals, Rosettes and
Diamond Forms (50×, Brightfield); (D) Uric Acid Crystals. Note the Layering or Lamination of the Structures (50×, Brightfield); (E)
Uric Acid Crystals, Rhomboid (50×, Brightfield); (F) Uric Acid Crystals, Barrel and Diamond Forms (50×, Brightfield)

Birefringent; crystals
exhibit layering or surface Diamonds or spades,
lamination. Cause of barrels, cubes, rosettes;
Uric acid Yellow to Yellow-Brown renal calculi. rarely six sided.

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Alkaline pH

White precipitate after

settling or centrifugation.
Large numbers can
obscure important
Amorphous phosphate Colorless elements.

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Most often found in urine

after prolonged storage; Round sphere. May have
Ammonium biurate Yellow to Yellow Brown however, may form in vivo. spicules (thorny apples).

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Forms Carbon Dioxide with

addition of acetic acid.
Large amounts may form
pseudocasts under Granular. Rarely,
Calcium carbonate Colorless coverslip. dumbbells.

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Form in alkaline to slightly

Calcium biphosphate Colorless acid pH. Large plates
Calcium di basic phosphate Colorless Rosettes

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Form in neutral to alkaline Coffin lids or, less commonly,

Triple phosphate Colorless pH. Cause renal calculi. in leaf-like form

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Other crystals

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Inherited amino
Colorless to brown to acid disorder. May Fine needles,
black depending on rarely be seen in sometimes
Tyrosine concentration. liver disease. forming sheaves.

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Medication Crystals

Bundles of wheat.
May cause kidney Brown
damage if formed rosettes or
Sulfonamides (UTI) Dark Orange in vivo. spheres.

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Ampicillin Colorless blank Fine needles

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Distinguished from
cholesterol by Flat plates,
absence of rectangles,
Radiographic dyes Colorless lipiduria. notched plates.

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Cellular elements in urine sediment

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• White blood cells (WBCs)

– Clinical significance
▪ Inflammation
▪ Pyelonephritis
▪ Glomerulonephritis

- Polymorphonuclear leukocytes (PMNs) are most common

– Eosinophils
▪ Hypersensitivity to drugs
▪ Renal transplant

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(A) Squamous Epithelial Cells (200×, Brightfield); (B) Squamous

Epithelial Sheet, Sternheimer Malbin Stain. (50×, Brightfield)

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• Epithelial cells
– Squamous epithelial cells (SQEP)
 Distal third of urethra
▪ Increased removal by catheterization, normal sloughing
▪ Fried-egg appearance

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• Epithelial cells
– Transitional, nonsquamous epithelial cells (NSE)
▪ Upper urethra
▪ Pear-shaped appearance
– No clinical significance of either SQEP or NSE

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(A) Renal Tubular Epithelial (Arrow) and Squamous Epithelial Comparison (200×,
Brightfield); (B) Renal Tubular Epithelia Cell, Calcium Oxalate Crystal (200×, Brightfield)

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Oval Fat Body (200×, Brightfield)

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(A) White Blood Cells, Bacteria, Yeast (Arrow) (200×, Brightfield); (B) Yeast, Hyphae (400×,
Brightfield)

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T. Vaginalis vs. WBC (round)

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• Hyaline casts
– Low refractive index
– Clinical significance
▪ Strenuous exercise
▪ Stress, dehydration, fever

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Cylindroid with Tapering Tail (Arrow) in a Field of Mucin Threads, Sternheimer-Malbin Stain
(50×, Brightfield)
Hyaline – Cylindroid cast

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• Casts
– Clinical significance
▪ Red cell cast
– Bleeding within the nephron
▪ White cell cast
– Infection within the kidney
▪ RTE cast
– Viral diseases, toxins, acute tubular necrosis

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(A) Granular Cast (50×, Brightfield); (B) Granular Casts (50×, Brightfield)

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• Casts
– Granular
▪ Granular inclusions within the matrix; degrading
cells
– Coarse or fine granular
▪ Can indicate urinary stasis

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(A) Waxy Cast (200×, Brightfield); (B) Waxy Cast, Sternheimer-Malbin Stain (200×,
Brightfield)

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• Casts
– Waxy
▪ Highly refractile
▪ Presence indicates urinary stasis and renal failure
– Fatty
▪ Oval fat bodies within the matrix

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• Crystals
– Amorphous urates

Amorphous Urates (200×, Brightfield)

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• Crystals
– Uric acid
▪ Most commonly observed
▪ Can be seen in patients with gout

Uric acid Crystals (50*, Brightfield); (B) Uric Acid Crystal, Barrel Form (50×, Brightfield); (C) Uric Acid Crystals, Rosettes
and Diamond Forms (50×, Brightfield); (D) Uric Acid Crystals. Note the Layering or Lamination of the Structures (50×,
Brightfield); (E) Uric Acid Crystals, Rhomboid (50×, Brightfield); (F) Uric Acid Crystals, Barrel and Diamond Forms (50×,
Brightfield)

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• Crystals
– Calcium oxalate
▪ Monohydrate form seen in ethylene glycol ingestion

• Calcium Oxalate Dihydrate Crystals, Granular Cast (200×, Brightfield); (B) Calcium Oxalate Crystals,
Amorphous Urates (50× Brightfield); (C) Calcium Oxalate Monohydrate Crystals, Dumbbell and Oval (Arrow)
Forms (50×, Brightfield); (D) Calcium Oxalate Crystal. Note that the Monohydrate (Arrows) and Dihydrate
Forms are Both Present (50×, Brightfield)

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Triple Phosphate Crystals (50×, Brightfield)

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Ammonium Biurate Crystals (200×, Brightfield)

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• Crystals
– Abnormal found in acid or neutral urine
▪ Cholesterol
– High concentration in nephrotic syndrome or
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▪ Bilirubin
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• Crystals
– Abnormal found in acid or neutral urine
▪ Cystine
– Can be confused with uric acid (hexagonal)
▪ Leucine or tyrosine
– In-born errors of metabolism
▪ Sulfonamides, ampicillin

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(A) Mucin Thread (Arrows) with Hyaline Casts in the

Same Field (50×, Brightfield); (B) Mucus

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Note the Small Indentations in the Center of the Granule (200×, Brightfield) – Starch granules

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Exercises

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CSF
Physical Examination

• Normal CSF is clear and colorless with a viscosity similar to water.

• Patient specimens may be cloudy or turbid, purulent (containing pus), or
colored.
• Each CSF specimen should be checked for discoloration by comparing the
color of the CSF sample to water.

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CSF Specimens
(A) Normal CSF is clear and colorless. (B) Xanthochromic with a RBC button at the bottom of
the tube.

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Causes of Abnormal CSF Supernatant Colors

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Microscopic Procedures
• Total cell counts
• Manual cell counting
– Procedure

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Improved Neubauer Hemocytometer

Yellow square = 1 m m 2, Red square = 0.0625 m m 2, Blue square = 0.04 m m 2 and Green square= 0.0025 m m 2.
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Differential Counts

• Cytocentrifugation
• Cellular components

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Components used for Preparation of Cytospin Slides

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Chemical Testing
• Proteins

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Conditions Associated with Increased Levels of CSF

Protein

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CSF Proteins and Their Respective

Diseases/Conditions

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Chemical Testing

• Albumin Index

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Chemical Testing

• CSF/serum IgG ratio

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Chemical Testing
• IgG synthesis rate

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Chemical Testing

• CSF IgG -albumin ratio

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Positive oligoclonal protein pattern in a spinal fluid sample using high-resolution electrophoresis (HRE). There are three
bands present in CSF adjacent to the arrows but not serum. (A) Serum, (B) CSF from same patient.

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Computation

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Lymphocyte pleocytosis refers to...

Please select the single best answer

a decreased number of lymphocytes in a CSF when compared to a normal sample.

an increased number of lymphocytes in a CSF when compared to a normal sample.
a decreased number of blasts in a CSF when compared to a normal sample.
an increased number of blasts in a CSF when compared to a normal sample.

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How many white blood cells (WBCs) would be considered normal for adult cerebrospinal fluid?

Please select the single best answer

Any number of WBCs is considered abnormal

0-5 WBCs/µL
6-10 WBCs/µL
up to 30 WBCs/µL

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In an adult, 0 - 5 WBC/µL is considered normal. Children will have slightly higher cell counts. Up to 30 WBC/µL is
within normal limits for newborns. Lymphocytes account for 60 - 100% of these cells.

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A technologist decides to make a 1:20 dilution of cerebrospinal fluid (CSF) after briefly evaluating a
portion of the sample microscopically. After making the dilution and charging the chambers, the
number of cells that are observed in each of the large squares of the hemocytometer is >100. What
should the technologist do to obtain the most accurate count?

Please select the single best answer

Count all four corner squares.

Make a smaller dilution.
Make a larger dilution.
None of the above

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Feedback
The best choice would be to make a larger dilution before performing the cell count.
This will provide the most accurate results.

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The appearance of the CSF suggests a traumatic tap. This is indicated by the fact that
there is decreasing amounts of blood in the sequentially collected tubes; tube #1
contains visible red blood cells, but the amount of blood decreases in the other tubes.
Normal CSF is clear and colorless. If the blood was the result of a subarachnoid
hemorrhage, all three tubes would contain blood and the amount of blood in each
tube would be consistent (unless a traumatic tap also occurred during collection of
the sample).

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