INTRODUCTION TO URINALYSIS

CHAPTER 2

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 Learning Objectives

Upon completing this chapter, the reader will be able to

1. List three major organic and three major inorganic chemical
constituents of urine.
2. Describe a method for determining whether a questionable fluid is
urine.
3. Recognize normal and abnormal daily urine volumes.
4. Describe the characteristics of the recommended urine specimen
containers.
5. Describe the correct methodology for labeling urine specimens.
6. State four possible reasons why a laboratory would reject a urine
specimen.

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 Learning Objectives (cont’d)
7. List 10 changes that may take place in a urine specimen that remains
at room temperature for more than 2 hours.
8. Discuss the actions of bacteria on an unpreserved urine specimen.
9. Briefly discuss five methods for preserving urine specimens, including
their advantages and disadvantages.
10. Instruct a patient in the correct procedure for collecting the following
specimens: random, first morning, 24-h timed, catheterized,
midstream clean-catch, suprapubic aspiration, three-glass collection,
and pediatric, and identify a diagnostic use for each collection
technique.
11. Describe the type of specimen needed for optimal results when a
specific urinalysis procedure is requested.

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 History
• The beginning of laboratory medicine
– Cavemen drawings and Egyptian hieroglyphics
• Color, clarity, odor, viscosity, sweetness
– Fifth century BC, Hippocrates wrote uroscopy book
– AD 1140 color charts
– 1694 albumin determination by “boiling”
– Charlatans/“pisse prophets”
• First medical licensure laws
– 17th century invention of the microscope
• Evaluation of sediment
– Part of a routine physical in 1827

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 Importance
• Readily available and easily collected specimen
• Urine contains information, which can be obtained by
inexpensive laboratory tests, to assess many metabolic
functions
• CLSI Urinalysis definition: “the testing of urine with
procedures commonly performed in an expeditious,
reliable, safe, and cost-effective manner”
• Reasons to perform
– Aid disease diagnosis, screen for asymptomatic diseases,
monitor disease progress and therapy effectiveness
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 Urine Formation

• Ultrafiltrate of plasma
• Kidneys converts approximately 170,000 mL of
filtered plasma
• Average daily urine output of 1200 mL

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 Urine Composition

• Normal 95% water, 5% solutes

• Solute variations: diet, activity, metabolism, endocrine,
body position
• Major organic solute is urea (protein, amino acid
breakdown); makes up approximately one half of the
dissolved solids
• Inorganic chloride, sodium, and potassium
• Urea and creatinine identify a fluid as urine
• May also contain cells, casts, crystals, mucus, and bacteria
– Increases indicative of disease

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 Urine Volume

• Determined by body’s state of hydration

• Influenced by fluid intake, nonrenal fluid loss,
antidiuretic hormone (ADH) variations, excretion
of large amounts of dissolved solids (e.g.,
glucose)
• Usual daily volume = 1200 to 1500 mL
• Normal range = 600 to 2000 mL

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 Definitions

• Oliguria: a decrease in urine output

– <1 mL/kg/h in infants <0.5 mL/kg/h in children, <400 mL/day
in adults
– Causes: vomiting, diarrhea, perspiration, severe burns
• Anuria: cessation of urine flow
– Severe kidney damage, decreased renal blood flow
• Nocturia: increased urine excretion at night
– Normally two to three times more excretion in the day
• Polyuria: >2.5 L/day in adults and >2.5 to 3 mL/kg/day in
children

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 Polyuria in Diabetes Mellitus versus Diabetes Insipidus

Diabetes mellitus Diabetes insipidus

• Increased volume caused • Decreased production or
by need to excrete the function of antidiuretic
excess glucose not hormone (ADH) causing
reabsorbed from the decreased reabsorption of
ultrafiltrate water from ultrafiltrate
• Patients exhibit polydipsia • Urine is dilute with low
• Urine appears dilute with specific gravity
a high specific gravity • Patients also exhibit
polydipsia

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 Specimen Collection
• Wear gloves when working with urine
• Clean, dry, leak-proof containers
• Disposable, wide-mouthed, and flat-bottom containers
with screw caps are recommended
• Clear containers/at least 50 mL capacity
– Sterile transfer devices and containers are available
– Facilitates automated analysis
– 12 mL minimum for analysis
• Adhesive bags for pediatrics
• Large plastic containers for 24-hour specimens

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 Specimen Labeling
• Information on label
– Patient’s name, ID number, date, time
• Additional information: age, location, health-care
provider’s name
• Place label on container, not lid
• Requisition form (manual or computerized)
– Must accompany specimen
– Information must match label
– Time of receipt is stamped on requisition
– Other information: type of specimen, interfering medications

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 Specimen Rejection

• Specimens in unlabeled containers

• Nonmatching labels and requisition forms
• Specimens contaminated with feces or toilet paper
• Containers with contaminated exteriors
• Specimens of insufficient quantity
• Specimens that have been improperly transported
• Labs must have written policies for rejection of
specimens
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 Specimen Integrity
• Changes in urine composition take place not only
in vivo but also in vitro
– Test within 2 hours of collection
– Refrigerate or chemically preserve if testing is
delayed
– Most problems are caused by bacterial multiplication
– Increased: color, turbidity, pH, nitrite, bacteria, odor
– Decreased: glucose, ketones, bilirubin, urobilinogen,
RBCs, WBCs, casts
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 Changes in Unpreserved Urine

Analyte Change Cause

Color Modified/darkened Oxidation or reduction of metabolites

Clarity Decreased Bacterial growth and precipitation of

amorphous material
Odor Increased Bacterial multiplication causing breakdown of
urea to ammonia
pH Increased Breakdown of urea to ammonia by urease-
producing bacteria/loss of CO2
Glucose Decreased Glycolysis and bacterial use

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 Changes in Unpreserved Urine (cont’d)

Analyte Change Cause

Ketones Decreased Volatilization and bacterial metabolism
Bilirubin Decreased Exposure to light/photooxidation to biliverdin

Urobilinogen Decreased Oxidation to urobilin

Nitrite Increased Multiplication of nitrate-reducing bacteria

RBCs, WBCs, Decreased Disintegration in dilute alkaline urine
and casts

Bacteria Increased Multiplication

Trichomonas Decreased Loss of motility, death

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 Specimen Preservation

• Routine is refrigeration at 2°C to 8°C

– Decreases bacterial growth and metabolism
– Must return to room temperature for chemical testing
• Chemical preservative
– Ideal is bactericidal: inhibits urease and preserves formed
elements
– Should not interfere with chemical tests
• Commercial transport tubes are available, but they
must be compatible with tests
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 Preservatives
Preservatives Advantages Disadvantages Additional Information
Refrigeration Does not interfere Precipitates amorphous Prevents bacterial growth
with chemical tests phosphates and urates for 24 h2
Boric acid Prevents bacterial Interferes with drug and Keeps pH at about 6.0
growth and hormone analyses Can be used for urine
metabolism culture transport

Formalin Excellent sediment Acts as a reducing agent, Rinse specimen container

(formaldehyde) preservative interfering with chemical with formalin to preserve
tests for glucose, blood, cells and casts
leukocyte esterase, and
copper reduction
Sodium fluoride Is a good Inhibits reagent strip tests
preservative for drug for glucose, blood, and
analyses leukocytes

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 Preservatives (cont’d)
Preservatives Advantages Disadvantages Additional Information
Commercial Convenient when Check tablet composition to
preservative tablets refrigeration not determine possible effects on
possible desired tests
Have controlled
concentration to
minimize interference
Urine Collection Contains collection cup,
4
Kits (Becton transfer straw, C&S
Dickinson, preservative tube or
Rutherford, NJ) UA tube
Light gray and gray Sample stable at room Do not use if urine is below Preservative is boric acid,
C&S tube temperature (RT) for 48 minimum fill line sodium borate, and sodium
h; prevents bacterial formate
growth and Keeps pH at about 6.0
metabolism

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 Types of Specimen

• The composition of urine depends on the

patient’s metabolic state and the timing and
procedure used for collection
• Time, length, and method of collection and the
patient’s dietary and medicinal intake
• Patients must be instructed when special
collection techniques are required

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 Random Specimen
• Most common type received
• Routine screening for obvious abnormalities
• May be collected at any time
• Collection time must be recorded
• Dietary intake and physical activity may alter
results
• Patients may have to collect an additional
specimen under controlled conditions
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 First Morning Specimen

• Ideal screening specimen

– Patient is in a basal state
• Use for orthostatic protein confirmation and
urine pregnancy tests
• More concentrated than a random specimen
• Patient collects immediately on arising, delivers
to lab within 2 hours
– Refrigeration is an alternative

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 Fasting Specimen

• Actually is second specimen voided, collected

after the first morning specimen
• Does not contain metabolites from evening meal
• Recommended for glucose monitoring

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 24-Hour (Timed) Specimen
• Carefully timed specimen will produce accurate quantitative
results
• Good for diurnal variation solutes
– Catecholamines, electrolytes
• The patient must remain adequately hydrated during short
collection period
• Patient must be instructed on the procedure for collecting a
timed specimen
• Concentration of a substance in a particular period must be
calculated from the urine volume produced during that time

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 24-Hour (Timed) Specimen (cont’d)

• 24-hour specimen must be thoroughly mixed and

the volume accurately measured and recorded
• Multiple containers of the same collection must
be combined and mixed thoroughly
• Additives should not interfere with the tests to
be performed

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 24-Hour (Timed) Specimen (cont’d)

• Required for quantitative results

• 24-hour specimens are needed for measuring
substances with diurnal variation (results differ in
a.m. and p.m.) and substances that vary with
meals, activity, and body metabolism
• Shorter timed specimens can be used for
substances with consistent levels
• Accurate timing is critical for accurate results
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 2-Hour Postprandial Specimen

• Patient voids before eating routine meal

• Eats meal
• Collects next specimen 2 hours after finishing
meal
• Monitors insulin therapy
• Results can be compared with fasting urine
specimen and blood test results

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 Glucose Tolerance Specimen

• Institutional option for collection with blood

glucose tolerance test, not frequently done
• Specimens are collected at the same intervals as
the blood samples
• Used to correlate renal threshold with patient’s
ability to metabolize glucose

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 Catheterized Specimens

• Sterile specimen collected from bladder with a

hollow tube (catheter)
• Most common test is bacterial culture

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 Midstream Clean-Catch Specimen

• Alternative to catheterized specimen

– Less traumatic method
• Less contaminated than routine collection
• Provide patient with mild cleansing material and
container and instructions
• Do not touch or contaminate inside of container

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 Suprapubic Aspiration

• Completely free of contamination for culture and

cytology
• External introduction of needle for aspiration
from the bladder
• Possible pediatric specimen

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 Prostatitis Specimen

• Collection similar to midstream clean-catch

• Three-glass collection
– Container 1: first urine passed
– Container 2: midstream urine
– Massage prostate to obtain prostatic fluid
– Container 3: remaining urine and fluid
– Quantitative cultures on all three specimens, examine
1 and 3 microscopically for WBCs

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 Prostatitis Specimen (cont’d)
• Prostatic infection = higher WBC/hpf count in
specimen 3 than specimen 1; bacterial count in
specimen 3 is 10 times higher than in specimen 1
• Specimen 2 is a control for bladder or kidney
infection
– Positive culture in specimen 2 invalidates positive
culture in specimen 3 (cannot differentiate urinary
tract infection from prostate infection)

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 Prostatitis Specimen (cont’d)

• Pre- and postmassage test

– Specimen 1: midstream clean-catch specimen
– Specimen 2: postmassage specimen
• Prostatitis is indicated by a quantitative culture
result in the second glass that is 10 times higher
than specimen 1

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 Pediatric Specimens

• Soft, clear, plastic bags, with hypoallergenic tape

applied to genital area
• Monitor bag frequently
• Clean-catch method with sterile bag can be used
– Cleaning for microbiology specimens
• Bags with tubes to a larger container are
available for timed specimens

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 Drug Specimen Collection

• Proper collection, labeling, and handling must be

documented
• Chain of custody: documentation from the time
of specimen collection until the time of receipt of
laboratory results
– Free of substitution, adulteration, or dilution
• Standardized form always accompanies specimen
• Specimen must withstand legal scrutiny
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 Drug Specimen Collection (cont’d)

• Points to consider
– Photo ID of urine donor or ID by employer
– No unauthorized access to specimen
• Witnessed versus unwitnessed collection
– Determined by test orderer
– Both specimens must be handed immediately to
collector

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 Drug Specimen Collection (cont’d)

• Adulteration tests
– Temperature taken within 4 minutes must be 32.5°C
to 37.7°C
– Report temperatures outside of range immediately
– Collect another specimen ASAP
– Inspect urine color for anything unusual
• Follow laboratory instructions for labeling,
packaging, and transport
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 PHYSICAL EXAMINATION
OF URINE
CHAPTER 4

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 Learning Objectives
Upon completing this chapter, the reader will be able to
1. List the common terminology used to report normal urine
color.
2. Discuss the relationship of urochrome to normal urine color.
3. State how the presence of bilirubin, biliverdin, uroerythrin,
and urobilin in a specimen may be suspected.
4. Discuss the significance of cloudy, red urine and clear, red
urine.
5. Name two pathologic causes of black or brown urine.

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 Learning Objectives (cont’d)
6. Discuss the significance of phenazopyridine in a specimen.
7. State the clinical significance of urine clarity.
8. List the common terminology used to report clarity.
9. Describe the appearance and discuss the significance of
amorphous phosphates and amorphous urates in freshly
voided urine.
10. List three pathologic and four nonpathologic causes of cloudy
urine.
11. Define specific gravity, and tell why this measurement can be
significant in the routine analysis.

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 Learning Objectives (cont’d)
12. Describe the principles of the refractometer, reagent strip, and
osmolality for determining specific gravity.
13. Given the concentration of glucose and protein in a specimen,
calculate the correction needed to compensate for these high-
molecular-weight substances in the refractometer specific
gravity reading.
14. Name two nonpathogenic causes of abnormally high specific
gravity readings using a refractometer.
15. Describe the advantages of measuring specific gravity using a
reagent strip and osmolality.
16. State possible causes of abnormal urine odor.
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 Introduction

• Physical examination of urine includes

– Color
– Clarity
– Specific gravity
• Results provide
– Preliminary information
– Correlation with other chemical and microscopic
results

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 Physical Characteristics

• Provides preliminary information concerning

disorders such as
– Glomerular bleeding
– Liver disease
– Inborn errors of metabolism
– Urinary tract infection
– Renal tubular function

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 Color
• Ranges from colorless to black
• Normal variations caused by
– Normal metabolic functions
– Physical activity
– Ingested materials
– Pathologic conditions
• Abnormal variations caused by
– Bleeding
– Liver disease
– Infection
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 Normal Urine Color

• Common terminology
– Pale yellow, yellow, dark yellow
– Should be consistent within institution
– Urochrome is pigment causing yellow color
• Normally excreted at a constant rate
• Increased in thyroid disorders and fasting
• Increases when specimen sits at room temperature
• Provides estimate of body hydration
• Pale yellow to dark yellow can be normal

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 Normal Urine Color (cont’d)
• Additional pigments uroerythrin, urobilin
– Color changes in older specimens
• Uroerythrin
– Pink pigment
– Attaches to amorphous urates formed in refrigerated
specimens
• Urobilin
– Oxidation of normal constituent, urobilinogen
– Orange-brown color in older specimens
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 Abnormal Urine Color

• Many colors and causes

• Often reason patient comes to the physician
• Common abnormal colors
– Dark yellow/amber/orange
– Red/pink/brown
– Brown/black
– Blue/green

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 Dark Yellow/Amber/Orange
• Dark yellow and amber
– Normal = concentrated urine
– Abnormal = bilirubin
• Bilirubin indicates possible hepatitis virus present
– Standard precautions
• Foam
– Bilirubin produces yellow foam when shaken
– Normal urine produces small amount of white foam
caused by protein
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 Dark Yellow/Amber/Orange (cont’d)

• Photooxidation of large amounts of urobilinogen

produces yellow-orange urine
– No yellow foam when shaken
• Photooxidation of bilirubin to biliverdin produces
yellow-green urine

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 Dark Yellow/Amber/Orange (cont’d)

• Phenazopyridine (pyridium) or Azo-Gantrisin for

urinary tract infection produces thick orange
pigment and yellow foam (no bilirubin)
– Thick pigment is noticeable, obscures natural color,
and interferes with reagent strips

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 Red/Pink/Brown
• Blood is a common cause of red urine
– Color can range from pink to brown
– Pink = small amount of blood
– Brown = oxidation of hemoglobin to methemoglobin
• Methemoglobin
– RBCs remaining in acid urine
– Fresh brown specimen can indicate glomerular bleeding
• Cloudy red urine = RBCs
• Clear red urine = hemoglobin/myoglobin
• Hemoglobin
– In vivo lysis of RBCs
– Patient’s plasma will also be red
– Consider in vitro lysis/specimen handling
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 Red/Pink/Brown (cont’d)
• Myoglobin
– Breakdown of skeletal muscle
– Fresh urine is often more reddish/brown
– Patient’s plasma is clear
• Port wine–colored urine
– Oxidation of porphobilinogen to porphyrias
• Nonpathogenic red urine
– Menstrual contamination
– Pigmented foods
– Medications (rifampin, pheno-compounds)
• Fresh beets
– Genetically susceptible people in alkaline urine
• Black raspberries in acid urine
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 Brown/Black

• Additional testing for specimens that

– Turn black after standing at room temperature
– Test negative for blood
• Melanin
– Excess in malignant melanoma
– Oxidation of melanogen to melanin
• Homogentisic acid
– Black color in alkaline urine
– Alkaptonuria
• Medications, levodopa, phenol derivatives, flagyl

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 Blue/Green
• Urinary and intestinal bacterial infections are the
pathogenic cause
– Urinary: pseudomonas infection
– Intestinal: infection causing increased urinary indican
oxidizing to indigo blue
• Catheter bags: purple color from Klebsiella,
Providencia, and indican
• IV phenol medications cause green
– Clorets (green) medications: Robaxin, methylene blue,
Elavil (blue)

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 Color and Clarity Procedure

• Use a well-mixed specimen

• View through a clear container
• View against a white background
• Maintain adequate room lighting
• Evaluate a consistent volume of specimen
• Determine color and clarity

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 Clarity

• Refers to the transparency or turbidity of a specimen

• Normal reporting
– Clear, hazy, cloudy, turbid, milky
• Visual examination
– Gently swirl specimen in a clear container in front of a
good light source
• Automated turbidity readings are available
• Fresh clean-catch urine is normally clear
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 Urine Clarity
• Clear: No visible particulates, transparent
• Hazy: Few particulates, print easily seen
through urine
• Cloudy: Many particulates, print blurred
through urine
• Turbid: Print cannot be seen through urine
• Milky: May precipitate or be clotted

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 Nonpathogenic Turbidity

• Hazy female specimens with squamous epithelial

cells and mucus
• Bacterial growth in nonpreserved specimens
• Refrigerated specimens with precipitated
amorphous phosphates (white) and urates (pink)
• Contamination: fecal, talc, semen, vaginal
creams, IV contrast media

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 Pathologic Turbidity

• Most common: RBCs, WBCs, bacteria

• Also: nonsquamous epithelial cells, yeast, abnormal
crystals, lymph fluid, lipids
• The extent of turbidity should correspond to the
amount of material observed in the microscopic
examination
• Clarity is one of the criteria considered in
determining the necessity of performing a
microscopic examination

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 Specific Gravity (SG)

• Evaluation of urine concentration

• Determines if specimen is concentrated enough
to provide reliable screening results
• Definition: the density of a solution compared
with the density of an equal volume of distilled
water at the same temperature

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 Specific Gravity (SG) (cont’d)

• Isosthenuric: SG of 1.010 (the SG of the plasma

ultrafiltrate)
• Hyposthenuric: SG lower than 1.010
• Hypersthenuric: SG higher than 1.010
• Normal random specimen range
– 1.003 to 1.035; most common 1.015 to 1.025
– Below 1.003 may not be urine
• Consistent low readings: further testing
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 Refractometer

• Measures velocity of light in air versus velocity of

light in a solution
• Concentration changes the velocity and angle at
which the light passes through the solution
• Prism in the refractometer determines the angle
that light is passing through the urine and
converts angle to calibrated viewing scale

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 Refractometer (cont'd)

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 Refractometer (cont’d)

• Advantages
– Temperature compensation not needed
• Light passes through temperature-compensating liquid
• Compensated between 15°C and 38°C
– Small specimen size: one or two drops

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 Glucose and Protein Corrections

• Subtract 0.003 for each gram of protein present

• Subtract 0.004 for each gram of glucose present
• Protein or glucose concentration can be
determined from the chemical reagent strip tests

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 Correction Example

• A specimen containing 1 g/dL protein and 1 g/dL

glucose has a specific gravity reading of 1.030
• Calculate the corrected reading
1.030 – 0.003 (protein) = 1.027 – 0.004 (glucose) =
1.023 corrected specific gravity

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 Methodology
• Drop of urine placed on prism
• Focus on light source, and read
scale
• Wipe off prism between
specimens
• Calibration
– Distilled water should read 1.000;
adjust set screw if necessary
– 5% NaCl should read 1.022 ± 0.001
– 9% sucrose should read 1.034 ±
0.001

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 Clinical Correlations

• Abnormally high results = >1.040

– Radiographic contrast media (IVP)
– Dextran, other IV plasma expanders
– Check patient’s clinical course/history
• Reagent strip readings and osmometry not
affected by high-molecular-weight substances
– Should be used as an alternative if possible

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 Osmolality

• A more representative measure of renal concentrating

ability can be obtained
• Specific gravity depends on the number of particles
present in a solution and the density of these particles
• Osmolality is affected only by the number of particles
present
– Substances of interest are small molecules
• Sodium
• Chloride
• Urea

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 Osmole

• 1 g molecular weight of a substance divided by

the number of particles into which it dissociates
(= to MW of substance)
– Glu = 180 g/osm (C + H + O)
– NaCl = 58.5 g/osm (Na + Cl)
• The unit of measure used in the clinical
laboratory is the milliosmole (mOsm)

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 Osmolarity
• Osmolality of a solution can be determined by
measuring a property that is mathematically related
to the number of particles in the solution
– Colligative property
• Changes in colligative properties
– Lower freezing point
– Higher boiling point
– Increased osmotic pressure
– Lower vapor pressure

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 Measuring Osmolality

• Measuring osmolality in the urinalysis laboratory

requires an osmometer
– Additional step in the routine urinalysis procedure
• Automated osmometer utilizes freezing point
depression to measure osmolality

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 Reagent Strip SG

• The reagent strip reaction is based on the change in pKa

(dissociation constant) of a polyelectrolyte in an alkaline
medium
– Releasing H ions in direct proportion to the number of ions in the
solution
– The more hydrogen ions released, the lower is the pH
– Indicator bromothymol-LS blue on the reagent pad measures the
change in pH
– Indicator changes from blue (1.000 [alkaline]), through shades of
green, to yellow (1.030 [acid])
– Not affected by nonionizing substances

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 Odor
• Not routinely reported
• Fresh urine: faintly aromatic
• Older urine: ammonia
• Metabolic disorders: maple syrup urine disease,
ketosis (fruity), infection (ammonia/unpleasant)
• Food: garlic, onions, asparagus (genetic: only
certain people can smell asparagus, but all
produce odor)
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 Common Causes of Urine Odor
Odor Cause
Aromatic Normal
Foul, Bacterial decomposition, urinary tract
ammonia-like infection
Fruity, sweet Ketones (diabetes mellitus, starvation,
vomiting)
Maple syrup Maple syrup urine disease
Mousy Phenylketonuria
Rancid Tyrosinemia
Sweaty feet Isovaleric acidemia
Cabbage Methionine malabsorption
Bleach Contamination

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 CHEMICAL EXAMINATION
OF URINE
CHAPTER 5

Copyright © 2014. F.A. Davis Company

 Learning Objectives
Upon completing this chapter, the reader will be able to
1. Describe the proper technique for performing reagent strip testing.
2. List four causes of premature deterioration of reagent strips, and describe how
to avoid them.
3. List five quality-control procedures routinely performed with reagent strip
testing.
4. List the reasons for measuring urinary pH, and discuss their clinical
applications.
5. Discuss the principle of pH testing by reagent strip.
6. Differentiate between prerenal, renal, and postrenal proteinuria, and give
clinical examples of each.

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 Learning Objectives (cont’d)
7. Explain the “protein error of indicators,” and list any sources of interference
that may occur with this method of protein testing.
8. Discuss microalbuminuria including significance, reagent strip tests, and their
principles.
9. Explain why glucose that is normally reabsorbed in the proximal convoluted
tubule may appear in the urine, and state the renal threshold levels for
glucose.
10.Describe the principle of the glucose oxidase method of reagent strip testing
for glucose, and name possible causes of interference with this method.
11.Describe the copper reduction method for detection of urinary reducing
substances, and discuss the current use of this procedure.
12.Name the three “ketone bodies” appearing in urine and three causes of
ketonuria.

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 Learning Objectives (cont’d)
13.Discuss the principle of the sodium nitroprusside reaction to detect ketones,
including sensitivity and possible causes of interference.
14.Differentiate between hematuria, hemoglobinuria, and myoglobinuria with
regard to the appearance of urine and serum and clinical significance.
15.Describe the chemical principle of the reagent strip method for blood testing,
and list possible causes of interference.
16.Outline the steps in the degradation of hemoglobin to bilirubin, urobilinogen,
and finally urobilin.
17.Describe the relationship of urinary bilirubin and urobilinogen to the diagnosis
of bile duct obstruction, liver disease, and hemolytic disorders.
18.Discuss the principle of the reagent strip test for urinary bilirubin, including
possible sources of error.

Copyright © 2014. F.A. Davis Company

 Learning Objectives (cont’d)

19.State two reasons for increased urine urobilinogen and one reason for a
decreased urine urobilinogen.
20.Discuss the principle of the nitrite-reagent-strip test for bacteriuria.
21.List five possible causes of a false-negative result in the reagent strip test for
nitrite.
22.State the principle of the reagent strip test for leukocytes.
23.Discuss the advantages and sources of error of the reagent strip test for
leukocytes.
24.Explain the principle of the chemical test for specific gravity.
25.Compare reagent strip testing for urine specific gravity with osmolality and
refractometer testing.
26.Correlate physical and chemical urinalysis results.

Copyright © 2014. F.A. Davis Company

 Reagent Strips
• Reagent strips provide a simple, rapid means for performing
routine chemical tests on urine
• Single and multitest strips available
• The brand and number of tests used are a matter of
laboratory preference
• Specified by urinalysis instrumentation manufacturers
• Strips consist of chemical-impregnated absorbent pads on a
plastic strip

Copyright © 2014. F.A. Davis Company

 Reagent Strips (cont’d)
• A color-producing chemical reaction takes place when the
absorbent pad comes in contact with urine
• The reactions are interpreted by comparing the color
produced on the pad within the required time frame with a
chart supplied by the manufacturer
• Color comparison charts are supplied by the manufacturer
• Several degrees of color are shown to provide
semiquantitative readings of neg, trace, 1+, 2+, 3+, and 4+
• Estimates of mg/dL are also provided for many of the test
areas
Copyright © 2014. F.A. Davis Company
 Reagent Strip Technique

• Dip strip briefly into well-mixed specimen at

room temperature
• Remove excess urine by touching edge of strip to
container as strip is withdrawn
• Blot edge of strip on absorbent pad
• Wait specified amount of time
• Read using a good light source
Copyright © 2014. F.A. Davis Company
 Improper Technique Errors

• Formed elements such as red and white blood cells sink to

the bottom of the specimen and will be undetected in an
unmixed specimen
• Allowing the strip to remain in the urine for an extended
period may cause leaching of reagents from the pads
• Excess urine remaining on the strip after its removal from
the specimen can produce a runover between chemicals
on adjacent pads, producing distortion of the colors

Copyright © 2014. F.A. Davis Company

 Improper Technique Errors
(cont’d)
• The timing for reactions to take place varies between tests and
manufacturers; the manufacturer’s stated time should be
followed
• A good light source is essential for accurate interpretation of
color reactions
• The strip must be held close to the color chart without actually
being placed on the chart; reagent strips and color charts from
different manufacturers are not interchangeable
• Specimens that have been refrigerated must be allowed to
return to room temperature prior to reagent strip testing

Copyright © 2014. F.A. Davis Company

 Handling and Storing
Reagent Strips
• Store with desiccant in an opaque, tightly sealed
container
• Remove strips immediately prior to use
• Do not expose to volatile fumes
• Store below 30°C
• Do not use past the expiration date
• Visually inspect for discoloration/deterioration
Copyright © 2014. F.A. Davis Company
 Quality Control of Reagent Strips
• Run positive and negative controls at least once per 24
hours
• Run additional controls
• When a new bottle of strips is opened
• When results are questionable
• When there are concerns over strip integrity
• Record control results
• Manufactured positive and negative controls are available
• Do not use distilled water as a negative control as
reactions are designed for urine ionic concentration
Copyright © 2014. F.A. Davis Company
 Quality Control of Reagent Strips
(cont’d)
• All negative control readings should be negative
• Positive control readings should agree with
published control values
• Be aware of manufacturer-stated limitations and
interfering substances
• Correlate chemical readings to each other and
physical and microscopic readings

Copyright © 2014. F.A. Davis Company

 Confirmatory Testing

• Confirmatory tests use different reagents or

methodologies to detect the same substances as
reagent strips with the same or greater sensitivity or
specificity
• Nonreagent strip testing procedures using tablets
and liquid chemicals may be available when
questionable results are obtained
• Chemical reliability of these procedures also must be
checked using positive and negative controls
Copyright © 2014. F.A. Davis Company
 Urine pH

• Lungs and kidneys are major regulators of acid-

base content
• They do this through the secretion of hydrogen
in the form of ammonium ions, hydrogen
phosphate, and weak organic acids, and by the
reabsorption of bicarbonate from the filtrate in
the convoluted tubules.

Copyright © 2014. F.A. Davis Company

 • First morning specimen slightly acidic at 5.0 to
6.0
• Postprandial specimen more alkaline
• Normal range is 4.5 to 8.0
• No absolute values are assigned ,it must be
considered in conjunction with other patient
information, such

Copyright © 2014. F.A. Davis Company

 Urine pH (cont’d)
• Considerations include
– Acid-base content of the blood
– Patient’s renal function
– Presence of a urinary tract infection
– Patient’s dietary intake
– Age of the specimen
• A pH above 8.5 is associated with an
aged/improperly preserved specimen, so a fresh
specimen should be obtained
Copyright © 2014. F.A. Davis Company
 Clinical Signifance

• The importance of urinary pH is primarily as an

aid in determining the existence :-

Copyright © 2014. F.A. Davis Company

 Summary of Clinical Significance
of Urine pH
• Respiratory or metabolic acidosis/ketosis
• Respiratory or metabolic alkalosis
• Defects in renal tubular secretion and reabsorption
of acids and bases—renal tubular acidosis
• Renal calculi formation
• Treatment of urinary tract infections
• Precipitation/identification of crystals
• Determination of unsatisfactory specimens

Copyright © 2014. F.A. Davis Company

 pH-Reagent Strip Reactions
• Needed to measure between 5.0 and 9.0 in one half or one unit
increments
• Double-indicator system reaction
– Methyl red = 4 to 6 red/orange to yellow
– Bromthymol blue = 6 to 9 green to blue

Methyl red + H+ → Bromthymol blue − H+

(Red/Orange → Yellow) (Green → Blue)

• Interference
– No known substances interfere with urinary pH measurements performed
by reagent strips

Copyright © 2014. F.A. Davis Company

 Protein
• Most indicative of renal disease
– Proteinuria seen in early renal disease
• Normal = <10 mg/dL or 100 mg/24 h
• Low-molecular-weight serum proteins are filtered;
many are reabsorbed
• Albumin is primary protein of concern
• Other proteins include
– Vaginal, prostatic, and seminal proteins
– Tamm-Horsfall (uromodulin)
Copyright © 2014. F.A. Davis Company
 Clinical Significance

• Presence requires determination of normal or

pathological condition
• Clinical proteinuria = 30 mg/dL, 300 mg/24 h
• Variety of causes
– Prerenal
– Renal
– Postrenal

Copyright © 2014. F.A. Davis Company

 Prerenal Proteinuria

• Conditions affecting the plasma, not the kidney

• Transient, increase levels of low-molecular-
weight plasma proteins, acute phase reactants,
exceed reabsorptive capacity
• Rarely seen on reagent strip (not albumin)

Copyright © 2014. F.A. Davis Company

 Bence Jones Protein (BJP)

• Multiple myeloma (plasma cell myeloma)

• Immunoglobulin light chains
• Multiple myeloma confirmation is serum
electrophoresis

Copyright © 2014. F.A. Davis Company

 Renal Proteinuria

• Glomerular or tubular damage

– Glomerular proteinuria
– Microalbuminuria
– Orthostatic (postural) proteinuria
– Tubular proteinuria

Copyright © 2014. F.A. Davis Company

 Glomerular Proteinuria
• Damage to glomerular membrane
• Impaired selective filtration causes increased
protein filtration leading to cellular excretion
• Abnormal substances deposit on the membrane
– Primarily immune disorders result in immune
complex formation
• Lupus erythematosus, streptococcal glomerulonephritis
– Amyloids and other toxins
Copyright © 2014. F.A. Davis Company
 Glomerular Proteinuria (cont’d)

• Increased pressure on the filtration mechanism

– Hypertension
– Strenuous exercise
– Dehydration
– Pregnancy
• Preeclampsia
• Benign proteinuria (transient)
– Strenuous exercise, high fever, dehydration, and exposure
to cold

Copyright © 2014. F.A. Davis Company

 Microalbuminuria

• Diabetic nephropathy with type 1 and type 2

diabetes mellitus
– Reduced glomerular filtration
– Eventual renal failure
• Also associated with an increased risk of
cardiovascular disease

Copyright © 2014. F.A. Davis Company

 Orthostatic (Postural) Proteinuria

• Increased pressure on the renal vein when in the

vertical position
• Occurs in vertical position, disappears in horizontal
position
• Collection instructions
– Empty bladder before bed
– Collect specimen immediately on arising
• Negative reading will be seen on the first morning specimen
• Positive result will be found on the second specimen

Copyright © 2014. F.A. Davis Company

 Tubular Proteinuria

• Tubular damage affecting reabsorptive ability

– Acute tubular necrosis
• Toxic substances, heavy metals, viral infections, Fanconi
syndrome (generalized proximal convoluted tubule defect)
• Amount of protein
– Glomerular disorders: up to 4 g/day
– Tubular disorders: much lower levels

Copyright © 2014. F.A. Davis Company

 Postrenal Proteinuria
• Protein added in the lower urinary and
genitourinary tract
• Microbial infections causing inflammations and
release of interstitial fluid protein
• Menstrual contamination
• Semen/prostatic fluid
• Vaginal secretions
• Traumatic injury
Copyright © 2014. F.A. Davis Company
 Protein-Reagent Strip Reactions

• Traditional principle
– Protein error of indicators
– Certain indicators change color in the presence of
protein at a constant pH
– Protein accepts H+ from the indicator, increased
sensitivity to albumin due to more amino groups to
accept H+ than other proteins

Copyright © 2014. F.A. Davis Company

 Reagent Strip Reactions

• Tetrabromophenol blue or tetrachlorophenol

tetrabromosulfonephthalein and an acid buffer
• pH level 3 both indicators are yellow
• Color progresses through green to blue
• Report: neg, trace, 1+, 2+, 3+, 4+, or 30, 100,
300, 2000 mg/dL
• Trace values are <30 mg/dL
Copyright © 2014. F.A. Davis Company
 Reagent Strip Reactions (cont’d)

pH 3.0
Indicator (H+) + Protein → Protein + H+
(Yellow) → Indicator – H+
(Green/Blue)

Copyright © 2014. F.A. Davis Company

 Reaction Interference

• Highly buffered alkaline urine overrides acid buffer

system (color change unrelated to protein
concentration)
– Leaving reagent pad in urine too long removes buffer
• False-positive
– Highly pigmented urine
– High SG
– Quaternary ammonium compounds, detergents, antiseptics,
chlorhexidine

Copyright © 2014. F.A. Davis Company

 Sulfosalicylic Acid (SSA)
Precipitation

• Confirmatory test for protein

• Cold precipitation test that reacts equally with all
forms of protein
• Must be performed on centrifuged specimens to
remove any extraneous contamination

Copyright © 2014. F.A. Davis Company

 Microalbuminuria

• Semiquantitative testing for patients at risk for

renal disease
• Immunochemical assays for albumin or albumin-
specific reagent strips
• Measure creatinine to produce an
albumin:creatinine ratio
• First morning specimens are recommended

Copyright © 2014. F.A. Davis Company

 Micral-Test
• Gold-labeled antihuman antibody-enzyme conjugate
• Dip strip in urine to marked level for 5 seconds
• Albumin binds to antibody
• Bound and unbound conjugates move up strip
• Unbound removed in captive zone containing albumin;
bound continues up strip
• Reaches enzyme substrate, reacts
• Colors from white (neg) to red (varying degrees)
• Compare color to chart
• Results read from 0 to 10 mg/dL
Copyright © 2014. F.A. Davis Company
 Immunodip Test
• Immunochromographic technique
• Specially designed container for strip
• Place container in controlled amount of specimen for 3 min, urine
enters container
• Albumin binds to blue latex particles coated with antihuman
albumin antibody
• Bound and unbound migrate up strip
• Unbound encounters area of immobilized albumin on strip—forms
blue band
• Bound continues migrating to an area of immobilized antibody and
forms blue band
• Color of band is compared with chart
Copyright © 2014. F.A. Davis Company
 Reagent Strip Microalbumin Tests

• Clinitek microalbumin reagent strips and

Multistix Pro reagent strips
• Simultaneous measurement of albumin and
creatinine
• Provide an estimate of the 24-hour albumin
concentrations from random urine
• Albumin pad uses dye-binding reaction for
specific albumin testing
Copyright © 2014. F.A. Davis Company
 Reagent Strip Reactions
• Albumin strip dye (DIDNTB)
– bis(3′,3″, diodo-4′,4″-dihydroxy-5′,5″-dinitrophenyl)-3,4,5,6-
tetra-bromo-sulfonphthalein
– Specific for albumin
– Sensitivity: 8 to 15 mg/dL (80 to 150 mg/L)
– Highly buffered alkaline urine interference is controlled by
treated paper
– Polymethyl vinyl glycol carbonate decreases nonspecific
binding of poly amino acids
• Visibly bloody urine elevates results
• Abnormally colored urines may interfere with readings
Copyright © 2014. F.A. Davis Company
 Creatinine Reagent Strip
• Principle: pseudoperoxidase activity of copper-creatinine
complexes
• Reagent strips contain copper sulfate (CuSO4, 3,3′,5,5′-
tetramethylbenzidine (TMB) and diisopropyl benzene
dihydroperoxide (DBDH))
• Creatinine in urine combines with copper sulfate to form
copper-creatinine peroxidase
• Peroxidase reacts with DBDH, releases oxygen ions that
oxidize TMB
• Colors change from orange to green to blue
Copyright © 2014. F.A. Davis Company
 Creatinine Reagent Strip (cont’d)

CuSO4 + CRE → Cu(CRE) peroxidase

Cu(CRE) peroxidase

DBDH + TMB → oxidized TMB + H2O

(peroxidase) (chromogen) (orange to blue)

Copyright © 2014. F.A. Davis Company

 Creatinine Reagent Strip (cont’d)

• Results: 10, 50, 100, 200, 300 mg/dL or

– 0.9, 4.4, 8.8, 17.7, 26.5 mmol/L
• Elevated results: bloody urine, tagamet
(cimetidine), abnormal urine color
• No creatinine results are abnormal
• Purpose is to correlate creatinine with albumin
results to determine the albumin:creatinine ratio

Copyright © 2014. F.A. Davis Company

 Albumin (Protein):
Creatinine Ratio
• Automated and manual methods available
• Clinitek microalbumin strips can be read only on
Clinitek instruments
• Instrument calculates A:C ratio and prints out
albumin, creatinine, and A:C results
• Results in conventional and SI units
• Abnormal A:C ratio: 30 to 300 mg/g or 3.4 to
33.9 mg/mol
Copyright © 2014. F.A. Davis Company
 Albumin (Protein):
Creatinine Ratio (cont'd)
• Bayer Multistix Pro 10 strips measure creatinine,
protein-high and protein-low
– Protein-high is protein error of indicators method
– Protein-low is dye-binding method
• Urobilinogen and bilirubin are not included on these
strips
• Read manually or on instrumentation
• Print-out is protein:creatinine ratio with albumin
result included on print-out
Copyright © 2014. F.A. Davis Company
 Albumin (Protein):
Creatinine Ratio (cont'd)
• Instrument automatically calculates
• A chart is available for manual ratio calculation
• Results are reported as Normal or Abnormal
• A result of normal dilute indicates that the
specimen should be recollected, making sure it is
a first morning specimen

Copyright © 2014. F.A. Davis Company

 Glucose

• The most frequent chemical analysis performed on

urine
• Blood and urine glucose tests are included in all physical
examinations
• Mass health screening programs

Copyright © 2014. F.A. Davis Company

 Glucose (cont’d)

• Clinical significance
– Major screening test for diabetes mellitus
– Renal threshold is 160 to 180 mg/dL
– Higher blood sugar = glycosuria
• Gestational diabetes
– Placental hormones block action of insulin
• High fetal glucose stresses baby’s pancreas
• Result is fat baby
• Mother prone to type 2 diabetes

Copyright © 2014. F.A. Davis Company

 Clinical Significance

• Elevated blood glucose, diabetes mellitus

• Renal threshold is ~160 to 180 mg/dL
• Higher blood sugar = glycosuria
• Collection under controlled conditions
– Fasting specimen
– “Second” collection
– 2 h postprandial

Copyright © 2014. F.A. Davis Company

 Nondiabetic Glycosuria
• Hormonal disorders: pancreatitis, pancreatic cancer,
acromegaly, Cushing’s syndrome, hyperthyroidism,
pheochromocytoma
• Hormones: glucagon, epinephrine, cortisol,
thyroxine, growth hormone oppose glucose
• Insulin: converts glucose to storage glycogen
• Hormones: glycogen back to glucose
• Epinephrine: inhibits insulin; seen with stress,
cerebral trauma, and myocardial infarction
Copyright © 2014. F.A. Davis Company
 Renal Glycosuria

• Tubular reabsorption disorder

• End-stage renal disease
• Cystinosis
• Fanconi syndrome
• Temporary lowering of renal threshold in
pregnancy

Copyright © 2014. F.A. Davis Company

 Reagent Strip Reactions
• Glucose oxidase reaction specific for glucose
• Glucose oxidase, peroxide, chromogen, buffer on test
pad
– Double sequential enzyme reaction
• Glucose oxidase catalyzes a reaction between glucose
and oxygen
– Produces gluconic acid and peroxide
• Peroxidase catalyzes the reaction between peroxide and
chromogen to form an oxidized colored compound
– Direct proportion to the concentration of glucose
Copyright © 2014. F.A. Davis Company
 Reagent Strip Reactions (cont’d)

Glucose oxidase
Glucose + O2 (air) → gluconic acid + H2O2

Peroxidase
H2O2 + chromogen → oxidized colored
chromogen + H2O

Copyright © 2014. F.A. Davis Company

 Reagent Strip

• Chromogens used
– Potassium iodide (green to brown) (Multistix)
– Tetramethylbenzidine (yellow to green) (Chemstrip)
• Reporting results
– Neg, trace, 1+, 2+, 3+, 4+
– 100 mg/dL to 2 g/dL
– 0.1% to 2%

Copyright © 2014. F.A. Davis Company

 Reaction Interference

• False-positive: only peroxide, oxidizing

detergents
• False-negative: enzymatic reaction interference
– Ascorbic acid and strong reducing agents
– High levels of ketones (unlikely)
– High specific gravity and low temperature
– Greatest source of error is old specimens
• Subjecting the glucose to bacterial degradation

Copyright © 2014. F.A. Davis Company

 Copper Reduction Test (Clinitest)

• Reduction of copper sulfate to cuprous oxide with

alkali and heat
• Clinitest tablets: copper sulfate, sodium carbonate,
sodium citrate, sodium hydroxide
• Sodium citrate + NaOH = heat
• Sodium carbonate = CO2 blocks room air
• Reducing substance + CuSO4
– Color change: negative blue (CuSO4) through green,
yellow, and orange/red (Cu2O)
Copyright © 2014. F.A. Davis Company
 Copper Reduction Test

Heat
CuSO4 (cupric sulfide) + reducing substance -----→

Alkali

Cu2O (cuprous oxide) + oxidized substance → color

(blue/green to orange/red)

Copyright © 2014. F.A. Davis Company

 Clinitest Procedure
• Pass through
– High levels of reducing substance
– Color from blue through red back to green-brown: rapid
reaction
– Repeat with two-drop procedure
• 10 drops water
• 2 drops urine
• Values up to 5 g/L versus 2 g/L
• Separate chart must be used
• Hygroscopic tablets: strong blue color and excess fizzing
= deterioration
Copyright © 2014. F.A. Davis Company
 Reducing Substances
• Not a specific test for glucose
– Sensitivity: 200 mg/dL (lower) than strip
• Clinitest does not provide a confirmatory test for
glucose
• Interference from reducing sugars
– Galactose, lactose, fructose, maltose, pentoses, ascorbic acid,
cephalosporins
• Major use is quick screen for “inborn error of
metabolism” in children up to 2 years old
– Newborn screening programs for galactosemia in all states

Copyright © 2014. F.A. Davis Company

 Ketones

• Three intermediate products of fat metabolism

– Acetone: 2%
– Acetoacetic acid: 20%
– β-hydroxybutyrate: 78%
• Appear in urine when body stores of fat must be
metabolized to supply energy

Copyright © 2014. F.A. Davis Company

 Clinical Significance
• Increased fat metabolism = inability to metabolize
carbohydrate
• Primary causes
• Diabetes mellitus
• Vomiting (loss of carbohydrates)
• Starvation, malabsorption, dieting (↓ intake)
• Ketonuria shows insulin deficiency
• Monitor diabetes
• Diabetic ketoacidosis = increased accumulation of ketones in
the blood
• Electrolyte imbalance, dehydration, and diabetic coma
Copyright © 2014. F.A. Davis Company
 Clinical Significance (cont’d)

• Ketonuria unrelated to diabetes

– Inadequate intake/absorption of carbohydrates
– Vomiting
– Weight loss
– Eating disorders
– Frequent strenuous exercise

Copyright © 2014. F.A. Davis Company

 Reagent Strip Reactions

• Primary reagent: sodium nitroprusside

– (Nitroferricyanide)
• Measure primarily acetoacetic acid
– Assumes the presence of β-hydroxybutyrate and
acetone
• Acetoacetic acid (alkaline) + nitroprusside →
purple color

Copyright © 2014. F.A. Davis Company

 Reagent Strip Reactions (cont’d)

• Report qualitatively • Semiquantitatively

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

Copyright © 2014. F.A. Davis Company

 Reagent Strip Reactions (cont’d)

acetoacetate (and acetone) + sodium nitroprusside

Alkaline
+ (glycine) ——————> purple color

Copyright © 2014. F.A. Davis Company

 Reaction Interference
• Levodopa in large dosage
• Medications containing sulfhydryl groups
– May produce atypical color reactions
• False-positive results from improperly timed
readings
• Falsely decreased values in improperly preserved
specimens
– Breakdown of acetoacetic acid by bacteria

Copyright © 2014. F.A. Davis Company

 Acetest

• Not a urine confirmatory test

• Tablet = sodium nitroprusside, glycine, disodium
phosphate, lactose (gives better color)

Copyright © 2014. F.A. Davis Company

 Blood
• Hematuria: intact RBCs
– Cloudy red urine
• Hemoglobinuria: product of RBC destruction
– Clear red urine
• Any amount of blood greater than five cells per
microliter of urine is considered clinically significant
• Chemical tests for hemoglobin provide the most
accurate means for determining the presence of blood
• The microscopic examination can be used to
differentiate between hematuria and hemoglobinuria
Copyright © 2014. F.A. Davis Company
 Blood (cont’d)
• Hematuria: intact RBCs, cloudy red urine
• Damage to renal system
– Renal calculi
– Glomerular disease
– Tumors
– Trauma
– Pyelonephritis
– Exposure to toxic chemicals
– Anticoagulants
Copyright © 2014. F.A. Davis Company
 Blood (cont’d)
• Hemoglobinuria: clear, red urine
– Transfusion reactions
– Hemolytic anemias
– Severe burns
– Infections/malaria
– Strenuous exercise/RBC trauma
– Brown recluse spider bites
• Hemoglobinuria may result from the lysis of red
blood in dilute, alkaline urine
• Hemosiderin: yellow brown granules in sediment

Copyright © 2014. F.A. Davis Company

 Blood (cont’d)
• Myoglobinuria: heme-containing protein in muscle
tissue; clear, red/brown urine
– Rhabdomyolysis: muscle destruction
• Muscular trauma/crush syndromes
• Prolonged coma
• Convulsions
• Muscle-wasting diseases
• Alcoholism
• Drug abuse
• Extensive exertion
• Cholesterol-lowering statin medications
Copyright © 2014. F.A. Davis Company
 Reagent Strip Reactions
• Principle pseudoperoxidase activity of
hemoglobin
• Catalyze a reaction between the heme
component
– Hemoglobin and myoglobin
– Chromogen tetramethylbenzidine
– Produce an oxidized chromogen
• Green-blue color

Copyright © 2014. F.A. Davis Company

 Reagent Strip Reactions (cont’d)
hemoglobin
H2O2 + chromogen ---------------→ oxidized chromogen + H2O
peroxidase
• Two charts corresponding to different reactions
• Free hemoglobin shows uniform color
• Intact RBCs show a speckled pattern on pad
– Report: trace, small (1+), moderate (2+), large (3+)
– Sensitivity 5 RBCs/μL
Copyright © 2014. F.A. Davis Company
 Reaction Interference
• False-positive
– Menstrual contamination, strong oxidizing agents,
bacterial peroxidases
• False-negative
– Ascorbic acid >25 mg/dL
– High SG/crenated cells
– Formalin
– Captopril
– High concentrations of nitrite
– Unmixed specimens
Copyright © 2014. F.A. Davis Company
 Bilirubin

• Urine bilirubin early indicator of liver disease

• Normal degradation product of hemoglobin
– RBCs destroyed by liver and spleen following 120-day life span
• Body recycles iron, protein
• Protoporphyrin is broken down into bilirubin
• Bilirubin is bound to albumin
– Kidneys cannot excrete
• Unconjugated bilirubin: water insoluble

Copyright © 2014. F.A. Davis Company

 Bilirubin (cont’d)

• Conjugated bilirubin: water soluble

• Unconjugated bilirubin to the liver
– Conjugated with glucuronic acid
• Forms conjugated bilirubin
– From liver to intestines
– Reduced to urobilinogen, stercobilinogen, and
urobilin by intestinal bacteria
• Excreted in feces

Copyright © 2014. F.A. Davis Company

 Bilirubin Metabolism

Copyright © 2014. F.A. Davis Company

 Clinical Significance

• Conjugated bilirubin appears in urine with bile duct obstruction,

liver disease or damage
• Obstruction: bilirubin backs up into circulation and is excreted in
urine
– No urobilinogen is formed
• Hepatitis, cirrhosis: conjugated bilirubin leaks back into circulation
from damaged liver; some bilirubin passes to intestine
• Hemolytic disease: increased unconjugated bilirubin, increased
urobilinogen

Copyright © 2014. F.A. Davis Company

 Reagent Strip Reactions

• Principle is a diazo reaction

• Report: neg, small (1+), moderate (2+), large (3+)
• Colors may be difficult to interpret
– Easily influenced by other pigments present in the urine
• Atypical colors can be problem for automated readers

Copyright © 2014. F.A. Davis Company

 Reagent Strip Reactions (cont’d)
acid
bilirubin glucuronide + *diazonium salt--------→ azodye
(tan or pink to violet)

* diazonium salt- (2,4-dichloroaniline diazonium salt or

2,6-dichlorobenzene-diazonium-tetrafluoroborate)
Copyright © 2014. F.A. Davis Company
 Reaction Interference
• False-positive
– Urine pigments
– Pyridium (phenazopyridine)
– Drugs indican, iodine
• False-negative
– Old specimens (biliverdin does not react)
– Ascorbic acid >25 mg/dL
– Nitrite
• Combine with diazonium salt and block bilirubin reaction

Copyright © 2014. F.A. Davis Company

 Ictotest

• Confirmatory for bilirubin

– Tablets containing p-nitrobenzene-diazonium-p-
toluenesulfonate, SSA, sodium carbonate, and boric acid
• Use specified mat for test; mat keeps bilirubin on
surface for reaction
• Positive reaction = blue-to-purple color
• Interfering substances are washed into the mat, and
only bilirubin remains on the surface

Copyright © 2014. F.A. Davis Company

 Urobilinogen

• Bilirubin in intestine converted to urobilinogen and

stercobilinogen
• Urobilinogen is reabsorbed into circulation and
stercobilinogen is not = urobilin
– Pigments responsible for the characteristic brown color of
feces
• There is always a small amount of urobilinogen filtered
by the kidneys and is found in the urine <1 mg/dL

Copyright © 2014. F.A. Davis Company

 Clinical Significance
• Early detection of liver disease, greater than 1
mg/dL
• Liver disorders, hepatitis, cirrhosis, carcinoma
• Hemolytic disorders
– Excess bilirubin being converted to urobilinogen and
↑ urobilinogen recirculated to liver
• Negative bilirubin and strong positive
urobilinogen are seen in hemolytic disorders

Copyright © 2014. F.A. Davis Company

 Clinical Significance (cont’d)

• 1% of the nonhospitalized population and 9% of

a hospitalized population exhibit elevated results
– This is frequently caused by constipation
• No urobilinogen is seen in the urine with bile
duct obstruction; strip will give a normal result
• Reagent strips cannot report a negative
urobilinogen reading

Copyright © 2014. F.A. Davis Company

 Urine Bilirubin and Urobilinogen
in Jaundice

Urine Bilirubin Urobilinogen

Bile duct obstruction +++ Normal

Liver damage + or − ++
Hemolytic disease Negative +++

Copyright © 2014. F.A. Davis Company

 Reagent Strip Reactions
• Different principles for Multistix and Chemstrip
• Multistix: Ehrlich’s aldehyde reaction
– p-dimethylaminobenzaldehyde (Ehrlich reagent); report in Ehrlich units
(EU) 1 EU = 1 mg/dL
– Normal readings 0.2 to 1, abnormal 2, 4, 8
– Light to dark pink
• Chemstrip: diazo (azo-coupling) reaction
– 4-Methoxybenzene-diazonium-tetrafluoroborate; more specific than
Ehrlich reaction; report in mg/dL
– White to pink

Copyright © 2014. F.A. Davis Company

 Reagent Strip Reactions (cont’d)
MULTISTIX:
acid
urobilinogen + p-dimethylaminobenzaldehyde --------------→ red color
* ERC (Ehrlich’s reagent)
CHEMSTRIP:
acid
urobilinogen + **diazonium salt ———-----→ red azodye

*(Ehrlich reactive compounds)

**(4-methyloxybenzene-diazonium-tetrafluoroborate)

Copyright © 2014. F.A. Davis Company

 Reaction Interference
• Ehrlich reactive compounds: porphobilinogen,
indican, sulfonamides, methyldopa, procaine,
chlorpromazine, p-aminosalicylic acid
• Both tests: urobilinogen is highest after meals
(increased bile salts), old specimens and formalin
preservation decrease results
• Chemstrip: false-negative with high nitrite
interferes with diazo reaction
Copyright © 2014. F.A. Davis Company
 Nitrite
• Clinical significance
• Rapid screening test for the presence of urinary tract
infection (UTI)
– Cystitis (initial bladder infection)
– Pyelonephritis (tubules)
– Evaluation of antibiotic therapy
– Monitoring of patients at high risk for urinary tract
infection
– Screening of urine culture specimens (in combination
with LE test)
Copyright © 2014. F.A. Davis Company
 Reagent Strip Reaction

• Tests ability of bacteria to reduce nitrate (normal

constituent) to nitrite (abnormal)
• Greiss reaction: nitrite reacts with aromatic amine to
form a diazonium salt that then reacts with
tetrahydrobenzoquinoline to form a pink azodye
• Correspond with a quantitative bacterial culture
criterion of 100,000 organisms/mL
• Results: negative and positive

Copyright © 2014. F.A. Davis Company

 Reagent Strip Reaction (cont’d)

Acid
para-arsanilic acid or sulfanilamide + NO2 —————→ diazonium salt

(nitrite)
Acid
diazonium salt + tetrahydrobenzoquinolin —————→ pink azodye

Copyright © 2014. F.A. Davis Company

 Reaction Interference
• 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
– Negative results in the presence of even vaguely suspicious
clinical symptoms should always be repeated or followed by a
urine culture
Copyright © 2014. F.A. Davis Company
 Reaction Interference (cont’d)

• False-positive
– Old specimens (bacterial multiplication)
– Highly pigmented urine
– Pink edge or spotting on reagent strip is considered
negative
– Check automated readers manually for color
interference

Copyright © 2014. F.A. Davis Company

 Leukocyte Esterase (LE)

• Standardized means for the detection of leukocytes

• Purpose is to detect leukocytes so as not to rely on
microscopic
• LE test detects the presence of esterase in the
granulocytes and monocytes
• Advantage: detects presence of lysed leukocytes
• Not considered a quantitative test: do microscopic if
positive

Copyright © 2014. F.A. Davis Company

 Clinical Significance

• Bacterial and nonbacterial urinary tract infection

• Inflammation of the urinary tract
• Screening of urine culture specimens in
conjunction with nitrite but a better predictor
than nitrite
• Also seen with Trichomonas, Chlamydia, yeast,
interstitial nephritis

Copyright © 2014. F.A. Davis Company

 Reagent Strip Reactions
• LE catalyzes hydrolysis of acid esterase on pad to
aromatic compound and acid; aromatic compound
reacts with diazonium salt on pad for purple color

Leukocyte
indoxylcarbonic acid ester —————→ indoxyl + acid indoxyl
Esterase

Acid
+ diazonium salt ————→ purple azodye
Copyright © 2014. F.A. Davis Company
 Reagent Strip Reactions (cont’d)
• LE reaction requires the longest time of all the reagent
strip reactions
– 2 minutes
• Reported as
– Negative
– Trace
– Small: 1+
– Moderate: 2+
– Large: 3+
• Trace readings may not be significant and should be
repeated on a fresh specimen
Copyright © 2014. F.A. Davis Company
 Reaction Interference
• False-positive
– Strong oxidizing agents
– Formalin
– Highly pigmented urine, nitrofurantoin
• False-negative
– High concentrations of protein, glucose, oxalic acid, ascorbic
acid
– Crenation from high specific gravity
– Inaccurate timing: must have 2 min
– Presence of the antibiotics; gentamicin, cephalosporins,
tetracyclines
Copyright © 2014. F.A. Davis Company
 Specific Gravity

• Based on pKa (dissociation constant) of a

polyelectrolyte in alkaline medium
• Polyelectrolyte ionizes releasing H+ in relation to
concentration of urine
• ↑concentration = more H+ released
• Indicator bromthymol blue measures pH change
• Blue (alkaline) through green to yellow (acid)
Copyright © 2014. F.A. Davis Company
 Reagent Strip-Specific Gravity
Reaction

Copyright © 2014. F.A. Davis Company

 Reaction Interference

• No interference from large molecules, glucose

and urea and radiographic dye and plasma
expanders
– Reason for difference in refractometer reading
• Slight elevation from protein
• Decreased readings: urine pH 6.5 or higher
– Interferes with indicator; add 0.005 to the reading;
readers automatically add this

Copyright © 2014. F.A. Davis Company

 Chapter 10
Cerebrospinal Fluid

Copyright ©2021 F.A. Davis Company

 Learning Outcomes

Upon completing this chapter, the reader will be

able to
1. Describe the formation of cerebrospinal fluid
(CSF), and state the three major functions of
cerebrospinal fluid (CSF).
2. Distribute CSF specimen tubes numbered 1,
2, 3, and possibly 4 to their appropriate
laboratory sections and correctly preserve
them.

Copyright ©2021 F.A. Davis Company

 Learning Outcomes (continued_1)

3. Describe the appearance of normal CSF and

the causes of CSF not appearing normally.
4. Define xanthochromia and state its
significance.
5. Differentiate between CSF specimens caused
by intracranial hemorrhage and a traumatic
tap.

Copyright ©2021 F.A. Davis Company

 Learning Outcomes (continued_2)

6. Calculate CSF total cell count as well as

counts for white blood cells, and red blood
cells, when given the number of cells seen,
amount of specimen dilution, and squares
counted in the Neubauer chamber.
7. Describe the leukocyte content of the CSF in
various forms of meningitis, including
bacterial, viral, tubercular, and fungal.
8. Describe and state the significance of
macrophages in the CSF.
Copyright ©2021 F.A. Davis Company
 Learning Outcomes (continued_3)

9. Differentiate between the appearance of

normal choroidal cells and malignant cells.
10. State the reference values for CSF total
protein and name three pathologic
conditions that produce an elevated CSF
protein.
11. Determine whether increased CSF albumin
or immunoglobulin is the result of damage
to the blood-brain barrier or central nervous
system production.
Copyright ©2021 F.A. Davis Company
 Learning Outcomes (continued_4)

12. Discuss the significance of CSF

electrophoresis, immunophoresis, and
isoelectric focusing findings in multiple
sclerosis and the identification of CSF.
13. State the reference values for CSF glucose
and name the possible pathologic
significance of a decreased CSF glucose.
14. Discuss the diagnostic value of CSF lactate
and glutamine determinations.

Copyright ©2021 F.A. Davis Company

 Learning Outcomes (continued_5)

15. Name the microorganism associated with a

positive India ink preparation.
16. Discuss the diagnostic value of the bacterial
and cryptococcal antigen tests.
17. Explain the advantage of molecular
diagnostic methods for determining the
causative organism in meningitis.

Copyright ©2021 F.A. Davis Company

 Learning Outcomes (continued_6)

18. Determine whether a suspected case of

meningitis is of bacterial, viral, fungal, or
tubercular origin, when presented with
pertinent laboratory data.
19. Describe the Venereal Disease Research
Laboratories test and the fluorescent
treponemal antibody-absorption test for
syphilis in CSF testing.

Copyright ©2021 F.A. Davis Company

 Introduction

▪ Cerebrospinal fluid is a major fluid of the body

▪ Physiological system
• Supplies nutrients to nervous tissue
• Removes metabolic wastes
• Maintains intracranial pressure
• Produces a mechanical barrier to cushion the brain
and spinal cord against trauma

Copyright ©2021 F.A. Davis Company

 Formation and Physiology

▪ Brain and spinal cord lined by meninges

▪ Three layers of meninges
• Dura mater: outer
• Arachnoid: middle
• Pia mater: surfaces of brain and spinal cord
▪ Cerebrospinal fluid (CSF) produced in choroid
plexuses of the four ventricles
• 20 mL/min produced in adults
• Volume adults 90 to 150 mL, neonates 10 to 60 mL

Copyright ©2021 F.A. Davis Company

 The Meninges

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 Formation and Physiology (continued_1)

▪ CSF flows through subarachnoid space

between arachnoid and pia mater
▪ Reabsorbed into blood in arachnoid
granulations/villae (one-way valves)
▪ Formation by selective filtration
• Hydrostatic pressure and active transport
• Not an ultrafiltrate
• Very tight-fitting endothelial cells, prevent passage
of many molecules—called the blood-brain barrier

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 Flow of Spinal Fluid Through the Brain

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 Formation and Physiology (continued_2)

▪ Blood-brain barrier
• Essential to protect brain
• Chemicals and harmful substances do not pass
• Antibodies and medications are excluded
• CSF composition differs from plasma
• Meningitis, multiple sclerosis disrupt membrane

Copyright ©2021 F.A. Davis Company

 Specimen Collection & Handling

▪ CSF collected between third to fifth lumbar

vertebrae
▪ Precautions including measurement of
intracranial pressure and careful technique
must be followed to prevent infection or
damage to neural tissue

Copyright ©2021 F.A. Davis Company

 Specimen Collection & Handling
(continued_1)
▪ Three sterile tubes in this order
• Chemistry/serology
• Microbiology (avoid skin contamination)
• Hematology (avoid cells from tap)
‒ Save leftover fluid/fourth tube for additional tests
‒ Volume removed based on patient volume and
opening pressure

Copyright ©2021 F.A. Davis Company

 CSF Specimen Collection Tubes

Copyright ©2021 F.A. Davis Company

 Specimen Collection & Handling
(continued_2)
▪ Usually STAT requests
▪ Handle carefully to avoid repeat taps
▪ Preservation
• Hematology=refrigerate up to four hours
• Microbiology=room temperature (cells must be
counted within 1 hour of collection)
• Chemistry/serology=frozen

Copyright ©2021 F.A. Davis Company

 Appearance

▪ Crystal clear, cloudy/turbid, milky,

xanthochromic, hemolyzed/bloody
▪ Cloudy = infection; milky = lipid or protein
▪ Xanthochromic
• Pink, orange, yellow
• RBC degradation products causes Xanthochromia
• Also jaundice, ↑ ↑ protein, carotene, melanoma
• Pathologic = cerebral hemorrhage if the patient
has bleeding in the brain.

Copyright ©2021 F.A. Davis Company

 Tubes of CSF

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 Traumatic Collection (Tap)

▪ Blood vessel punctured during tap

▪ Three visual examinations can differentiate
from cerebral hemorrhage

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 Traumatic Collection (Tap) (continued_1)

▪ Blood will be evenly distributed throughout

the three CSF tubes bleeding.
▪ Traumatic tap will leave the heaviest
concentration of blood in tube 1
▪ Amounts in tubes 2 and 3 will gradually
diminish
▪ RBC counts on all three tubes not always
reliable

Copyright ©2021 F.A. Davis Company

 Traumatic Collection Tap (continued_2)

▪ Clot formation
• Clots present = traumatic tap (plasma)
• Hemorrhage does not have enough fibrinogen
• Other causes of clot formation
‒ Nonbloody CSF = damage to blood-brain barrier
‒ TB meningitis: web-like pellicle after refrigeration
▪ Xanthochromia
• Not present in a recent traumatic tap
• Indicates older hemorrhage
• Testing for differentiation includes microscopic
examination and D-dimer
Copyright ©2021 F.A. Davis Company
 Cell Count

▪ White blood cell (WBC) routinely

performed on CSF specimens
▪ Red blood cell (RBC) count seldom done
▪ Granulocytes lyse within 1 hour; STAT
▪ 40% of the leukocytes disintegrate after
2 hours
▪ Specimens should be refrigerated up to
4 hours if not analyzed immediately you
DON’T leave them for one day
Copyright ©2021 F.A. Davis Company
 Methodology

▪ Normal adult CSF 0-5 WBCs/µL

▪ Number is ↑with children
• Neonates up to 30 mononuclear cells/µL
▪ Neubauer counting chamber routinely used
• Automated cell counters can be used
▪ Automation ↑ precision, standardization,
TAT for results

Copyright ©2021 F.A. Davis Company

 Neubauer Counting Chamber

Copyright ©2021 F.A. Davis Company

 Calculating CSF Cell Count

▪ Standard Neubauer calculation formula

(cells/µL)
Number of cells counted × dilution
Number of cells counted × volume of 1 square
= cells/µL
▪ Can be used for diluted and undiluted samples

Copyright ©2021 F.A. Davis Company

 Calculating CSF Cell Count (continued)

▪ Example
# cells counted × dilution × 1 μL = cells/µL
1 μL (0.1 × 10)
(volume counted)

Copyright ©2021 F.A. Davis Company

 Total Cell Count

▪ Clear specimens count undiluted unless

overlapping cells are seen
• Load with transfer pipette
• Dilute with normal saline if necessary
▪ Cells are counted in the four corner squares,
and the center square on both sides of the
hemocytometer

Copyright ©2021 F.A. Davis Company

 WBC Count

▪ RBC lysis must be obtained before

performed the WBC count
▪ Dilute with 3% glacial acetic acid
▪ Methylene blue helps to see cells
▪ Differentiation between neutrophils and
mononuclear cells

Copyright ©2021 F.A. Davis Company

 Quality Control of CSF and Other Body
Fluid Cell Counts
▪ Commercial cell controls are available
▪ Check diluents for contamination biweekly
▪ Monthly check on cytocentrifuge speed and
timing
▪ Soak nondisposable chambers in bactericidal
solution for 15 minutes; rinse; clean with
isopropyl alcohol

Copyright ©2021 F.A. Davis Company

 Differential Count on a CSF Specimen

▪ Valuable diagnostic aid

▪ Stained smear only
▪ Must concentrate specimen
• Sedimentation, filtration, centrifugation, and
cytocentrifugation
▪ 100 cells should be counted, classified, and
reported in terms of percentage
▪ Report only the numbers of the cell types seen
if cell count is low

Copyright ©2021 F.A. Davis Company

 Cytocentrifugation

▪ Cytocentrifuge
• Forces cells onto a slide in a monolayer
• Filter paper absorbs moisture
• 0.1 mL CSF to 1 drop 30% albumin
‒ Albumin increases the cell yield and decreases the
cellular distortion
• Positively charged slides to attract cells
• Daily control of 0.2 mL saline and two drops of
albumin stained for bacterial contamination

Copyright ©2021 F.A. Davis Company

 Cellular Constituents

▪ Normal lymphocytes and monocytes

▪ Adults: normal lymphocytes:monocytes =70:30
• Children’s ratio is reversed
• Occasional neutrophils are normal
▪ Pleocytosis: increased amounts of normal cells
▪ Pleocytosis of normal cells is valuable in
determining the cause of meningitis
• Neutrophils = bacterial
• Lymphocytes = viral, tubercular, fungal, parasitic

Copyright ©2021 F.A. Davis Company

 Prominent Cells Seen in CSF
Table 10-3 Prominent Cells Seen in CSF
Type of Cell Major Clinical Significance Microscopic Findings
Lymphocytes Normal All stages of
Viral, tubercular, and fungal development may be
meningitis found
HIV infection and AIDS
Multiple sclerosis
Degenerative disorders
Parasitic infections
Neutrophils Bacterial meningitis Granules may be less
prominent than in blood

Copyright ©2021 F.A. Davis Company

 Prominent Cells Seen in CSF (continued_1)
Table 10-3 Prominent Cells Seen in CSF (continued)
Type of Cell Major Clinical Significance Microscopic Findings
Monocytes Early cases of viral, tubercular, and Found mixed with
fungal meningitis lymphocytes
Cerebral hemorrhage
Cerebral abscess
CNS infarction
Injection of medications or
radiographic
dye into the subarachnoid space
Metastatic tumors
Repeated lumbar punctures
Normal
Viral, tubercular, and fungal
meningitis
Multiple sclerosis

Copyright ©2021 F.A. Davis Company

 Prominent Cells Seen in CSF (continued_2)
Table 10-3 Prominent Cells Seen in CSF (continued)
Type of Cell Major Clinical Significance Microscopic Findings
Eosinophils Parasitic infections May contain phagocytized
Fungal infections RBCs appearing
Coccidioidal meningitis as empty vacuoles or
Introducing medications and shunts ghost cells,
in the CNS hemosiderin granules, and
hematoidin
crystals
Macrophages RBCs in spinal fluid
Blast forms Acute leukemia Lymphoblasts,
myeloblasts, or
monoblasts
Lymphoma cells Disseminated lymphomas Resemble lymphocytes
with cleft nuclei

Copyright ©2021 F.A. Davis Company

 Prominent Cells Seen in CSF (continued_3)
Table 10-3 Prominent Cells Seen in CSF (continued)
Type of Cell Major Clinical Significance Microscopic Findings
Plasma cells Multiple sclerosis Traditional and classic
Guillain-Barre syndrome forms seen
Sarcoidosis
Parasitic infections
Syphilitic meningitis
Tuberculous meningitis
Lymphocyte reactions Reactive lymphocytes
Ependymal, choroidal, Diagnostic procedures Seen in clusters with
and spindle-shaped cells distinct nuclei and distinct
cell walls
Malignant cells Metastatic carcinomas Seen in clusters with
fusing of cell
borders and nuclei
Primary central nervous system
carcinoma

Copyright ©2021 F.A. Davis Company

 Neutrophils
▪ Primarily in bacterial
meningitis
▪ Often contain
phagocytized bacteria
▪ Increased early viral,
fungal, tubercular,
parasitic
▪ Vacuoles may be
present

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 Neutrophils (continued_1)

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 Neutrophils (continued_2)
▪ Neutrophils with
pyknotic nuclei may
resemble NRBCs
▪ Seen with bone marrow
contamination from tap
in 1% of specimens
▪ Capillary structures and
epithelial cells from
traumatic taps

Copyright ©2021 F.A. Davis Company

 Cellular Constituents (continued)

Copyright ©2021 F.A. Davis Company

 Lymphocytes and Monocytes
▪ Lymphs and monos in
viral, fungal, tubercular
▪ Reactive lymphocytes
with viral
▪ Multiple sclerosis has 50
or fewer
lymphocytes/μL, both
normal and reactive
▪ Seen in HIV and AIDS

Copyright ©2021 F.A. Davis Company

 Eosinophils
▪ Parasitic and fungal
infections
• (primarily Coccidioides
immitis)
▪ Medications and
shunts into the
central nervous
system

Copyright ©2021 F.A. Davis Company

 Macrophages
▪ Purpose is to remove
cellular and other debris
▪ May be seen after
repeated taps
▪ Hemorrhage: enter CSF
within 2 hours to
phagocytize RBCs
▪ RBCs degraded to
hematoidin crystals
representing
unconjugated bilirubin

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 Macrophages (continued)

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 Nonpathologically Significant Cells

▪ Seen after diagnostic procedures

▪ Choroidal cells
▪ Epithelial lining of choroid plexus, singular
and in clumps, uniform cells
▪ Ependymal cells lining ventricles and neural
canal; less defined cell membranes in clumps
▪ Spindle cells lining arachnoid seen in clusters

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 Nonpathologically Significant Cells
(continued)

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 Malignant Cells of Hematologic Origin

▪ Leukemias
• Lymphoblasts, myeloblasts, and monoblasts
• Nucleoli may be more prominent than in blood
▪ Lymphomas
• Dissemination from lymph organs
• Cleaved nucleii and prominent nucleoli

Copyright ©2021 F.A. Davis Company

 Malignant Cells of Hematologic Origin
(continued_1)

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 Malignant Cells of Hematologic Origin
(continued_2)

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 Malignant Cells of Hematologic Origin
(continued_3)

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 Malignant Cells of Nonhematologic Origin
▪ Metastatic carcinoma
cells
• Lung, breast, renal, and
gastrointestinal
▪ Primary tumors
• Astrocytomas,
retinoblastomas,
medulloblastomas
▪ Fused cells walls,
nuclear irregularities,
and hyperchromatic
nucleoli

Copyright ©2021 F.A. Davis Company

 Chemistry Tests

▪ CSF formed by plasma filtration

▪ Normal values differ from plasma because of
selectivity of blood-brain barrier
▪ Abnormal values result
• Alterations in the permeability of the blood-brain
barrier
• Increased production or metabolism by the
neural cells in response to a pathologic condition

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 Cerebrospinal Protein

▪ Total protein is the most common test

• Normal 15 to 45 mg/dL (mg, not grams)
‒ Method dependent
‒ Increased in infants and persons >40
• Albumin is predominant, prealbumin is second
• Alpha globulins-haptoglobin and ceruloplasmin
• Transferrin is major beta globulin
• TAU, carbohydrate-deficient transferrin seen in
CSF, not in blood; used to identify CSF
• IgG major gamma globulin
Copyright ©2021 F.A. Davis Company
 Clinical Significance

▪ Decreased protein levels = fluid leakage

▪ Elevated levels = damage to blood-brain
barrier, IG production within CNS, decreased
clearance, degeneration of neural tissue
▪ Meningitis/hemorrhage most common
causes of increased damage to blood-brain
barrier
▪ Find abnormal results on clear fluid with low
cell counts from neurologic disorders

Copyright ©2021 F.A. Davis Company

 Clinical Significance of Elevated Protein
Values
▪ Elevated Results
• Meningitis
• Hemorrhage
• Primary CNS tumors
• Multiple sclerosis
• Guillain-Barré syndrome
• Neurosyphilis
• Polyneuritis
• Myxedema
• Cushing disease
Copyright ©2021 F.A. Davis Company
 Clinical Significance of Elevated Protein
Values (continued_1)
▪ Elevated Results (continued)
• Connective tissue disease
• Polyneuritis
• Diabetes
• Uremia

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 Clinical Significance of Elevated Protein
Values (continued_2)
▪ Decreased Results
• CSF leakage/trauma
• Recent puncture
• Rapid CSF production
• Water intoxication
*Reference values for protein are usually 15 to 45 mg/dL, but
are method dependent, and higher values are found in infants
and people older than 40 years of age.

Copyright ©2021 F.A. Davis Company

 Methodology (continued)

▪ Turbidity production and dye-binding ability

▪ Automated instrumentation available
• Nephelometry

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 Protein Fractions

▪ Comparisons between serum and CSF levels

of albumin and IgG
▪ CSF/serum albumin index
• Blood-brain barrier integrity
▪ CSF IgG index
▪ Comparison of the CSF/serum albumin index
with the CSF/serum IgG index
▪ Values for CSF albumin and globulin adapted
for automated instruments
Copyright ©2021 F.A. Davis Company
 Protein Fractions (continued)

IgG index =
CSF IgG (mg/dL)/serum IgG (g/dL)
CSF albumin (mg/dL)/serum albumin (g/dL)
▪ Values >0.70 indicate IgG production within
the CNS

Copyright ©2021 F.A. Davis Company

 Electrophoresis and Immunophoretic
Techniques
▪ Detection of oligoclonal bands
• Represent inflammation within the CNS
• Located in the gamma region of the protein
electrophoresis
• Simultaneous serum electrophoresis must be
performed

Copyright ©2021 F.A. Davis Company

 Electrophoresis and Immunophoretic
Techniques (continued)
▪ Multiple sclerosis (MS) = no bands in serum,
bands in CSF
▪ Leukemia, lymphoma, viral, HIV: bands in both
▪ Primary purpose for MS, compare also to IgG
index
▪ Encephalitis, neurosyphilis, Guillain-Barré, and
neoplasms may give same pattern
▪ Consider symptoms

Copyright ©2021 F.A. Davis Company

 Myelin Basic Protein

▪ Presence in CSF indicates demyelination of

myelin sheath around axons of neurons
▪ Monitors the course of multiple sclerosis
▪ Effectiveness of treatment
▪ Immunoassay procedures available

Copyright ©2021 F.A. Davis Company

 CSF Glucose

▪ Selective transport across blood-brain barrier

▪ Approximately 60% to 70% plasma glucose
• Plasma = 100 mg/dL; CSF = 65 mg/dL
• Draw blood 2 hours before spinal tap
▪ Significance
• Values that are decreased relative to plasma values
• Elevated CSF glucose values are always a result of
plasma elevations

Copyright ©2021 F.A. Davis Company

 CSF Glucose (continued)

▪ Markedly decreased with increased

neutrophils in bacterial meningitis
▪ Tubercular meningitis decreased with
increased lymphocytes
▪ Viral/fungal meningitis, normal glucose and
increased lymphocytes

Copyright ©2021 F.A. Davis Company

 CSF Lactate

▪ Diagnosis and management of meningitis

• Bacterial, TB, and fungal levels >25 mg/dL
• Viral <25 mg/dL
▪ Levels remain elevated until treatment
becomes effective, then fall rapidly
▪ Can result from any condition that decreases
oxygen flow to the tissues
• Monitor severe head injuries

Copyright ©2021 F.A. Davis Company

 CSF Glutamine

▪ Produced by brain cells from ammonia and α-

ketoglutarate to remove toxic ammonia
▪ Indirect test for the presence of excess
ammonia in the CSF
▪ Normal: 8 to 18 mg/dL
• Elevated in liver disease
• Elevated in children with Reye syndrome
• Disturbance of consciousness when glutamine
levels are more than 35 mg/dL
▪ More reliable than direct CSF ammonia
Copyright ©2021 F.A. Davis Company
 Microbiology Tests

▪ Identify the causative agent of meningitis

▪ Positive identification
• Microorganism must be recovered from the fluid
• Use appropriate culture medium
• 24 hrs. for bacterial meningitis
• Up to 6 weeks for tubercular meningitis
• Confirmatory, not diagnostic

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 Gram Stains

▪ CSF centrifuged at 1500 g for 15 minutes;

Gram stain and cultures must be
performed on sediment from centrifuged
CSF; cytocentrifuge helps Gram stains
▪ Blood cultures also must be drawn
▪ Difficult to interpret Gram stains, few
organisms and often debris
▪ Organisms: S. pneumocystis, Haemophilus
influenza, Escherichia coli, Neisseria
meningitidis, Listeria monocytogenes, S.
agalactiae
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 SCF Glutamine (continued)

▪ Acid-fast or fluorescent antibody stains

usually not performed unless tubercular
meningitis is suspected
▪ India ink preparation detects presence of
Cryptococcus neoformans
▪ Special attention should be paid to Gram
stain for a classic starburst pattern produced
by Cryptococcus

Copyright ©2021 F.A. Davis Company

 Immunologic Assays

▪ Latex agglutination test provide a more

sensitive method for detecting C. neoformans
than an India ink preparation
• Confirmation by culture due to false-positive
reactions occuring
▪ Latex agglutination and enzyme-linked
immunosorbent assay are available for

Copyright ©2021 F.A. Davis Company

 Immunologic Assays (continued_1)

• Group B streptococcus, Haemophilus influenzae, S.

pneumoniae, Neisseria meningitidis,
Mycobacterium tuberculosis, C. immitis, and
Escherichia coli
▪ Gram stain is the best for detection
▪ Compare with hematology and chemistry
results

Copyright ©2021 F.A. Davis Company

 Immunologic Assays (continued_2)
▪ Naegleria fowleri
• Found in ponds, lakes, and
some pools
• Enters nasal passages and
migrates to the brain
• Motile amoeba seen in
wet preps, nonmotile in
cytospin preps
• Elongated with tapered
posterior

Copyright ©2021 F.A. Davis Company

 PCR Molecular Diagnostic Testing

▪ Nucleic acid amplification tests can detect the

cause of meningitis with small amounts of the
pathogens’ DNA
▪ Universal PCR detects pathogens with
↑sensitivity and specificity
▪ Assays based on amplifications of ribosomal
RNA genes to detect and differentiate
causative pathogens of meningitis

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 Serologic Testing

▪ Primary test is for neurosyphilis

▪ Performed less now that people have been
treated early with penicillin
▪ Detection of antibodies remains a necessary
diagnostic procedure
▪ The Venereal Disease Research Laboratories
(VDRL) produces the recommended test for
specificity
▪ Should be accompanied by a positive serum
Fluorescent Treponemal Antibody Absorption
(FTA-ABS)
Copyright ©2021 F.A. Davis Company
 SEROUS FLUID
CHAPTER 12

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 Learning Objectives

Upon completing this chapter, the reader will be able to

1. Describe the normal formation of serous fluid.
2. Describe four primary causes of serous effusions.
3. Differentiate between a transudate and an exudate,
including etiology, appearance, and laboratory tests.
4. Differentiate between a hemothorax and a hemorrhagic
exudate.
5. Differentiate between a chylous and a pseudochylous
exudate.
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 Learning Objectives (cont’d)
6. State the significance of increased neutrophils,
lymphocytes, eosinophils, and plasma cells in pleural
fluid.
7. Describe the morphologic characteristics of mesothelial
cells and malignant cells.
8. List three common chemistry tests performed on pleural
fluid, and state their significance.
9. State the common etiologies of pericardial effusions.
10. Discuss the diagnostic significance of peritoneal lavage.

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 Learning Objectives (cont’d)

11. Calculate a serum-ascites gradient, and state its

significance.
12. Differentiate between ascitic effusions of hepatic and
peritoneal origin.
13. State the clinical significance of the carcinoembryonic
antigen and CA 125 tests.
14. List four chemical tests performed on ascitic fluid, and
state their significance.

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 Serous Membranes

• Line the closed body cavities

– Pleural
– Pericardial
– Peritoneal
• Two membranes
– Parietal: lines cavity wall
– Visceral: lines organs in cavity
• Fluid between membranes
– Serous fluid: named for each location

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 Formation
• Purpose: provide lubrication between the two membranes
• Ultrafiltrate of plasma
• Produced by hydrostatic and oncotic (protein) pressure in
the capillaries lining the membranes
• Normally, oncotic pressure is the same on both sides of
the membrane; hydrostatic pressure causes the
production
• Small amounts of excess fluid are absorbed by lymphatic
capillaries
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 Formation and Absorption
of Serous Fluid

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 Formation

• Disruption causes fluid buildup; termed effusion

– Causes: hydrostatic pressure increased from
congestive heart failure; oncotic pressure decreased
from hypoproteinemia; increased capillary
permeability from inflammation, infection,
malignancy; lymphatic obstruction from tumors

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 Pathological Causes of Effusions
• Disruption of the mechanisms of serous fluid formation and
reabsorption
• Increased capillary hydrostatic pressure
– Congestive heart failure, salt and fluid retention
• Decreased oncotic pressure
– Nephrotic syndrome, hepatic cirrhosis
– Malnutrition, protein-losing enteropathy
• Increased capillary permeability
– Microbial infections, membrane inflammations
– Malignancy
• Lymphatic obstruction
– Malignant tumors, lymphomas, infection and inflammation, thoracic duct
injury
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 Specimen Collection
and Handling
• Collected by needle aspiration
– Thoracentesis: pleural cavity
– Pericardiocentesis: heart cavity
– Paracentesis: peritoneal cavity
• Abundant fluid collected >100 mL
• EDTA tube: hematology
• Sterile heparinized or polyanethol sulfonate (SPS):
microbiology, cytology
• Clotted or heparin: chemistry, serology

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 Specimen Collection
and Handling (cont'd)

• Specimens for pH must be kept on ice

• Concentrate fluid for microbiology and cytology
by centrifuging 100 mL
• Chemical tests compared with blood tests drawn
at the same time

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 Transudates and Exudates
• Primary classification of serous fluids
• Transudates: systemic disorder disrupts filtration
and reabsorption, congestive heart failure,
nephrotic syndrome
• Exudates: conditions affecting membranes,
inflammation, infection, malignancy
• Differentiation important for further testing
• Transudates = little further testing
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 Laboratory Differentiation of
Transudates and Exudates
Transudate Exudate
Appearance Clear Cloudy
Fluid:serum protein ratio <0.5 >0.5
Fluid:serum LD ratio <0.6 >0.6
White blood cell count <1000/µL >1000/µL
Spontaneous clotting No Possible
Pleural fluid cholesterol <45 to 60 mg/dL >45 to 60 mg/dL
Pleural fluid:serum cholesterol ratio <0.3 >0.3
Pleural fluid:bilirubin ratio <0.6 >0.6
Serum-ascites albumin gradient >1.1 <1.1

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 General Laboratory Tests
• Appearance
• Transudate versus exudate tests first
– Transudates: seldom tested further
– Exudates: microbiology, cytology, and tests for
general symptoms
– Differentials performed routinely on cytocentrifuged
specimens
• White blood cells (WBCs), normal tissue cells, malignant
cells

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 Pleural Fluid
• Additional transudate versus exudate tests
– Pleural fluid cholesterol <60 mg/dL versus >60 mg/dL
– Fluid-serum cholesterol ratio <0.3 versus >0.3
– Fluid-serum bilirubin ratio <0.6 versus >0.6
• Appearance
– Normal: clear, pale yellow
– Turbid: WBCs, inflammation, infection
– Milky: chylous material from thoracic duct leakage,
pseudochylous material from chronic inflammations

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 Pleural Fluid (cont’d)
• Bloody: hemothorax (trauma), hemorrhagic effusion
(membrane) damage
• Differentiate: do a hematocrit on fluid
– >50% blood HCT is hemothorax: more blood
– Membrane damage: low blood
• Differentiate chylous and pseudochylous
– Chylous is triglycerides; stain with Sudan III
– Pseudochylous is cholesterol; polarize and crystals also
seen in wet bright-field view

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 Correlation of Pleural Fluid
Appearance and Disease
Appearance Disease
Clear, pale yellow Normal
Turbid, white Microbial infection (tuberculosis)
Bloody Hemothorax, hemorrhagic effusion, pulmonary
emboli, tuberculosis, malignancy
Milky Chylous material from thoracic duct leakage
Pseudochylous material from chronic inflammation
Brown Rupture of amoebic liver abscess
Black Aspergillus
Viscous Malignant mesothelioma (increased hyaluronic acid)

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 Hematology Tests
• Differential: primary cells are neutrophils,
lymphocytes, macrophages, eosinophils, mesothelial
cells, plasma cells, and malignant cells
• Macrophages (scavengers) often the highest
• ↑ Neutrophils: bacterial infection, pancreatitis,
pulmonary infarction
• ↑ Lymphs: TB, viral infections, autoimmune
disorders, malignancy
• Eosinophils: trauma introducing air and blood,
allergic reactions, parasites
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 Hematology Tests (cont’d)
• Mesothelial cells: single cell
layer lines membranes,
common to see in serous
fluid, pleomorphic, dark
blue cytoplasm, round
nuclei, normal and reactive;
“fried egg appearance”
• Reactive cells may be
multinucleated

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 Hematology Tests (cont’d)

• Lack of mesothelial
cells is significant in
tuberculosis (TB),
exudate covers
membranes

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 Hematology Tests (cont’d)
• Pleural fluid: primary
cancer cells
• Adenocarcinoma cells
are large and irregular;
small cell or oat cell
carcinoma cells are
small like lymph cells,
mesothelioma cells are
large
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 Hematology Tests (cont’d)

• Metastatic breast
carcinoma cells seen in
clumps

• Small cell carcinoma

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 Chemistry Tests
• Glucose: decreased in inflammations and infections,
have blood comparison
• pH: <7.0 indicates need for chest tubes, <6.0
indicates esophageal rupture (gastric fluid)
• Adenosine deaminase elevated in TB and
malignancy
• Amylase: elevated in esophageal rupture and
malignancy
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 Significance of Chemical Testing
of Pleural Fluid
Test Significance
Glucose Decreased in rheumatoid inflammation
Decreased in purulent infection
Lactate Elevated in bacterial infection
Triglyceride Elevated in chylous effusions
pH Decreased in pneumonia not responding to antibiotics
Markedly decreased with esophageal rupture
ADA Elevated in tuberculosis and malignancy
Amylase Elevated in pancreatitis, esophageal rupture, and
malignancy

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 Microbiology and Serology Tests

• Gram stains and aerobic and anaerobic cultures

on fluid, TB smears, and cultures
• Serology tests for autoimmune disorders
• Tumor markers for metastatic malignancy
– Carcinoembryonic antigen: gastrointestinal
malignancy
– CA125: uterine and ovarian malignancy

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 Pleural Fluid Testing Algorithm

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 Pericardial Fluid
• Normally small amount: 10 to 50 mL
• Permeability of membranes from infection
(pericarditis, endocarditis), malignancy, trauma
produces exudates
• Transudates: hypothyroidism, uremia, immune
disorders
• Detect by cardiac tamponade (compression)
heard by physician
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 Pericardial Fluid (cont’d)
• Appearance: normal clear, pale yellow
• Turbid: infection, malignancy (also blood
streaked)
• Bloody: accidental puncture, anticoagulants
• Milky: chylous and pseudochylous material
• Fluid-serum protein and LD ratios for transudate
versus exudate differentiation
• WBCs >1000/μL is bacterial endocarditis
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 Pericardial Fluid (cont’d)

• Malignant cells are metastatic from lung and

breast
• Gram stains and cultures for endocarditis often
caused by previous respiratory infections
• TB smears and cultures done in AIDS

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 Pericardial Fluid (cont’d)

• Metastatic giant
mesothelioma cell also
seen in pleural fluid as a
primary malignancy in
persons with asbestos
contact

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 Significance of Pericardial
Fluid Testing
Test Significance
Appearance
Clear, pale yellow Normal, transudate
Blood streaked Infection, malignancy
Grossly bloody Cardiac puncture, anticoagulants
Milky Chylous and pseudochylous material
Differential
Increased neutrophils Bacterial endocarditis
Malignant cells Metastatic carcinoma
Carcinoembryonic antigen Metastatic carcinoma
Gram stain and culture Bacterial endocarditis
Acid-fast stain Tubercular effusion
Adenosine deaminase Tubercular effusion

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 Peritoneal Fluid

• Effusion between the peritoneal membranes is

called ascites
• Fluid is often called ascitic fluid
• Transudates: hepatic origin (cirrhosis)
• Exudates: bacterial peritonitis from intestinal
perforation, ruptured appendix, and malignancy

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 Peritoneal Lavage

• Performed to detect early abdominal bleeding

and need for surgery
• Blunt trauma injuries
• Normal saline injected into cavity, withdrawn,
and red blood cell (RBC) count performed
• RBC count >100,000 indicates blunt trauma case
• Radiographic procedures also available

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 Transudates and Exudates
• More difficult than pleural and pericardial fluids
• Serum-ascites gradient is best differentiation
• Serum and fluid albumin levels are measured;
fluid level is subtracted from serum level;
difference (gradient) > than 1.1 is a transudate
(hepatic origin)
• Serum albumin 3.8 – fluid albumin 1.2 = 2.6
= hepatic transudate

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 Appearance
• Normal: clear, pale yellow
• Turbid: bacterial and fungal infections
• Green-brown color = bile
• Milky: chylous and pseudochylous with trauma and
lymphatic blockage
• WBC count: normal 350 cells/μL
• Absolute neutrophil count: >50% of total WBC count or
greater than 250 cells indicates infection
• Lymphocytes elevated in TB
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 Cellular Examination

• Cells: WBCs,
mesothelial cells,
macrophages
(lipophages)
• Yeast cells and
Toxoplasma gondii
• Malignant cells, often
contain mucin vacuoles

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 Cellular Examination (cont’d)

• Malignant cells of
prostate,
gastrointestinal, and
ovarian origin

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 Cellular Examination (cont’d)

• Psammoma bodies
benign or cancer of
thyroid and ovaries

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 Chemical Tests

• Glucose: below plasma levels = peritonitis and

malignancy
• ↑ Amylase: pancreatitis, gastrointestinal
perforation
• ↑ Alkaline phosphatase: intestinal perforation
• ↑ BUN, creatinine: ruptured bladder, accidental
perforation

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 Microbiology Tests

• Gram stains and aerobic and anaerobic cultures

• Anaerobic cultures: inoculate blood culture
bottle at bedside
• Acid-fast smear, adenosine deaminase and
culture for TB

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 Serological Tests

• Tumor markers
– CEA
– CA 125
• Presence of CA 125 antigen with a negative CEA
suggests the source is from the ovaries, fallopian
tubes, or endometrium
• Presence of CEA antigen suggests source is
gastrointestinal
Copyright © 2014. F.A. Davis Company
 SEMEN
CHAPTER 10

Copyright © 2014. F.A. Davis Company

 Learning Objectives

Upon completing this chapter, the reader will be able to

1. State the structures involved in sperm production and their function.
2. Describe the four components of semen with regard to source and
function.
3. Explain the procedures for collecting and handling semen specimens.
4. Describe the normal appearance of semen and three abnormalities in
appearance.
5. State two possible causes of low semen volume.
6. Discuss the significance of semen liquefaction and viscosity.

Copyright © 2014. F.A. Davis Company

 Learning Objectives (cont’d)
7. Calculate a sperm concentration and count when provided with the
number of sperm counted, the dilution, the area of the counting
chamber used, and the ejaculate volume.
8. Define round cells, and explain their significance.
9. State the two parameters considered when evaluating sperm motility.
10. Describe the appearance of normal sperm, including structures and
their functions.
11. Differentiate between routine and strict criteria for evaluating sperm
morphology.
12. Given an abnormal result in a routine semen analysis, determine
additional tests that might be performed.

Copyright © 2014. F.A. Davis Company

 Learning Objectives (cont’d)

13. Describe the two methods routinely used to detect antisperm

antibodies.
14. List two methods for identifying a questionable fluid as
semen.
15. State the World Health Organization (WHO) reference values
for routine and follow-up semen analysis.
16. Discuss the types and significance of sperm function tests.
17. Describe methods of quality control appropriate for semen
analysis.

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 Andrology

• Assisted reproductive technology (ART)

• Abnormal results on the routine semen analysis
– Specialized andrology laboratories
• Fertility testing
• In vitro fertilization (IVF)

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 Physiology

• Semen consists of four components

– Contributed separately by the
• Testes and epididymis
• Seminal vessels
• Prostate
• Bulbourethral glands
– Normal semen specimen must have all four
components

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 Physiology (cont’d)
• Paired testes: located in scrotum
– Lower scrotum temperature optimal for sperm development
• Seminiferous tubules of testes
– Produce spermatozoa
• 5% volume
– Sertoli cells provide support and nutrients for the germ cells as they
undergo spermatogenesis
– Mature and stored in the epididymis
• Develop flagella
• Seminal vessels
– Produce majority of fluid (60% to 70%); transport medium
– Provide fructose and flavin for sperm metabolism
– Sperm are not motile without this fluid

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 Physiology (cont’d)
• Prostate gland
– Produce acidic fluid (20% to 30%)
– Contains acid phosphatase, citric acid, zinc, and
proteolytic enzymes
– Enzymes coagulate semen prior to ejaculation and cause
liquefaction after ejaculation
• Bulbourethral glands
– Produce thick, alkaline fluid (5%) to neutralize acid from
prostate and acid pH of vagina
– Absence of fluid = diminished sperm motility

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 Summary of Semen Production

Structure Function
Seminiferous Spermatogenesis
tubules of testes
Epididymis Sperm maturation
Ductus deferens Propel sperm to ejaculatory ducts
Seminal vesicles Provide nutrients for sperm and fluid
Prostate gland Provide enzymes and proteins for
coagulation and liquefaction
Bulbourethral Add alkaline mucus to neutralize
glands prostatic acid and vaginal acidity

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 The Male Genitalia

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 Specimen Collection
• Must be a complete specimen
• First portion of the ejaculate is missing, then
– Sperm count will be decreased
– pH is falsely increased
– Specimen will not liquefy
• Last portion of ejaculate is missing, then
– Semen volume is decreased
– Sperm count is falsely increased
– pH is falsely decreased
– Specimen will not clot
• Detailed instructions to patients

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 Specimen Collection (cont’d)
• 2 to 3 days abstinence; no longer than 7 days
– WHO recommends that two or three samples be collected not less
than 7 days or more than 3 weeks apart
• Laboratory provides containers and ideally a room for
collection
• Home: deliver to laboratory within 1 hour; keep specimen 37°C
• Record
– Patient name and date of birth
– Period of abstinence
– Time of collection and receipt
• Collected by masturbation
– If not possible, no lubricated, antispermicidal condoms
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 Specimen Handling

• Standard precautions must be observed at all

times during analysis.
• Specimens are discarded as biohazardous waste.
• Sterile materials and techniques must be used.

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 Fertility Evaluation
• Macroscopic and microscopic
• Appearance
• Volume
• Viscocity
• pH
• Sperm concentration and count
• Motility
• Morphology
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 Appearance
• Normal is gray-white, translucent
• Musty odor
• White turbidity = infection
– Culture
– LE reagent strip
• Red = blood cells, abnormal
• Yellow = urine, prolonged abstinence, medications
• Urine is toxic to sperm: no motility
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 Liquefaction

• Fresh semen specimen is clotted and should liquefy

within 30 to 60 minutes after collection
– Deficiency in prostatic enzymes
• Analysis cannot begin until liquefaction has occurred
• Enzymes added to induce liquefaction
– Document
– May affect biochemical tests, motility, and
morphology

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 Volume and Viscosity
• Normal: 2 to 5 mL
• Measure in a graduated cylinder
• Increased volume may be seen following periods of
extended abstinence
• Decreased = infertility, incomplete collection
• Viscosity: droplets with thin threads from a pipette
are normal
– Form threads longer than 2 cm = highly viscous
• Rate 0 (watery) to 4 (gel-like) or low, normal, high
Copyright © 2014. F.A. Davis Company
 pH

• Measure within 1 hour of ejaculation

• Normal: 7.2 to 8.0
• Over 8.0 = infection
• Decreased pH = increased prostate fluid,
ejaculatory duct obstruction, or poorly
developed seminal vesicles
• Check with pH pad of a urinalysis reagent strip

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 Sperm Concentration and Count
• Valid measurement of fertility
• Concentration = number sperm/mL
• Count = number sperm per ejaculate
• Total sperm count
– Sperm concentration × specimen volume
• Reference value: >20 to 250 million sperm per milliliter
– 10 to 20 million borderline
• Normal count = concentration × volume
– >40 million/ejaculate

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 Sperm Concentration and Count (cont’d)

• Concentration performed in Neubauer chamber

– Five small squares, corners, and center of large center
square
• Common dilution: 1 to 20
• Diluting fluid = sodium bicarbonate and formalin,
must immobilize sperm
• Count both sides of chamber, sides must agree
within 10%; use the average
• With this method, the number of sperm counted are
multiplied by 1,000,000 = sperm/mL
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 Areas of the Neubauer Counting Chamber
Used for Red and White Blood Cell Counts

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 Sperm Concentration and Count
• Must multiply sperm per μL by 1000 to reach
sperm/mL
• Sperm count = sperm/mL × volume
– Example
- 60,000,000 sperm concentration × 3 mL volume =
180,000,000 sperm count/ejaculate
• Instrumentation has been developed; used
primarily in fertility clinics

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 Sperm Motility

• Need to have sperm with forward, progressive movement

• Well-mixed, liquefied semen specimen
• Examine within 1 hour; evaluate undiluted on glass slide with cover slip
• Estimate percentage with progressive, forward motion in 20 HP fields
• Or, examine 200 sperm per slide and count the percentages of the
different motile categories using a manual cell counter
• Grading can be done using a scale of 0 to 4, with 4 indicating rapid,
straight-line movement and 0 indicating no movement
• A minimum motility of 50% with a rating of 2.0 after 1 hour is
considered normal

Copyright © 2014. F.A. Davis Company

 Sperm Motility (cont’d)

• World Health Organization (WHO)

recommendation
– Motility is graded as progressive motility (PM),
nonprogressive motility (NP), and immotility (IM);
motility must be specified as total motility (PM and
NP) or progressive motility (PM)
• Instrumentation available
– Computer-assisted semen analysis (CASA)

Copyright © 2014. F.A. Davis Company

 Sperm Morphology
• Critical to fertilization
• Evaluate head, neck, midpiece, tail
– Head: oval with acrosomal cap at end and covering half of
the head
• 5 µm long and 3 µm wide
• Contains enzymes for ovum penetration
– 7.0 µm long neckpiece attaches head to midpiece and tail
• Midpiece surrounded by sheath of mitochondria for tail
movement
– Flagellar tail approximately 45 µm
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 Spermatozoon Structure

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 Sperm Morphology

• Observe on thin smear under oil immersion

• Wright’s, Giemsa, Shorr, or Papanicolaou stain
• Count 200 and report number of abnormal
• Routine criteria measures
– Abnormalities in head structure include double heads,
giant and amorphous heads, pinheads, tapered heads,
and constricted heads
– Abnormal sperm tails are frequently doubled, coiled, or
bent

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 Sperm Morphology (cont’d)
• Kruger’s strict criteria include
– Measuring head, neck, and tail size
– Measuring acrosome size
– Evaluating for the presence of vacuoles
• Strict criteria evaluation is an integral part of
assisted reproduction evaluations
• >30% normal forms when using routine criteria
• >14% normal forms when using strict criteria
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 Sperm Morphology (cont’d)

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 Sperm Morphology (cont’d)

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 Sperm Morphology (cont’d)
• Round bodies
– White blood cells (WBCs) and spermatids (immature sperm)
– Differentiate on morphology smear
– Count number of each separately in 100 cells
• N is the number of spermatids or neutrophils counted per 100 mature
sperm,
• S is the sperm concentration in millions per milliliter

C= N×S
100

Normal: <1,000,000

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 Sperm Vitality

• Eosin-nigrosin stain
• Dead cells stain red;
normal are blue-white
• Count number/100 cells
• Normal: 75% living
• Corresponds to motility
• Seminal fructose tests

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 Seminal Fluid Fructose

• Low sperm concentration

• Low to absent fructose level in the semen
• Lack of the support medium
– Abnormalities of the seminal vesicles
– Bilateral congenital absence of the vas deferens
– Obstruction of the ejaculatory duct
– Partial retrograde ejaculation
– Androgen deficiency

Copyright © 2014. F.A. Davis Company

 Antisperm Antibodies
• Present in both men and women
• Male more common: surgery, vasectomy
reversal, trauma
• Sperm normally do not encounter the immune
system, so body considers them foreign
• Damaged sperm create female antibodies
• Suspect male antibodies when clumps of sperm
are seen; female no clumping
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 Antisperm Antibodies (cont’d)
• Female: mix female serum with sperm and check for
agglutination
• Immunoassays: males
• Mixed agglutination reaction (MAR) test
– Incubate sperm with antihuman globulin (AHG) and IgG-
coated latex particles
– AHG combines with particles and antibody-coated sperm
forming clumps
• Normal: <10% motile sperm attach to particles
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 Antisperm Antibodies (cont’d)

• Immunobead test
– Demonstrates antibodies to head, neck, midpiece,
and tail
– Beads are coated with antihuman globulin
– Microscopic examination shows where on sperm
antibodies are attacking
– Tail = movement; head = penetration
– Normal: beads on <20% of sperm
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 Microbial and Chemistry Testing
• >1 million WBCs usually indicate prostate
infection
– Culture and test for Mycoplasma hominis, Chlamydia
trachomatis, Ureaplasma urealyticum
• Chemistry tests: neutral α-glucosidase, zinc, citric
acid, acid phosphatase
– ↓ Neutral α -glucosidase = epididymis
– ↓ Zinc, citric acid, acid phosphatase =↓ prostatic
fluid
Copyright © 2014. F.A. Davis Company
 Normal Semen Chemical Values

• Neutral a-glucosidase ≥20 mU/ejaculate

• Zinc ≥2.4 µmol/ejaculate
• Citric acid ≥52 µmol/ejaculate
• Acid phosphatase ≥200 units/ejaculate

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 Semen Detection

• Semen present in specimen?

• Observe microscopically for sperm
– Enhance with xylene; use phase microscopy
• Specimen acid phosphatase content
• Detection of seminal plasma glycoprotein, p30
• DNA analysis

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 Postvasectomy Analysis
• Only concern is presence of sperm
• Takes several months for all sperm to be gone,
based on time and ejaculations
• Begin in 2 months; continue until 2 months are
negative
• Wet preparation under phase; if negative,
centrifuge for 10 minutes, examine again
• Only one sperm is required for fertilization
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 Semen Analysis Quality Control

• Clinical Laboratory Improvement Amendments

(CLIA) rates semen analysis as high complexity
• Commercial controls and training materials are
available
• CAP offers proficiency testing

Copyright © 2014. F.A. Davis Company