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.
Copyright © 2014. F.A. Davis Company
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.
Copyright © 2014. F.A. Davis Company
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 acidbase content
• 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
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
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
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Prerenal Proteinuria
• Conditions affecting the plasma, not the kidney
• Transient, increase levels of low-molecularweight plasma proteins, acute phase reactants,
exceed reabsorptive capacity
• Rarely seen on reagent strip (not albumin)
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Bence Jones Protein (BJP)
• Multiple myeloma (plasma cell myeloma)
• Immunoglobulin light chains
• Multiple myeloma confirmation is serum
electrophoresis
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Renal Proteinuria
• Glomerular or tubular damage
– Glomerular proteinuria
– Microalbuminuria
– Orthostatic (postural) proteinuria
– Tubular proteinuria
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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
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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 albuminspecific reagent strips
• Measure creatinine to produce an
albumin:creatinine ratio
• First morning specimens are recommended
Copyright © 2014. F.A. Davis Company
Micral-Test
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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)-3,4,5,6tetra-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)
Copyright © 2014. F.A. Davis Company
(orange to blue)
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)
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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
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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
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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
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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)
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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
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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
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Negative
Trace
Small (1+)
Moderate (2+)
Large (3+)
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• Semiquantitatively
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Negative
Trace (5 mg/dL)
Small (15 mg/dL)
Moderate (40 mg/dL)
Large (80 to 160 mg/dL)
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)
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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
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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
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Blood (cont’d)
• Myoglobinuria: heme-containing protein in muscle
tissue; clear, red/brown urine
– Rhabdomyolysis: muscle destruction
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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
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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
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Bilirubin Metabolism
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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-ptoluenesulfonate, 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:
urobilinogen +
* ERC
acid
p-dimethylaminobenzaldehyde --------------→ red color
(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