Clinical Urinalysis Review
Austin Community College
Medical Laboratory Technology
Clinical II Spring 07
Urine Blood Testing
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http://library.med.utah.edu/WebPath/TUTORIAL/URINE/URINE.html
Chemical Exam of Urine
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Chemical Exam of Urine
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Chemical Exam of Urine
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Reagent strip manufactures

Bayer Corporation- Diagnostics Division
(formerly Ames) produces Multistix

Boehringer-Mannheim Corporation which
produces Chemstrip

Behring Diagnostics which produces Rapignost
Chemical Exam of Urine
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Chemical Exam of Urine

Reagent strip precautions and source of errors

Normal dipstick procedure:


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Dip strip briefly, but completely into well mixed,
room temperature urine sample.
Withdraw strip, blot briefly on its side
Keeping the strip flat, read results at the
appropriate times by comparing the color to the
appropriate color on the chart provided.
Chemical Exam of Urine


Sources of error (& preventions)
Testing cold specimens

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Inadequate mixing of specimen

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would result in a slowing down of reactions; test
specimens when fresh or bring them to RT before
testing
could result in false reduced or negative reactions to
blood and leukocyte tests; mix specimens well before
dipping
Over-dipping of reagent strip

will result in leaching of reagents out of pads; briefly,
but completely dip the reagent strip into the urine
Chemical Exam of Urine

Inadequate blotting & Failure to keep strip horizontal


will result in over-run or mixing of reagents between
the different reaction pads; blot excess urine off the strip
and keep strip horizontal. If dipping from the tube, can
run the side of the strip along the rim to remove excess
urine.
Improper timing of tests

over development of reagent pad colors leading to
falsely increased results; follow manufacturer’s
recommendations
Chemical Exam of Urine

Inadequate light

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Mis-using color chart

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misinterpretation of results; use good lighting
misinterpretation of results; hold strip just over color
chart and match colors as close as possible, consider
use of back-up tests, if needed, especially if urine’s
color masks reaction colors.
Chemical Exam of Urine
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Handling and Storage
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Keep strips in original container, stored at RT
Protect from moisture and volatile fumes
Use before expiration date
Do not touch reagent pad areas
Chemical Exam of Urine
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Quality Control - use appropriate,
commercially prepared positive and negative
controls.

Use commercially prepared pos and neg
controls, at least once per 24 hours, and
anytime a new bottle is opened, or question of
validity of results. Readings should agree with
published results ± one color block.
Urine Glucose Testing
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Normal : no glucose detected

Clinical significance of abnormal results
(Glucosuria)
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Plasma glucose level exceeds renal threshold
(160-189 mg/dL)

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Diabetes mellitus
Renal tubular dysfunction

Filtered glucose not being reabsorbed in tubules
Urine Glucose Testing

Dipstick Testing Method
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Glucose initiates reaction

Coupled reaction

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Glucose oxidase – oxidizes glucose to gluconic acid and
concurrently reduces oxygen to hydrogen peroxide.
Hydrogen peroxide in presence of the enzyme peroxidase
will oxidize an indicator, giving a colored reaction.
Chromogens


Potassium iodide or
Tetramethylbenzindine
Urine Glucose Testing
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Sensitivity

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@ 50-100 mg (compared to Clinitest’s 250) SO- Can
have a positive dipstick but a neg Clinitest
Specificity - is specific for glucose only.
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not affected by other sugars or reducing substances.
Urine Glucose Testing
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Interfering substances
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High specific gravity and high pH may depress color.
Ascorbic acid-false neg
Bleach or peroxide may give false positive
Urine Bilirubin Testing
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Normal : no bilirubin detected
Clinical significance of abnormal results
(Bilirubinuria)

Jaundice - Condition when serum bilirubin
becomes greater than the liver can handle, and
there is an abnormal collection of bilirubin in the
tissues giving them a yellow color
Urine Bilirubin Testing
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Prehepatic / Hemolytic jaundice


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Excessive hemolysis of RBC; beyond what the
liver can process
Type of bilirubin?
Is bilirubin found in the urine? YES/NO? Explain.
Urine Bilirubin Testing
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Prehepatic / Hemolytic jaundice
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Type of bilirubin? – indirect, insoluble,
unconjugated
Is bilirubin found in the urine? – No, the bilirubin
is not water soluble
Urine Bilirubin Testing
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Hepatic jaundice
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Liver’s cells malfunctioning

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Ie. viral hepatitis, cirrhosis etc.
Both (direct) bilirubin and urobilinogen found in
urine.
Urine Bilirubin Testing
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Post hepatic (regurgative or obstructive)
hepatitis
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Obstruction to outflow of bile – some type of
blockage
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Gall stones
Tumor
Edema
Conjugated bilirubin backed up into blood
(Bilirubinuria) and passes into urine
Urine Bilirubin Testing
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Testing method
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Urine dipsticks for bilirubin – a diazo reaction

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Impregnated with stabilized diazotized 2,4 dichloraniline
Color goes from buff to brown also shades of pink –
violet
If urine is strongly colored, look for change in
pad color after dipping. Use Ictotest for
backup.
Urine Bilirubin Testing

Interfering substances


Medication metabolites, pigments and indican may
obscure readings
False negatives due to aged specimens, especially
those exposed to light and oxidation.
Urine Ketone Testing
 Ketone Bodies
 Origin - not normally present
 Products of fat catabolism - breakdown of
fat into CO2 and H2O
 What are the 3 ketone bodies?
Urine Ketone Testing
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Acetone

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Diacetic Acid (Acetoacetic)
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2%. -Acetone is volatile, & excreted primarily through the
lungs
the first formed,
20 % of the total
the form detected by most ketone test procedures
Beta hydroxybutyric Acid


majority formed, but not detected by routine tests.
Only Hart’s test, an old ‘wet chemical’ test will detect this one.
Urine Ketone Testing
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Definitions
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Ketonuria - ketones in the urine
Ketonemia - ketones in the blood
Ketosis - disease state, when patient has
increased amount of ketones.
Acidosis - state when blood pH is decreased,
an accumulation of acids; commonly occurs
as a result of ketosis
Urine Ketone Testing
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Clinical significance
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Health – formed in liver and completely metabolized
Disease – excessive formation and accumulation
 Disturbance of carbohydrate metabolism
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when there is a decrease of carbohydrate metabolism, then the
body stores of fat must be metabolized to supply energy.
As a result of this increased fat metabolism ketones will be found
in the urine. Ex. low carbohydrate diets, diabetes
Starvation
Vomiting and diarrhea in children
Van Gierke's Disease – glycogen storage disease
High fat diet
Urine Ketone Testing
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Clinical effects

Metabolic acidosis
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Lowering of blood & urine pH
Brain toxicity
Urine Ketone Testing
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Testing

most use nitroprusside
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detects diacetic acid and a small amount of acetone, but
does not detect β-hydroxybutyric acid.
Produces purple color
Can be used on urine or blood
Urine Specific Gravity Testing

The specific gravity is a measure of the
weight of urine compared to an equal amount
of water.

Specific gravity it proportional to urine
osmolality which is a measure of
concentration.
Urine Specific Gravity Testing
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
The specific gravity will always be greater
than 1.000 and will increase as more
materials are dissolved in the urine.
The value changes throughout the day
depending on fluid intake.
Urine Specific Gravity Testing
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Specific gravity between 1.002 & 1.035 on a
random sample is normal if kidney function is
normal.

Specific gravity in Bowman’s capsule fluid is @
1.007
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Any reading below this indicates hydration
Any reading above this indicates some degree of
dehydration
Urine Specific Gravity Testing
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Again dilute urine will have values less than
1.010.
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Fixed specific gravity = 1.010; isothenuria
Diabetes insipidus
End-stage renal disease
And concentrated urine will have values
usually over 1.020.

Usually due to dehydration and can be seen in
well population as well as sick.
Urine Specific Gravity Testing
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Increased urine specific gravity may indicate / be seen in:
* Dehydration
* Diarrhea
* Excessive sweating
* Glucosuria
* Heart failure (related to decreased blood flow to the kidneys)
* Renal arterial stenosis
* Syndrome of inappropriate antidiuretic hormone secretion
(SIADH)
* Vomiting
* Water restriction
Urine Specific Gravity Testing

Decreased urine specific gravity may indicate
/ be seen in:
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* Excessive fluid intake
* Diabetes insipidus – central or nephrogenic
* Renal failure (that is, loss of ability to
reabsorb water)
* Pyelonephritis
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Urine Specific Gravity Testing
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Specific gravity > 1.035 (refractometer)
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Could have very high glucose levels
Could contain radiographic dye
Urine Specific Gravity Testing
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Testing

Polyelectrolytes , pH indicator (bromthymol blue
measures the pH change), and alkaline buffer
Urine Specific Gravity Testing
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Interfering substances
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False elevation of results may be seen in samples
with increased protein concentration.
Some reports of reduced specific gravity results
on alkaline specimens.
Lipids may also effect results
Urine Blood Testing
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Normally not found in urine
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Hemoglobinuria – free hemoglobin in urine
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Circulating free hemoglobin normally picked up by
haptoglobin preventing loss in urine
When serum levels of hemoglobin > 100 mg/dL
threshold is exceeded
Hematuria – RBCs in the urine

Trauma / irritation of renal organs
Urine Blood Testing
Urine Blood Testing

Testing dipstick reaction
HGB  H2 O2
peroxidase
 Oxygen
Oxygen + Gum guaiac, benzidine or orthotolidine  green or blue
oxidation products
Urine Blood Testing

‘Blood’ test detects
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Free Hemoglobin
RBCs – get lysed on the pad & their hemoglobin
reacts
Myoglobin – muscle hemoglobin
Principle based on the peroxidase-like activity
of the heme portion of the molecule
Urine Blood Testing
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Sensitivity – can detect at levels of 5-10
cells/uL
Interfering substances
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Ascorbic acid
Nitrates
Oxidizing agents (ie bleach)
Contaminate blood (menstrual)
Urine pH Testing
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Normal: kidneys capable of 4.5 – 8.0
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Factors effecting pH
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Diet – general & specific foods
Time of day
Metabolic disorders
Drugs / medications
Dipstick capable: 4.5 – 9.0
Urine pH Testing
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Test method
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Dipstick indicators – methyl red and bromthymol blue
Range 4.5-9.0
Caution – other chemicals on dipstick can effect
pH reading
Urine Protein Testing
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Normally not found in measurable amounts
on dipstick (<150 mg/dL /day)

Permeability of glomerulus
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Damage to glom capularies
Changes in glom blood flow
Albumin excretions may be increased temporarily
due to exercise, uti, and acute illness with fever.
Dipstick results of >@ 1+ (30mg/dL) would equal
to @ 500 mg/dL (clinical proteinuria)
Urine Protein Testing
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Only albumin detectable by dipstick
Sensitivity (@15-30 ml/dL)
Urine Protein Testing
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New testing for microalbumin & creatinine
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Results:
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Protein 20-200 mg/dL (30-300 mg/dL /24 hr)
Creatinine 10-300 mg/dL
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Albumin/creatinine ratio

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Normally albumin in the urine is less than 30 mg/ gram
creatinine
Urine Protein Testing

Principle - Protein error of indicators
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at fixed pH, certain indicators show one color in the
presence of protein and another in absence of protein the “error” of the indicator.
Indicator – tetrabromphenol blue - can be hard to
read at the trace end
Citrate Buffer – maintains pH 3 -quite acid
Urine Protein Testing
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Sources of error
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Sensitive only to albumin
Urine with strong / unusual color makes reading
difficult
Highly alkaline or buffered urine will neutralize
acid buffer and lead to increased erroneous
results.
Urine container contamination would interfere
Urine Protein Testing
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Urine back up test
3% sulfosalicylic acid

Added to the supernatant to detect any kind of protein.
Urine will turn cloudy if protein is present.
Urine Urobilinogen Testing
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Normally found in small amounts, especially
in early afternoon
Increased amounts may indicate liver disease
or be seen as result of hemolytic disorders
Decreased amounts:
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If intestinal bacteria destroyed
Liver doesn’t conjugate bilirubin
Biliary obstruction – failure of bilirubin to reach
small intestine
Urine Urobilinogen Testing
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Test principle based on Ehrilich’s reaction


Para-dimethylaminobenzaldehyde = Ehrlich's
reagent.
Must protect specimen from light and test
immediately
Urine Nitrate Testing
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Nitrate

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Detects presence of certain types of bacteria
screening for presence of UTI.
Certain species of bacteria convert nitrate (normal
constituent of urine) to nitrite
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Escherichia - most common cause of UTI
Klebsiella
Proteus
Pseudomonas
Enterobacter
Citrobacter
Urine Nitrate Testing

Aromatic amine in reagent strip reacts with nitrite;
producing a diazonium salt

The diazonium salt reacts with sulfanilic acid and
acetic acid to produce a pink azo dye
Urine Nitrate Testing

Limitations

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reported as positive or negative
Not all UTI causing bacteria convert nitrate to nitrite
 Haemophilus
 Staphylococcus
 Streptococcus
Urine Nitrate Testing

Fresh first morning specimen is preferred - besides
being the most concentrated specimen, the urine
has been in the bladder longer, allowing bacteria
time and opportunity to convert the nitrates to
nitrites.
Urine Leukocyte Testing
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
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Leukocyte esterase testing is another test
used as a means of screening for urinary tract
infection.
Does not measure concentration of leukocytes
Will detect presence of lysed leukocytes as well
as intact WBCs
Urine Leukocyte Testing
test principle:

Leukocyte esterase, an enzyme present in
granulocytes, hydrolyzes indoxylcarbonic acid
esterase to produce indoxyl, which reacts with a
diazonium salt to create a purple color usually in 2
min.
Urine Leukocyte Testing

Reaction interference


False positives - oxidizing detergents
False negatives - greatly increased glucose, protein,
or specific gravity- increased sp gr could cause
WBC to crenate preventing their releasing their
esterase, So it is possible for the dipstick to be
negative when there are WBCs present.