DIAGNOSTIC IN CLINICAL

CHEMISTRY I
MKEB 2404
Title:
Examination of body fluids - urinalysis

Objective:
 To learn the principle of urinalysis

Principle:
A urinalysis is an examination of the urine by physical or chemical means.
Urinalysis comprises a battery of physical, chemical and microscopic tests
that help to screen for urinary tract infections, renal disease, and disease of
other organs that result in abnormal metabolites (breakdown products)
appearing in the urine. In physical examination the color, smell and clarity
of urine is analyzed. Chemical examination is the ‘dipstick’ method, which
contains little pads of chemicals that change color when they come in
contact with the substances of interest. Microscopic examination can be
used to detect microscopic components such as casts, crystals, leukocytes
and red blood cells. Microorganisms such as fungi, bacteria and parasites
can also be observed

Procedure:
1. Physical examination
• Examine urine sample by its color, clarity and odor

2. Chemical test (dipstick test)

• Pour 10 ml of well – mixed urine sample into a centrifuge tube
• Dip the strip briefly (max 1 sec.) into the urine so as to wet all the test
areas
• Wipe the test strip along the rim of the reaction color with the color
scales on the label

3. Microscopic examination
• Centrifuge the tube containing urine sample (from 2) at 1500 rpm for
5minutes
• After centrifugation, decant 9ml of urine. Reconstitute the remaining
1ml thoroughly
• By using a Pasteur pipette, fill the counting chamber with urine and
examine it microscopically

1|Page
Results:
Physical characteristics, chemical test using dipstick method and
microscopy:

Reference FIRST MORNING AFTER LUNCH

Physical characteristics
Bright yellow with little
Color Dark yellow
turbidity
Clarity Turbid Clear
Chemical test (Dipstick method)
Specific Gravity 1.010 1.020
pH 6 5
Leukocyte Negative Negative
Nitrite Negative Negative
Protein Negative Negative
Glucose Normal +1
Ketone Negative Negative
Urobilinogen Normal Normal
Bilirubin Negative Negative
Blood Negative Negative
Microscopy examination and WBC count
Formation of some Formation of some
Microscopy
artifacts artifacts
WBC count No WBC No WBC

Discussion and conclusion:

Urinalysis by definition refers only to the chemical analysis of urine.
Urinalysis can reveal diseases that have gone unnoticed because they do
not produce striking signs or symptoms. Examples include diabetes
mellitus, various forms of glomerulonephritis, and chronic urinary tract
infections.

Macroscopic analysis:
Normal, fresh urine is pale to dark yellow or amber in color and clear.
Turbidity or cloudiness may be caused by excessive cellular material or
protein in the urine or may develop from crystallization or precipitation of
salts upon standing at room temperature or in the refrigerator. Clearing of
the specimen after addition of a small amount of acid indicates that
precipitation of salts is the probable cause of turbidity. A red or red-
brown (abnormal) color could be from a food dye, eating fresh beets, a
drug, or the presence of either hemoglobin or myoglobin. If the sample

2|Page
 contained many red blood cells, it would be cloudy as well as red. Some
conditions associated with abnormal urine color are:
 Black - Melanin, alkaptonuria
 Brown to red - Free hamoglobin, myoglobin, blood
 Orange to yellow - Bile pigment
 Green urine - Pseudomonas infection
 Dark urine - Acute intermittent porphyria

pH:
The glomerular filtrate of blood plasma is usually acidified by renal
tubules and collecting ducts from a pH of 7.4 to about 6 in the final urine.
However, depending on the acid-base status, urinary pH may range from
as low as 4.5 to as high as 8.0. The change to the acid side of 7.4 is
accomplished in the distal convoluted tubule and the collecting duct.
Usually pH of urine ranges between 5.0 and 6.0, due to obligatory
excretion of acid produced every day. pH tends to be lower in first void
specimen and tends to increase after a meal (alkaline tide due to acid
secretion in the stomach). Very alkaline urine is suggestive of infection
with a urea splitting organism. Prolonged storage can lead to overgrowth
of urea splitting organism and falsely cause high urine pH. Rarely alkaline
pH may be due to metabolic alkalosis or acidification defect. Low urine
pH (<5.0) is most commonly due to metabolic acidosis. Acid urine may
also be associated with ingestion of large amounts of meat.

Specific Gravity:
Specific gravity which is directly proportional to urine osmolality that
measures solute concentration use to measures urine density,or the ability
of the kidney to concentrate or dilute the urine over that of plasma.
Specific gravity between 1.002 and 1.035 on a random sample should be
considered normal if kidney function is normal. Since the sp gr of the
glomerular filtrate in Bowman's space ranges from 1.007 to 1.010, any
measurement below this range indicates hydration and any measurement
above it indicates relative dehydration. Measurement of specific gravity
by dipstick depends on the ionic strength of the urine and the fact that
there is a linear correlation between ionic strength and osmolality in urine.
The strip contains a polyionic polymer with binding sites saturated with
hydrogen ions; the release of hydrogen ions when they are competitively
displaced by urinary cations causes a change in the pH sensitive indicator
dye. Hence specific gravity tends to be falsely high at urine pH < 6 and
falsely low if the urine pH >7.0. Moreover, increase in non-ionic
substances like albumin, glucose, and urea does not increase specific
gravity by this method.

3|Page
 Bilirubin and urobilinogen:
False negative results are obtained if there are large amounts of ascorbic
acid and nitrites, or if urine is tested after prolonged exposure, since
bilirubin degenerates with exposure to light. Urobilinogen is formed by
the action of intestinal bacteria on bilirubin. Some of this is reabsorbed in
the intestine and excreted in the urine. in conditions with
hyperbilirubinemia (except when there is obstruction preventing bilirubin
from reaching intestine), there is increase in urobilinogen.
Leukocyte:
A positive leukocyte esterase test results from the presence of white blood
cells either as whole cells or as lysed cells. Pyuria can be detected even if
the urine sample contains damaged or lysed WBC's. A negative leukocyte
esterase test means that an infection is unlikely and that, without
additional evidence of urinary tract infection, microscopic exam and/or
urine culture need not be done to rule out significant bacteriuria
The esterase method is used to detect leucocytes in the urine by dipstick.
The esterase released from the neutrophils reacts with the ester on the
reagent strip releasing 3-0H-5-phenyl pyrrole that reacts with a diazonium
salt to produce pink to purple color. Allowing the urine to stand promotes
lysis of leucocytes and hence more intense reaction. Since intact leucocytes
are not required for this test it may be positive even when microscopy is
negative for leucocytes. False positives can occur with vaginal
contamination. Presence of glucose, albumin, ascorbic acid, tetracycline,
cephalexin, cephalothin, or large amounts of oxalic acid can inhibit the
reaction.

Nitrite:
A positive nitrite test indicates that bacteria may be present in significant
numbers in urine. Gram negative rods such as E. coli are more likely to
give a positive test. False negative results are common and may be due to:
 Low level of nitrate in the urine as a result of low dietary intake
 Inadequate bladder retention; since it may take upto 4 hours to convert
nitrate to nitrite
 Prolonged storage of test sample, which can lead to degradation of nitrites
 Some urinary pathogens such as Streptococcus faecalis, other gram
positive organisms, Neisseria gonorrhoeae, and Mycobacterium
tuberculosis which do not convert nitrates to nitrites
 Presence of ascorbic acid that can inhibit the reaction.

4|Page
Ketone:
Ketones (acetone, aceotacetic acid, beta-hydroxybutyric acid) resulting
from either diabetic ketosis or some other form of calorie deprivation
(starvation), are easily detected using either dipsticks or test tablets
containing sodium nitroprusside

Protein:
Normally charge and size selective properties of the capillary wall prevent
all but a tiny fraction of protein from crossing. Smaller proteins (< 20,000
daltons) pass readily across the capillary wall; however, the amount
filtered is small due to less plasma concentration and moreover most of
these are normally reabsorbed by proximal tubule. The very small amount
of proteins that normally appears in the urine is a result of normal tubular
secretion. Abnormal amounts of protein can appear in the urine as a result
of 3 mechanisms:
 Disruption of the capillary wall barrier
 Tubular damage that inhibits the normal resorptive capacity
 Increased production of normal or abnormal plasma proteins. Rarely,
increased tubular protein production can cause increased protein
excretion.

Glucose:
This test is specific for glucose and does not react with other reducing
substances. Ascorbic acid, ketones, and aspirin in urine can cause false
negative reaction. Increase in specific gravity also decreases reactivity of
glucose test. False positive test can occur with peroxide contamination.
Glycosuria (excess sugar in urine) generally means diabetes mellitus.
However the best result to determine whether the person have diabetes or
not is depends on oral glucose tolerance test (OGTT).

Red blood cells:

Occult blood can be detected by use of dipsticks impregnated with
orthotoluidine and peroxidase. Hemoglobin, either free in urine or from
RBC's catalyze an oxidation reaction resulting in colour change. A positive
dipstick test for occult blood in the absence of RBC's on the microscopic
examination is an important clue in differentiating between hematuria
and hemoglobinuria. Elevated specific gravity and captopril may reduce
the reactivity of the test. Certain oxidizing substances like hypochlorite,
povidone-iodine, and microbial peroxidase can cause false positive
results. Increased levels of ascorbic acid may cause a false negative result.

5|Page
 Tamm horsfall protein:
Tamm-Horsfall (TH) protein is a high molecular weight glycoprotein that
is formed on the epithelial surface of the thick ascending limb of the loop
of Henle. It is a protein that normally appears in the urine is a result of
normal tubular secretion. Casts are elements of the urinary sediment,
formed by the polymerization of the Tamm-Horsfall fibrils, taking the
shape of the site of its formation (casting). Casts are formed, after the loop,
in the late section of the distal tubules and the early section of the
collecting tubes. Factors known to be promoters of cast formation are:
 Albumin
 Urinary stasis
 Cellular debris
 Low glomerular filtration rate
 Acid pH
 Presence of certain proteins ( Bence-Jones, myoglobin,
hemoglobin...)
 Osmolality between 200 and 400 mOsm/Kg
1. Hyaline cast
Hyaline casts, is a hyaline matrix casts without inclusion, are seen in
numerous conditions. Some of these conditions are the results of a
pathological process, but many are results of a normal physiological
activity. Hyaline casts can be seen after a strenuous exercise. Hyaline casts
are considered as physiological cast

2. Waxy cast
The waxy casts owe their name to the opaque waxlike matrix. Waxy casts
have square ends and have frequent notch like cracks perpendicular to the
long axis. This matrix is related to a long period urinary stasis affecting
some nephrons. Waxy casts are usually without inclusion or slightly
granular, but waxy casts with inclusions are occasionally seen.

3. Granular cast
The causes of this degeneration are unknown, but a proteinuria is a usual
finding. A protein overload could be responsible for the granular
degeneration of the tubular cells. Usually it to glomerular and tubular
disease. Also related viral infection and lead poisoning.

4. Fatty cast
The fatty casts are casts that contain lipid droplets sequestrated in a
hyaline matrix. The oval fat body casts are associated with the nephrotic
syndrome. The urinary context will be one of a high proteinuria. If the
specimen has a dipstick reaction positive for blood, then, the presence of
blood casts is expected. Maybe linked to hypothyroidism

6|Page
5. Red Blood Cell cast
Red blood cells casts are described as hyaline casts, containing ghost red
blood cells, or as hyaline casts filled with numerous orange red
erythrocytes. Usually related to glomerular disease or glomerular damage

6. Epithelial Cell cast

Usually use to differentiate from leukocyte cast, supravital staining and
phase -contrast microscopy is helpful. This cast usually associated with
tubular necrosis, viral disease (CMV), heavy metal ingestion

7. White Blood Cell cast:

WBCs enter the tubular lumen through and between tubular epithelial
cells. Usually it is associated with pyelonephritis and tubulointerstitial
disease

Crystals:
Crystals are common findings in urine especially if urine is allowed to
stand long before examination. The type of crystal depends on the pH and
constituents of the urine. Certain crystals like cystine, tyrosine, and
leucine are always abnormal if detected. But other crystals are not
abnormal, unless when associated with stone disease, in which case the
type of crystal can give a clue to the origin of the stone. Exception is
presence of large number of calcium oxalate crystals in association with
ethylene glycol overdose. Ammonium biurate and calcium carbonate are
other crystals sometimes seen in alkaline urine. Common crystals seen
even in healthy patients include calcium oxalate, triple phosphate crystals
and amorphous phosphates. Very uncommon crystals include: cystine
crystals in urine of neonates with congenital cystinuria or severe liver
disease, tyrosine crystals with congenital tyrosinosis or marked liver
impairment, or leucine crystals in patients with severe liver disease or
with maple syrup urine disease.

7|Page