Urinalysis Lab
G.Brady & G.Blevins
SFCC Anatomy/Physiology
A/P 243 / 2007
Note:
Adapted from “The Internet Pathology Laboratory” See Link on SFCC online syllabus.
Urinalysis can reveal diseases
such as diabetes mellitus, various forms of glomerulonephritis,
and chronic urinary tract infections.
The most cost-effective device
used to screen urine is a paper or plastic dipstick. This microchemistry system
has been available for many years and allows qualitative and semi-quantitative
analysis within one minute by simple but careful observation. The color change
occurring on each segment of the strip is compared to a color chart to obtain
results. Microscopic urinalysis
requires a light microscope and a centrifuged urine sample.
MACROSCOPIC URINALYSIS
The first part of
a urinalysis is direct visual observation.
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 color could
be from a food dye, eating fresh beets, a drug, or the presence of either
hemoglobin or myoglobin. If the sample contained many
red blood cells, it may be cloudy as well as red.
URINE DIPSTICK CHEMICAL
ANALYSIS
(Record your urine dipstick
results on the Urinalysis Results Form provided by your instructor)
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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. In
general, people with high protein (meat) diets will have a urine pH in the acid
range and a person who is a vegetarian will have a urine pH in the alkaline
range.
Specific Gravity (sp gr):
Specific gravity (which is
directly proportional to urine osmolality which measures
solute concentration) measures urine density, or the ability of the kidney to
concentrate or dilute the urine over that of plasma. Dipsticks are available
that also measure specific gravity in approximations. Most laboratories measure
specific gravity with a refractometer. In this SFCC lab we will use a urinometer/hydrometer to measure specific gravity as
described by your instructor.
Specific gravity between 1.001
and 1.030 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. Dilute urine may
result when a person drinks excessive amounts of fluids, uses diuretics, or
suffers from diabetes insipidus or has chronic renal
failure. High specific gravity may
be due to limited fluid intake, fever, or inflammation of the kidneys such as
in pyelonephritis. Excessively concentrated urine may
precipitate or crystallize and form renal calculi (kidney stones).
If sp gr
is not > 1.022 after a 12 hour period without food or water, renal
concentrating ability is impaired and the patient either has generalized renal
impairment or nephrogenic diabetes insipidus. In end-stage renal disease, sp gr tends to become 1.007 to 1.010.
Any urine having a specific
gravity over 1.035 may be contaminated or possibly contains very high levels of
glucose or other dissolved substances.
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Protein:
Dipstick screening for
protein: Normally, only small
plasma proteins filtered at the glomerulus are
reabsorbed by the renal tubule. However, a small amount of filtered plasma
proteins and protein secreted by the nephron
Dipsticks detect protein by
production of color with an indicator dye, Bromphenol
blue, which is most sensitive to albumin.
Trace positive results (which
represent a slightly hazy appearance in urine) are equivalent to 10 mg/100 ml
or about 150 mg/24 hours (the upper limit of normal). 1+ corresponds to about
200-500 mg/24 hours, a 2+ to 0.5-1.5 gm/24 hours, a 3+ to 2-5 gm/24 hours, and
a 4+ represents 7 gm/24 hours or greater.
Glucose:
Less than 0.1% of glucose
normally filtered by the glomerulus appears in urine
(< 130 mg/24 hr). Glycosuria (excess sugar in
urine) generally means diabetes mellitus. Dipsticks employing the glucose oxidase reaction for screening are specific for glucose but
can miss other reducing sugars such as galactose and
fructose. For this reason, most newborn and infant urines are routinely
screened for reducing sugars by methods other than glucose oxidase
(such as the Clinitest, a modified Benedict's copper
reduction test).
Ketones:
Ketones (acetone, aceotacetic
acid, beta-hydroxybutyric acid) resulting from either
diabetic ketosis or some other form of calorie deprivation (starvation), are
easily detected using urine dipsticks.
Ketonuria may result from starvation or diets
low in carbohydrates since the body is forced to use fat stores. Ketonuria and glycosuria together, is generally diagnostic for diabetes
mellitus.
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. Presence of bacteria in urine (bacteriuria) may be confirmed by observing the urine
microscopically.
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Leukocyte Esterase:
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.
Blood /
Hemoglobin:
The urine dipstick can detect whole red blood cells as well as lysed RBC’s. A positive result for blood may be
verified by observing the urine specimen under the microscope.
MICROSCOPIC URINALYSIS
Methodology
A sample of well-mixed urine
(usually 10-15 ml) is centrifuged in a test tube at relatively low speed (about
2-3,000 rpm) for 5-10 minutes until a moderately cohesive button is produced at
the bottom of the tube. The supernate is decanted and
a volume of 0.2 to 0.5 ml is left inside the tube. The sediment is resuspended in the remaining supernate
by flicking the bottom of the tube several times. A drop of resuspended
sediment is poured onto a glass slide and coverslipped. A drop of methylene
blue stain may be added if staining of the cells is desired.
Examination
The sediment is first examined
under low power to identify most crystals, casts, squamous
cells, and other large objects. The numbers of casts seen are usually reported
as number of each type found per low power field (LPF). Example: 5-10 hyaline
casts/L casts/LPF. Since the number of elements found in each field may vary
considerably from one field to another, several fields are averaged.
Next, examination is carried out
at high power to identify crystals, cells, and bacteria. The various types of
cells are usually described as the number of each type found per average high
power field (HPF). Example: 1-5 WBC/HPF.
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Red Blood Cells
Hematuria is the presence of abnormal numbers of
red cells in urine due to: glomerular damage, tumors
which erode the urinary tract anywhere along its length, kidney trauma, urinary
tract stones, upper and lower urinary tract infections, nephrotoxins,
and physical stress. Red cells may also contaminate the urine from the vagina
in menstruating women or from trauma produced by bladder catherization.
Theoretically, no red cells should be found, but some find their way into the
urine even in very healthy individuals. However, if one or more red cells can
be found in every high power field, and if contamination can be ruled out, the
specimen is probably abnormal.
RBC's may appear normally shaped, swollen by dilute urine (in fact,
only cell ghosts and free hemoglobin may remain), or crenated
by concentrated urine. Both swollen, partly hemolyzed
RBC's and crenated RBC's are sometimes difficult to distinguish from WBC's in the urine. In addition, red cell ghosts may
simulate yeast. The presence of dysmorphic RBC's in urine suggests a glomerular
disease such as a glomerulonephritis. Dysmorphic RBC's have odd shapes
as a consequence of being distorted via passage through the abnormal glomerular structure.
White Blood Cells
Pyuria refers to the presence of abnormal
numbers of leukocytes that may appear with infection in either the upper or
lower urinary tract or with acute glomerulonephritis.
Usually, the WBC's are granulocytes. White cells from
the vagina, especially in the presence of vaginal and cervical infections, or
the external urethral meatus in men and women may
contaminate the urine.
If two or more leukocytes per each high power
field appear in non-contaminated urine, the specimen is probably abnormal.
Leukocytes have lobed nuclei and granular cytoplasm.
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Epithelial Cells
Renal tubular epithelial cells,
usually larger than granulocytes, contain a large round or oval nucleus and
normally slough into the urine in small numbers. However, with nephrotic syndrome and in conditions leading to tubular
degeneration, the number sloughed is increased.
Transitional epithelial cells
from the renal pelvis, ureter, or bladder have more
regular cell borders, larger nuclei, and smaller overall size than squamous epithelium. Renal tubular epithelial cells are
smaller and rounder than transitional epithelium, and their nucleus occupies
more of the total cell volume.
Squamous epithelial cells from the skin surface
or from the outer urethra can appear in urine.
Their significance is
that they represent possible contamination of the specimen with skin flora.
Casts:
Urinary casts
are formed only in the distal convoluted tubule (DCT) or the collecting duct
(distal nephron). The proximal convoluted tubule
(PCT) and loop of Henle are not locations for cast
formation. Hyaline casts are composed primarily of a mucoprotein secreted by tubule cells.
Even with glomerular injury causing increased glomerular
permeability to plasma proteins with resulting proteinuria,
most matrix or "glue" that cements urinary casts together is mucoprotein,
although albumin and some globulins are also incorporated.
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The factors which favor protein
cast formation are low flow rate, high salt concentration, and low pH, all of
which favor protein denaturation and precipitation,
particularly that of the mucoprotein. Protein casts
with long, thin tails formed at the junction of Henle's
loop and the distal convoluted tubule are called cylindroids. Hyaline casts can
be seen even in healthy patients.
Red blood cells may stick
together and form red blood cell casts. Such casts are indicative of glomerulonephritis, with leakage of RBC's
from glomeruli, or severe tubular damage.
White blood cell casts are most
typical for acute pyelonephritis, but they may also
be present with glomerulonephritis. Their presence
indicates inflammation of the kidney, because such casts will not form except
in the kidney.
When cellular casts remain in
the nephron for some time before they are flushed
into the bladder urine, the cells may degenerate to become a coarsely granular
cast, later a finely granular cast, and ultimately, a waxy cast. Granular and
waxy casts are be believed to derive from renal
tubular cell casts. Broad casts are believed to emanate from damaged and
dilated tubules and are therefore seen in end-stage chronic renal disease.
Bacteria
Bacteria are common in urine
specimens because of the abundant normal microbial flora of the vagina or
external urethral meatus and because of their ability
to rapidly multiply in urine standing at room temperature. Therefore, microbial
organisms found in all but the most scrupulously collected urines should be
interpreted in view of clinical symptoms.
Diagnosis of bacteriuria
in a case of suspected urinary tract infection requires culture. A colony count
may also be done to see if significant numbers of bacteria are present.
Generally, more than 100,000/ml of one organism reflects significant bacteriuria. Multiple organisms reflect contamination.
However, the presence of any organism in catheterized specimens should be
considered significant.
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Yeast
Yeast cells may be contaminants or
represent a true yeast infection. They are often difficult to distinguish from
red cells and amorphous crystals but are distinguished by their tendency to
bud. Most often they are Candida albicans species, which may colonize bladder, urethra,
or vagina.
Crystals
Common crystals seen even in
healthy patients include calcium oxalate and amorphous phosphates.
Miscellaneous:
Unidentifiable objects may find
their way into a specimen, particularly those that patients bring from
home. “Colored worms”
are often cloth fibers from the person’s underwear. Pubic hairs may also appear wormlike.
Spermatozoa can sometimes be
seen in both male and female urine specimens.
METHODS OF URINE COLLECTION
1. Random collection taken at any time of
day with no precautions regarding contamination. The sample may be dilute,
isotonic, or hypertonic and may contain white cells, bacteria, and squamous epithelium as contaminants. In females, the
specimen may cont contain vaginal contaminants such as trichomonads,
yeast, and during menses, red cells.
2. Early morning collection of the sample
before ingestion of any fluid. This is usually hypertonic and reflects the
ability of the kidney to concentrate urine during dehydration which occurs
overnight. If all fluid ingestion has been avoided since
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Lab
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3. Clean-catch, midstream urine specimen
collected after cleansing the external urethral meatus.
A cotton sponge soaked with benzalkonium
hydrochloride is useful and non-irritating for this purpose. A midstream urine
is one in which the first half of the bladder urine is discarded and the
collection vessel is introduced into the urinary stream to catch the last half.
The first half of the stream serves to flush contaminating cells and microbes
from the outer urethra prior to collection. This sounds easy, but it isn't (try
it yourself before criticizing the patient).
4. Catherization of the bladder through the urethra for
urine collection is carried out only in special circumstances, i.e., in a
comatose or confused patient. This procedure risks introducing infection and
traumatizing the urethra and bladder, thus producing iatrogenic infection or hematuria.
5. Suprapubic transabdominal
needle aspiration of the bladder. When done under ideal conditions, this
provides the purest sampling of bladder urine. This is a good method for
infants and small children.
Summary
To summarize, a properly
collected clean-catch, midstream urine after cleansing of the urethral meatus is adequate for complete urinalysis. In fact, these
specimens generally suffice even for urine culture. A period of dehydration may
precede urine collection if testing of renal concentration is desired, but any
specific gravity > 1.022 measured in a randomly collected specimen denotes
adequate renal concentration so long as there are no abnormal solutes in the
urine.
Another important factor is the
interval of time which elapses from collection to examination in the
laboratory. Changes which occur with time after collection include: 1)
decreased clarity due to crystallization of solutes, 2) rising pH, 3) loss of ketone bodies, 4) loss of bilirubin,
5) dissolution of cells and casts, and 6) overgrowth of contaminating
microorganisms. Generally, urinalysis may not reflect the findings of
absolutely fresh urine if the sample is > 1 hour old. Therefore, get the
urine to the laboratory as quickly as possible.
G.Brady&G.Blevins
SFCC A/P 243
URINALYSIS TEST RESULTS
Test
Color
Lt > Dk Yellow
Transparency Clear
Specific Gravity 1.001 – 1.030
DIP STICK:
Student Specimen
Unknown #1
Unknown #2
Leukocytes Negative
pH
4.5
– 8.0
Nitrite
Negative
Protein
Negative
Glucose
Negative
Ketones
Negative
Blood
Negative
Hemoglobin Negative
Bilirubin
Negative
MICROSCOPIC EXAM: Student Specimen Results:
WBC’s
0 – 5 / hi pwr field
RBC’s
0 – 5 / hi pwr field
Bacteria
small numbers present
Epithelial Cells Present
Casts
None