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King Saud University
College of Science
Department of Biochemistry
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Chapter 6
Urinalysis-2
Professor A. S. Alhomida
1
Examination of Urine Sediment
Macroscopic Screening
• Abnormalities in the
physical and chemical
portion of urinalysis play
a particular role in the
decision to perform a
microscopic examination
• Parameters considered
significant vary among
Lab but include
macroscopic screening
2
Examination of Urine Sediment,
Cont’d
Correlation of Results
• Microscopic results
should be correlated with
the physical and chemical
findings
• Results of Specimen don’t
correclate must be
rechecked for both
technical and clerical
errors
3
Examination of Urine Sediment,
Cont’d
General Aspects
1. Preservation
• Cells and casts begin to disintegrate in 1 - 3 hr at room
temperature
• Refrigeration for up to 48 h (little loss of cells)
2. Specimen Concentration
• Ten to twenty-fold concentration by centrifugation
4
Examination of Urine Sediment,
Cont’d
General Aspects
3. Types of Microscopy
•
•
•
Phase contrast microscopy
Polarized microscopy
Bright field microscopy with special staining (e.g.,
Sternheimer-Malbin stain)
5
Examination of Urine Sediment,
Cont’d
Microscopic Techniques
6
Examination of Urine
Sediment, Cont’d
Procedure
1. 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
2. The supernate is decanted and a volume of 0.5 to 1.0
mL is left inside the tube
3. The sediment is resuspended in the remaining
supernate by flicking the bottom of the tube several
times
4. A drop of resuspended sediment is poured onto a glass
slide and coverslipped
7
Preparing the Sediment for
Analysis
• Tubes, pipets and slides
used for standardizing
urine sediments
8
Preparing the Sediment for
Analysis
• Mix the urine well and pour 12
milliliters of urine in a clean
centrifuge tube
• Cap the tube and centrifuge
the urine for five minutes at
450 g. RPMs
• At this speed the sediment will
be optimally concentrated
without destroying any of the
fragile elements in the urine
9
Preparing the Sediment for
Analysis, Cont’d
• After centrifugation,
remove the supernatant
fluid with a polyethylene,
transfer pipet leaving 0.5
to 1.0 mL urine on the
sediment
• Resuspend the sediment
in the remaining urine by
flicking the bottom of the
tube or by running the
tube across a test tube
rack
10
Preparing the Sediment for
Analysis, Cont’d
11
Preparing the Sediment for
Analysis, Cont’d
12
Preparing the Sediment for
Analysis, Cont’d
• Using a plastic or
polypropylene transfer
pipet, mount a drop of
the urine in the counting
chamber of the prepared
commercial plastic slides
• Avoid using glass pipets
when mounting the urine
sediment as elements like
casts tend to cling to the
glass surface
13
Preparing the Sediment for
Analysis, Cont’d
• Exam the sediment using
the microscope
• After counting the
elements in the
sediment, you may
calculate the number of
elements per mL instead
of lpf (low- or hight
power field or hpf), to
provid standardization
among the various
techniques
14
Preparing the Sediment for
Analysis, Cont’d
1. Calculate the area of an lpf or hpf using the
diameter:
2
2. Area = pr
3. For example:
4. r for hpf 0.35 m= 0.175 m
2
2
5. Area = 3.14 X (0.175) = 0.096 mm
15
Preparing the Sediment for
Analysis, Cont’d
6. Calculate the maximum number of lpf or hpf in
the viewing area under
area
22
X
22
mm
for
2
cover slip = 484 m
7. hpf = 484 / 0.096 = 5040
8. Convert hpf into per mL
9. 5040/ 0.02 mL X 12 = 21,000 hpf/mL
•
where is 0.02 the volume of sediment and 12 is
concentration factor
16
Preparing the Sediment for
Analysis, Cont’d
• Special stains can be
used to enhance the
elements in the sediment
• Sternheimer-Malbin stain,
tolui Papanicolau
• Wright’s
• Immunoperoxidase
• Immunofluorescence dine
blue
• They will enhance the
internal structures of the
cells making them easier
to identify
17
Preparing the Sediment for
Analysis, Cont’d
18
Preparing the Sediment for
Analysis, Cont’d
19
Examination of Urine
Sediment, Cont’d
At Low Power
1. To identify most crystals, casts, squamous cells, and
other large objects
2. The numbers of casts seen are usually reported as
number of each type found per low power field (LPF)
3. Example: 5-10 hyaline casts/L casts/LPF
4. Since the number of elements found in each field may
vary considerably from one field to another, several
fields are averaged
20
Examination of Urine
Sediment, Cont’d
At High Power
1. To identify crystals, cells, and bacteria
2. 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
21
Examination of Urine Sediment,
Cont’d
Abnormal Findings
1. Per High Power Field (HPF) (400x)
•
•
•
•
>
>
>
>
3 erythrocytes
5 leukocytes
2 renal tubular cells
10 bacteria
22
Examination of Urine Sediment,
Cont’d
Abnormal Findings
2. Per Low Power Field (LPF) (200x)
•
•
•
> 3 hyaline casts or > 1 granular cast
> 10 squamous cells (indicative of contaminated
specimen)
Any other cast (RBCs, WBCs)
23
Examination of Urine Sediment,
Cont’d
Abnormal Findings
3. Presence of:
•
•
•
Fungal hyphae or yeast, parasite, viral inclusions
Pathological crystals (leucine, tyrosine, cystine)
Large number of uric acid or calcium oxalate crystals
24
Components of
Urine Sediment
Organized Components
1.
2.
3.
4.
5.
6.
7.
8.
Red blood cells
White blood cells
Epithelial cells
Casts
Bacteria
Yeast
Parasites
Spermatozoa
25
Components of
Urine Sediment, Cont’d
Unorganized Components
1. Crystals
• Mostly insignificant
• Exceptions
• Cystine
• Uric acid
• Sulfa drug crystals
2. Chemical elements
26
Examination of Urine Sediment,
Cont’d
Cells
1.
Erythrocytes
•
2.
3.
•
•
•
•
•
•
“Dysmorphic”
vs. “normal”
Leukocytes
Neutrophils (glitter cells)
Eosinophils
Epithelial Cells
Renal tubular epithelial cells
Oval fat bodies
Transitional epithelial cells
Squamous cells
(> 10 per HPF)
More than 1 per 3 HPF
Hansel test (special stain)
Few are normal
Abnormal, indicate Nephrosis
Few are normal
Indicate level of contamination
27
Examination of Urine Sediment,
Cont’d
Cells
1. Erythrocytes (GN, stone, tumor, exogenous)
2. Renal tubular epithelial cells (many renal
diseases)
•
Oval Fat bodies (Fat means nephrotic syndrome)
3. PMNs (Infection or sterile inflammation)
4. Eosinophils (Some allergic interstitial
nephritis)
5. Squamous cells (CONTAMINATION)
28
Examination of Urine Sediment,
Cont’d
Red Blood Cells
1. 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, renal infarcts, acute tubular
necrosis, upper and lower uri urinary tract infections,
nephrotoxins, and physical stress
2. Red cells may also contaminate the urine from the vagina
in menstruating women or from trauma produced by
bladder catherization
3. Theoretically, no red cells should be found, but some find
their way into the urine even in very healthy individuals
29
Examination of Urine Sediment,
Cont’d
Red Blood Cells
4.
5.
6.
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
30
Examination of Urine Sediment,
Cont’d
Red Blood Cells, Cont’d
7. In addition, red cell ghosts may simulate yeast. The
presence of dysmorphic RBC's in urine suggests a
glomerular disease such as a glomerulonephritis
8. Dysmorphic RBC's have odd shapes as a consequence
of being distorted via passage through the abnormal
glomerular structure
9. Dysmorphic RBCs
Irregular shape, size, density
Indicate glomerular bleeding
31
Examination of Urine Sediment,
Cont’d
RBCs
• There are many RBCs in
this field
• Note the center pallor of
most of the cells
especially the one labeled
with a "B"
• Can you picture the donut
shape or biconcave disc
form of the cell?
• “A" points to a red cell on
its side
32
Examination of Urine Sediment,
Cont’d
RBCs
• This is another field of
RBCs
• Note that some of them
look granular
• That is because they are
crenated or puckered
• The spicules make the
cell look granular
33
Examination of Urine Sediment,
Cont’d
RBCs
• Note the irregular outlines of
many of these RBC's,
compared to two relatively
normal RBC's at the center left
of the right panel. These
abnormal RBC's are
dysmorphic RBC's.
• Dysmorphic RBC's have odd
shapes as a consequence of
being distorted via passage
through the abnormal
glomerular structure
34
Examination of Urine Sediment,
Cont’d
Summary of RBC
35
Examination of Urine Sediment,
Cont’d
WBCs
1. 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
2. Usually, the WBC's are granulocytes
3. 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
36
Examination of Urine Sediment,
Cont’d
WBCs
4. If two or more leukocytes per each high power field
appear in non-contaminated urine, the specimen is
probably abnormal
5. Leukocytes have lobed nuclei and granular cytoplasm
37
Examination of Urine Sediment,
Cont’d
WBCs
• WBC in urine are most
neutrophils
• Like erythrocytes, WBC
may lyse in very dilute or
highly alkaline urine;
WBC cytoplasmic
granules released into
the urine often resemble
cocci bacteria
38
Examination of Urine Sediment,
Cont’d
WBCs
• In this field, "C" points to
a white cell and "B"
points to two transitional
epithelial cells
• Note that you can
distinguish a nucleus in
the white cell if you look
very closely
• Also note the granular
appearance
39
Examination of Urine Sediment,
Cont’d
Summary of WBCs
40
Examination of Urine Sediment,
Cont’d
Renal Tubular Cells
1. Usually larger than granulocytes, contain a large round
or oval nucleus and normally slough into the urine in
small numbers
2. In nephrotic syndrome and in conditions leading to
tubular degeneration, the number sloughed is
increased
3. When lipiduria occurs, these cells contain endogenous
fats. When filled with numerous fat droplets, such cells
are called oval fat bodies
4. Oval fat bodies exhibit a "Maltese cross" configuration
by polarized light microscopy
41
Examination of Urine Sediment,
Cont’d
Squamous Cells
• This image provides a
good example of two
unstained squamous
epithelial cells
• Note the size of the cell,
its nucleus and its
irregular shape
• These two cells look
somewhat granular which
is an artifact of the urine
preservation
42
Examination of Urine Sediment,
Cont’d
Renal Tubular Cells
• This frame compares a
WBC ("B") and the renal
tubular cells ("A")
• Note the size comparison
• Also note how prominent
the nucleus is in the renal
tubular cells
• It is much easier to see
than the nucleus of the
WBC.
43
Examination of Urine Sediment,
Cont’d
Transitional Cells
• They originate from the
renal pelvis, ureter, or
bladder
• It shows a transitional
epithelial cell at "B"
• The three cells just above
this one are also
transitional epithelials
• "A" points to a squamous
epithelial cell
44
Examination of Urine Sediment,
Cont’d
Oval Fat Body
• Oval fat bodies
consist of
degenerated tubular
cells containing
abundant lipid, which
appears refractile
• Indicate heavy
proteinuria
45
Examination of Urine Sediment,
Cont’d
Oval Fat Body
• Under polarized light,
oval fat bodies
demonstrate the
"Maltese cross"
appearance
46
Examination of Urine Sediment,
Cont’d
LE Cell
• 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
47
Examination of Urine Sediment,
Summary of Epithelial Cells Cont’d
48
Examination of Urine Sediment,
Cont’d
Bacteria and Yeasts
1. Bacteria
•
Bacteriuria
2. Yeasts
•
Candidiasis
3. Viruses
•
CMV inclusions
More than 10 per HPF
Most likely a contaminant
but should correlate with
clinical picture
Probable viral cystitis.
49
Examination of Urine Sediment,
Cont’d
Bacteria
1. They 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
2. Diagnosis of bacteriuria in a case of suspected urinary tract
infection requires culture
3. A colony count may also be done to see if significant
numbers of bacteria are present
50
Examination of Urine Sediment,
Cont’d
Bacteria
4. More than 100,000/ml of one organism reflects significant
bacteriuria
5. Multiple organisms reflect contamination. However, the
presence of any organism in catheterized or suprapubic
tap specimens should be considered significant
51
Examination of Urine Sediment,
Cont’d
Bacteria
• This is an excellent
example of a mixed field
of red blood cells and
bacteria
• "C" is pointing to the
bacteria
• Note how small they are
compared to the RBC
52
Examination of Urine Sediment,
Cont’d
Yeasts
1. Yeast cells may be contaminants or represent a true
yeast infection
2. They are often difficult to distinguish from red cells
and amorphous crystals but are distinguished by their
tendency to bud
3. Most often they are Candida, which may colonize
bladder, urethra, or vagina
53
Examination of Urine Sediment,
Cont’d
Yeasts
• This frame shows an
excellent example of
yeast ("B")
• Note the budding and the
more oval appearance of
each cell
• "A" is a crenated red
blood cell
• "C" is also a red cell
54
Examination of Urine Sediment,
Trichomonas Parasite Cont’d
• Trichomonas vaginalis is
the most common
parasite in urine in USA
and is found in
approximately 25% of
women in casual vaginal
cervical examination.
• The presence of this
organism in urine
specimens of females is
due to the contamination
of the urine with vaginal
secretions
55
Examination of Urine Sediment,
Cont’d
Cytomegalovirus
• CMV is a common virus that
infects people of all ages and
once CMV is in a person’s body,
it stays there for life
• Most infections with CMV are
“silent,” no signs or symptoms
• It is rarely causes serious
consequences except in people
with suppressed or impaired
immune systems
• It is found in saliva, urine, and
other bodily fluids
• Because it is often found in
semen as well as in cervical
secretions, the virus can be
spread by sexual contact
56
Examination of Urine Sediment,
Cont’d
57
Significance of Cellular Casts
Erythrocyte Casts
Leukocyte Casts
Bacterial Casts
Individual Erythrocytes
Individual Leukocytes
Individual Bacteria
58
Renal Tubular Cast Formation
59
Renal Tubular Cast Formation,
Cont’d
60
Examination of Urine Sediment,
Cont’d
Casts, Cont’d
1.
2.
3.
4.
Casts are cylinders of Tamm-Horsfall protein that
solidified in distal tubules
Cellular casts most commonly result when disease
processes such as ischemia, infarction or
nephrotoxicity cause degeneration and necrosis of
tubular epithelial cells
A common scenario is the patient with decreased
renal perfusion and oliguria secondary to severe
dehydration
Ischemic injury results in degeneration and sloughing
of the epithelial cells
61
Examination of Urine Sediment,
Cont’d
Casts, Cont’d
5.
6.
7.
8.
The resulting casts often are prominent in urine
produced following rehydration with fluid therapy
The restoration of urine flow flushes numerous casts
out of the tubules
Leukocytes can also be incorporated into casts in
cases of tubulo-interstitial inflammation (eg.,
pyelonephritis)
Red Cell - Proliferative GN or GBM defect
62
Examination of Urine Sediment,
Cont’d
Casts, Cont’d
9. Urinary casts are formed only in the distal convoluted
tubule (DCT) or the collecting duct (distal nephron)
10. WBC - Intrarenal infection or inflammation
11. The proximal convoluted tubule (PCT) and loop of
Henle are not locations for cast formation
12. Hyaline casts are composed primarily of a mucoprotein
(Tamm-Horsfall protein) secreted by tubule cells
63
Examination of Urine Sediment,
Cont’d
Casts, Cont’d
13. Even with glomerular injury causing increased
glomerular permeability to plasma proteins with
resulting proteinuria, most matrix or "glue" that
cements urinary casts together is Tamm-Horsfall
mucoprotein
14. The Tamm-Horsfall protein secretion (green dots) is illustrated in
the diagram below, forming a hyaline cast in the collecting duct
15. Granular - Many types of renal disease
64
Examination of Urine Sediment,
Cont’d
Casts, Cont’d
16. Although albumin and some globulins are also
incorporated
17. An example of glomerular inflammation with leakage of
RBC's to produce a red blood cell cast is shown in the
diagram below
18. Hyaline - may be normal unless numerous
19. Waxy - Chronic renal disease
65
Factors Favoring Protein Cast
Formation
1.
2.
3.
4.
Low flow rate
High salt concentration
Low pH
All of which favor protein denaturation and
precipitation, particularly that of the Tamm-Horsfall
protein
5. Protein casts with long, thin tails formed at the junction
of Henle's loop and the distal convoluted tubule are
called cylindroids
6. Hyaline casts can be seen even in healthy patients
66
Examination of Urine Sediment,
Cont’d
Casts, Cont’d
Tamm-Horsfall protein
Red blood cell cast
67
Examination of Urine Sediment,
RBCs Cast - Histology Cont’d
• The presence of this red
blood cell cast on urine
microscopic analysis
suggests a glomerular or
renal tubular injury
68
Examination of Urine Sediment,
RBCs Cast - Histology Cont’d
• This histologic section at
medium power with
trichrome stain highlights
red blood cells grouping
together in tubules to
form casts
• The tubular epithelium is
also damaged, with a
foamy appearance, and is
the basis for the
appearance of oval fat
bodies in urine in this
case
69
Examination of Urine Sediment,
RBCs Cast - Histology Cont’d
• 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
70
Examination of Urine Sediment,
Cont’d
WBCs Cast
• 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
71
Examination of Urine Sediment,
Cont’d
Tubular Epith. Cast
• Renal Tubular Cells are
originally cubic in shape; but
once exfoliated, they adopt a
rounded shape
• These cells are slightly larger
than leukocytes (10-14 um)
with lightly granular cytoplasm
• The nucleus is round, well
defined and usually centric.
• The cytoplasm often shows a
perinuclear halo when stained.
Note the "glitter" cell (fatty
degenerated WBC) in the
lower-left corner
72
Examination of Urine Sediment,
Tubular Epith. Cast
Cont’d
• This renal tubular cell
cast suggests injury
to the tubular
epithelium
73
Examination of Urine Sediment,
Cont’d
Granular Cast
• 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
• Casts which persist may
break down, so that the
cells forming it are
degenerated into
granular debris
74
Examination of Urine Sediment,
Cont’d
Granular and waxy Cast
• This slide shows waxy
cast and granular casts
• Granular casts have a
textured appearance
which ranges from fine to
coarse
• Since they usually form
as a stage in the
degeneration of cellular
casts, the interpretation
is similar to that for
cellular casts
75
Examination of Urine Sediment,
Cont’d
Waxy Cast
• They have a smooth
consistency but are more
refractile and easier to
see compared to hyaline
casts
• They commonly have
squared off ends, as if
brittle and easily broken
• They are found especially
in chronic renal diseases
• Diabetic nephropathy
• Malignant hypertension
• Glomerulonephritis
76
Examination of Urine Sediment,
Cont’d
Hyaline Cast
• Hyaline casts, which
appear very pale and
slightly refractile, are
common findings in urine
• Greater numbers of
hyaline casts may be seen
associated with
proteinuria of renal
glomerular disease or
extrarenal (overflow
proteinuria as in
myeloma)
77
Examination of Urine Sediment,
Cont’d
Fatty Cast
• They are identified by the
presence of refractile lipid
droplets
• The background matrix of
the cast may be hyaline
or granular
• Interpretation of the
significance of fatty casts
should be based on the
character of the cast
matrix, rather than on
the lipid content
78
Examination of Urine Sediment,
Cont’d
Summary of Urine Casts
79
Examination of Urine Sediment,
Cont’d
Summary of Urine Casts
80
Examination of Urine Sediment,
Cont’d
Crystals
1. Urate
•
•
Ammonium biurate
Uric acid
2. Triple Phosphate
3. Calcium Oxalate
4. Amino Acids
•
•
•
Leucine
Cystine
Tyrosine
5. Sulfonamide
81
Examination of Urine Sediment,
Cont’d
Dihydrated Calcium Oxalate Crystals
• These are oxalate
crystals
• They look like little
envelopes (or
tetrahedrons, depending
upon your point of view)
• Oxalate crystals are
common
82
Examination of Urine Sediment,
Cont’d
Monohydrated Calcium Oxalate Crystals
• Rarely found, vary in size
and may have a spindle,
oval, or dumbbell shape
• Appear as flat, elongated,
six-sided crystals ("fence
pickets")
• The arrow indicates
"daughter" crystal
forming on the face of a
larger underlying crystal
83
Examination of Urine Sediment,
Cont’d
Triple Phosphate Crystals
• Usually appear as colorless,
prism-like "coffin lids"
• Often seen in urine from
normal individuals.
• Can be found in urine of
any pH, their formation is
favored in neutral to
alkaline urine
• Urinary tract infection
with urease producing
bacteria can promote it
(and urolithiasis) by
raising urine pH and
increasing free ammonia
84
Examination of Urine Sediment,
Cont’d
Urate Crystals
• Uric acid crystals may
appear as yellow to
brown rhombic or
hexagonal plates, needles
or rosettes
• With rare exceptions, the
finding of uric acid
crystals in urine is of little
clinical value
85
Examination of Urine Sediment,
Cont’d
Leucine Crystals
• Leucine crystals are seen
as yellow spheres with
concentric and radial
strias
• These crystals can
sometimes be mistaken
for cells, with the center
resembling a nucleus
86
Examination of Urine Sediment,
Cont’d
Cystine Crystals
• These cystine crystals
are shaped like stop
signs
• Cystine crystals are seen
as flat colorless
hexagonal plates
• They often aggregate in
layers, and their
formation is favored in
acidic urine
87
Examination of Urine Sediment,
Cont’d
Ammonium Biurate Crystals
• These crystals generally
appear as yellow-brown,
radially-striated spheres
with irregular "thornapple" or "ox-horn"
projections
• They may be seen in acid
urine, their formation is
favored in neutral to
alkaline urine
88
Examination of Urine Sediment,
Cont’d
Cholesterol Crystals
• The cholesterol crystals
appear to be needleshaped
• They are actually
rectangles in crosssection
• The surrounding tissue is
all necrotic (dead) debris
89
Examination of Urine Sediment,
Cont’d
Sulfonamide Crystals
• Sulfonamide crystals are
typically yellow in color and
often resemble uric acid
crystals. However, sulfa
crystals are easily
distinguished from uric acid by
confirmatory tests
• Sulfa crystals are readily
soluble in acetone and exhibit
a positive dextrine/sulfuric acid
test ("old yellow newspaper"
test)
90
Examination of Urine Sediment,
Cont’d
91
Examination of Urine Sediment,
Cont’d
92
THE END
Any questions?
93