Microscopic examination of Urine sediments.ppt

CHAPTER FIVE
Microscopic Examination Of Urine
1
12/22/2022 By Sintayehu Ambachew Wondemagegn

Chapter Objective
At the end of this chapter the students will be able to:
 Describe microscopic examination for urine sediment
 Describe normal and abnormal urine sediments with their
diagnostic features
 Describe formation and significance of casts
 Normal and abnormal crystals encounter in urine
sediments
 Describe relationship between sediments, chemical,
physical findings in urine
 Describe reporting of urinary sediments
 Describe quality control in urinalysis
2
12/22/2022 By Sintayehu Ambachew

Chapter Outline
 Microscopic Examination of Urine
• Materials & Procedure for microscopic examination
• Urinary Sediments
 Organized Urinary Sediments
 Non-Organized Urinary Sediments
• Methods of reporting formed elements
• Source of errors in the microscopic examination of
urine
• Quality control in urinalysis
3

Microscope
4

Introduction
• Microscopic examination of the urinary sediment
– Most time-consuming part of the routine urinalysis
– To detect and identify insoluble materials present in the
urine
These include RBCs, WBCs, epithelial cells, casts,
bacteria, yeast, parasites, mucus, spermatozoa, crystals,
and artifacts
– Some of these components are of no clinical significance
and others are considered normal unless they are
present in increased amounts
– Both identification and quantitation of the elements must
present
5

CONT’….
Valuable information can be obtained from
microscopic examination of urine specimen
 If the urine sample is properly collected
 If the test is carefully performed, and
 If the person performing the test is
knowledgeable, skillful
6

Standardization
• Aspects of microscopic examination that should be
standardized:
1. Volume of urine analyzed
2. Length and force of centrifugal
3. Re-suspending volume and concentration of
sediment
4. Volume and amount of sediment examined
5. Terminology and reporting format
7

Principles of method
• Centrifuge 10-12 ml urine specimen at 1500 – 2000 rpm
for 3-5 minutes
– Discard the supernatant by quick inversion of the tube,
suspend
– Take the sediment by Pasteur pipette from the tube
– Transfer a drop into the clean and dry slide
– Finally examine under the microscope
• Repeated evaluation of urine sediment is frequently
valuable in following the course and management of
urinary tract disorders
• The appearance of cellular elements, and casts in the
urine is a reflection of changes that take place in the
kidney 8

Reagents and equipment
• Assemble all necessary materials used for the collection,
centrifugation and examination
– Specimen collecting cup
– Centrifuge
– Conical centrifuge tubes
– Pasture pipette with rubber fit or automatic pipettes
if possible
– Slides and cover slides 22 x 22 mm.
– Microscope
– Some staining reagents if needed (10% KOH and
crystal violet, safranin stain, etc)
9

Cont’…
10

Specimen requirement
• Preparation of patient
– Instruct the patient how to collect the specimen
– Explain the purpose of the test by using simple language
Do not use medical terms or try to explain details of the
procedure
– If the patient is female, advice her to wash her genital organ
before giving the specimen
This is because bacteria that are normally found on the genital
tract may contaminate the sample and affect the result
• The first morning urine or mid-stream urine specimen is
more preferable, because it is more concentrated and
formed elements are less likely disintegrated
11

Cont’ .…
• The collected urine sample should arrive at a diagnostic
laboratory as soon as possible
• If the urine sample is delayed by more than 2 hours, without
preservation, urine sediment appearance and constituent may
be changed and false results may be obtained and reported
• If it is difficult to deliver within 2 hrs, it is better to preserve
specimen in the refrigerator at the temperature between 2-60C
or use chemical preservatives
What are the changes that occur in old urine specimen?
12

13

Source of Errors
• Drying of the specimen on the slide
• If the supernatant fluid after centrifugation is not poured
off properly
• If the whole sediment with supernatant is discarded
during inverting down the tube for long period, the
whole sediments will be discarded and so again false
negative result will be reported
– Thus another sample should be collected and the test
should be repeated
14

Classification of Urinary Sediments
• Urine sediments can grossly be categorized into 2
Based on the substances they are composed
1. Organized
2. Non-organized sediments
15

Organized (Formed ) elements
RBCs/HPF
WBCs/HPF
Epithelial cells / LPF
Casts / LPF
Parasites/LPF
 T. vaginalis/HPF
Bacteria / HPF
Yeast Cells /HPF
Spermatozoa
Miscellaneous substances 16

Non-organized (Non-living Material)
• Slightly acidic urine
• Acidic, Neutral, or slightly alkaline Urine crystal
• Alkaline, Neutral, or Slightly acidic urine
– Triple phosphates
– Amorphous Urates
– Calcium carbonate
– Calcium phosphate
– Calcium Oxalate crystals
17

Organized Urinary Sediments: RBCs
• RBCs not usually present in normal urine
• Appearance:
– RBCs:- Smooth, non-nucleated, biconcave disks
– Measure 7-8 m
– Normally RBCs appear in the fresh sample as intact, small and
faint yellowish discs, darker at the edges
• In concentrated urine may be crenated and became small (5-6
m)
• In diluted urine, RBCs may be turbid and increase in size (9-10
m)
• In alkaline urine, they may be small or entirely destroyed forming
massive of brownish granules
• In diluted and alkaline urine, the red cell will rupture and release
the hemoglobin, leaving faint colorless cell membrane, and are
known as “ghost” cells (shadow of original cells) 18

19
• This field contains mostly
RBC’s, (hpf)
• Notice many of them show
biconcavity
• Some show variability in
shape
• Some times it is easy to
mistake fungi for RBC’s
• To get rid of RBC’s so that
WBC’s are more visible –
acetic acid is very helpful,
Why?

Clinical significance of RBCs
When the number of RBCs is found more than their normal range,
usually greater than 5 RBCs/HPF it may indicate:
• Presence of disease conditions in the urinary tract, such as:
– Acute and chronic glomerulonephritis
– Tumor that erode any part of the urinary tract
– Renal stone
– Cystitis (Inflammation of bladder)
– Prostitis
– Trauma of the kidney
– Traumatic catheterization
20

Substances confusing with RBCs
 Yeast cells, leukocytes, and bubbles may confuse with RBCs
Differentiate by:-
 Yeast cells:
– Smaller and are oval in shape flattened
– Vary considerably in size in one specimen
– Have budding at the surface
– Upon addition of 2-5% acetic acid the RBCs will disappear
 Bubbles (oil droplets)
– Vary considerably in size,
– Are extremely refractive or shiny
 Leukocytes
– Larger and have granular appearance
– Upon addition of 2-5% acetic acid the RBCs will disappear
21

22
• These can easily be mistaken
for RBC’s
• They are budding yeast,
notice the almost cactus like
appearance of those in the box
• They will not rupture in
acetic acid, RBC’s will
• These may truly be from the
bladder or they may be a
contamination
Yeast cells
cactus

Interfering factors
• Factors that may result falsely in high number of RBCs,
i.e without the presence of actual renal or other normal
physiological disturbances included:
– Menstrual bleeding
– Vaginal bleeding
– Trauma to perianal area in female patients
– Following traumatic catheterization
• Some drugs:
 Aspirin ingestion or over dose
 Anticoagulant therapy over dose
23

24
and Leukocytes

LEUKOCYTES (WBCs)
• Normal range: 0-4 WBC/HPF
• Appearance: normally, clear granular disc shaped
– Measure 10-15 m, the nuclei may be visible
• In alkaline urine, they may increase their size and become
irregular
• Predominantly, polymorph nuclear neutrophils are seen
• Sometimes because of predominance of neutrophils and the
occurrence of bacterial cell together with polymorphonuclear
cells, WBCs are called pus cells
• WBCs (pus cells) may be seen in clumps
25

Cont’…..
• Neutrophils exposed to hypotonic urine absorb
water and swell
26

Microscopic Exam
• White blood cells
 A few are normal
 High numbers indicate
inflammation or infection
somewhere along the
urinary or genital tract
27
40x objective

Clinical significance of WBC
• Increased number of urine leukocyte are seen in case of:
– Urinary tract infection such as renal tuberculosis
– All renal disease
– Bladder tumor
– Cystitis
– Prostatis
• Temporarily increased number of leukocytes are also
seen during:
– Fever
– After strenuous exercise
28

How to report the result of WBCs
• After observing the distribution of leukocytes under 40x
objective, at least 10 fields of microscope
• When 0-5 leukocytes / HPF are seen-- normal
• 5-10 WBCs / HPF are seen-- few leukocytes / HPF
• 10-20 WBCs /HPF are seen--->moderate WBCs/ HPF
• 20-30 WBCs /HPF are seen ----> many WBCs / HPF
• Above 30 WBCs / HPF are seen  full of WBCs /HPF
29

30

Epithelial cells
• It is not unusual to find epithelial cells in the urine
• Unless they are present in large numbers or in
abnormal forms, they represent normal sloughing
of old cells
• Three types of epithelial cells are seen in urine:
1. Squamous
2. Transitional (urothelial), and
3. Renal tubular
31

EPITHELIAL CELLS
Those coming from renal cells: RTE cells
– Size is small as compared to other epithelial cells
– It measures 10 to 18 m in length, i.e., slightly larger than
leukocytes
– Very granular
– Have refractive and clearly visible nucleus
– Usually seen in association with proteins or casts
32

EPITHELIAL CELLS …
Cells from renal pelvis and urethra of the kidney
• Size is larger than renal epithelia’s
• Those from pelvis area are granular with sort of tail,
while those from urethra are oval in shape
• Most of the time urethral epithelia is seen with
leukocytes
33

EPITHELIAL CELLS…
Bladder cells
– Are squamous epithelial cells
– Very large in size
– Shape seems rectangular and often with irregular
border
– Have single nucleus
34

35
• These are 2 Squamous
Epithelial cells shown on
high power (hp)
• They are usually large,
flat, colorless cells
• However, there can be
some granularity to the
cytoplasm & the edges
may be rolled
• The nucleus is usually
distinct & centered

Microscopic Exam
• Epithelial cells are large
and flat
• Normal cells that line
the urinary and genital
tract or renal tubules
36
40x objective

37
• The details of these Transitional
Epithelial cells (3) are somewhat
obscured by the large number of
bacteria present
• They are usually round with a
large round nucleus
• Note: They are smaller than the
Squamous Epithelial cell

Clinical significance
• Presence of epithelial cells in large number, mostly
renal types may indicate:
– Acute tubular damage
– Acute glomerulonephritis
– Silicate over dose
Note:- The presence of large number of epithelial cells
with large number of Leukocytes may indicate urinary
tract infections (UTI)
38

Reporting of epithelial cells
• Epithelial cells distribution reported after looking under
10x objective of the microscope.
• Usually they are reported semi quantitatively by saying
– 1-3 epithelial cells /LPF
– 3-5 epithelial / LPF
– 6-14 epithelial / LPF
– 15-25 epithelial/ LPF
– Full of epithelial cells / LPF when the whole field of
10 x objective covered by epithelial cells
39

40

Casts
• The only elements unique to the kidney
• Long cylindrical structures
• Result from the solidification of material within the lumen
of the kidney tubules
• Formed by precipitation of proteins, and aggregation of
cells within the renal tubules
• Most urinary casts are formed either in the distal
convoluted tubules or in the collecting ducts
• But rare conditions, casts may be found in the proximal
convoluted tubules
41

Cont’….
• Casts formed in the collecting tubules tends to be:
– Very broad
– Usually indicates the significant reduction in the
functional capacity of the nephron and indicate
severe renal damage
• Most of casts dissociate in alkaline urine, and diluted
urine (specific gravity  1.010) even in the presence of
proteinuria
• Most of them are transparent
• Detected using lower power magnification
42

• Condition that favor formation of casts are:
 Proteinuria：albumin, mucoprotein (Tamm-
Horsfall protein) (THP)
 High salt concentration
 Low PH
 Low flow rate
Cont’…
43

Cast Composition and Formation
1. Aggregation of Tamm-Horsfall protein into individual protein
fibrils attached to the RTE cells
2. Interweaving of protein fibrils to form a loose fibrillar
network
3. Further protein fibril interweaving to form a solid structure
4. Possible attachment of urinary constituents to the solid
matrix
5. Detachment of protein fibrils from the epithelial cells
6. Excretion of the cast
• Any elements present in the tubular filtrate, including cells,
bacteria, granules, pigments, and crystals, may become
embedded in or attached to the cast matrix. The types of
casts found in the sediment represent different clinical
conditions 44

Cont’...…
• Major casts types:
– Hyaline casts
– Epithelial casts
– White blood cell, and Red blood cell casts
– Granular (coarse and fine)
– Waxes casts
– Fatty casts 45

Cont’……
• Casts in urinary sediment is an important aid in the
differential diagnosis of renal disease
• Pure Hyaline casts may be seen in proteinuria from a
variety of causes
• Small Hyaline cast seen transiently may occur with marked
exercise or febrile conditions
46

Hyaline Casts
• All hyaline cast have a precipitated protein matrix, so
there has to be renal proteinuria for these to be formed
• The proteinuria is predominately Tamm-Horsfall
mucoprotein
• Low pH & increased electrolyte concentration readily
precipitate Tamm-Horsfall protein
47

Hyaline Casts
48
Hyaline cast in urine as seen with the 40 objective.

Hyaline Casts
• Changes in PH and osmolality
• Arising from normal renal tubular secretion of mucoproteins
• Basic matrix of all casts
• Mild renal disease
• The most frequent casts and clear cylinders
• Difficult to visualize by bright-field microscopy
49

Hyaline Casts
50

Clinical Implication
• Presence of large number of hyaline casts may show
possible damage of glomerular capillary membrane
• This damage permits leakage of protein through
glomerulus and result in precipitate and gel formation
(i.e. hyaline casts) in the tubule
• Thus this may indicate:
– Nephritis
– Meningitis
– Chronic renal disease
– Congenital heart failure
– Diabetic nephropathy
51

Granular cast
• More similar in appearance with hyaline casts and in
which homogenous, course granules are seen
• More dense (opaque) than hyaline cast
• Shorter and broader than hyaline casts
• May represent the first stage of epithelial cell cast
degeneration
• Based on the amount and type of granules, divided into:
 Fine (which may appear grey or pale yellow in color)
 Course granular casts ( which may appear as darker)
52

Granular Casts
• If cellular casts stay within the tubules of the nephron due to
prolonged stasis, the cells will begin to degenerate
• These casts are referred to as Finely Granular or Coarsely
Granular Casts
• Usually the original type of cell entrapped cannot be
determined unless the cells were RBC & hgb remains
53

54

• Granular cast under
bright light microscopy
• If this were on your
scope you would want
to reduce the amount
of light by closing the
substage condenser,
this really shows the
importance of your
lighting!
55

• Mixed Cellular
Granular Cast,
high power
• Notice that the
cells are
degenerating
• This would tend
to be a Course
Granular
56

• Notice the
coarse
granularity is
very noticeable
57

• Coarse
Granular Cast
next to 2
Epithelial
Cells, high
power
58

• Granular casts may be seen in:
 Acute tubular necrosis
 Advanced glomerulonephritis
 Pyelonephrites
 Malignant nephrosis
 Chronic lead poisoning
 In healthy individuals these casts may be seen after
strenuous exercise
59

Cellular & Other Cast
• As the protein concentrates in the distal tubule & becomes
stickier, cells can become trapped
• These become Hyaline Casts with Inclusions & while the formal
name would be for example Hyaline-WBC Cast, they are
frequently simply referred to as WBC Cast
60

White blood cell casts
• Formed by aggregates of white blood cells that trapped
in protein matrix in the renal tubular lumen
• An excess of white blood cells, singly or in clamps, in the
urine may indicate inflammation
• White blood cell casts definitely are renal origin
• They characteristically seen in acute pyelonephritis and
occasionally in glomerulonephritis
61

White blood cell casts
• Upper urinary tract Infection
• Inflammation of the kidney
• Pyelonephritis
• Interstitial nephritis
62

63

• WBC cast,
high power
field
• Some of the
nuclear lobes
can be seen
64

Red blood cell casts
• Normal range:- normally not seen in normal individual
• Usually, they are found in hematuria
• Appear brown to almost colorless
• Usually diagnostic of glomerular diseases
• Formed usually after accumulation of cellular element
in the renal tubules
65

Red blood cell casts…
• Indicate glomerulonephritis
• Primary glomerular disease with RBC’s passing the damaged
glomeruli in large quantities
• Lupus nephritis
• Rapidly progressive glomerulonephritis
66

• RBC cast, high
power
• Notice the cell
membranes are
clearly visible, but
there does not
appear to be a
nucleus
• Notice how difficult
the mucous
threads are to see,
this might be
improved by
reducing the light a
bit
67

68

Epithelial Casts
• Epithelial Casts are composed largely of tubular
epithelial cell desquamated within the tubule
• They often appear as two rows if cells in protein cast
matrix
• Inflammation of the kidney may cause greater sloughing
of renal epithelial cells, so large number of epithelial
casts is indicative of renal parenchymal disease with
tubular damage
69

70

• Renal Tubular Cast, high
power
• Can be difficult to
differentiate from other
cellular casts & at times
the decision must be
made on other cells in
the sample
• Notice in this cast the
cells have large nuclei
71

Waxy Casts (Renal Failure Casts)
Not seen in normal individuals
Appearance
• Shorter and broader than hyaline casts
• Composed of homogeneous, yellowish materials
• Broad waxy casts
 Are from two to six times the width of ordinary waxy casts
 Appear waxy and granular
 Have high refractive index
 May occur from cells (WBC, RBC, or Epithelial) casts, hyaline
casts
72

• This is a Waxy
Cast, on high
power
• Notice the
crack in the
side of the cast,
which is
frequently seen
in Waxy Casts
73

74

Waxy Casts
• Waxy casts are found in:
 Chronic renal disease
 Tubular inflammation and degeneration
 Localized nephron obstruction
 Malignant hypertension, in diabetic diseases (nephropathy)
• The presence of waxy casts indicates severity of renal
disease
75

Fatty Casts
• Normally not seen in health individuals
Appearance:
• These are casts, which contain fat droplets inside them
• Fat droplets are formed after accumulation of fat in the
tubular vessels, especially tubular epithelial and finally
disintegrated
• Clinical Implication:
• The occurrence of fat droplets, oval fat bodies, or fat casts
is:
 Very important sign of nephritic syndrome
 Chronic renal disease
 Inflammation and degeneration of renal tubules
 lupus and toxic renal poisoning
76

• This is a cast
containing ‘fat’
bodies, high
power
• On wet mount
the droplets are
highly refractile
[they bounce the
light back]
77

Reporting of casts
• Casts are examined under 10x objective of the
Microscope
• Casts are reported quantitatively by saying:
– Few casts / LPF
– Moderate casts / LPF and
– Many casts / LPF
• During the report the, type of cast that is seen should also
be mentioned
• Example: few hyaline casts / LPF are seen
78

PARASITES
• Parasites that can be seen in urine microscopy are:
 Trichomonas vaginalis
 Schistosoma haematobium
 Wuchereria bancroftie
• Other parasites such as Entrobious vermicularies also
may occur due to contamination of the urine with stool.
79

80
• Here is another frame of the
Trichomonas (hp), both of
these are shown with phase
microscopy which enhances
the details of cells with low
refractive indices

81
Yeast cells and Trichomonas
vaginalis in urine sediment as
seen with the 40 objective.

82
• Trichomonas (hp) is a small
parasite that is very active in
a fresh specimen
• They have multiple flagella
(white pointers) as well as an
undulating membrane which
contribute to movement
• In the absence of movement
they can be mistaken for
other cells

83
Schistosoma haematobium and Enterobius vermicularis
Egg

YEAST CELL
• Yeast cells are fungi that are not normally seen in health
individuals.
• Appearance
– Variable in size
– Colorless.
– Oval in shape, and usually form budding.
– Have high refractive index.
– Usually confused with Red Blood Cells.
84

85
Yeast cells and pseudo hyphae of Candida
albicans in urine sediment as seen with the
40x objective.

86
• These can easily be mistaken for
RBC’s
• They are budding yeast, notice the
almost cactus like appearance of
those in the box
• They will not rupture in acetic acid,
RBC’s will
• These may truly be from the bladder
or they may be a contamination

87
• These are branching
pseudohyphae of a
fungus
• Their main significance is
that they obscure more
important features of the
specimen & may indicate
that the specimen is not
a clean catch

Clinical Significance
• They are usually of candida species (candida
albicans) and are common in patients with
 Urinary tract infection
 Vaginitis
 Diabetic mellitus
 Intensive antibiotic
 immunosuppressive therapy
88

BACTERIA
• Bacteria are commonly found in urine specimen
because of abundant normal microbial flora of the vagina
or external urinary meatus
• Most common cause of UTI dipstick test can give
indirect clue
• Further the observed bacterial cell can be identified by
bacteriological culture
89

BACTERIA …
• Usually only a single type of organism is present in
uncomplicated acute urinary infections.
• More than one type of organism is often seen in chronic
and recurring infections.
• Vaginal contamination of the specimen is indicated by a
mixed bacterial flora (including Gram positive rods) and
often the presence of epithelial cells.
90

Clinical Significance
• Presence of bacteria may indicate the presence of UTI
or contamination by genital or intestinal microflora.
• Report of the Result
– Few bacteria / HPF
– Moderate bacteria / HPF
– Many bacteria / HPF
– Full of bacteria / HPF
91

Bacteria…
• Neisseria gonorrhoeae in urine
– In male patients with acute urethritis, it is often
possible to make a presumptive diagnosis of
gonorrhoea by finding Gram negative intracellular
diplococci in pus cells passed in urine
92

93
Crystals in Sediment

94
Crystals in Sediment
Microscopic solids usually composed of a small
number of different ions/molecules
Formed by precipitation of urine solutes including
Inorganic salts
Organic compounds, and
Medications (iatrogenic compounds)
Small amount of most type of crystals are not
necessarily pathologic
• High specific gravity specimens crystals

95
Cont’……
• Formation of crystals is most dependent up on:
 Concentration of ions and molecules
 Urine PH
 Decreased flow of urine through tubules
Solutes precipitate more readily at low temperatures

Urinary crystals identification
• Liver disease
• Inborn errors of metabolism
• Renal damage caused by crystallization of iatrogenic
compounds within the tubules
Crystals are usually reported as rare, few, moderate,
or many per HPF
Abnormal crystals may be averaged and reported per
LPF
96

General Identification Techniques
Characteristic shapes and colors
Urine PH
Polarized microscopy
Solubility characteristics of the crystals
(temperature, chemical)
97

98
Crystals solubility in urine PH
Solubility of solutes differ at different PH
• Inorganic salts: oxalate, phosphate, calcium,
ammonium & magnesium less soluble in neutral or
alkaline urine
• Organic solutes: uric acid, bilirubin, & cystine less
soluble in acidic urine
• N.B:- Most clinically significant crystal are found in acid
urine
– Include: cystine, tyrosine, leucine & iatrogenic
crytsals: sulfonamide & ampicillin

Crystals Seen in Acidic Urine
• The most common crystals seen in acidic urine are
urates:
Amorphous urates
Uric acid
Acid urates and
Sodium urates
• Calcium oxalate crystals are frequently seen in acidic
urine, but they can be found in neutral urine and
even rarely in alkaline urine
99

100
Amorphous Urates
• Amorphous urates
– Non crystalline urate salts of sodium, potassium,
magnesium & calcium
– Small & yellow-brown granules - similar to sand
– Enhanced by refrigeration
– Can be in acidic or neutral urine (pH greater than 5.5)

101
Amorphous Urates
– Will dissolve in alkaline or heated to 600C
– If acetic acid added, uric acid crystals will precipitate
out
– Uroerythrin deposits on urate crystals giving pink-
organish color - referred to as “brick dust”

Amorphous urates
102

103
Uric Acid Crystals
• Seen in a variety of shapes, including rhombic,
four-sided flat plates (whetstones), wedges, and
rosettes
• Most common form is diamond shape but may be
cube shaped or cluster in rosettes
• Appear yellow-brown
• Urine pH usually around 5.0 to 5.5

104
Uric Acid Crystals
• Diamond shape may cluster in rosettes
• Sometimes 6 sided & must be differentiated from
clinically significant cysteine
• Highly birefringent under polarized light, which aids in
distinguishing them from cystine crystals

105
Uric Acid Crystals and Pathology
• Usually yellow to orange-brown
• Are birefringent under polarizing
light
• Can appear normally BUT
– Seen in large #s in gout &
increased purine metabolism
such as cytotoxic drugs
• E.g. patients with Leukemia who
are receiving chemotherapy

106
Acid Urine: Calcium Oxalate Crystals
1. Dihydrate form :- colorless, octahedral envelope or
as two pyramids joined at their bases
2. Monohydrate form:- oval or dumbbell shaped
 Rare & can mistake for RBC’s
• Both the dihydrate and monohydrate forms are
birefringent under polarized light

Acid Urine: Calcium Oxalate Crystals
• Normally associated with foods high in oxalic acid, such as
tomatoes and asparagus, and ascorbic acid
• The most common form of calcium oxalate crystals is the
dehydrate forms
• Clumps of calcium oxalate crystals in fresh urine may be
related to the formation of renal calculi
• Monohydrate crystals are pathologically present in ethylene
glycol poisoning & severe chronic renal disease
107

108

Crystals Seen in Alkaline Urine
• Phosphates represent the majority of the crystals
seen in alkaline urine
– Amorphous phosphate
– Triple phosphate and
– Calcium phosphate
• Other normal crystals associated with alkaline
urine are calcium carbonate and ammonium
biurate
109

Amorphous phosphates
• Alkaline or neutral urine
• Microscopically not distinguishable from amorphous
urates
– Distinguishable on urine PH & solubility
– Precipitate white rather than pink-orange of
amorphous urates
– Are soluble in acid & will not dissolve when heated
to 60C
• Fine colorless grains with tendency to obscure other
more significant sediment
• Presence enhanced by refrigeration
110

Amorphous phosphates
111

Triple phosphate
(Ammonium Magnesium Phosphate)
112
• Colorless & in different forms
– most common are 3 & 6 sided
‘coffin lids’
– vary greatly in size
– may also see a ‘fern leaf’ form,
feathery
• See in normal healthy individuals but
are often present in formation of
calculi & are associated with UTI
(urea-splitting bacteria)

Triple phosphate
Triple Phosphate Crystals
prism
12/22/2022 113
By Sintayehu Ambachew

114
Calcium Phosphate
• In 2 forms dicalcium & calcium
• Dicalcium colorless thin prisms in rosettes or star-
shaped ‘stellar phosphates’
– Tend to have tapered or pointed end & the other
squared off
– Calcium phosphates are irregular granular sheets or
plates often resemble degenerating squamous
epithelial cells

Calcium phosphate
12/22/2022 115

116
Calcium Carbonate
• Very small granular
crystals
• colorless, Usually
found in pairs
‘dumbbell shape’, or
spherical shapes
• Birefringent with
polarizing light
• Form gas after the
addition of acetic acid

117
Ammonium Biurate
• Yellow brown spheres with
striations
• Can have irregular spicules
‘thorny apple’
• In alkaline or neutral urine
• Not significant unless seen in
fresh urine
• Usually seen in old specimens
• Dissolve in acetic acid or
heating to 600C
12/22/2022 thorny apple

Abnormal Urine Crystals
• Abnormal urine crystals are found in acidic urine or
rarely in neutral urine
Bilirubin
Tyrosine
Leucine
Cystine
Cholesterol
119

120
Bilirubin
• Appear as fine needles, granules, or plates
– Urine is acidic
– Always yellow-brown
– The bile stains the other components of the
sediment
– Presence of the crystals indicate high concentrations
of bilirubin in the urine

121
Bilirubin Crystals: Abnormal State
• If you suspect bilirubin crystals are present, the strip reaction
must confirm the presence of bilirubin
– Otherwise the identification is incorrect
• The presence of the positive bilirubin strip &/or the crystals
indicate a pathologic process - are always considered an
abnormal crystal
• May be seen in liver disease

122
Amino Acid Crystals
• Tyrosine
– fine, delicate needles,
colorless or yellow
– frequently in clusters or
sheaves [as in stacks of
wheat]
– seen singly or in small
groups
– in acidic urine
– less soluble than leucine, so
found more often

123
Leucine
• Highly refractile yellow to brown
spheres in acid urine.
• Have concentric/radial striations
on their surface
• Can be mistaken for fat globules
[or vice versa]
• But will not stain with fat stains
or appear as maltese cross under
polarization
• Can be seen in urine containing
tyrosine crystals if use alcohol to
‘precipitate’ Bactrim has similar appearance
check patient history

124
Amino Acid Crystals and Pathology
• Amino acid crystals are abnormal & seen in
overflow amino aciduria
– Can be seen in rare cases of liver disease, more
likely to reflect inherited metabolic disorder
– Before reporting should be confirmed by
confirmatory tests such as chromatography

125
Cystine: Always Abnormal
• Colorless hexagonal plates
– sides may be uneven
• Crystals appear layered
– tend to clump
– primarily seen in acidic urine
– Must be counted
• Can be confused with uric acid
crystals, must confirm
identification with sodium
cyanide

126
Cystine: Always Abnormal
• Clinically significant, seen in congenital cystinosis or
cystinuria
– Deposit out in tubules as calculi/stone causing damage

127
Cholesterol • Clear flat rectangular plates with
notched corners
– in acidic urine
– are soluble in chloroform & in
ether
• Rarely seen
• Presence indicates both ideal
conditions for precipitation &
supersaturating:
• Always seen with positive protein
+ fat droplets, fatty casts or oval
fat bodies
• Seen in nephrotic syndrome &
other renal damage

128
Confounding Conditions
• Radiopaque contrast medium [diatrizoate meglumine ] can be
mistaken for cholesterol
– Contrast medium will give abnormally high S.G. >1.040
– Not associated with proteinuria or lipiduria
– Cholesterol crystals found with normal S.G.
• Medications
– Can be excreted in high concentrations, resulting in precipitation
– These crystals are termed ‘iatrogenic’
– Proper identification of drug crystals important in alerting to
potential renal tubular damage

129
Ampicillin
• Appear as long thin colorless
prisms or needles
• May aggregate in small clusters or
if refrigerated may form large
clusters
• Appear in acidic urine
• Require large dosage for formation,
so rarely seen

130
Sulfonamides
• Highly refractile & birefringent
• In acidic urine,
• Should be confirmed before reporting
• Closely resemble ammonium biurate but
differentiated on
– pH & solubility
– chemical confirmatory test
• Type varies with form of drug prescribed
• Sulfa drugs have been modified to be
more soluble & so crystals rarely seen

MISCELLANEOUS
Spermatozoa
• Are small structures consisting of a head and tail,
connected by a short middle piece (neck)
• Easily recognized especially if they are motile
• Frequently seen in the urine of males
• They may see in the urine of females, when the urine
collected after coitus usually not reported, unless the
physician has special interest in it
131

132
Spermatozoa and occasional pus cell in urine sediment as seen
with the 40 objective

133
• There are 2 sperm in
this frame
• In a fresh specimen,
they are visible due to
their movement
• In an older urine
specimen, they may be
difficult to visualize

Mucus threads
• Formed by the precipitation of mucoprotein in cooled urine
• Have fine, fiber like appearance
• Wavy in shape and tapered at ends
• If not examined carefully may confuse with hyaline casts
134

Contaminates and Artifact Structure
• Muscle fibers
• Vegetable cells
• Structure from slide or cover slide
• Fat droplets (other bubbles)
• Oil droplets
• Pollen greens
• Starch granules
135

Methods for Examining Urine Sediments
• Unstained Urine Sediment Preparation
– Bright field microscopy
– Phase Contrasts (PC) microscopy
• Stained Preparation
136

1. A crystal violet safranin stain (sternheimer and
malbin)
• Useful in the identification of cellular elements
• Staining reaction to crystal – violet safranin stain:
RBC – Purple to dark purple
WBC – Cytoplasm -violet to blue
Nucleus – reddish purple
Glitter cells – blue
137
Stained preparation

Cont’…..
2. Toluidine blue (0.5%)
A metachromatic stain
Provides enhancement of nuclear detail
Differentiate WBCs and RTE cells
3. Lipid Stains
Oil Red O and Sudan III
Triglycerides and neutral fats stain orange-
red, whereas cholesterol does not stain but
is capable of polarization
138

Cont’….
4. Gram Stain
Differentiate between gram-positive (blue) and
gram negative (red) bacteria
A dried, heat-fixed preparation of the urine
sediment must be used
5. Hansel Stain
Consists of methylene blue and eosin Y
Preferred stain for urinary eosinophils
However, Wright’s stain can also be used
139

Cont’….
6. Prussian Blue Stain
Used and stains the hemosiderin granules a blue
color
7. CytoDiachrome stains
Papanicolaou stain
Detection of malignancies of the lower urinary
tract
140

141

Automations in Urinalysis
• Automations are utilized in urinalysis laboratories
• These machines can be applied for physical, chemical, and
microscopical analysis of urine
• Reflectance photometry
UF-100 Automated Urine Cell
Analyzer
142

Cont’…….
Advantages of automations:
 The readings are more reproducible and unbiased
 Help to analyze a great number of specimen in less
time
 Help to develop standards about the sediments
 Give better interpretation about the sediments in
close agreement between laboratories
143

Quality assurance in urinalysis
Quality lab results require
following procedures in
many areas.
 Pre-analytic
 Analytic
 Post-analytic
Quality
 Requirements
 Standards
12/22/2022 144

Definition of Terms
Quality Assurance (QA) – steps taken to assure reliable
laboratory results
Quality control (QC) – procedure, samples and rules to
determine if analysis is acceptable
Pre-analytic: steps before testing the analyte
Analytic: steps of testing the analyte
Post-analytic: steps after testing is complete
12/22/2022
145

Quality Assurance?
Assuring quality laboratory service requires Monitoring
and Tracking all aspects:
Pre-analytical
Analytical
Post-analytical
Internal quality control and External quality
assessment
12/22/2022 146

Key Components of QA
Internal quality assessment (IQA)
External quality assessment (EQA)
Standardization of processes and procedures (pre-
analytic, analytic and post-analytic phases)
Management and organization
12/22/2022 147

1. Internal Quality Assessment
 Monitoring lab procedures
 Track lab processes
Instrument calibration
Equipment maintenance
 Tracking patient test results
Tracking the reports and archiving
Senior technologist
12/22/2022 148

2. External Quality Assessment
 External agency for
1. Proficiency testing
2. On-site evaluation
3. Retesting (rechecking
samples)
12/22/2022 149
On-site evaluation

Quality Assurance
3. Standardization of Processes and
Procedures
 Reproducible lab results
 Uniform activities
Standard operating procedures
(SOP)
• Pre-analytical
• Analytical
• Post-analytical
12/22/2022 150

151
Summary
• You should be able to describe:
– Appearance and clinical significance of RBC and WBC.
– Appearance and clinical significance of three types of
epithelial cells.
– Formation, composition and clinical significance of the
different types of urinary casts. types of crystals,
identify them and state clinical significance of each.
– Other formed elements to include: bacteria, fat, fibers,
mucous, parasites, sperm, starch, trichomonas and
yeast.
– Types of quality assurance in urinalysis

Exercise
1. State two technical errors in sediment preparation that
could produce decreased sediment constituents ?
2. The finding of yeast cells in the urine is commonly
associated with____
3. Why do casts vary in size and composition? What is the
primary constituents that all casts have in common?
4. State three factors that contribute to the formation
urinary casts, and explain the significance of each
5. State two methods for enhancing nuclear detail and
two methods for detecting lipids
6. State the advantage of checking complete urinalysis
correlation
12/22/2022 152

References:
• District laboratory practice in tropical countries. 2nd ed. Part I.
Monica Cheesbrough, 2005
• Text book of urinalysis and body fluids. Doris LR, Ann EN, 1983
• Urinalysis and body fluids: A color text and atlas. Karen MR, Jean
JL. 1995
• Clinical chemistry: Principles, procedures, correlation. 3rd ed.
Michael L. Bishop et al. 1996
• Tietz Text book of clinical chemistry. 3rd ed. Carl AB, Edward RA,
1999
• Clinical chemistry: Theory, analysis, correlation 4th ed. Lawrence
AK. 2003
• Urinalysis lecture note . Mistire W. , Dawite Y.
• Urinalysis and body fluids / Susan King Strasinger, 5th ed. 2008
153

154

Case Studies and Clinical Situations
1. An 85-year-old women with diabetes and a broken hip has been confined
to bed for the past 3 months. Results of an ancillary blood glucose test
are 250 mg/dL, and her physician orders additional blood tests and a
routine urinalysis.
 The urinalysis report is as follows:
COLOR: Pale yellow, KETONES: Negative
CLARITY: Hazy, BLOOD: Moderate
SP. GRAVITY: 1.020, BILIRUBIN: Negative
pH: 5.5, UROBILINOGEN: Normal
PROTEIN: Trace, NITRITE: Negative
GLUCOSE: 100 mg/dL, LEUKOCYTES: 2
 Microscopic:
20 to 25 WBCs/hpf
Many yeast cells and hyphae
12/22/2022 155

Cont’….
A. Why are yeast infections common in patients with diabetes
mellitus?
B. With a blood glucose level of 250 mg/dL, should glucose be
present in the urine? Why or why not?
C. Is there a discrepancy between the negative nitrite and the
positive leukocyte esterase results? Explain your answer.
D. What is the major discrepancy between the chemical and
microscopic results?
E. Considering the patient’s history, what is the most probable
cause for the discrepancy?
12/22/2022 156

2. A 2-year-old left unattended in the garage for 5 minutes is
suspected of ingesting antifreeze (ethylene glycol).
 The urinalysis has a pH of 6.0 and is negative on the chemical
examination.
 Two distinct forms of crystals are observed in the microscopic
examination.
A. What type of crystals would you expect to be present?
B. What are the two crystal forms present?
C. Describe the two forms.
D. Which form would you expect to be predominant?
12/22/2022 157

3. As supervisor of the urinalysis section, you are reviewing results.
State why or why not each of the following results would concern
you.
A. The presence of waxy casts and a negative protein in urine from a 6-
month-old girl
B. Increased transitional epithelial cells in a specimen obtained
following cystoscopy
C. Tyrosine crystals in a specimen with a negative bilirubin test result
D. Cystine crystals in a specimen from a patient diagnosed with gout
E. Cholesterol crystals in urine with a specific gravity greater than 1.040
F. Trichomonas vaginalis in a male urine specimen
G. Amorphous urates and calcium carbonate crystals in a specimen
with a pH of 6.0
12/22/2022 158

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