for clinical laboratory professionals

1. Mercury Y. Lin, MD FCAP
LCDR MC USN
Staff Pathologist
NHCP

2.  Overview of laboratory examination for urine
and body fluid crystals with emphasis on
morphologic properties.
 “Piss for path”: helpful information for
pathologist-clinical lab consultants. Inherent
limitation of urine examination.
 Most importantly, enable board exam
candidates to rock the clinical path portion of
ABP examination. (continue our tradition of
near 100% passing rate for 1st time exam
takers).

4.  5th century BC: Hippocrates described fluid intake
and urine output. DM urines taste sweet.
 Byzantium and Middle Ages: Uroscopy, “piss
prophets” and pseudo-science (“Uromancy”)
 1820’s: Richard Bright noted relationship
between edema and proteinuria. (archaic term
“Bright’s Disease”)
 1837: Medical urine microscopy being taught in
Paris by Alfred Donné.
 1844: Golding Bird publishes “Urinary Deposits”
and described urine casts in pts with “Bright’s
Dz.”

5. Sweet urine
I agree, it is sweet with a
fruity odor, which may
present ketone bodies.
Let’s pass it around so
everyone can get a sip.
pathology consults in Byzantium
Urine Wheel from
“Epiphaniae Medicorum”

6.  NCCLS definition: “testing of urine with
procedures commonly performed in an
expeditious, reliable, and cost effective
manner…”
 So routine UA protocols look for
- Physical : (color, transparency, odor, foam, spec
gravity)
- Chemical: (pH, protein, blood, nitrite, leuk
esterase, glucose, ketone, bilirubin, urobilinogen)
- Microscopic: (RBC, WBC, Epithelial cells, casts,
microrganisms, crystals)

7.  Typically 96% water + 4 % solutes (of which
50% is urea, there is also creatinine).
 Na,Cl,Ca,Mg,K,Phos, are also present in urine.
 1st morning void (8hr specimen)
 Random urine
 Timed urine (24hr urine)
 Double voided urine (glucose comparison blood vs. urine)
 Mid-stream “clean catch” (for bacterial culture).
 Catheterized urine
 Suprapubic collectionl

8.  Routine UA (room temp specimens) shall be
performed within 2 hrs of voiding. (CAP and
NCCLS requirement).
 Refrigeration may reduce bacterial growth, but
may result in precipitation of amorphous urates
and phosphates (interfere with microscopy).
 For 24hr urine collection, preservative maybe
required, depending on analyte of interest.
 UA specimen in room temp >2hr should be
rejected. (refer to CAP checklist and local lab
policy for exceptions).

9. Constituent Changes Mechanism
pH increase urea breakdown
cells decrease/disappear lysis
casts decrease/disappear dissolved
sugar decrease bacteria mediated
glycolysis
acetone decrease evaporation
acetoacetic acid decrease converted to
acetone
bilirubin decrease oxidized to
biliverdin
(yellow->green)
urobilinogen decrease oxidize to uribilin
(colorless ->orange)
Adopted from Ringsrud and Linne “Urinalysis and Body Fluids:
A Colortext and Atlas” 1st Ed.

10.  Color: normal is pale yellow to straw colored.
 if clear redhemoglobin
 cloudy redRBCs
 brownmyoglobin, hemoglobin, bilirubin
 blackmelanin, homogentisic acid.
 Foam: normal is small amt of white foam, if shaken.
 if abundant foam proteinuria
 yellow foambilirubin.
 Specific Gravity: normal is 1.001 – 1.035 (water is 1.000)
 spec grav is dependent on mass and amount of particles in
urine.
 “spec grav” reported by reagent strips is actually a gestimation
by measures ionic concention (since most urine solutes are
ionized).

11.  A refractometer can be used for more
accurate measurement of urine specific
gravity.
 Usually, it doesn’t matter, but if there is
prominent glucosuria.
 Spec grav by refractometer will be >1.035
 yet
 Spec grav by reagent strip will be WNL (b/c
glucose is not ionized).


12.  Urine pH: normal is usually slightly acidic
(about pH of 6 but can range from 4.5 to 8 in
healthy population).
 Other effects:
 high protein diet, cranberries, morning void
 more acidic urine.
 Citrus fruits, postprandial, urine sample left
in room temp more alkaline urine.

13.  Reagent strips are the most common way of
analyzing chemical properties of urine.
 Reagent strips are simple and low cost with
relatively good shelf life, but they suffer from
 - variable interpretation
 - inadequate sensitivity for some analytes
 - susceptible to interfering substances.

16.  Urine is also been used to screen for
metabolic disease in babies.
 But reagent strip (glucose oxidase reaction)
will only detects glucose.
 Clinitest will detect reducing substances
(including glucose, galactose, fructose,
lactose, and pentose).

17.  If regagent strip is negative (for glucose) but Clinitest is positive.
Additonal testing is needed to identify the specific non-glucose
reducing sugars.
 Ascorbic acid, nalidixic acid, cephalosporins and probenecid in large
quantities may cause false positive results with Clinitest.

18.  Most of the time, examination is performed
by lab instruments
This is an Iris iQ®200SPRINT™ Automated Urine Microscopy Analyzer.
By flow cell digital imaging, it can analye 101 samples/hr and reports RBC
WBC, WBC clumps, squamous epithelial cells, renal epithelial cells, urothelial
cells, hyaline casts, other casts, crystals (9 subtype), yeasts, bacteria, sperm
mucus, oval fat bodies, trichomans
But remember, without an astute human operator, even the
most advanced lab instruments is no more than a machine.
Auto “TAR” by lab techs will report out false results.

19.  Urine microscopy are often unstained, so it will
look slightly different to us (anatomic focus
pathologists).
RBCs
Crenulated RBCs due to urine
hypertonicity

20. These pale RBCs have
“ghost cell” appearance.
This is due to loss of Hb
which occurs in hypotonic
urine specimen. This
doesn’t necessarily mean
patient is anemic.

21.  These are dysmorphic RBCs (cytoplasmic blebs) and may mimic budding yeast.
Note the “Micky Mouse” morphology. Right hand side is differential interference
contrast microscopy which accentuates membrane distortion.

22. Dysmorphic RBCs are
associated with glomerular
hematuria. (glomerulonephritis)
But, elevated uric acid content
and hypotonicity can also show
similar morphologic changes.

23. WBCs
In fresh urine sample, the cytoplasmic
granules induce a sparkling appearance.
Squamous cells. Urothelial cells
(tadpole form)

24.  Urine eosinophils
(to identify eos,
the urine needs
to be stained
with either
Wright Giemsa or
Hansel stain).
Eosinophiluria: Top 3 drug inducted acute intersitital nephritis,
hypersensitivity, parasitic infections. Also transplant rejection, RPGN,
Post infectious GN, eosphilic cystitis…etc.
The reagent strip will show ++WBC, but negative leukocyte esterase.

25.  Oval Fat Bodies (renal tubular cells with fat
globules). Show maltese cross under cross
polarization. Associated with nephrotic
syndrome.

26.  True maltese cross (arms of equal length): lipids (e.g. oval fat
bodies)
 vs.
 Pseudo maltese cross(arms of unequal length): starch, leucine,
tiamterene diuretics, adenine phosphoribosyltransferase [APRT]
deficiency

27. Spermatozoa in urine.
Some labs may not report it.
Considered “critical value” in
Pre-adolescent children and
vulnerable adults. Huge
medical-legal implication.
Need to be sure!

28. Now we turn our attention towards urinary casts (cylinders). Casts in
the urine mirrors that physiologic state of the kidney.
Casts are classified into 2 broad groups.
Acellular Casts Cellular Casts
- Hyaline - RBC
- Granular - WBC
- Waxy - RTE
- Fatty - Bacterial

29.  Hyaline casts and granular casts are the only 2
types of cast that should be seen in normal
population.
 Hyaline casts can be difficult to visualize by
bright field microscopy, but (fortunately) are also
clinically least important.
 Vigorous exercise can increase hyaline cast in
urine. Granular casts are less commonly seen.
Hyaline casts are composed of
Tamm-Horsfall proteins. The
matrix contains few granular
material. Hyaline casts do not
polarize.

30. Granular casts are refractile and can have fine or coarse granules. The
granules are thought to derive from degenerative products of renal
tubular epithelium. Can be seen in both healthy population and in
patients with intrinsic renal disease.

31. Waxy casts are broad (>40um), often short, with sqare ends. They also
tend to show cracks and indentation. They from within dilated renal
tubules and are not seen in normal healthy population. Waxy casts are
associated with ESRD, and glomerulonephritis.

32.  Fatty casts show surface fat globules and produce
maltese cross under polarization. They are seen in
nephrotic syndrome, ATN, and other renal dz.

33.  RBC casts are rarely seen. RBCs are stuck
together in a matrix of Tamm-Horsfall protein.
But RBC casts are fragile and will degenerate after
voiding. Reddish appearance due to Hb. Seen in
acute glomerulonephritis, renal neoplasms, and
malignant HTN.

34.  The so-called “Muddy Brown Cast.” There is usually a dirty background
composed of degnerated epithelial cells. A clinical nephrology buzzword in
1990’s, this has been equated to acute tubular necrosis(ATN).
The brownish pigment seen is either myoglobin or hemoglobin. This
But pts with ATN can also have non-pigmented casts in their urine.
So, this term is not endorsed by clinical laboratory community.

35.  WBC casts: highly refractive, look for multilobed nuclei and
single WBCs in surrounding area. WBC casts are seen
pyelonephritis, interstitial nephritis, renal transplant
rejection, sepsis, lupus nephritis…etc.

36.  Renal Tubular Epithelial (RTE) Casts: Mimics WBC
cast and at times can be difficult to
differentiated. So the term “Cellular Cast” can be
used to include both RTE and WBC casts.
 RTE casts are seen in ATN, renal transplant
reject, heavy metal poisoning…etc.

37.  Renal stones form because of undesired
phase change of a substance.
 Normal urine is in a shifting state of
oversaturation. Small crystals are constantly
forming and dissolving.
 Stones develop when there is an unstable
oversaturation which leads to precipitation.
 Many different crystals have been described
in urine. We will mainly focused on ones that
are either clinically important or IMHO,
morphologically peculiar

38.  Amorphous urate
and phosphate are
similar/essentially
identical under
light microscopy.
Difference are:
Amorphus urate: seen in pH <5.8, show birefringence in large clumps,
associated with dehyration, fever. But usually no significant
clinical implication.
Amorphus phosphate: seen in pH >6.3, no birefringence, clinically insignificant.

39.  Alternatively,
urine can also be
spun and the
pellet is examined
macroscopically.
 Urate is pink.
 Phosphate is
white.

40.  Triple phosphate/Struvite (Ammonium magnesium
phosphate) crystals. Left is the prototypical coffin lid
morphology. But, note that triple phosphate crystals
can be seen (less commonly) in “fern” form. These
crystals develop via biofilm produced by urease
producing bacteria.
 Associated with staghorn calculi, UTI, vericoureteral
reflux.

41.  Calcium oxalate: Left side is the classic “square
envelope” (large 8 faced bi-pyramids). Right side is
the monohydrate with “dumbbell” and oval shapes,
which can cluster into microlith.
 Calcium oxalate crystals form in pH <5.4 and is
associated with oxalate stones, ethylene glycol
poisoning, oxaluria, s/p ileal resection, Crohn’s Dz.
dihydrate form (Weddellite) monohydrate form (Whewellite)

42.  2/3 of all renal calculi are calcium oxalate
stones.
 S/P small bowel resection leads to
malabsorption of fat. Ca selectively binds fat
and leave free oxalate in the colon to be
absorbedoxalate goes to the kindey and
stones develop.
 Ethylene glycol ingestionmetabolized to
glycoaldehydeglycolic acidglyoxylic
acidoxalic acidadd Ca to form calcium
oxalate (more crystals in monohydrate form).

43.  Uric acid crystals: has many morphologic appearance; most commonly
rhomboid plates, but also hexagonal, barrel, clubs/short rode/blade (not
illustrated).
 More about uric acid cystals in joint fluid examination section

44.  Cystine: variably sized hexagonal plates.
 Associated with cystinuria.

45.  While hexagonal form of uric acid is less
common, it mimics cystine crystals.
 They can be differentiate by polarized light and
cyanide nitroprusside test.
Uric acid hexagons show
Polychromasia.
Nitroprusside negative.
Cystine crystals lack
polychromasia
Nitroprusside positive.

46.  Cholesterol crystals: overlapping rectangular plates
 Associated with nephrotic syndrome, chyluria.
 Should also be accompanied by oval fat bodies and
fatty casts.
 Radiologic dye can mimic cholesterol crystals.

47.  Leucine crystals: concentric rings, like tree’s
annual rings. Very rare. Seen in severe liver
disease.

48.  Tyrosine crystals: tufts of fine silky needles.
 Seen in severe liver dz, tyrosinemia.

49.  sulfonamide crystals: “sheafs of
wheat”
ammonium biurate crystals: “thorn apples”
Seen in prolonged storage of urine. No
clinical significance
ampicillin crystals

50.  Should be examined with compensated
polarized light microscopy.
 Also consider other clinical laboratory values
(e.g. cell count)
 First, lets review compensated polarized light
microscopy.

51.  Don’t always expect clinical hx when an
orthopod ask you to look at a joint fluid.

52.  Since I barely recall being awake during undergrad
physics, this is my fraudulent technician’s version of
polarized microscopy.
 Most illumination transmits light waves whose electric
field vectors vibrate in all perpendicular planes with
respect to the direction of propagation. With
polarized light, filtration is used to restrict vibration
to a single plane.
http://www.olympusmicro.com

53.  Crystals can be either
 - isotropic: index of refraction is equal in all
directions.
 - anisotropic: refraction differs when light
enters at non-equivalent axis.
http://www.olympusmicro.com

54.  When placing anisotropic crystals between cross polarization. From the
left, polarized light (P) enter the anisotropic crystal and is refracted into
2 component waves. This light then passes through the analyzer (A).
One component wave is now “retarded.”
 After passing through the analyzer, light interference between 2
component waves acquire a spectrum of color under usual bright field
microscopy.
http://www.olympusmicro.com

55.  In this case, light reflections from outer- and inner-
surfaces of the bubble (separated by a few micros)
interfere with each other and result in this colorful display.

56.  Above is a Michel-Levy chart used to quantify
birefringence. The most sensitive area for
detection of birefringence is approx 550nm (1st
order red). With slight change in retardation,
there will be a dramatic color shift into either
blue or yellow.
 This is achieved by using a red compensator.

57. From: Bullough- Orthopedic Pathology 5th ed.
arrow is the “slow wave”
“U PAY PEB”
Urate PArallel Yellow, PErpendicular Blue

58.  Gout: monosodium urate (MSU) crystals are
needle shaped, negatively birefringent,
(parallel yellow, perpendicular blue).

60.  Calcium pyrophosphate crystals are
rhomboid, positively birefringent (parallel
blue, perpendicular yellow).

61.  Now the fun parts, lets walk over to the core
lab and get some hands on experience with
operating a compensated polarized light
microscope.
 Questions?