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WEEK: 4 ANALYSIS OF URINE AND OTHER BODY FLUIDS (LABORATORY)

 Provides clearer delineation of cells


Microscopic Analysis of  Provides contrasting color of the nucleus and cytoplasm

Urine 2. 0.5% Solution of Toluidine Blue


 Metachromatic stain
Microscopic Urine Analysis  Enhances nuclear detail of cells
 Third part of routine urinalysis  Differentiates WBCs from RTE cells
 Used to detect/ identify/ quantify insoluble materials  Useful for cell identification of other body fluids
present in urine (sediments, crystals, cells)
 Most time consuming part of routine urinalysis 3. 2% Acetic Acid
 LEAST STANDARDIZED among all analyses conducted  Lyses RBCs
in urine sample.  Enhances nuclear detail WBCs.
 Also known as chemical seiving.
4. Hansel Stain
Urine Sediment Preparation and Examination  Composed of methylene blue and eosin Y
 Standardization started in 1926 by Thomas Addis (Addis’  Preferred stain for urinary eosinophls
Count)  Dried smear of centrifuged urine is used
- uses hemocytometer
- 12 hour urine sample 5. Wright’s Stain
- Used to monitor the course of diagnosed cases of RENAL  Primarily used for peripheral blood smear but can also be
DISEASE. use as a substitute for urinary staining (particularly
 Commercialized methods have been developed throughout the eosinophil)
years.  Not as specific as Hansel stain for eosinophil
e.g.
KOVA CenSlide 6. Prussian Blue Stain
Urisystem R/S Workstation  Stains hemosiderin granules as seen in hemoglobinuria
Count-10 Quick-Prep  Hemosiderin granules stain BLUE

CenSlide Lipid Stains


 Doesn’t require manual charging of urine sample on slides 1. Oil red O, Sudan III & Sudan IV
 Closed-system procedure to eliminate exposure  For the identification of lipid containing cells and free fat
 Especially designed tube to facilitate urine microscopy bodies.
 Triglycerides and neutral fats stains well with a ORANGE-
R/S Workstation RED color
 Glass flow cell through which urine sediment is pumped,
examined and discarded from the system. Bacterial Stain
1. Gram’s Stain
Specimen Preparation  Differentiates Gram positive from Gram negative bacterial
 Freshly collected then thoroughly mixed, midstream clean- infection
catch sample  For bacterial cast identification
 10-15 mL (standard amount of urine )
 Centriguation time: 5 minutes at 400 RCF 2. Ziehl Neelsen Stain
 Decantation (rapid pouring of centrifuged urine sample)  Differentiates acid fast organisms from non acid fast
 Charging the sediment on the slide (20uL/ 0.02 mL) organisms.
 Examination under the microscope  For urinary tubercular infection
 Reporting the microscopic exam semiquantitatively (rare,
few, moderate, many) Cytodiagnostic Testing
1. Papanicolaou’s Stain
Checking the Correlation…  Used for preparing fixed slides for urine sediment
Correlation between physical and chemical exam plus the identification and cytology.
microscopic exam must be observed to ensure accuracy and  For the detection of malignancies in lower urinary tract.
reliability of reports.  Detects and monitors renal diseases
 Provides more definitive information about: transplant
Sediment Examination Techniques rejection, inflammatory conditions, pathologic casts
STAINS  First morning urine sample or suprapubic aspirate.
 Increases the overall visibility of sediments being analysed.
 Imparts identifying characteristics to cellular structures: MICROSCOPE
nuclei, cytoplasm, and inclusions. Bright-field Microscopy
 Most common type of microscopy done in the lab
Sediment/ Cellular Stain  Object appears dark against light background
1. Sternheimer-Malbin Stain  When using this method, decreased light level must be
 Supravital stain (stains living cells) employed. (Adjust the rheostat level)
 Made up of crystal violet and safranin O
 Absorbed well by WBC, EC, Cast Phase-Contrast Microscopy
WEEK: 4 ANALYSIS OF URINE AND OTHER BODY FLUIDS (LABORATORY)

White Blood Cells


 Larger than RBCs
 12 mm in diameter
 Stains well with Sternheimer-Malbin Stain
 NEUTROPHIL- predominating urinary WBC
At HYPOTONIC ENVIRONMENT = SWELL
BROWNIAN MOVEMENT = GLITTER CELLS

Eosinophil
 Stains well with Hansel Stain
Polarizing Microscopy  Seen in:
 Confirms the identification of fat droplets, oval fat bodies - Drug- induced interstitial nephritis (allergy)
and fatty casts that produce MALTESE CROSS - UTI (parasitic in origin)
APPEARANCE - Renal transplant rejection (anaphylactic reaction)

Interference Contrast Microscopy NOT NORMALLY SEEN IN URINE= (at least 1%)
 Provides a three dimensional image by showing very fine CLINICALLY SIGNIFICANT!
structural detail Mononuclear cells
 ADVANTAGE: object appears dark against light  Not identified in wet mount preparation of urine
background without any halo associated with phase  LYMPHOCYTES
contrast microscopy - resembles RBCs
- increased in early stages of renal rejection
Dark-Field Microscopy  MONOCYTES, MACROPHAGE, HISTIOCYTES
 Enhances visualization of specimens that cannot be seen
- large cells with vacuoles
easily with bright field microscopy
 For identification of spirochetes (Treponema, Borrelia, etc) The primary concern of mononuclear cells is to differentiate
them from RTE
Fluorescence Microscopy
 Used to detect bacteria and viruses within cells and tissues CLINICAL SIGNIFICANCE
through immunofluorescence.  Pyuria (increased WBC in urine)
 FLUORESCENCE: property by which an atom absorbs  Pyelonephritis (ascending UTI; involve the kidneys)
light of a particular wavelength and emit light of a longer
 Bacteuria (increased bacterial multiplication)
wavelength, fluorescence lifetime
 Cystitis (inflammation of the bladder)
Urine Sediments
Epithelial Cells
 Normally seen in urine except in large amounts or
Red Blood Cells
abnormal forms
 Smooth, non-nucleated, biconcave discs
 7um in diamter
3 Types of EC:
 MOST DIFFICULT to recognize by students because of 1. Squamous EC
variation in sizes (anisocytes), characteristic structures and 2. Transitional EC (urothelial cells)
close resemblance to YEAST, BUBBLES, OIL 3. Renal Tubular Cells
DROPLETS and other microscopic sediments.
Squamous Epithelial Cells
HYPERSTHENURIC SAMPLE:  Largest cells found in urine sediment
 RBCs shrink= CRENATED= loss of water
 Originates from the linings of vagina and female urethra,
HYPOSTHENURIC SAMPLE: and the lower portion of the male urethra.
 RBCs swell, lyse, relase Hgb= GHOST CELLS
CLUE CELLS
DYSMORPHIC RBC  infected epithelial cells
 Aids in determining the site of renal bleeding
 Seen in bacterial vaginosis
 Associated with glomerular bleeding
 Gardnerella vaginalis
 ACANTHOCYTES
Transitional Epithelial Cells
CLINICAL SIGNIFICANCE  Originates from the lining of renal pelvis, calyces and
 Glomerular bleeding bladder
 Lower urinary tract bleeding  Smaller than epithelial cells
 Trauma  Spherical, polyhedral or caudate in shape due to the ability
 Acute infection/ inflammation to absorb water.
 Coagulation disorder  Pathologically seen in Malignancy and Viral Infection
MANNER OF REPORTING: SYNCTIA
 Hpf/ HIGH POWER FIELD  increased numbers of transitional cells seen in clumps.
 For cast: LOW POERT FIELD
WEEK: 4 ANALYSIS OF URINE AND OTHER BODY FLUIDS (LABORATORY)

 Seen in catheterization collection  Major component of mucus and casts.


 Considered as NORMAL
Casts
Renal Tubular Epithelial cells  The only element found in the urinary sediments
 Slightly larger than WBC  that are unique to the kidney
 May be flat, cuboidal, columnar  Made up of TAMM-HORSFALL PROTEIN
 Must be identified under HPO  Cannot be detected by reagent strips, therefore, increased or
 THE MOST CLINICALLY SIGNIFICANT URINARY positive CHON strip is frequently associated with renal
EPITHELIAL CELL (e.g. oval fat bodies) disease.

CLINICAL SIGNIFICANCE CYLINDURIA


 Malignancy  Presence of casts in urine
 Exposure to heavy metals
 Tubular damage  Type: Hyaline
 Pyelonephritis  Description: Homogenous with parallel sides and rounded
 Kidney transplant rejection ends
 Significance: 0-2/low-power field (LPF) are normal.
Oval Fat Bodies Increased with stress. Fever, trauma, exercise, renal disease
 RTE cells that absorb lipid  Comments: Most common type. Least significant. Contain
 “lipid containing RTEC” Tamm-Horsfall protein only . May be overlooked if light is
 Lipiduria too bright
- presence of lipid/ fat in urine
- Most frequently associated with nephrotic syndrome  Type: Granular
 Description: Same as hyaline, but contains granule
Bacteria  Significance: 0-1/LPF is normal. Increased with stress,
exercise, glomerulonephritis, Pyelonephritis.
 Not normally present in urine especially if specimen is  Comments: May originate from disintegration of cellular
taken under normal condition. casts.
 Most indicative of urinary tract infection  Type: RBC
 Most common cause of UTI are the Enterobacters  Description: RBCs in cast matrix. Yellowish to orange
color or orange-red color
Yeast  Significance: Acute glomerulonephritis. Strenous exercise
 Small, refractile oval structures, usually mistakes as RBC  Comments: Pinpoints source of bleeding in kidney. Most
 Candida albicans is the most common cause of yeast UTI. fragile of casts. Often in fragments.
CLINICAL SIGNIFICANCE
 Diabetes  Type: Blood
 Immunocompromised patients  Description: Contain hemoglobin. Yellowish to orange
 Vaginal Moniliasis color.
 Significance: Same as RBC cast
Parasites  Comments: From disintegration of RBC casts
Trichomonas vaginalis
 The most frequent parasite encountered in urine samples  Type: WBC
 Resmbles WBC, transitional cells or RTE; hard to identify  Description: Leukocytes incorporated into cast matrix.
when not moving. Irregular in shape.
 Can be seen in wet mount preparation  Significance: Pyelonephritis
 Comments: Pinpoints kidneys as the site of infection
Schistosoma haematobium
 Parasite of the urinary bladder that can be excreted in urine.  Type: Epithelial cell
 Description: Renal tubular epithelial cells incorporated
Enterobius vermicularis into cast matrix
 Most common ova contaminant in urine  Significance: Renal tubular damage
 Comments: Transitional and squamous epithelial cell casts
Spermatozoa do not exist. These cells are found distal to renal tubules
 Oval, slightly tapered heads with long, flagella- like tails. and collecting ducts where casts are formed.
 Occasionally found in both male and female following
sexual intercourse  Type: Waxy
 Not usually reported in the laboratory due to possible legal  Description: Homogenous, opaque, notched edges, broken
consequences ends
 Significance: Urinary stasis
Mucus  Comments: From degeneration of cellular and granular
 Thread-like structure that is made of protein materials casts. Unfavorable sign.

TAMM-HORSFALL PROTEIN  Type: Fatty


 Produced at the distal convoluted tubule by RTE excretion  Description: Casts containing lipid droplets
WEEK: 4 ANALYSIS OF URINE AND OTHER BODY FLUIDS (LABORATORY)

 Significance: Nephrotic syndrome Crystals Found in Alkaline Urine


 Comments: Maltese crosses with polarized light. Stain
with Sudan and oil red O.  Crystal: Amorphous phosphates
 Description: Irregular granules
 Type: Broad  Significance: None
 Description: Wide. May be cellular, granular, or waxy.  Comments: Form white precipitate in bottom of tube.
 Significance: Advanced renal disease Dissolve with 2 acetic acid.
 Comments: Formed in dilated distal tubules and collecting
ducts. “Renal failure casts.”  Crystal: Triple phosphate
 Description: “Coffin-lid”crystal
Crystals in Acidic/Neutral Urine  Significance: None

 Crystal: Amorphous urates  Crystal: Ammonium biurate


 Description: Irregular granules  Description: Yellow-brown “thorn apples” and spheres
 Significance: None  Significance: None
 Comments: From pink precipitate in bottom of tube. May  Comments: Seen in old specimens
obscure significant sediment. Dissolved by warming to
60ºC.  Crystal: Calcium phosphate
 Description: Needles, rosettes, “pointing finger”
 Crystal: Uric acid  Significance: None
 Description: Very pleomorphic. Four-sided, six-sided, star-  Comments: Only needle form seen in alkaline urine
shaped, rosettes, spears, plates. Colorless, red brown, or
yellow.  Crystal: Calcium carbonate
 Significance: Usually normal  Description: Colorless dumbbells
 Comments: Birefringent. Polarized light.  Significance: None

 Crystal: Calcium oxalate


 Description: Octahedral (eight-sided) envelope form is
most common. Also dumbbell and ovoid forms.
 Significance: Normal
 Comments: Occasionally found in slightly alkaline urine.
Monohydrate form may be mistaken for RBCs. Most
common constituent of renal calculi.

 Crystal: Leucine
 Description: Yellow, oily-looking spheres with radial and
concentric striations
 Significance: Severe liver disease
 Comments: Often accompanied by tyrosine

 Crystal: Tyrosine
 Description: Fine yellow needles in sheaves or rosettes
 Significance: Severe liver disease
 Comments: Often accompanied by leucine

 Crystal: Cystine
 Description: Hexagonal (six-sided)
 Significance: Cystinuria
 Comments: Must be differentiated from uric acid. Does
not polarize light.

 Crystal: Cholesterol
 Description: Flate plate with notched out corner. “Star-
step.”
 Significance: Nephrotic syndrome
 Comments: Birefringent

 Crystal: Bilirubin
 Description: Yellowish-brown needles, plates, and
granules
 Significance: Liver disease
 Comments: Reagent strip or Ictotest should be positive for
bilirubin.