Lecture 5: Microscopic Examination of Urine

Microscopic Examination of Urine

Done to detect and identify formed elements found in urine

Thomas Addis

Developed the first procedure to standardize the quantitation of formed elements in urine

examined fresh or adequately preserved

How should urine specimen be examined?

Refrigeration

It causes precipitation of amorphous urates and phosphates and other nonpathologic crystals.

Warm the specimen to 35 degrees Celsius prior to centrifugation

How to remove crystals from refrigeration?

Midstream clean catch specimen

Minimizes external contamination of the sediment

10 - 15 mL

Specimen volume Range

12mL

Frequently used urine volume

6 mL

Urine volume for Pediatric patients

5 minutes at 400 RCF

How many minutes and at what Relative Centrifugal Force

Should a urine be centrifuged?

1.118 X 10-5 X radius in centimeters X RPM^2

RCF formula

Radius (cm)

Refers to the distance from the center of the rotor to the outermost point of the cup, tube or trunnion when the rotor is in motion

0.5 - 1 mL

How many mL of urine should remain after decantation

20 UL

Volume of sediment in sediment preparation

Gentle agitation

How do you resuspend urine sediment?

Casts

Usually found near the edges of the cover slip

A minimum of 10 LPF and 10 HPF

Examination of sediment

Average number per LPF

How to report casts ?

Average number per 10 HPFs

How to report RBCs and WBCs?

Semi quantitative terms (rare, few, moderate and many or as 1+, 2+, 3+ and 4+) per LPF/HPF

How to report Epithelial cells, crystals and other elements?

Rare, 1+

Present, but hard to find

Few, 1+

One (or more) present in almost every

field of view

Moderate, 2+

Easy to find; number present in FOV

varies; "more than few, less than many"

Many, 3+

Prominent; large number present in all

FOV

Packed , 4+

FOV is crowded by or overwhelmed with

the elements

Red blood cells

PHYSICAL : Turbidity ; Red color

CHEMICAL : + Blood

EXCEPTIONS : Number ; Hemolysis

White Blood cells

PHYSICAL : Turbidity

CHEMICAL : + Protein ; + Nitrite ; + Leukocytes

EXCEPTIONS : Number ; Lysis

Epithelial cells

PHYSICAL : Turbidity

CHEMICAL :

EXCEPTIONS : Number

Casts

CHEMICAL : + Protein

EXCEPTIONS : Number

Bacteria

PHYSICAL : Turbidity

CHEMICAL : pH ; + Nitrite ; + Leukocytes

EXCEPTIONS : Number and type

Crystals

PHYSICAL : Turbidity ; color

CHEMICAL : + pH

EXCEPTIONS : Number and type

• Sternheimer-Malbin stain

• 0.5% Toluidine Blue

• 2% Acetic acid

• Fat or Lipid stains

• Gram stain

• Hansel stain

• Prussian blue stain

Sediment stains

Sternheimer-Malbin stain

Most frequently used stain in urinalysis, supravital stain. This is commercially available as Sedi stain and Kova stain.

• Absorbed well by WBCs , Epithelial cells and casts.

• Safranin O

• Crystal violet

What is Sternheimer-Malbin stain consist of?

May precipitate in strongly alkaline in urine

What is the disadvantage of Sternheimer-Malbin stain?

0.5% Toluidine Blue

• Metachromatic stain

• Provides enhancement of nuclear detail

• Differentiates WBCs from Renal Tubular Epithelial Cells

2% Acetic Acid

• Identification of WBCs enhances nuclear detail

• Lyses RBCs

Fat or Lipid stains

Stains triglycerides, neutral fats and cholesterol

• Under this stain is Oil Red O and Sudan III

Cholesterol

Does not stain in Fat or lipid stains but is capable of Polarization

Gram stain

It is used primarily in the Microbiology section for the differentiation between Gram-positive and Gram-negative bacteria.

Hansel stain

Preferred stain for urinary eosinophils

• Consists of methylene blue or eosin Y

Prussian Blue Stain

Stain that detects the presence of Iron

• Oculars

• Objective lenses

• Coarse adjustment knob

• Fine adjustment knob

Lens system of the microscope

• Light source

• Condenser

• Field

• Iris diaphragm

Illumination system of the microscope

Brightfield microscopy

Most commonly type of microscopy used in urinalysis.

Objects appear dark against a light background

Phase-contrast microscopy

identifying low refractive hyaline casts or mixed cellular casts, mucous threads, and Trichomonas

Polarizing Microscopy

Uses polarized light in the identification of crystals and Lipids

Birefringent

a property indicating that the element can refract light in two dimensions at 90 degrees to each other.

Isotopic substances

Do not have refractive property

Interference - contrast Microscopy

Provides a three-dimensional image showing very fine structural detail by splitting the light ray.

Not Routinely Used in urinalysis

Dark field Microscopy

Used to enhance visualization of specimens that cannot be easily viewed with a bright-field microscope.

• Used to Identify the spirochete Treponema pallidum

Fluorescence Microscopy

Used to detect bacteria and virus within cells and tissues through a technique called immunofluorescence

Red Blood Cells

smooth, non-nucleated, biconcave disks measuring approximately 7 um in diameter.

• Identified using High Power Objective (HPO)

• Routinely reported as the average number seen in 10

High Power Fields (HPFs)

Hypersthenuric urine

RBCs shrink due to loss of water and may appear crenated or Irregularly shaped.

Hyposthenuric Urine

Cells absorb water, swell an lyse rapidly releasing the hemoglobin and leaving only the cell membrane called ghost cells

• Damage to the glomerular membrane

• Vascular injury within the genitourinary tract

RBC clinical significance is associated with?

• yeast cells, oil droplets and air bubbles

What is RBCs frequently confused with?

Add acetic acid

How to differentiate RBC from yeast cells?

Dysmorphic RBCs

RBCs that vary in size, have cellular protrusions or are fragmented, associated primarily with glomerular bleeding

Macroscopic Hematuria

• Frequently associated with advanced glomerular damage

• Reported in terms of greater than 100 RBCs per HPF

• Cloudy with red to brown color urine

Microscopic Hematuria

• Critical to the early diagnosis of glomerular disorders and malignancy of the urine tract and to confirm the presence of renal calculi (kidney stones)

White Blood Cells

• Larger than RBCs, measuring an average of about 12 um in diameter

• Identified using HPF

• Reported as average number seen in 10 HPF

Glitter cells

These are neutrophils exposed to hypotonic urine which causes the cells to swell.

• No pathological significance

Eosinophils

Urinary presence is associated primarily with drug-induced interstitial nephritis .

Stained with Hansel’s stain

Mononuclear cells

Mononuclear cells: lymphocytes, monocytes, macrophages and histiocytes

These cells are present in small numbers and usually not

identified in the wet preparation.

Lymphocytes

smallest WBCs

Monocytes, Macrophages, Histiocytes

large WBCs,

may appear vacuolated or contain inclusion

Supravital stain

Used to differentiate mononuclear cells and disintegrating neutrophils from renal tubular epithelial cell.

Pyuria

Term used if there is an increase in urinary WB

• pyelonephritis

• Cystitis

• Prostatitis

• Urethritis

Bacterial Infection Cause of pyurie

• glomerulonephritis

• Lupus

• Erythematosus

• Interstitial nephritis

• Tumors

Non-bacterial Disorders

Epithelial cells

Derived from the linings of the genitourinary system

Squamous Epithelial Cells

• Largest cells found in the urine sediment

• Originate from the linings of the vagina, female urethra, and the lower portion of the male urethra

• First structures observed when examined under LPO

Rare, few, moderate , or many per LPF or HPF

How are squamous epithelial cells reported?

Midstream clean catch specimen

In order to to prevent contamination or increased number of epithelial cells in the sample we make use of what urine specimen?

Clue cells

• A variation of squamous epithelial cells found in with pathological significance

Gardnerella vaginalis

Clue cells are Indicative of vaginal infection by what bacteria?

Vaginal wet preparation

Clue cells are performed by what preparation?

Transitional / Urothelial Epithelial cells

Smaller than squamous epithelial cells and appear in several forms including spherical, polyhedral, and caudate

• Centrally located nucleus

• No clinical significance

Renal Tubular Epithelial Cells

most clinically significant epithelial cell

• Proximal Convoluted Tubule

• Distal Convoluted Tubule

• Collecting Duct

  Renal Tubular Epithelial cells

Types of Renal Tubular Epithelial cells

Proximal convoluted tubule cells

Larger cells than RTE cells, rectangular, columnar / convoluted cells

Distal convoluted tubule cells

Smaller than cells of PCT, round / oval, mistaken for WBCs

Collecting Duct cells

Cuboidal, never round, large sheets of cells

Tubular injury

Presence of more than 2 RTE cells per high power field, indicates?

Necrosis

Increased amount of RTE cells is indicative of ?

Oval Fat Bodies

Lipid-containing RTE cells

• Confirmed by staining with Sudan III or Oil Red O Fat stains

Bubble cells

RTE cells containing large, no lipid-filled vacuoles. They represent injured cells in which the endoplasmes reticulum has dilated before cell death.

Bacteria

Nor normally present in urine

• few may be present as a result of contamination from the vagina, urethra, external genitalia or collection container.

Cocci or bacilli

Bacteria may be present in what form?

Few, moderate, or many per HPF

How is bacteria reported?

White Blood Cells

To be considered significant for Urinary Tract Infection, what other urine sediment should be present along with bacteria?

Enterobacteriaceae

• Escherichia coli

• Proteus

• Klebsiella

Bacteria most frequently associated with UTI

Yeast

Appear as small , refractive oval structure that may or may not contain a bud.

• Its infection should be accompanied with the presence of WBCs

Candida albicans

Is usually the yeast seen in urine or during urinalysis

Trichomonas vaginalis

The parasite most frequently encountered in urine, and is sexually transmitted, causing vaginal inflammation

• White Blood Cells

• Tranditional or Renal Tubular Epithelial cells

If Trichomonas vaginalis is not moving, it can be confused with what other urine sediments?

Schistosoma haematobium , Ova

• Bladder parasite

• Appears in urine

• Associated with bladder cancer

Enterobius vermiculares, Ova

Most commonly intestinal parasite that can contaminate the urine specimen (contaminated with feces)

Spermatozoa

This is easily identified by their oval, slightly tapered heads, and long, flagella-like tail.

• occasionally found in urine of both males and females after sexual intercourse, masturbation, or nocturnal emission.