AUBF 15: MICROSCOPIC EXAMINATION OF URINE

Addis

DEVELOPED THE MICROSCOPIC EXAMINATION OF URINE

1926

MICROSCOPIC EXAMINATION OF URINE WAS DEVELOPED WHEN?

MICROSCOPIC EXAMINATION OF URINE

First procedure to standardize the quantitation of formed elements in the urine.

hemocytometer to count the number of RBC, WBC, cast and epithelial cells present in a 12- hour specimen

MICROSCOPIC EXAMINATION OF URINE USED A:

slide system

conventional method of placing a drop of centrifuged urine on a glass slide with coverslip

10-15ml (12 ml)

Specimen (Urine) Volume for the multi- parameter reagent strip testing

Centrifuge tube

ideal container of urine prior to centrifugation

fresh or when properly preserved

Examine the urine while ?

two or three

The microscopic examination must be analyzed immediately or refrigerated for not more than ___________ hours.

12 mm

Mix the urine well and pour ___________ of urine in a clean centrifuge tube.

5 minutes ; 400 relative centrifugal force (rcf).

Cap the tube and centrifuge the urine for _________ mins. And __________rpm

0.5 to 1.0ml

Amount of sediment at the bottom of the tube

supernatant fluid

After centrifugation, remove the _______________.

gentle agitation

Re-suspend the sediment in the remaining urine through ________________ commonly performed by flicking the bottom of the tube or by running the tube across a test tube rack.

SEDIMENT PREPARATION

Using a plastic or polypropylene transfer pipet, aspirate 20ml (0.02ml) sediment and place it in a slide and cover it with a 22x22mm glass cover slip

plastic or polypropylene transfer pipet; 20ml (0.02ml); 22x22mm

SEDIMENT PREPARATION : Using what ___________, aspirate __________ sediment and place it in a slide and cover it with a ___________ glass cover slip

glass pipets

Avoid using ______________ when mounting the urine sediment as elements like casts tend to cling to the glass surface.

casts

Avoid using glass pipets when mounting the urine sediment as elements like ________ tend to cling to the glass surface.

• Casts • Amorphous materials • Squamous epithelial cells

Sediment must first be observed using the LP0 to observe for the presence of:

LP0

Sediment must first be observed using the_______to observe for the presence

HP0

After (LPO); shift to ______ then observe and quantitate cells and crystals.

cells and crystals

After (LPO); shift to HP0 then observe and quantitate:

HPO

If one needs to confirm the identity of the casts,______ is commonly used.

10

Minimum of ____ fields per objective

low- power field (lpf)

casts are reported as the average number per _________

high-power fields (hpf's)

RBC and WBCs as the average number per __________

semi- quantitative terms such as rare, few, moderate, and many, or as 1,2,3, and 4.

Epithelial cells, crystals, and other elements are frequently reported in _______________

• Stern Heimer -Malbin -Crystal violet and Safranin O • Toluidine blue • Lipid stains: Oil Red O and Sudan III • Gram Stain • Hansel Stain

• Methylene Blue and Eosin Y - Eosinophils': sig.> 1% • Prussian Blue Stain

Sediments Stains:

1. Crystal violet

2. Safranin O

Stern Heimer -Malbin is comporised of:

1. Methylene Blue

2. Eosin Y

Hansel Stain is comprised of:

• Bright-field Microscopy • Phase-contrast Microscopy • Polarizing Microscopy • Dark-field Microscopy • Fluorescence Microscopy • Interference- Contrast Microscopy A. Hoffman - modulation contrast B. Nomarski- Diff. Interference Contrast

Microscopic Techniques:

Sternheimer-Malbin

Delineates structure and contrasting colors of the nucleus and cytoplasm

Sternheimer-Malbin

Identifies WBCs, epithelial cells, and casts

Toluidine blue

Enhances nuclear detail

Toluidine blue

Differentiates WBCs and renal tubular epithelial (RTE) cells

2% acetic acid

Lyses RBCs and enhances nuclei of WBCs

2% acetic acid

Distinguishes RBCs from WBCs, yeast, oil droplets, and crystals

Lipid stains: Oil Red O and Sudan III

Stain triglycerides and neutral fats orange-red Do not stain cholesterol

Lipid stains: Oil Red O and Sudan III

Identify free fat droplets and lipid-containing cells and casts

1. Oil Red O

2. Sudan III

Types of Lipid stains:

Gram stain

Differentiates gram-positive and gram-negative bacteria

Gram stain

Identifies bacterial casts

Hansel stain

Methylene blue and eosin Y stains eosinophilic granules

Hansel stain

Identifies urinary eosinophils

Prussian blue stain

Stains structures containing iron

Prussian blue stain

Identifies yellow-brown granules of hemo- siderin in cells and casts

Bright-field microscopy

Urinalysis Microscopic Technique Used for routine urinalysis

Phase-contrast microscopy

Enhances visualization of elements with low refractive indices, such as hyaline casts, mixed cellular casts, mucous threads, and Trichomonas

Polarizing microscopy

Aids in identification of cholesterol in oval fat bodies, fatty casts, and crystals

Dark-field microscopy

Aids in identification of Treponema pallidum

Fluorescence microscopy

Allows visualization of naturally fluorescent microorganisms or those stained by a fluorescent dye including labeled antigens and antibodies

Interference- contrast

Produces a three-dimensional microscopy image and layer- by-layer imaging of a specimen

Hoffman

Used for modulation contrast

Nomarski

Used for Diff. interference contrast

Treponema pallidum

causative agent of syphilis

• CELLS • CASTS • CRYSTALS • ARTIFACTS

Microscopic Structures Present in Urine:

RED BLOOD CELLS (RBC's)

Appearance: Smooth, non-nucleated, biconcave disc approximately 7um in diameter

7um

size of RBC

hypersthenuria

high specific gravity of urine

crenated

In concentrated urine specimen (hypersthenuria) the cells shrink due to loss of water therefore the appearance of RBC's are __________.

hyposthenuria

Low urine concentration

GHOST CELLS

In dilute specimens (hyposthenuria) the cells absorb water, swell and then lyse rapidly releasing their hemoglobin leaving only their cell membrane; thus the empty cells are termed as _____________.

• Yeast cells • Oil droplets • Air bubbles • Monohydrate Calcium oxalate crystals

RBC's are frequently confused with:

Acid acetic acid

RBC's are frequently confused with Yeast cells , Oil droplets, Air bubbles, Monohydrate Calcium oxalate crystals: To resolve what should be added to a portion of sediment?

yeasts, air bubbles, CaOx and oil droplets

To resolve RBC confusion Acid acetic acid to a portion of the sediment, if the suspected cells are RBC's they will be lysed and therefore will disappear leaving the ___________ intact.

supravital stains

RBCs can also use ________ stains

0-2/ hpf

Normal RBC in urine

Renal or Genitourinary organ bleeding primarily due to glomerular membrane or vascular injury

RBC Clinical significance:

Dysmorphic RBC's

RBC's with irregular cellular protrusions, with blebs and punch-out centers are indicative of GLOMERULAR BLEEDING

• Urine Color

• Strip Reaction (Blood)

Dysmorphic RBC's Clinical Correlations:

red blood cells

The number of __________ present is indicative of the extent of the damage or injury

WHITE BLOOD CELLS (WBC's)

Appearance: Larger than RBc's 12um, with visible cytoplasmic granules

Neutrophil

Most predominant WBC is ____________ containing granules and nuclei.

GLITTER CELLS

When exposed to hypotonic urine the WBC absorb water and swell & when subjected to Brownian motion, they sparkle, and their granules become more prominent and they are termed as ___________

Eosinophils

Not normally seen in the urine; thus the finding of more than 1% ___________ is considered significant.

Hansel stain

stain used for eosinophils

• Methylene blue - Eosin Y

Hansel stain 2 dyes.

1. Drug- Induced Interstitial Nephritis

2. UTI and renal transplant rejection

Eosinophils Clinical Significance:

1. Lymphocytes

2. monocytes

3. macrophages

4. histiocytes

Types of Mononuclear Cells:

Mononuclear Cells

Increased in renal transplant rejections

Lymphocytes

the smallest WBC's they may resemble RBCs.

Monocytes, macrophages, and histiocytes

are large cells and may appear vacuolated or contain inclusions.

RTE

Sources of Error: Differentiate WBC from ________

PYURIA (increased urinary WBC's) indicating infection or inflammation of the genitourinary system.

WBC Clinical significance:

PYURIA

increased urinary WBC's

PYURIA

increased urinary WBC's

-Leukocyte Esterase -Nitrite

Clinical correlations of WBC:

0-5/hpf or 0-8 hpf

WBC NORMAL results:

acetic acid

If you still want to confirm if the cell is White Blood Cell, the med tech can add ____________ to the sediment.

acetic acid

will make the granules of the WBC's more visible

1. Squamous

2. Transitional ( Urothelial)

3. Renal Tubular Epithelial

4. (RTE) Cell

TYPES OF EPITHELIAL CELLS :

SQUAMOUS EPITHELIAL CELL

Appearance: Largest cell in urine sediment usually flattened with abundant irregular cytoplasm and prominent nuclei

rare, few moderate, many/low power field (e.g. many/low power field (e.g. many/lpf)

SQUAMOUS EPITHELIAL CELL Reporting:

1. lining of the vagina and female urethra

2. lower portion of the male urethra.

SQUAMOUS EPITHELIAL CELL Originate from the:

Represent normal sloughing and pose no clinical significance

SQUAMOUS EPITHELIAL CELL Clinical significance:

Female

SQUAMOUS EPITHELIAL CELL is most seen with what gender

Midstream Clean Catch collection (MSCC)

SQUAMOUS EPITHELIAL CELL remedy for contamination in female specimen

Squamous epithelial cell studded with coccobacillus

Variation of SEC Significance:

coccobacillus

the bacteria almost cover the surface of the cell and extend beyond the edges

Gardnerella vaginalis

Variation of SEC are Clue cells for ______