UA- SOLO 3- microscopic analysis

purpose of microscopic examination

to detect and ID insoluble materials present in urine

items that contribute to formed elements include:

• blood

• kidney

• lower genitourinary tract

• external contamination

the formed elements within urine can include:

• RBCs

• WBCs

• epithelial cells

• bacteria

• yeast

• parasites

• spermatozoa

• mucus

• casts

• crystals

• artifacts

what is microscopic examination of urine MOST COMMONLY used to detect?

renal disease and/or urinary tract disease

when is microscopic examination MOST necessary? q

• abnormal findings present on chemical/physical analysis

• requested by physician based on patient’s condition/history

• lab specified population being tested

  • pregnant women

  • pediatric or geriatric

  • immunocompromised

  • renal patients

first procedure used to standardize quantitation of formed elements in urine; utilized a hemocytometer to count numbers of cells in a 12 hr urine specimen

Addis count

specimen requirements for microscopic examination

freshly voided specimens are preferred; 10-15 mL is optimal; formed elements can disintegrate rapidly in dilute or alkaline urines (RBCs, WBCs, and hyaline casts)

what should you do if microscopic examination of urine is delayed?

• refrigerate if it cannot be performed within 2 hours

• refrigeration may cause precipitation of amorphous urates and phosphates and nonpathogenic crystals; warm specimen back to RT to dissolve crystals before examination

specimen prep for microscopic analysis

centrifuge specimen in the conical tube at 2,000 RPM for 5 min; decant supernatant with a pipette; leave 1 mL of sediment; resuspend for equal distribution by gentile agitation; examine sediment

microscopic examination of urine:

• count at least 10 fields on both low (10X) and high (40X) power

• examine under LPF first to detect/count casts and obtain general composition of sediment

• NEXT, examine/enumerate other formed elements on HPF

how are RBC reported?

#RBCs/hpf

how are WBCs reported?

#WBCs/hpf

how are casts reported?

#casts/lpf (notice, low power field is where casts are counted)

how are epithelial cells, crystals, and other elements reported that aren’t RBCs/WBCs/casts?

semiquantitative terms (rare, few, moderate, many OR 1+, 2+, 3+, 4+)

what should be done w/ microscopic results that do not correlate w/ physical/chemical tests?

results should be rechecked for technical/clerical errors

color signifiance

usually pertaining to blood

clarity signifiance

can help distinguish hematuria vs hemoglobinuria/myoglobinuria; can also help confirm pathological vs nonpathological cause of turbidity

.

significance of blood in screening tests

presence of blood/RBC casts

protein significance

can indicate casts/cells present

nitrite significance

bacteria/WBCs present

leukocyte esterase significance

WBCs/WBC casts/Bacteria present

glucose significance

yeast (among other things)

purpose of sediment stains

increase overall visibility of sediment elements being examined using bright-field microscopy — can impart identifying characteristics to cellular structures, such as nuclei, cytoplasm, and inclusions

stains — action/function: sternheimer-malbin (made of crystal violet and safranin O)

delineates structure/ contrasting colors of nucleus and cytoplasm; ID’s WBCs, casts, and epithelial cells

stains — action/function: toluidine blue

enhances nuclear detail; differentiates WBCs from renal tubular epithelial cells

stains — action/function: 2% acetic acid

lyses RBCs and enhances nuclei of WBCs; distinguishes RBCs from WBCs, yeast, oil droplets, and crystals

stains — action/function: lipid stains — oil red O and sudan III

stains trigs and neutral fats orange-red; ID’s free fat droplets and lipid-containing cells/casts (does NOT stain cholesterol)

stains — action/function: gram stain

differentiates GP and GN bacteria; ID’s bacterial casts

stains — action/function: hansel stain

methylene blue and eosin Y stains eosinophilic granules; ID’s urinary eosinophils

stains — action/function: prussian blue stain

stains structures containing iron; ID’s yellow-brown granules of hemosiderin in cells/casts

phase contrast purpose in UA

enhances visualization w/ low refractive indices

polarizing microscopy purpose in UA

aids in ID of oval fat bodies, fatty casts, and crystals

bright field microscopy purpose in UA

used for routine UA, must examine the sediment under reduced light

dark field microscopy purpose in UA

enhances visualization of specimens that cannot be easily seen w/bright field microscope; aids in the ID of Treponema pallidum

Fluorescence microscopy purpose in UA

allows visualization of naturally fluorescent microorganisms or those stained by fluorescent dye

interference-contrast microscopy purpose in UA

produces a 3D image and layer-by-layer imaging

normal RBC urine sediment

0-3/hpf

normal WBC urine sediment

0-8/hpf

hyaline casts normal urine sediment

0-2/lpf

what factors must be considered when looking at microscopic results?

• recent stress

• exercise

• menstrual contamination

• presence of other sediment constituents

• etc.

the primary stain of UA sediments

Sternheimer-malbin stain

RBCs appear as…

smooth, nonnucleated, biconcave disks measuring about 7 microns in diameter; they are ID’d using hpf objective and reported as an average of 10 fields

concentrated (hypersthenic) urine may demonstrate RBCs that appear _______ due to loss of water/shrinkage

crenated

in dilue (hyposthenuria) urine, RBCs absorb water, swell, and lyse rapidly, releasing _____ and leaving only the cell membrane. these largely empty cells are called _____ cells — these cells can be easily missed if specimens are not examined under reduced light

hemoglobin; ghost (cells)

hematuria causes what color of urine?

red to brown cloudy urine

what are RBCs easily confused with?

yeast cells, oil droplets, and air bubbles

RBCs are susceptible to lysis w/ addition of which substance?

acetic acid

yeast

oil droplet

air bubble

dysmorphic RBCs…

• vary in size; have cellular protrusions or fragments

• large number assoc w/ glomerular bleeding

  • acanthocyte w/ multiple protrusions is most associated

• demonstrated after strenuous exercise, indicating glomerular origin

• caused by differences in urine conc…

RBCs lyse most rapidly in what type of urine?

dilute, alkaline urine

normal vs ghost RBC

created RBC vs. normal RBC from a side view

what are ways RBCs can be differentiated from other sediment features?

• refractivity of oil droplets/air bubbles — can be seen w/ fine focus adjustment up and down; they may also appear in a different plane from other constituents

• rough, crenated RBCs may resemble WBCs, but they are generally much smaller

• acetic acid to lyse RBCs — everything else remains in tact

• budding of yeast present

• supravital staining

presence of RBCs in urine is associated w/ damage to…

• the glomerular membrane

• vascular injury within the genitourinary tract

true or false: number of RBCs present in urine indicates the extent of damage/injury

TRUE

RBCs detected in urine can aid in early diagnosis of what? what else is it associated with?

early diagnosis for:

• glomerular damage

• malignancy of the urinary tract

• confirm presence of renal calculi

also assoc. w/:

• trauma

• acute infection

• inflammation

• coagulation disorders

• contamination

• exercise

characteristics of MACROscopic hematuria:

• urine appears cloudy w/ red to brown color

• >100 RBCs/hpf

• associated w/ advanced glomerular damage

• also seen with damage to vascular integrity of the urinary tract caused by trauma, acute infection, or inflammation

• also associated with coagulation disorders

characteristics of MICROscopic hematuria:

• can be critical to the early diagnosis of glomerular disorders and malignancy of the urinary tract as well as to confirm the presence of renal calculi

• may also see hyaline, granular and RBCs casts following strenuous exercise

• can be seen with nonpathological or pathological origins

• menstrual contamination can also be associated

predominant WBC found in urine

neutrophils

WBCs vs RBCs

larger than RBCs, measuring an average of 12 microns in diameter; nucleated

what type of urine can lyse neutrophils?

dilute, alkaline urine

what type of movement is seen in the granules of neutrophiles when they swell from hypotonic urine

Brownian movement

what are glitter cells?

when the neutrophils produce a ‘sparkling appearance’ from the Brownian movement of their granules in hypotonic urine; not clinically significant

what stain can be used for urine eosinophils?

Hansel stain (wright’s or Giemsa can also be used, but are not preferred)

are eosinophils normally found in urine?

NO

what % of urine eos is signifiant?

more than 1% out of 100-500 cells counted is considered significant

when can urine eosinophils be seen?

• drug-induced interstitial nephritis

• UTI

• renal transplant rejection

what can be done to differentiate mononuclear cells from RTE cells in urine sediment?

• supravital staining

• addition of acetic acid for nuclear clarity

• cytodiagnostic urine testing

higher amounts of WBCs are seen in men or women?

women

WBCs and RBCs may enter the urine through ____ or _____ trauma, but they can also enter through amoeboid action…

glomerular or capillary

what type of action do WBCs use to migrate through tissues to sites of infection?

amoeboid action

pyuria

increased WBCs in urine; indicates infection or inflammation in the genitourinary system

bacterial infections associated w/ pyruria include:

• pyelonephritis (kidney)

• cystitis (bladder)

• prostatitis

• urethritis

non-bacterial inflammation associated with pyruria includes:

• glomerulonephritis (post-strep infection antibody complexes deposit)

• lupus erythematosus

• interstitial nephritis

• tumors

epithelial cells can be found in the urine normally, as long as they are not present in large numbers or abnormal forms; they represent normal sloughing of old cells. the three types of epithelial cells seen in urine include:

1. squamous

2. transitional (urothelial)

3. renal tubular

squamous epithelial cells

largest found in urine sediment; may contain abundant, irregular cytoplasm and a prominent nucleus about the size of an RBC; may be folded; may resemble casts; disintegrate in old urine; a few can normally be found in urine; usually reported as rare, few, moderate, or many

where do squamous epithelial cells originate from?

linings of the vagina and female urethra and the lower portion of the male urethra; represent normal cell sloughing and have no pathological significance; inc amounts seen more frequently in female urine — this is why clean catch urine is preferred…

what form of squamous epithelial cells HAS clinical significance?

clue cells

clue cell…

variation of squamous epithelial cells; clinical significance: indicative of vaginal infection by a bacterium known as Gardnerella vaginalis.

Appears as squamous epithelial cell covered w/ coccobacillus organism; routine testing is ID’d by vaginal wet preparation; small numbers of clue cells may be present in urinary sediment

AKA urothelium or uroepithelium; this type of epithelium that lines much of the urinary tract, including the renal pelvis, the ureters, the bladder, and parts of the urethra; they are smaller than squamous cells and appear in various forms due to their ability to absorb large amounts of water. the three forms include: ________,________, and _________. all forms have a distinct, ______ located nuclei.

1. spherical

2. polyhedral

3. caudate

CENTRALLY (located nuclei)

transitional epithelial cells presence in urine

present in small numbers in normal urine — represent normal cell sloughing; increased numbers seen singly, in pairs, or in clumps — present following invasive urologic procedures, such as cetherization or cytoscopy

syncytia

group of cells with/ continually joining cell walls

transitional epithelial cells have no clinical significance unless morphologically abnormal, such as…

vacuoles or irregular nuclei that may indicate malignancy or viral infection

polyhedral transitional epithelial cell

spherical transitional epithelial cell

caudate transitional epithelial cell

syncytia (transitional)

are RTEs normally found in urine in any circumstances?

NO

what is the criterial for calling something an RTE?

• nucleus present

• large and eccentric nucleus

RTE cells come from various areas of the ______, and they vary on size and shape depending on which area they come from

tubules

RTEs that originate from the proximal convoluted tubule

large and rectangular w/ coarse granular cytoplasm; referred to as columnar or convoluted cells often resembling casts

RTEs from the distal convoluted tubule

cells are smaller and round to oval; mistaken for WBCs and spherical transitional epithelial cells

RTEs of the collecting duct

cuboidal and are NEVER round; has at LEAST one straight edge; may occur in groups of three or more called renal fragments

more than 2 RTEs/hpf indicates what type of injury?

tubular injury

increased numbers of RTEs indicate necrosis of the renal tubules with the possibility of it affecting overall renal function:

• heavy metal exposure

• drug-induced toxicity

• hemoglobin/myoglobin toxicity

• viral infections (i.e., hep b)

• pyelonephritis

• allergic reactions

• malignant infiltrations

• salicylate poisoning

• acute allogenic transplant rejection

• also seen as secondary effects of glomerular disorders

renal fragments (groups of RTEs) represent…

severe tubular injury w/ basement membrane disruption

since RTEs funciton to reabsorb glomerular filtrate, it can absorb things such as…

• bilirubin

• hemoglobin —> hemosiderin granules (prussian blue can pick up)

• lipids

oval fat bodies are..

RTEs that have absorbed lipids present in the glomerulus…