Ch. 7 - Microscopic Examination of Urine

what is the least standardized and most-time consuming portion of urinalysis?

microscopic urinalysis

what could be the significance of color in macroscopic examination that could relate to microscopic examination

blood, infections

what could be the significance of clarity in macroscopic examination that could relate to microscopic examination

hematuria vs. hemoglobinuria/myoglobinuria

confirm pathologic or nonpathologic cause of turbidity

what could be the significance of blood in macroscopic examination that could relate to microscopic examination

RBCs/ RBC casts

what could be the significance of protein in macroscopic examination that could relate to microscopic examination

casts/cells

what could be the significance of nitrite in macroscopic examination that could relate to microscopic examination

bacteria/ WBCs

what could be the significance of leukocyte esterase (LE) in macroscopic examination that could relate to microscopic examination

WBCs/ WBC casts/ bacteria

what could be the significance of glucose in macroscopic examination that could relate to microscopic examination

yeast

Clinical and Laboratory Standards Institute (CLSI) recommended reasons to perform microscopic analysis

requested by clinician

laboratory-specified population

• pregnant, pediatric, geriatric, diabetic, immunocompromised or renal patients

any abnormal physical or chemical results

reflex criteria

microscopic screening

identification and quantitation of insoluble substances (formed elements)

-ex. RBCs, WBCs, epithelial cells, bacteria, yeast, parasites, mucus, spermatozoa, casts, crystals, artifacts

how should the specimen be prepared for microscopic analysis

midstream clean catch - less contamination

examine when fresh or preserved

• rbcs, wbcs, and casts lyse quickly in dilute, alkaline urine

• refrigeration precipitates urates, phosphates and crystal

measure and record urine volume

• too little volume will yield fewer formed elements

examination of sediment in urine

occurs after centrifugation

low power (10x): casts and general composition

• initial focusing on low power w/ reduced light

• helpful hint- focus on epithelial cells

high power (40x): everything else

minimum 10 lpfs and 10 hpfs

• use fine adjustment continuously for best view

what are common microscopy techniques

brightfield

phase-contrast

polarizing

bright field microscopy

most commonly used microscopic technique

phase-contrast micrscopy

allows more detailed visualization of translucent or low-refractile components and living cells

polarizing light microscopy

confirms presence of cholesterol

lipids from a maltese cross pattern w/ polarized light

also helpful for evaluating crystals

• cholesterol and uric acid

how are casts reported in microscopy

avg. # seen per lpf

how are RBC/WBCs reported in microscopy

avg. number seen per hpf

number per lpf per facility

semiquantitative terms

• rare, few, moderate, or many

• 1+, 2+, 3+, 4+

how are epithelial cells and crystals reported in microscopy

how are trans/renal cells reported in microscopy

number per hpf per facility

semiquantitative terms

• rare, few, moderate, or many

• 1+, 2+, 3+, 4+

sediment stains

low refractive index elements are often difficult to see under bright-field microscopy

stains improve visibility

Sternheimer-Malbin stain

Lipid stains

Gram stain

Hansel stain

Prussian blue stain

Sternheimer-Malbin stain

sediment stain

crystal violet & safranin O

most commonly used

increases refractive index

absorbed well by WBCs, epithelial cells, and casts

• stains nuclei, cytoplasm and inclusions

Lipid stain

sediment stain

Oil Red O and Sudan II

Triglycerides, neutral fats and cholesterol

Gram stain

identification of bacterial casts

hansel stain

urinary eosinophils

methylene blue and eosin Y

Prussian blue stain

Hemosiderin granules seen w/ hemoglobinuria

Formed elements in sediment

small amounts can be normal or pathogenic based on the clinical picture

large numbers of abnormal formed elements is diagnostically significant

many urines have just a rare epithelial cell or mucus strand

some cellular elements are easily distorted

RBCs

small, smooth, non-nucleated, biconcave disks

identify and quantitate using high power (40x)

shape or appearance can aid in diagnosis

• crenated in concentrated urine (shrink)

• ghost cells (no hgb) in dilute urine (lyse)

• dysmorphic - usually benign

  • acanthocytic, fragmented or hypochromic

  • acanthocytes - glomerular disorder

what can RBCs be mistaken for when identifying elements microscopically

Yeast - look for buds

oil droplets - refractility

air bubbles - refractility and possibly in a different plane

starch - refractile, polarizes

RBCs - reagent strip correlation - positive strip for blood w/ no RBCs seen microscopically

RBCs lysed; released hemoglobin tests positive

false-positive test - detects myoglobin, peroxidases

RBCs - reagent strip correlation - negative strip for blood w/ RBCs seen microscopically

ascorbic acid interference causes false-negative result

RBC look-alikes present, such as yeast or crystals

RBC clinical significance and reference value

ref value= 0-3 or 5/hpf

damage to glomerular membrane or vascular injury to the genitourinary tract - # cells correlate with damage

• along w/ RBC casts - renal bleeding

• no casts or protein - bleeding below the kidney or caused by contamination (menstruation)

macroscopic versus microscopic hematuria

• cloudy, red urine

  • advanced disease, trauma, acute infection or coagulation disorders

• clear urine

  • early glomerular disease, malignancy, strenuous exercise or renal calculi confirmation

early glomerular disease, malignancy, strenuous exercise or renal calculi confirmation

what can clear red urine indicate

early glomerular disease, malignancy, strenuous exercise or renal calculi confirmation

what can cloudy red urine indicate

WBCs

neutrophil is predominant → visible lobes and granules

glitter cells

• hypotonic urine creates cell swelling of neutrophil

• Brownian movement of granules → cells appear to sparkle

Eosinophils

Mononuclear cells (lymphocytes, monocytes, macrophages, or histiocytes are rare)

WBCs - Eosinophils

• associated with: drug-induced nephritis and renal transplant rejection

• evaluation:

  • concentrate sediment (centrifuge or cytocentrifuge)

  • Hansel stain

  • report % eosinophils per 100-500 cells (>1% significant, seen or not seen)

WBCs - Mononuclear cells

lymphocytes, monocytes, macrophages, or histiocytes are rare

lymphocytes may resemble RBCS

• seen in early transplant rejection

differentiate from renal tubular epithelial (RTE) cells

• may need to refer to cytodiagnostic testing

WBC clinical significance and ref value

ref value= <5 per hpf

• more in females

may enter thru glomerulus or trauma but also be amoeboid migration

increased = pyuria

• bacterial infections (cystitis, pyelonephritis, prostatitis, or urethritis

• nonbacterial disorders (glomerulonephritis, lupus, interstitial nephritis, or tumors

possible presence of bacteria

squamous epithelial cells

vagina, female urethra and lower portion of male urethra

largest cell in urine

• good for focusing microscope

rare, few, mod, or many (lpf or hpf per laboratory)

Normal sloughing

clue cells

squamous cell with pathologic significance

Gardnerella vaginalis - causes bacterial vaginosis infection

• Coccobacillus covers most of the cell and extends over the edges of the squamous cell

seen in urine but more common in vaginal wet preparation

transitional epithelial cells

line renal calyces, pelvis, ureters, bladder, part of male urethra

vary in size due to separate layers

provides integrity and flexibility

centrally located nucleus

clinical significance: inflammation, infection, urinary procedure, carcinoma

what are the three forms of the transitional epithelial cells in bladder

spherical → superficial layer

caudate → intermediate layer

polyhedral → deep basal layer

clumps (malignancy) and sheets (instrumentation)

renal tubular epithelial cells

located in the kidney

involved in kindey function

each portion of the tubule is lined with different types of cell. shape varies based on location

maintain passage of water and electrolytes

clinical significance: kidney damage and renal tubular disease

PCT cells

larger than other RTEs

columnar, convoluted and/or rectangular

eccentric nucleus

DCT cells

round or oval-shaped

smaller

may resemble WBCs or spherical transitional cells

eccentrically placed nucleus

• differentiates them from spherical transitional

collecting duct RTEs

cuboidal, never round

at least one straight edge

eccentric nucleus

renal fragment

• 3 or more cuboidal cells in a group

• often large sheets

• PCT and DCT not seen in clumps

clinical significance of RTE cells

most clinically significant urine epithelial cells

indicate tubular necrosis

Renal fragments indicate basement membrane destruction

• at least 3 cells are still intact

• heavy metals, drug toxicity, hemoglobin, myoglobin, viral infections, pyelonephritis, transplant rejection, salicylate poisoning

casts usually present

Oval fat bodies

often seen with free-floating refractile droplets

renal tubular cells w/ absorbed fat

confirm w/ sudan III or Oil red O stain

• stains tryglycerides and neutral fats

view w/ polarizing light

• cholesterol polarizes

• “maltese cross”

lipiduria

• nephrotic syndrome

• acute tubular necrosis

• diabetes

• crush syndromes

bacteria

urine is usually sterile

• contaminates during voiding

• contaminants multiply fast

rods and cocci may be seen

• rods most common

nitrite helps to confirm rods, not cocci

report few, moderate, or many per hpf

WBCs should accompany bacteria in urinary tract infection (UTI)

yeast

single, refractile, budding structures or hyphae

• discern frm RBCs

few, moderate, or many per hpf

Clinical significance

• diabetic urine w/ increased glucose

• immunocompromised

• Vaginal Candida species

nitrite negative w/ WBCs present

parasites

most common: Trichomonas vaginalis

• pear shaped flagellate

• swims across field very rapidly

few, moderate, or many per hpf

may resemble WBC, transitional, or RTE cells

spermatozoa

oval, tapered heads and long tail

urine is toxic to sperm

• immobile

rarely significant

• infertility

• sperm expelled into bladder instead of urethra

• legal consequences

may cause positive protein

reporting varies w. laboratories

mucus

protein produced from RTE, glands, and epithelial cells

• composed of Tamm-Horsfall protein (uromodulin)

Threadlike w/ low refractive index

confused w/ casts

• irregular shape

UTIs, STDs, inflammation

mostly insignificant

rare, few, moderate, or many per hpf

casts

from the kidney

• formed in distal convoluted tubules and collecting ducts

parallel sides, rounded ends, inclusions

• reflect conditions of the nephron

detect under low power and identify under high power

• report avg number per 10 lpf

• scan edges of glass coverslip

composition, formation, and flow of casts

Tamm Horsfall (TH) protein secreted by renal tubular epithelial cells of the ascending loop of Henle, the distal convoluted tubule and upper portion of the collecting ducts

• normally consistent excretion

• increases w/ stress and exercise

TH protein gels

• urine stasis, acid pH, Na and Ca

TH protein not detected by reagent strips

aggregated TH structure attached to RTEs

interweaving loose network which traps elements

more interweaving to form solid matrix

attachment of elements to matrix

detachment of structure from RTEs

excretion of cast

Hyaline casts

low refractive index

colorless when unstained

use low light or phase

normal parallel sides or convoluted, wrinkled, cylindroid, occasional adhering cell or granule

clinical significance of hyaline casts

most frequently seen cast

0-2 per lpf is normal

nonpathologic

• stress, excercise, fever, heat exposure, dehydration

pathologic

• glomerulonephritis, pyelonephritis, chronic renal disease, congestive heart failure

RBC casts

orange-red color

look for cast matrix to avoid mistaking a RBC clump for a cast

confirm seeing free RBCs and positive reagent strip for blood

report number seen under lpf

clinical significance of RBC casts

bleeding w/i the nephron

glomerular damage or nephron capillary damage

• acute glomerulonephritis, nephritic syndrome

may be seen following strenuous exercise

cells begin to disintegrate w/ stasis of urine flow

hemoglobin degraded to methemoglobin

• muddy brown casts

• acute tubular necrosis

WBC casts

mostly neutrophils with multilobed nucleus and granules are seen

staining helps differentiate from RTE cells

may be tightly packed

• look for cast matrix to distinguish from WBC clump

clinical significance of WBC casts

seen w/ infection and inflammation of the tubules

pyelonephritis

• WBC casts and bacteria

acute interstitial nephritis

• WBC casts and no bacteria

may accompany RBC casts

fatty casts

seen with oval fat bodies and free fat droplets

highly refractile

• identify w/ polarized microscopy and lipid stains

nephrotic syndrome, diabetes mellitus, crush trauma, or tubular necrosis

granular casts

coarse and fine granular

detect with low power and identify with high power

pathologic or nonpathologic origin

granules disintegrate to form waxy casts

differentiate granular casts from clumps of debris and crystals

• look for cast matrix

waxy cast

brittle, highly refractile

• often fragmented with jagged ends and notches

• well visualized w/ stain

degenerated hyaline and granular casts

represent extreme urine stasis

• chronic renal failure

broad casts

chronic renal failure casts

• destruction and widening of the tubule walls

• formation in the upper collecting duct

all types of casts may be broad

material fibers, meat and vegetable fibers, hair, powder, pollen, oil droplets, fecal contamination

casts do not polarize except fatty casts

• many fibers polarize

what artifacts resemble casts and how might they be distinguished

Lower UTI

what disease state do these results indicate:

RBC: ++

WBC: +++

Trans Epi: ++

Bacteria: +

upper UTI

what disease state do these results indicate:

RBC: +

WBC: +++

Renal epi: +

WBC cast: ++

RTE cast: +

Gran. cast: ++

acute glomerulonephritis

what disease state do these results indicate:

RBC: +++

WBC: +

Renal epi: +

RBC cast: +++

WBC cast: +

RTE cast: +

Gran. cast: ++

presence of dysmorphic RBCs (acanthocytes)

chronic glomerulonephritis

what disease state do these results indicate:

RBC: +

WBC: +

Renal epi: +

RTE cast: ++

Gran. cast: ++

waxy cast: ++

presence of broad casts

nephrotic syndrome

what disease state do these results indicate:

RBC: +

renal epi: +

gran cast: +

waxy cast: +

fatty cast: +++

presence of cholesterol crystals

acute tubular necrosis

what disease state do these results indicate:

RBC: ++

WBC: ++

renal epi: +++

RTE cast: ++

gran cast: ++

waxy cast: ++

presence of proximal RTE and collecting duct RTE

renal transplant

what disease state do these results indicate:

RBC: ++

WBC: ++

renal epi: +++

RTE cast: ++

gran cast: ++

waxy cast: ++

presence of lymphocytes

urinary crystals

significant in freshly voided urine

• formed in vivo and can cause tubular damage

identified by microscopic appearance and pH

some indicate a pathologic process

report as rare, few, moderate, or many per hpf

crystal formation

precipitation of urine solutes

• salts, organic compounds, and medications

formation based on:

• temperature

• solute conc.

• urine pH

• old urine

crystals in fresh specimens are associated w. high specific gravity

general identification of crystals

most have characteristic shapes and colors

most valuable identification is urine pH

polarized microscopy characteristics are valuable in identification

solubility characteristics of crystals

temperature and pH contribute to formation and solubility

amorphous urates form in refrigerated acid urine

• dissolved w. heat

amorphous phosphates form in refrigerated alkaline urine

• dissolve in acetic acid as will RBCs

amorphous urates

yellow-brown granules microscopically

urine sediment has pink color

often in clumps

pH is usually greater than 5.5

uric acid crystals

shapes

• rhombic, whetstones, wedges, and rosettes

yellow-brown or colorless

• may resemble cystine crystals but always polarize

seen in specimens from patients

• with gout

• undergoing chemotherapy for leukemia

calcium oxalate crystals

acid and neutral pH

colorless

envelope or two-pyramid-shaped

• most common

monohydrate is oval or dumbbell-shaped

• antifreeze poisoning

is a major component of renal calculi

Triple phosphate

colorless

prism or coffin-lid shaped

polarize

no clinical significance

• highly alkaline urine and urinary tract infections (UTIs)

amorphous phosphates

may appear similar to amorphous urates

differentiate

• alkaline pH

• heavy white precipitate after refrigeration

ammonium biurate crystals

yellow-brown, spicule-covered spheres

• “thorny apples”

only urate in alkaline urine

found in old specimens and with urea-splitting bacteria producing ammonia

abnormal crystals - cystine

hexagonal, thin and thick plates

• similar to uric acid

• uric acid polarizes, but only thick cystine crystals polarize

seen in cystinuria

• inherited ability to reabsorbed cystine

• form renal calculi

abnormal crystals - cholesterol crystals

seen in refrigerated specimens

rectangular plates w. characteristic notched corners

identify by polarized light

seen in disorders w/ lipiduria

• nephrotic syndrome

• fatty casts and oval fat bodies

liver disease crystals

bilirubin crystals

tyrosine crystals

leucine crystals

bilirubin crystals

clumped needles or granules

characteristic yellow color

viral hepatitis w. tubular damage

positive reagent strip for bilirubin

liver disease crystal

tyrosine crystals

fine yellow needles in clumps or rosettes

seen w. leucine crystals

inherited amino acid disorders

liver disease crystals

will test positive on the bilirubin reagent strip

leucine crystals

yellow-brown spheres w/ concentric circles and radial striations

will test positive on the bilirubin reagent strip

ampicillin crystals

colorless needles that form bundles after refrigeration

massive dosage with inadequate hydration

sulfa crystals

most common to precipitate

variety of shapes

patient treated for UTI

radiographic dye

can form crystals in urine