MICROSCOPIC URINALYSISURINARY SEDIMENTS

Importance integral part of the urinalysis (UA). Sediment findings often are necessary for the proper interpretation of results of the

physicochemical portion of the UA Detect and identify insoluble materials in the urine.

Limitations Least standardized Time consuming Expensive Requires Technical expertise

“ The decision to perform microscopic examinations should be made by each individual laboratory based on its specific patient population.”

-CLSI

Specimen consideration Fresh or adequately preserved Mid-stream clean catch First morning specimen Thoroughly mixed

VOLUME: 10-15 mL Indicate if lesser volume is used Correct for volumes

Specimen Centrifugation 5 minutes 400 RCF (relative centrifugal force) Braking mechanism is not recommended

SEDIMENT PREPARATION 0.5 – 1.0 mL Aspirate off the supernatant Thorough resuspension of the sediment

SEDIMENT VOLUME: 20 uL (0.02 mL) Cover slip overflow of sediment not allowed

RCF Centrifugal force – how many times greater than gravity

- expressed as relative centrifugal force (RCF) or g- depends on three variablesi. speed- expressed as revolutions per minute (rpm)

**related to RCF by the following equation:RCF = 1.118 x 10-5 x r x (rpm)2

ii. mass iii. radius(r)- measured from the center of the centrifugal axis to the bottom of the test tube shield.

Constituents of urine sediment Organized sediment – biological source

RBC WBC Epithelial cells Fats Casts Bacteria Yeast Fungi Parasite spermatozoa

Unorganized sedimentchemical source

Normal acid crystals Normal Alkaline crystals Abnormal crystals of metabolic origin Abnormal crystals of iantrogenic origin

Red Blood Cells (RBC) Smooth, non-nucleated, biconcave disk 7 um in diameter Examined under HPO Reported as average in 10 HPFs

Variations:1. Crenated – found in concentrated urine2. Ghost cells – cell membrane found in dilute urine3. Dysmorphic – cells of varying sizes, have cellular protrusions or are fragmented

Normal and Crenated RBC

Normal and Dysmorphic RBC

Clinical Significance Damage to the glomerular membrane Vascular injury within the genito-urinary tract Increased following strenuous exercise Contamination with menstrual blood

GROSS HEMATURIA: Advanced glomerular damage Trauma damaging vascular integrity Acute inflammation and infection Coagulation disorder

White Blood Cells (Neutrophils) Larger than RBCs measuring 12 um in diameter. Predominantly neutrophils Contains granules and multi-lobed nuclei Reported as average number per 10 hpf “Glitter Cells” – disintegrated neutrophils Easily lyses in dilute alkaline urine

Uncommon WBC

Eosinophils – associated with drug induced interstitial nephritis, UTI and renal transplant rejection

Reported as a percentage in 100 to 500 WBC 1 % eosinophil is significant Preferred stain – Hansel

Mononuclear Cells – increased in the early stages of transplant rejection Appears vacuolated and contains inclusions Diagnosed by cytocentrifugation and Wright’s stain

Clinical SignificancePyuria – increase in urinary WBCs

Indicates: -infection or inflammation in the genitourinary system-Bacterial infection: pyelonephritis, cystitis, prostatitis and urethritis -Non-bacterial disorders – glumerulonephritis, LE, Interstitial nephritis and tumors

Epithelial Cells Represents the normal sloughing of old cells Clinically insignificant in small numbers May be contamination from the genitalia Three types are seen in urine classified according to their site of origin in the genitourinary

tract.- Squamous epithelial cells- Transitional epithelial (Urothelial) cells- Renal Tubular Epithelial Cells

Squamous epithelial cells Largest cells found in the urine sediment Contains abundant cytoplasm with a prominent nucleus Originates from the linings of the vagina and female urethra and the lower portion of the

male urethra. No clinical significance Usually increased in females Reported in words as rare, few, moderate or many

Clue Cells Indicates infection vaginal infection with Gardnerella vaginalis Squamous cells covered with the bacteria

Transitional Epithelial Cells(Urothelial) Smaller than squamous cells Appears in several forms (polyhedral, spherical, caudate) due to its ability to absorb water. Originates from the lining of the renal pelvis, calyces, ureters and bladder and the upper

portion of the male urethra No clinical Significance Increased in invasive urologic procedures such as catheterization Presence of vacuoles and irregular nuclei may indicate viral infection or malignancy

Renal Tubular Epithelial Cells The most clinically significant of the epithelial cells. Morphology varies depending on the site of origin

PCT – largest of the RTE, rectangular, coarsely granular cytoplasm.DCT – smaller, round or oval, with eccentrically placed round nucleusCollecting ducts – cuboidal, never round, eccentrically placed nucleus, one side is straight,

appears in sheets

RTE Clinical Significance Increase indicate necrosis of the renal tubules

- exposure to heavy metals- drug induced toxicity- hemoglobin and myoglobin toxicity- viral infection-pyelonephritis - allergic reaction- malignant infiltration-salicylate poisoning-acute allogenic transplant rejection

Oval Fat Bodies RTE cells that absorb lipids present in the glomerular filtrate Highly refractile Seen along with free-floating fat droplets Stains well with Sudan III and Oil Red O Composed of triglycerides, neutral fats and cholesterol “Maltese Cross” – observed in the presence of cholesterol under polarized light

Maltese Cross

Casts Unique to the kidney Most difficult to recognize and most important sediment Represent a biopsy of the tubules Must be observed under subdued light because of the low refractive index of the cast

matrix Reported as the average number in 10 lpfs Disintegrates in dilute alkaline urine.

Composition of Casts Major constituent: Tamm-Horsfall protein The glycoprotein gels easily under conditions of urine stasis, acidity and the presence of

sodium and calcium Width of the cast depends on the size of the tubule

Cast Formation Formed in the lumen of the DCT and collecting ducts

Hyaline Cast The most commonly seen cast in the urine

Consist almost entirely of Tamm-Horsfall protein Colorless, homogenous, non-refractive, semi-transparent 0-2/lpf is normal Seen in strenous exercise, dehydration, heat exposure, and emotional stress Increased in acute glomerulonephritis, pyelonephritis, chronic renal disease and

congestive heart failure

RBC Cast

Indicates bleeding within the nephron Primarily associated with damage to the glomerulus. Also associated with proteinuria and dysmorphic RBC Orange-red in color Dirty brown cast indicates hemoglobin degradation and associated with acute tubular

necrosis.

WBC Casts Indicates infection or inflammation within the nephron. Associated with pyelonephritis and differentiates upper UTI from lower UTI Also seen in acute interstitial nephritis and glomerulonephritis. Appears granular and multilobed

Epithelial Cell Casts

Contains RTE cells Indicates advanced tubular destruction Seen in heavy-metal and drug induced toxicity, viral infections, allograft rejections and

pyelonephritis.

Fatty Casts

Associated with oval fat bodies and free fat droplets in cases of lipiduria Indicates nephrotic syndrome, toxic tubular necrosis, DM and crush injuries Highly refractile, confirmed with Sudan III and Oil Red O using polarized light

Mixed Cellular Casts

Contains more than one type of cell Usual combinations: WBC and RBC in glomerulonephritis, WBC and RTE or WBC and

bacteria in pyelonephritis

Makes identification difficult

Granular Casts

May appear finely of coarsely Non-pathologic increase in strenuous exercise In diseases, it indicates disintegration of cellular casts

Waxy Casts

Represents extreme urine stasis indicating chronic renal failure. Presents brittle, highly refractile cast matrix due to disintegration of hyaline and other

cellular components of the cast Appears fragmented with jagged edges and notches on their sides

Broad Casts

Also referred to as renal failure casts Represents extreme urine stasis Indicates destruction of the tubular walls Commonly of the granular and waxy types.

Cast Formation

Other Casts

Rare incorporation of other structures in the urine sediments Pigmented Casts – hemoglobin, myoglobin and certain drugs Hemosiderin casts Crystal casts – urates, calcium oxalates and sulfonamides

Cylindroids Resemble casts but have one end that tapers to a tail Found in conjunction with casts and have same significance

Mucus Threads

Long thin waxy threads, very transparent Can be found in small number in normal urine

Increased numbers indicate inflammation or irritation of the urinary tract

Bacteria

Not normally seen in urine Results from vaginal, urethral, external genitalia or specimen container contamination Presents as cocci or bacilli Usually motile May Indicate UTI if seen in freshly voided urine and correlated with WBCs

Yeast

Small, refractile, oval structures which may show budding In severe in fections, mycelium may be seen Most common: Candida albicans Seen in DM, immunocompromised patients and women with vaginal moniliasis Accompanied by WBCs

Fungi

In severe infections May include appearance of mycelium

Parasites

Most frequent: Trichomonas vaginalis – pear shaped flagellate with undulating membrane In fresh wet preparations, usually motile with rapid darting movements Other parasites: Schistosoma haematobium, Enterobius vermicularis, other parasite

contaminants from the feces

T. vaginalis

S. haematobium

E. Vermicularis

Spermatozoa

Oval, slightly tapered heads and long flagella like tails, usually non-motile Seen in urine of both female and male after intercourse and in male urine after

masturbation and nocturnal emission Not clinically significant except in cases of male infertility and retrograde ejaculation Also important in medico-legal cases

Urinary Crystals

Formed by the precipitation of urine solutes Rarely of clinical significance Reported in words Identified in order to detect the few abnormal crystals

Crystal Formation

In vivo factors include: the concentration and solubility of crystallogenic substances contained in the specimen,

the urine pH the excretion of diagnostic and therapeutic agents.

In vitro factors include: temperature (solubility decreases with temperature), evaporation (increases solute concentration), urine pH (changes with standing and bacterial overgrowth).

Amorphous Urates

Amorphous urates appear as aggregates of finely granular material without any defining shape

Amorphous urates (Na, K, Mg, or Ca salts) tend to form in acidic urine

May have a yellow or yellow-brown color. Common in refrigerated specimens wit

pink sediments

Uric Acid

May appears as Rhombic, foursided flat planes, wedges, and rosettes Usually yellow-brown but may appear colorless Highly birefrigent under polarized light Increased in high levels of purines and nucleic acids Seen in patients with leukemia undergoing chemotherapy, Lesch-Nyhan syndrome and

gout

Calcium Oxalate Dihydrate

Calcium oxalate dihydrate crystals typically are seen as colorless squares whose corners are connected by intersecting lines (resembling an envelope).

They can occur in urine of any pH.

The crystals vary in size from quite large to very small.

In some cases, large numbers of tiny oxalates may appear as amorphous unless examined at high magnification.

Increased in high intake of oxalic acid and ascorbic acid

Calcium Oxalate Monohydrate

Less frequently seen Oval or dumbbell shaped Birefrigent Indicates ethylene glycol poisoning

Normal Crystals seen in neutral to alkaline urine

Amorphous phosphate Triple Phosphate Calcium Phosphate Calcium Carbonate Ammonium Biurate

Amorphous Phosphates

Morphologically resemble amorphous urates Increased in refrigerated sample but gives a white color Can be differentiated from urates by the pH of the urine and its non-dissolution on

warming.

Triple Phosphate, Struvite, Ammonium Magnesium phosphate

appear as colorless, 3-dimensional, prism-like crystals ("coffin lids"). Occasionally, they instead resemble an old-fashioned double-edged razor blade Birefrigent on polarized light

Calcium Phosphate

Colorless Shape: long, thin prisms with one pointed and arranged as rosettes or clusters of needles Thin irregular plates that float on surface of urine Associated with renal calculi Dissolves in dilute acetic acid

May be confused with sulfonamide crystal

Calcium Carbonate

Calcium carbonate crystals usually appear as large yellow-brown or colorless spheroids with radial striations.

They can also be seen as smaller crystals with round, ovoid, or dumbbell shapes Liberates gas on addition of acetic acid

Ammonium Biurate

Color: yellow to brown Shape: Spherical bodies with long irregular spicules Often described as thorn- apple Associated with the presence of ammonia from urea-splitting bacteria

Soluble in acetic acid and Heat

Abnormal Urine crystals of Metabolic Origin

Seen in acidic to neutral urine Requires chemical confirmation

- Cystine -Tyrosine-Leucine -Cholesterol- Bilirubin

Cystine

Colorless, refractile, hexagonal plates that are often laminated Seen in patients with cystinuria Disintegrates in alkaline urine Soluble in ammonia and dilute HCl Confirmed by the cyanide nitroprusside reaction

Tyrosine

Colorless, fine, silky needles arranged in sheaves or clumps Seen in hereditary tyrosinosis, oasthouse urine disease and with leucine in massive liver

failure Confirmed by the nitrosonaphthol test or HPLC

Leucine

Yellow, oily looking spheres with radial and concentric striations Extremely rare Seen in severe liver damage with tyrosine

Cholesterol

Color: transparent Shape: regular to irregular flat plates with one corner notched out, may be single or in

larger numbers Most often found after refrigeration Indicates Excessive tissue breakdown Seen in nephritis and nephritic syndrome Soluble in chloroform

Bilirubin

Bilirubin crystals tend to precipitate onto other formed elements in the urine. fine needle-like crystals can form on an underlying cell. This is the most common

appearance of bilirubin crystals. cylindrical bilirubin crystals can form in

association with droplets of fat, resulting in a "flashlight" appearance. This form is less commonly seen.

Seen in Obstructive jaundice Bilirubin must be present in urine

Abnormal Crystals of Iantrogenic origin

Caused by increased amount of drugs Important because of the likelihood of renal damage and bleeding leading to renal failure

-Sulfonamides-Ampicillin -Radiographic contrast media

Sulfonamides

Color: brown to yellow Shape: needle-like shapes seen in bundles or

sheaves; Stacks of wheat Common forms: sulfamethoxazole,

acetylsulfadiazine and sulfadiazine

Ampicillin

Long, thin, colorless needles in acidic urine Very rarely seen Seen in Administration of large parenteral doses

Radiographic contrast media

Color: opaque , appear dark and thick Shape: pleomorphic needles, single or sheaves May be mistaken for cholesterol crystals Significant in elderly patients Intravenous injection for radiography Can appear up to 3 days after injection

Contaminants and Artifacts Usually easy to see Causes distraction on the observer

- Starch-Fibers-Air bubbles-Oil droplets-Glass Fragments- Stains- Pollen grains- Fecal contamination