MICROSCOPIC EXAMINATION OF URINE

MICROSCOPIC EXAMINATION OF URINE
CHAPTER 6

Learning Objectives Upon completing this chapter, the reader will be able to List the physical and chemical parameters included in macroscopic urine screening, and state their significance. Discuss the advantages of commercial systems over the glass-slide method for sediment examination. Describe the recommended methods for standardizing specimen preparation and volume; centrifugation; sediment preparation, volume, and examination; and reporting results. State the purpose of Sternheimer-Malbin, acetic acid, toluidine blue, Sudan III, Gram, Hansel, and Prussian blue stains in the examination of urine sediment. Identify specimens that should be referred for cytodiagnostic testing.

Learning Objectives (cont’d)
Describe the basic principles of bright-field, phase-contrast, polarizing, dark-field, fluorescence, and interference-contrast microscopy, and their relationship to sediment examination. Differentiate between normal and abnormal sediment constituents. Discuss the significance of red blood cells (RBCs) in urine sediment. Discuss the significance of white blood cells (WBCs) in urine sediment. Name, describe, and give the origin and significance of the three types of epithelial cells found in urine sediment. Discuss the significance of oval fat bodies.

Learning Objectives (cont’d)
Describe the process of cast formation. Describe and discuss the significance of hyaline, RBC, WBC, bacterial, epithelial cell, granular, waxy, fatty, and broad casts. List and identify the normal crystals found in acidic urine. List and identify the normal crystals found in alkaline urine. Describe and state the significance of cystine, cholesterol, leucine, tyrosine, bilirubin, sulfonamide, radiographic dye, and ampicillin crystals. Differentiate between actual sediment constituents and artifacts. Correlate physical and chemical urinalysis results with microscopic observations and recognize discrepancies.

Introduction Microscopic examination of the urinary sediment
Identification of insoluble substances (formed elements) Red blood cells (RBCs) White blood cells (WBCs) Epithelial cells Casts Bacteria Yeast Parasites Mucus Spermatozoa Crystals Artifacts Least standardized, most time consuming

Macroscopic Screening
Microscopic is performed based on physical and chemical results Color, clarity, blood, protein, nitrite, leukocyte esterase, and possibly glucose Special populations: pregnant women; pediatric, geriatric, diabetic, immunocompromised, and renal patients

Clinical and Laboratory Standards Institute (CLSI)
Requested by the physician Laboratory-specified population Any abnormal physical or chemical result

Specimen Preparation Examine when fresh or preserved
RBCs, WBCs, casts disintegrate in dilute, alkaline urine Refrigeration precipitates crystals Can obscure other elements Less contamination (epithelial cells) from a midstream clean-catch specimen Thoroughly mix specimen before decanting to the centrifuge tube

Specimen Volume Centrifuge 10 to 15 mL urine (reagent strips fit into 12 mL) Quantities <12 mL should be documented Too little volume = fewer formed elements Some laboratories correct for volume

Centrifugation Standardize speed and time of centrifugation
5 min at relative centrifugal force (RCF) of 400 is ideal RCF corrects for variations in the diameter of centrifuge heads; revolutions per minute does not RCF = × 10−5 × radius in centimeters × RPM2 Do not brake the centrifuge Cap all specimens

Sediment Standardization
Preparation of sediment Volume of sediment examined 0.5 to 1.0 mL Methods of visualization Reporting of results Commercial systems: KOVA Calibrated centrifuge tubes, special slides to control volume, decanting pipettes, grids for better quantitation

Postcentrifuge Sediment
0.5 to 1.0 mL after decantation Concentration factor: volume of urine centrifuged/sediment volume Probability of detecting low quantities of formed elements Aspirate rather than pour off urine (pipettes available for this) Mix sediment gently, not vigorously

Volume of Sediment Examined
Be consistent Commercial systems control this Glass slide method 20 μL 22 × 22 glass cover slip Do not overflow cover slip Heavier elements (casts) flow outside

Commercial Systems Chambers capable of containing a standardized
Chamber volume Size of the viewing area Approximate number of low-power and high-power viewing areas Based on the area of the field of view using a standard microscope CLSI recommends these systems together with standardization of all phases of the methodology

Commercial Systems (cont’d)
Capped, calibrated centrifuge tubes Decanting pipettes to control sediment volume Slides that Control the amount of sediment examined Produce a consistent monolayer of sediment for examination Provide calibrated grids for more consistent quantitation

Examination of Sediment
Be consistent Minimum 10 low (10×) and 10 high (40×) fields Low power: casts, general composition Scan edges for casts with glass slide method High power: identification of type Initial focusing: low power, reduced light Focus on epithelial cell, not artifacts that are in a different plane Use fine adjustment continuously for best view

Reporting the Microscopic Examination
Consistent within laboratory Casts: average per lpf RBCs, WBCs: average per hpf Epithelial cells, crystals, etc., in semiquantitative terms Few, moderate, many 1+, 2+, 3+, 4+ Follwed by /lpf or /hpf

Reporting the Microscopic Examination (cont'd)
Converting the average number of elements per lpf or hpf to elements per mL 1. Calculating the area of an lpf or hpf for the microscope in use using the manufacturer-supplied field of view diameter and the formula πr2 = area Diameter of hpf = 0.35 mm 3.14 × = mm2 2. Calculating the maximum number of lpfs or hpfs in the viewing area; area under a 22 mm × 22 mm cover slip = 484 mm2 484 = 5040 hpfs

Reporting the Microscopic Examination (cont'd)
3. Calculating the number of hpfs per milliliter of urine tested using the concentration factor and the volume of sediment examined 5040____ = 5040 = 21,000 hpf/mL of urine 0.02 mL x Calculating the number of formed elements per milliliter of urine by multiplying the number of hpfs per milliliter by the average number of formed elements per field 4 WBC/hpf × 21,000 = 84,000 WBC/mL

Correlating Results Microscopic Elements Physical Chemical Exceptions
RBCs Turbidity Red color + Blood + Protein Number Hemolysis WBCs + Nitrite Lysis + LE Epithelial cells Casts Bacteria pH Number and type + Leukocytes Crystals Color + Bilirubin Table 6-2 Routine Urinalysis Correlations

Sediment Examination Techniques
Sediment appearance Cells and casts in various stages of development and degeneration Distortion of cells and crystals by the chemical content of the specimen The presence of inclusions in cells and casts Contamination by artifacts

Sediment Stains Low refractive index elements are often difficult to see under bright-field microscopy Sternheimer-Malbin stain: crystal violet /Safranin O Increases refractive index Stains nuclei, cytoplasm, inclusions Sedi-Stain, KOVA stain, etc. 0.5% solution of toluidine blue enhancement of nuclear detail Acetic acid will enhance WBC nuclei RBCs are lysed by this

Sediment Stains (cont’d)
Lipid stains Oil Red O and Sudan III for triglycerides and neutral fats; cholesterol polarizes Gram stain Identification of bacterial casts Hansel stain Urinary eosinophils Methylene blue and eosin Y: better than Wright stain Prussian blue stain Hemosiderin granules seen with hemoglobinuria

Cytodiagnostic Urine Testing
Cytodiagnostic urine testing is frequently performed to detect and monitor renal disease/malignancies Preparation of permanent slides using cytocentrifugation Papanicolaou stain Transplant rejection Viral, fungal, and parasitic infections Cellular inclusions Pathologic casts Inflammatory conditions

Microscopy Bright field most common in urinalysis
Reduced light is essential Magnification is 10× and 40× Par focal means minimal adjustment when changing objectives (use fine adjustment) Lower light using the rheostat Condenser can be raised up and down Do not use the aperture diaphragm Others include phase contrast, polarizing, dark field, fluorescence, and interference contrast

Microscopy (cont’d) Phase-contrast microscopy Polarizing microscopy
Increases refractive index Polarizing microscopy Crystals and lipids Ability to split light into two beams Crystals are multicolored Cholesterol produces Maltese cross formations Interference-contrast microscopy Three-dimensional images

The Microscope Compound bright-field microscope Two-lens system
In the oculars, the objectives The coarse- and fine-adjustment knobs Illumination system Light source, condenser, and field and iris diaphragms Body consisting of Base Body tube Nosepiece Mechanical stage

The Microscope (cont’d)

Binocular 10× Adjusts for interpupillary distance Field of view is determined by the eyepiece and is the diameter of the circle of view when looking through the oculars Objectives: near specimen UA sediment magnifications of 10× (low power, dry), 40× (high power, dry) Final magnification of an object is the product of the objective magnification times the ocular magnification

Objective characteristics Type of objective, magnification, numerical aperture, microscope tube length, and cover-slip thickness to be used Length of the objectives attached to the nosepiece varies with magnification Changing the distance between the lens and the slide when they are rotated Parfocal Only minimum adjustment when switching among objectives

The distance between the slide and the objective is controlled by the coarse and fine focusing knobs Coarse focus: initial focusing Fine focus: sharpen image, focusing after changing magnification

Illumination Base Equipped with rheostat Regulates intensity Filters vary illumination and wavelength Diaphragm contained in the light source controls the diameter of the light beam Condenser located below the stage to focus the light All have adjustments for optimal lighting

Köhler illumination: provide optimal viewing of the illuminated field

Care of the Microscope 1. Carry microscope with two hands, supporting the base with one hand. 2. Always hold the microscope in a vertical position. 3. Only clean optical surfaces with a good quality lens tissue and commercial lens cleaner. 4. Do not use the 10× and 40× objectives with oil. 5. Clean the oil immersion lens after use. 6. Always remove slides with the low-power objective raised. 7. Store the microscope with the low-power objective in position and the stage centered.

Urine Sediment Constituents
Small amounts of constituents can be normal or pathogenic based on the clinical picture Many urines have just a rare epithelial cell Some constituents are easily distorted Concentrations, pH, and presence of metabolites Normals are not clearly defined

RBCs Identification difficulties Yeast: look for buds
Oil droplets: refractility Air bubbles: refractility and possibly in a different plane Starch: refractile, polarizes Reagent strip correlation

RBCs (cont’d) Smooth, nonnucleated, biconcave disks ~7 µm
Crenated in hypersthenuric urine Ghost cells in hyposthenuric urine Identify using high power

RBCs (cont’d) Air Bubble Oil Droplets

RBCs (cont’d) Dysmorphic RBCs Glomerular bleeding Strenuous exercise
Acanthocytic, blebs Fragmented, hypochromic Aid in diagnosis

Clinical Significance
Normal value: 0–3 to 5/hpf Damage to glomerular membrane or vascular injury to the genitourinary tract Number of cells = extent of damage Macroscopic versus microscopic hematuria Cloudy, red urine, advanced disease, trauma, acute infection, coagulation disorders Clear urine, early glomerular disease, malignancy, strenuous exercise, renal calculi confirmation

WBCs 12 µm Neutrophil is predominant Identify under high power
Glitter cells Hypotonic urine Brownian movement Swell; granules sparkle Pale blue if stained Nonpathologic

WBCs (cont’d) Glitter cell

WBCs (cont’d) Eosinophils Hansel stain
Drug-induced interstitial nephritis Renal transplant rejection Hansel stain Percent per 100 to 500 cells >1% significant Concentrate sediment, centrifuge, or cytocentrifuge

WBCs (cont’d) Mononuclear cells
Lymphocytes, monocytes, macrophages, histiocytes are rare Differentiate from renal tubular epithelial (RTE) cells Staining Lymphocytes may resemble RBCs; seen in early transplant rejection May need to refer to cytodiagnostic testing

Normal = <5 per hpf, more in females May enter through glomerulus or trauma but also by amoeboid migration Increased WBCs = pyuria Infections: cystitis, pyelonephritis, prostatitis, urethritis Glomerulonephritis, lupus erythematosus, interstitial nephritis, tumors Report presence of bacteria

Epithelial Cells Three types Classification Squamous
Transitional (urothelial) RTE Classification Squamous: vagina, male and female urethra First structures observed Transitional: bladder, renal pelvis, calyces, ureters, upper male urethra RTE: renal tubules

Squamous Epithelial Cells
Largest cell in urine Good for focusing microscope Rare, few, moderate, many lpf or hpf per laboratory Normal sloughing Contamination if not midstream clean-catch

Squamous Epithelial Cells (cont’d)

Clue Cells Squamous cell with pathologic significance
Gardnerella vaginalis: vaginal infection Coccobacillus sp. covers most of the cell and extends over the edges Seen in urine but more common in vaginal wet preparation

Transitional Epithelial (Urothelial) Cells
Three forms Spherical: absorb water in bladder and become large and round Caudate: appear to have a tail Polyhedral: multiple sides Differentiate from RTE Centrally located nucleus Syncytia = clumps Catheterization Malignancy

Transitional Epithelial (Urothelial) Cells (cont'd)

Renal Tubular Epithelial Cells
Size and shape vary with renal tubular area Columnar = proximal convoluted tubule (PCT) Round, oval = distal convoluted tubule (DCT) Cuboidal = collecting duct Three or more cuboidal cells = renal fragment

PCT Cells Larger than other RTEs Columnar, convoluted, rectangular
May resemble casts Coarsely granular cytoplasm Notice presence of nucleus

DCT Cells Round or oval shaped, smaller
May resemble WBCs or spherical transitional cells Observe the eccentrically placed nucleus to differentiate from spherical transitional

Collecting Duct RTEs Cuboidal, never round
At least one straight edge Eccentric nucleus Three or more cells in clump is renal fragment; often large sheets PCT and DCT not seen in clumps

RTE cells are the most clinically significant urine epithelial cells; indicate tubular necrosis; fragments indicate severe destruction Heavy metals, drug toxicity, hemoglobin, myoglobin, viral infections, pyelonephritis, transplant rejection, salicylate poisoning Single cuboidal cells = salicylate poisoning Absorb: bilirubin, hemoglobin, lipids Hemosiderin stains with Prussian blue

RTE cells

Oval Fat Bodies RTE cells that have absorbed lipid in the filtrate
Also free-floating refractile droplets Maltese cross formation with polarized light If negative check with Sudan III or oil red O stain

Oval Fat Bodies (cont’d)
Stain polarizing negative structures Cholesterol polarizes Triglycerides and neutral fats stain Lipiduria: nephrotic syndrome, acute tubular necrosis, diabetes, crush syndromes

Bacteria Urine is usually sterile, contaminated on the way out; contaminants multiply fast WBCs should accompany bacteria in UTI Report few, moderate, many per hpf Rods and cocci may be seen; rods most common Nitrite helps to confirm rods, not cocci

Yeast Single, refractile, budding structures
Mycelial forms may be present Report: few, moderate, many Diabetic urine: ↑ glucose and acid ideal for yeast growth Immunocompromised, vaginal moniliasis Nitrite negative, WBCs present Confuse with RBCs

Yeast (cont’d)

Parasites Most common: Trichomonas vaginalis
Pear-shaped flagellate Swims across field rapidly Report few, moderate, many If not moving, may resemble WBC, transitional, or RTE cells Also Schistosoma haematobium and Enterobius vermicularis

Parasites (cont’d)

Spermatozoa Oval, tapered heads and long tail
Urine is toxic to sperm, so they are immobile Rarely significant, infertility: sperm expelled into bladder instead of urethra May cause positive protein Reporting varies with laboratories Lack of clinical significance, legal consequences

Mucus Protein from RTE, glands, squamous cells
Threadlike, low refractive index Confuse with casts Irregular, composed of uromodulin protein Female specimens, no clinical significance

Casts Elements unique to the kidney Formed in DCT and collecting duct
Parallel sides, rounded ends, inclusions Detect under low power, ID high power Scan edges of glass cover slip Low light is essential Report number per lpf Many pathologic and nonpathologic causes

Composition and Formation
Uromodulin protein secreted by RTE of DCT and collecting duct Consistent excretion normally ↑ stress and exercise Formation of protein fibrils into matrix Urine stasis, acid pH, Na, and Ca Uromodulin protein not detected by reagent strips ↑ protein is from renal disease

Composition and Formation (cont’d)
Aggregated uromodulin fibrils attached to RTEs Interweaving to loose network, traps elements More interweaving to form solid matrix Attachment of elements to matrix Detachment of fibrils from RTEs Excretion of cast Cylindroids Tapered ends, one or both Same significance as cast

Hyaline Casts Low refractive index Colorless when unstained
Uromodulin protein Use low light or phase Normal parallel sides or convoluted, wrinkled, cylindroid, occasional adhering cell or granule

Most frequently seen 0 to 2 is normal Nonpathologic: stress, exercise, fever, heat exposure, dehydration Pathologic: glomerulonephritis, pyelonephritis, chronic renal disease, congestive heart failure

Clinical Significance (cont’d)

RBC Casts Orange-red color Embedded and adhering cells
May be fragmented Confirm seeing free RBCs and positive reagent strip for blood Look for cast matrix to avoid mistaking a RBC clump for a cast

Bleeding within the nephron, casts are more specific than free RBCs in urine Glomerular damage or nephron capillary damage Glomerular damage: dysmorphic RBCs and elevated protein May be seen following strenuous exercise

Clinical Significance (cont’d)
Cells begin to disintegrate with more stasis of urine flow Hemoglobin and myoglobin damage tubules Hemoglobin degraded to methemoglobin = dirty brown casts Look for RTE cells to confirm tubular necrosis

WBC Casts Mostly neutrophils and lobed nucleus and granules are seen
Staining helps differentiate from RTE cells May be tightly packed; look for cast matrix to distinguish from WBC clump

WBC Casts (cont’d) WBC casts are seen with infection and inflammation of the tubules Pyelonephritis: WBC casts, bacteria Acute interstitial nephtitis: WBC casts, no bacteria May accompany RBC casts

Bacterial Casts May be pure bacteria or mixed with WBCs
Resemble granular casts Look for free WBCs and bacteria Confirm with Gram stain Seen in pyelonephritis Mixed cellular casts Glomerular nephritis: RBCs and WBCs Look for predominant type of cell

Epithelial (RTE) Casts
Formed in DCT = small, round cells Fibrils forming cast pull cells from damaged tubules Majority of cells are on the cast matrix Differentiate from WBCs: stain to show single nucleus

Tubular damage, heavy metals, viral infections, drug toxicity, graft rejection, pyelonephritis Cells may appear bilirubin stained Look for matrix to distinguish fragments

Fatty Casts Seen with oval fat bodies (OFBs) and fat droplets
Highly refractile, OFBs may attach to matrix Polarized microscopy and lipid stains Nephrotic syndrome, diabetes, crush trauma, tubular necrosis

Mixed Cellular Casts RBC and WBC casts in glomerulonephritis
WBC and RTE cell casts, or WBC and bacterial casts in pyelonephritis Identification difficult Staining or phase microscopy aids in the identification

Granular Casts Coarse and finely granular Granule origin
RTE lysosomes, excreted in normal metabolism, more after exercise and activity Disintegration of cellular casts and free cells

Granular Casts (cont’d)
Detect with low power, ID with high power Granules disintegrate to form waxy casts Differentiate granular casts from clumps of debris and crystals; look for matrix

Waxy Casts Brittle, highly refractile
Often fragmented with jagged ends and notches Well visualized with stain Degenerated hyaline and granular casts Extreme urine stasis Renal failure

Broad Casts Renal failure casts Destruction and widening of the DCTs
Formation in the upper collecting duct All types of casts may be broad Most common are granular and waxy Bilirubin stained from viral hepatitis

Urinary Crystals Most are not clinically significant but are reported
True geometrically formed structures or as amorphous material Must differentiate from the few abnormal crystals indicating liver disease, inborn errors of metabolism, and damage to tubules Iatrogenic: caused by medications or treatments Report: rare few, moderate, many

Crystal Formation Precipitation of urine solutes: salts, organic compounds, and medications Formation based on temperature, solute concentration, and pH Many crystals in refrigerated specimens High specific gravity needed in fresh specimens

General Identification Techniques
Most have characteristic shapes and colors Most valuable ID is urine pH Classification: normal acid, normal alkaline All abnormal crystals are found in acid urine Polarized microscopy characteristics are valuable in ID

Solubility Characteristics
Temperature and pH contribute to formation and solubility Amorphous urates form in refrigerated acid urine; will dissolve with heat Amorphous phosphates form in refrigerated alkaline urine; will dissolve in acetic acid; so will RBCs

Normal Crystals in Acid Urine
Amorphous urates Yellow-brown granules microscopically Urine sediment has pink color due to the pigment uroerythrin attaching on surface of granules Often in clumps; may resemble casts pH usually greater than 5.5

Uric Acid Crystals Rhombic, whetstones, wedges, rosettes
Yellow-brown color May resemble cystine crystals but always polarize ↑ purines, nucleic acids Chemotherapy for leukemia, gout

Calcium Oxalate Crystals
Acid and neutral pH Dihydrate is envelope or two pyramid–shaped Most common Monohydrate is oval or dumbbell shaped Antifreeze poisoning Calcium oxalate is a major component of renal calculi

Amorphous Phosphates May appear similar to amorphous urates
Differentiate Alkaline pH and heavy white precipitate after refrigeration

Normal Crystals in Alkaline Urine
Triple phosphate Colorless, prism, or coffin-lid shaped Highly alkaline urine and urinary tract infections (UTIs) Polarize No clinical significance

Calcium Phosphate and Carbonate
Flat rectangles and thin prisms in rosettes No clinical significance Carbonate Small, dumbbell, and spherical shapes Gas produced with addition of acetic acid

Ammonium Biurate Crystals
Yellow-brown, spicule-covered spheres; “thorny apples” Only urates in alkaline urine Old specimens and with urea-splitting bacteria

Abnormal Crystals Cystine crystals Hexagonal, thin and thick plates
Similar to uric acid UA polarizes but only thick cystine crystals polarize Seen in cystinuria: inability to reabsorb cystine Confirm: cyanide nitroprusside

Cholesterol Crystals Refrigerated specimens
Rectangular plates with characteristic notched corners Highly birefringent Nephrotic syndrome accompanying fatty casts and OFBs

Radiographic Dye Crystals
Similar to cholesterol crystals, polarize Patient history Very high SG with refractometer

Liver Disease Crystals
Tyrosine crystals Fine yellow needles in clumps or rosettes Seen with leucine crystals Inherited amino acid disorders Leucine crystals Yellow-brown spheres with concentric circles and radial striations

Liver Disease Crystals (cont’d)
Bilirubin crystals Clumped needles or granules Characteristic yellow color Viral hepatitis with tubular damage Positive reagent strip for bilirubin

Sulfonamide Crystals Possibility of tubular damage if crystals are forming in the nephron Shapes most frequently encountered include needles, rhombics, whetstones, sheaves of wheat, and rosettes with colors ranging from colorless to yellow-brown

Ampicillin Crystals Ampicillin crystals appear as colorless needles that tend to form bundles following refrigeration

Urinary Sediment Artifacts
Material fibers, meat and vegetable fibers, and hair Starch, oil droplets, air bubbles, pollen grains, vegetable fiber, hair, diaper fiber

Urinary Sediment Artifacts (cont’d)

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