Urinalysis and Body Fluids, 5th Edition

Urinalysis and Body Fluids, 5th Edition: Microscopic Examination of Urine

Introduction to Microscopic Examination

• Identification of insoluble substances (formed elements) involves:
  • Red blood cells (RBCs)
  • White blood cells (WBCs)
  • Epithelial cells
  • Casts
  • Bacteria
  • Yeast parasites
  • Mucus
  • Spermatozoa
  • Crystals
  • Artifacts
• Microscopic examination is noted as the least standardized and most time-consuming step in urinalysis.

Macroscopic Screening / Chemical Sieving

• The microscopic examination is performed following macroscopic and chemical analyses, which involve:
  • Color
  • Clarity
  • Presence of blood
  • Protein
  • Nitrite
  • Leukocyte esterase
  • Possibly glucose
• Special populations needing attention during examination include:
  • Pregnant women
  • Pediatric patients
  • Geriatric patients
  • Diabetic individuals
  • Immunocompromised individuals
  • Renal patients

Clinical and Laboratory Standards Institute (CLSI)

• Utilization by physicians to request examinations.
• Laboratory-specified populations must have abnormal physical or chemical results flagged.
• Laboratory criteria integrated into automated instrumentation for efficiency.

Sediment Standardization

• Preparation of sediment includes:
  • Volume of sediment examined
  • Methods of visualization
  • Reporting results
  • Availability of commercial systems (e.g., KOVA) which provide:
  • Calibrated centrifuge tubes
  • Special slides to control volume
  • Decanting pipettes
  • Grids for improved quantitation

Macroscopic Screening Correlations

• Screening tests relate specific findings to significance:
  • Color: Indicates blood presence.
  • Clarity: Differentiates between hematuria, hemoglobinuria, and myoglobinuria; confirms causes of turbidity.
  • Blood: Presence of RBCs or RBC casts.
  • Protein: Indicates casts or cells.
  • Nitrite: Presence of bacteria or WBCs.
  • Leukocyte esterase: Indicates WBCs, WBC casts, or bacteria.
  • Glucose: Indicates yeast presence.

Specimen Preparation

• Specimens should be examined when fresh or preserved.
• RBCs, WBCs, and casts may lyse in dilute, alkaline urine.
• Refrigeration can lead to precipitation of crystals.
• Aim for reduced contamination of epithelial cells from a midstream clean-catch specimen.
• Mix specimens prior to decanting into centrifuge tubes.

Specimen Volume

• Typically centrifuge 10–15 mL of urine, as reagent strips fit into 12 mL.
• Always cap tubes to prevent contamination or evaporation.
• Too small a volume can reduce the number of detectable formed elements; some laboratories may correct for volume variations.

Centrifugation Details

• Standardization of centrifugation speed and time is crucial; ideal conditions are:
  • 5 minutes at a relative centrifugal force (RCF) of 400.
• RCF accounts for variations in centrifuge head diameters, unlike revolutions per minute (RPM).
• It is important not to use the brake of the centrifuge during operation.

Post-centrifuge Sediment

• Aim for 0.5–1.0 mL after decantation.
• Concentration factor can be calculated as: ext{concentration factor} = rac{ ext{volume of urine centrifuged}}{ ext{sediment volume}}
• This factor increases the probability of detecting low quantities of formed elements.
• Aspirate urine rather than pouring it off; use pipettes for this task.
• Carefully mix sediment to avoid excessive disruption.

Volume of Sediment Examined

• Consistency in examining volumes is key; commercial methods often provide this control.
• The glass slide method generally involves examining 20 μL and using a 22 x 22 mm cover slip, ensuring not to overflow it.
• Heavier elements or casts tend to migrate outside the cover slip.

Examination of Sediment

• Consistent examination involves:
  • A minimum of 10 low power fields (lpfs) and 10 high power fields (hpfs).
  • Use low power for a general overview including casts and general composition; edges are scanned for casts.
  • High power is required for detailed identification, starting with low light, focusing on epithelial cells while avoiding artifacts.
  • Continuous fine adjustment is recommended for clarity.

Artifact Interference

• Larger particles, such as pollen grains, may be mistaken for sediment elements due to differences in liquid planes caused by size.

Microscopic Reporting

• Maintain consistency within the laboratory; report findings in semiquantitative terms, such as:
  • Rare
  • Few
  • Moderate
  • Many
• Report RBCs and WBCs averages per high power field, while expressing epithelial cells, crystals, etc., in a semiquantitative manner.

Sediment Stains

• Low refractive index elements can be hard to visualize under bright-field microscopy. Common stains include:
  • Supravital stains: crystal violet and Safranin O (most common being Sternheimer-Malbin stain).
  • Increases refractive index and stains nuclei, cytoplasm, and inclusions.
  • Brand names include Sedi-Stain and KOVA stain.
  • Acetic acid enhances WBC nuclei visibility.

Specific Staining Techniques

• Lipid Stains:
  • Oil Red O and Sudan III for triglycerides and neutral fats; cholesterol may not stain but can polarize under polarized light (exhibiting a Maltese cross pattern).
• Gram Stains:
  • Identification of bacterial casts.
• Hansel Stain:
  • For urinary eosinophils.
• Methylene Blue and Eosin Y Stains:
  • Better than Wright stain for visual clarity.
• Prussian Blue Stain:
  • Used for identifying hemosiderin granules amidst hemoglobinuria.

Microscopy Techniques

• Bright field microscopy is the most prevalent used in urinalysis; important points include:
  • Require reduced light for effective viewing.
  • Common magnification levels: 10x and 40x.
  • Par focal capability minimizes adjustment when changing objectives; always use fine adjustment.
  • Lower light by using the rheostat and manipulate the condenser for optimal light focus; do not utilize the aperture diaphragm.

Care of the Microscope

• Proper handling involves:
  1. Carry with two hands, supporting the base.
  2. Hold in a vertical position.
  3. Clean optical surfaces with quality lens tissue and designated commercial lens cleaner only.
  4. Avoid using the 10x and 40x objectives with oil.
  5. Clean oil immersion lens post-use.
  6. Remove slides with the low-power objective raised.
  7. Store with low-power objective in place and centered stage.

Advanced Microscopy Techniques

• Phase-Contrast Microscopy:
  • Enhances the refractive index of casts, mucus threads, and Trichomonas.
• Polarizing Microscopy:
  • Visualizes crystals and lipids by splitting light into dual beams; multi-colored crystals and Maltese cross formations from cholesterol are notable.
• Interference-Contrast Microscopy:
  • Provides three-dimensional images of samples.

Sediment Constituents

• Many urines may show only rare epithelial cells; the clinical implication of small constituents can vary based on context.

Red Blood Cells (RBCs)

• Normal appearance: smooth, non-nucleated, and biconcave disc shape.
• In hypersthenuric urine: RBCs appear crenated.
• In hyposthenuric urine: referred to as ghost cells.
• RBC identification is performed using high power magnification.

Identification Challenges for RBCs

• Distinguishing RBCs from:
  • Yeast (look for buds)
  • Oil droplets (due to refractibility)
  • Air bubbles (similar characteristics)
  • Starch (also refractile and polarizes)
• Correlation with reagent strips confirms the presence of yeast or other contaminants.

Dysmorphic RBCs

• Indicate:
  • Glomerular bleeding
  • Strenuous exercise effects
  • Characteristics include acanthocytic forms and blebs, which may be fragmented or hypochromic, aiding in diagnosis.

Clinical Significance of RBCs

• Normal range is 0–3 or up to 5 per hpf.
• Elevated levels indicate potential damage to the glomerular membrane or vascular injury to the genitourinary tract.
  • The quantity of cells usually reflects the degree of damage.
  • Distinction between macroscopic and microscopic hematuria includes visual observations such as cloudy red urine indicating advanced glomerular disease, trauma, or acute infection vs. clear red suggesting early glomerular disease, malignancy, or renal calculi.

White Blood Cells (WBCs)

• Primarily neutrophils, which are identified under high power.
• Appearance of glitter cells indicates they exist in hypotonic urine demonstrating Brownian movement, sparkling granules, and a pale blue coloration if stained.

Eosinophils

• Associated with conditions such as:
  • Drug-induced interstitial nephritis
  • Renal transplant rejection
• Identified using Hansel stain; significant if over 1% in a sample, typically evaluated per 100-500 cells.
• Enhanced detection may necessitate concentrating sediment, centrifuging, or employing cytocentrifuge techniques.

Mononuclear Cells

• Includes lymphocytes, monocytes, macrophages, and histiocytes, which are rare in urine.
• Differentiating from renal tubular epithelial (RTE) cells can be tricky; lymphocytes may bear resemblance to RBCs.
• Presence in early transplant rejection may necessitate referral to cytodiagnostic testing for accurate identification.

Clinical Significance of WBCs

• Normal values indicate less than 5 per high power field; more common in females.
• WBCs may enter the urine via trauma, migration (amoeboid), and elevated quantities lead to pyuria.
• Increased WBCs can indicate various infections:
  • Cystitis
  • Pyelonephritis
  • Prostatitis
  • Urethritis
• Associated with conditions like glomerulonephritis, lupus erythematosus, interstitial nephritis, and tumors.

Epithelial Cells

• Classified into three types:
  • Squamous cells (from vagina, male, and female urethra)
  • Transitional (urothelial) cells (from bladder, renal pelvis, calyces, ureters, upper male urethra)
  • Renal tubular epithelial (RTE) cells (from renal tubules)

Squamous Epithelial Cells

• They are the largest cells found in urine, useful for focusing in microscopy.
• Reporting terms include rare, few, moderate, many as per laboratory standards and are considered normal sloughing; contamination is possible if not collected via midstream clean-catch techniques.

Clue Cells

• Squamous cells with clinical significance; associated with Gardnerella vaginalis infections.
• Identified by their morphology, where the coccobacillus covers most of the cell’s surface.
• While they can be found in urine, they are more frequently observed in vaginal wet preparations.

Transitional (Urothelial) Cells

• Has three forms: spherical (absorbs water in bladder), caudate (tail-like), polyhedral (multiple sides).
• Characterized by centrally located nuclei and syncytia, which are clumps often found during catheterization or may signal malignancy.

Renal Tubular Epithelial (RTE) Cells

• Cell size and shape various by renal tubular area:
  • Columnar in proximal convoluted tubule (PCT)
  • Round/oval in distal convoluted tubule (DCT)
  • Cuboidal in collecting duct;
  • Three or more cuboidal cells may form renal fragments indicating damage.

PCT Cells

• Larger than other RTE cells; appear columnar, convoluted, and rectangular.
• Possess coarsely granular cytoplasm with visible nuclei.

DCT Cells

• Smaller, round or oval-shaped, may resemble WBCs or spherical transitional cells.
• Differentiation requires observation of eccentrically placed nuclei.

Collecting Duct RTEs

• Characterized as cuboidal and never round, possessing at least one straight edge.
• The presence of three or more cells in a clump suggests renal fragment status, while PCT and DCT are not typically seen in clumps.

Clinical Significance of RTE Cells

• RTE cells are of primary clinical significance, indicating tubular necrosis, particularly when fragments are observed, which points towards severe damage.
• Potential causes include:
  • Heavy metals
  • Drug toxicity
  • Hemoglobin and myoglobin toxicity
  • Viral infections
  • Pyelonephritis
  • Transplant rejection
  • Salicylate poisoning; identification aided by the presence of single cuboidal cells.
  • RTEs may absorb bilirubin, hemoglobin, and lipids, with hemosiderin staining observable using Prussian blue techniques.

Oval Fat Bodies

• Observed as RTE cells that have absorbed lipids from the filtrate.
• Free-floating refractile fat droplets may also be present, identifiable via Maltese cross formations under polarized light.
• Negative results should be checked with Sudan III or oil red O staining methods.

Staining Properties of Oval Fat Bodies

• Fat-related structures polarize, revealing cholesterol can present as polarizing substances; triglycerides and neutral fats stain positively in appropriate preparations.
• Associated conditions include:
  • Lipiduria from nephrotic syndrome
  • Acute tubular necrosis
  • Diabetes
  • Crush syndrome.

Bacteria in Urine

• Typically, urine is sterile; contaminants may form during urination; such contaminants tend to multiply rapidly.
• Presence of WBCs alongside bacteria signifies potential urinary tract infections (UTIs).
• Report observations in terms of few, moderate, or many per high power field; rods are most commonly found compared to cocci.
• Nitrite levels can help confirm presence of rods, as cocci usually do not correlate with nitrite.

Yeast Identification

• Yeast appears as single, refractile, budding structures, sometimes showing mycelial forms.
• Reporting includes assessments of few, moderate, or many.
• Diabetic urine conditions create ideal environments for yeast proliferation due to high glucose and acidic pH levels; immunocompromised patients may also show increased levels.
• Yeast presence can potentially confuse WBC and RBC identification.

Parasites in Urine

• The most common urinary parasite is Trichomonas vaginalis, noted for its pear-shaped flagellate appearance and rapid movement across microscopic fields.
• Reporting follows similar patterns of few, moderate, or many; if a parasite appears dormant, it may be confused with WBCs, transitional, or RTE cells.
• Other urinary parasites may include Schistosoma haematobium and Enterobius vermicularis.

Spermatozoa

• Characterized by oval, tapered heads and long tails; typically immobile in urine due to its toxic environment for sperm.
• Rarely of clinical significance, but their presence may indicate infertility due to expulsion into the bladder.
• Reporting standards vary; consideration must be given to potential legal implications regarding findings.

Mucus in Urine

• Mucus is a protein derivative from RTE and glands within the lower genitourinary tract; appears threadlike and has a low refractive index.
• Mucus can be confused with casts; it is irregularly composed and arises from Tamm-Horsfall (TH) protein presence.
• Typically, mucus's presence in female specimens is of low clinical significance.

Casts in Urine

• Casts are unique elements formed within the kidney and are produced predominantly in the distal convoluted tubule (DCT) and collecting duct.
• Characteristics include parallel sides, rounded ends, and may contain various inclusions.
• Detection generally uses low power, while identification requires high power; it is critical to scan the edges of the glass cover slip under low light.
• Report findings based on quantity per low power field; numerous pathological and non-pathological causes need evaluation.

Composition and Formation of Casts

• Casts are primarily composed of Tamm-Horsfall protein (THP) secreted by RTE cells of DCT and collecting duct.
• Consistent excretion occurs normally; increased levels may arise from stress or exercise.
• Cast formation includes protein fibrils aggregating into a gel-like matrix influenced by urinary stasis, acidic pH, sodium, and calcium presence.
• TH protein does not get detected by reagent strips; elevations in protein levels suggest renal disease.

Formation of Casts (Continued)

• Formation occurs through:
  • Aggregation of TH fibrils adjacent to RTE cells
  • Interweaving into a loose network that traps elements
  • Developing into a solid matrix through further interweaving
  • Elements attaching to the formed matrix, followed by detachment of fibrils from RTE cells
  • Final excretion of formed casts, referred to as cylindroids when they have tapered ends.

Hyaline Casts

• Exhibits low refractive index and a colorless appearance when unstained.
• Best viewed under low light or phase contrast microscopy; shapes appear as parallel sides or may be convoluted, wrinkled, or cylindroid, potentially containing cells or granules adhering to their surface.

Clinical Significance of Cast Types

• Hyaline casts are the most frequently observed, with normal values being 0–2 per hpf.
• Non-pathologic causes include:
  • Stress
  • Exercise
  • Fever
  • Heat exposure
  • Dehydration
• Pathological contexts include:
  • Glomerulonephritis
  • Pyelonephritis
  • Chronic renal disease
  • Congestive heart failure.

RBC Casts

• Distinctive orange-red coloration is noted; they may contain embedded or adhering cells which could fragment.
• To confirm, cross-check for free RBCs and positive blood reactions via reagent strips; also observe for cast matrix to avoid confusing with RBC clumps.

Clinical Significance of RBC Casts

• RBC casts are indicative of bleeding within the nephron; they offer specificity lacking in mere presence of free RBCs.
• Confirm conditions include glomerular damage or nephron capillary injury, characterized by:
  • Dysmorphic RBC formation
  • Elevated protein levels
• May also appear post-exercise as a temporary response.
• Continuous urine stasis leads to cellular disintegration; hemoglobin and myoglobin can damage renal tubules, with hemoglobin degradation yielding dirty brown casts.
• Presence of RTE cells verifies tubular necrosis.

WBC Casts

• Primarily composed of neutrophils demonstrating lobed nuclei and granules visible upon staining; they may compact tightly, requiring matrix identification for accurate differentiation from WBC clumps.

Clinical Significance of WBC Casts

• WBC casts indicate infection or inflammation within the tubules; common associations include:
  • Pyelonephritis (with bacteria presence)
  • Acute interstitial nephritis (without bacteria)
• Can be present alongside RBC casts under certain conditions.

Bacterial Casts

• These may present purely as bacteria or mixed with WBCs; they are similar to granular casts.
• Confirmation requires identification of free WBCs and bacteria in the sample, supplemented by Gram staining; associated with pyelonephritis.
• Mixed cellular casts can reflect glomerular nephritis status, highlighting predominant cell types present during evaluation.

Epithelial (RTE) Casts

• Created in DCT with small, round cellular structures; distinguished by their fibrils which extract cells from damaged tubules.
• Majority of cells will rest on the cast matrix; capture differentiation from WBCs involves staining to reveal single nuclei.

Clinical Significance of Epithelial (RTE) Casts

• RTE casts correlate with tubular damage from sources such as:
  • Heavy metal exposure
  • Viral infections
  • Drug toxicity
  • Graft rejection
  • Pyelonephritis
• Cells may stain positively for bilirubin; identification of the matrix becomes essential for differentiating fragments during analysis.

Fatty Casts

• Observed alongside oval fat bodies and free fat droplets; they exhibit high refractivity and can be attached to the cast matrix.
• Use polarized microscopy and lipid staining for assessment.
• Associated with:
  • Nephrotic syndrome
  • Diabetes
  • Crush trauma
  • Tubular necrosis.

Granular Casts

• Can appear coarse or finely granular:
  • Originating from RTE lysosomes during normal metabolism and have increased presence post-exercise or activity (nonpathological).
  • Disintegration of cellular casts and free cells correlates with disease states.

Identification of Granular Casts

• Detection requires low power, while identification is performed under high power.
• Granules may further disintegrate to create waxy casts; it is crucial to differentiate granular casts from debris clumps and crystals by looking for the underlying matrix.

Waxy Casts

• Exhibiting brittle composition and high refractility, often appearing fragmented with jagged ends and notches; they are ideally visualized using staining techniques.
• This represents degenerated hyaline and granular casts and indicates severe urine stasis, typically seen in renal failure.

Broad Casts

• Associated with renal failure, these casts represent destruction and dilation of the DCTs and can form in the upper collecting ducts.
• Types of casts visible include granular and waxy, with bilirubin-stained characteristics stemming from viral hepatitis.

Urinary Crystals

• Most urinary crystals are clinically insignificant but still need to be reported. Differentiation is required for abnormal crystals indicative of liver disease, genetic metabolism disorders, or tubular damage.
• Iatrogenic factors can also induce crystal formation due to medications or treatments.
• Reporting protocols include noting: rare, few, moderate, or many occurrences.

Crystal Formation

• Crystals arise from precipitation of urine solutes including salts, organic compounds, and medications. Key factors influencing formation include:
  • Temperature
  • Solute concentration
  • Urine pH
• Notably, many crystals may form in refrigerated specimens, while high specific gravity is needed for fresh specimens.

General Identification of Crystals

• Most crystals have distinct shapes and colors, offering valuable identification pathways; urine pH serves as the most significant identifying factor, categorizing crystals into:
  • Normal acid
  • Normal alkaline
• All abnormal crystals are identified within acidic urine; polarized microscopy can enhance identification accuracy.

Solubility Characteristics of Crystals

• Formation and solubility traits hinge on temperature and pH, outlined as follows:
  • Amorphous urates form in refrigerated acidic urine, dissolving when heated.
  • Amorphous phosphates form in alkaline refrigerated urine and dissolve in acetic acid; RBCs will share similar characteristics.

Normal Crystals in Acid Urine

• Amorphous Urates: Present as yellow-brown granules microscopically; result in a pink coloration due to pigment uroerythrin on surfaces of granules; can form clusters resembling casts; commonly seen at a pH value over 5.5.

Uric Acid Crystals

• Can take rhombic, whetstone, wedge, or rosette forms; exhibiting a yellow-brown color.
• Often polarized, they signify elevated purines and nucleic acids, especially in contexts of chemotherapy for leukemia or gout treatment.

Calcium Oxalate Crystals

• These may form in acid or neutral pH urine and appear in two shapes:
  • Dihydrate: enveloped or two-pyramid shaped (most common form).
  • Monohydrate: oval or dumbbell-shaped; significant in antifreeze poisoning.
  • Calcium oxalate is a major component of renal calculi formation.

Normal Crystals in Alkaline Urine

• Triple Phosphate Crystals: Appear colorless and take on prism or coffin-lid shapes; correlate highly alkaline urine conditions and urinary tract infections (UTIs), usually not clinically significant but observable under polarizing microscopy.

Amorphous Phosphates

• Can resemble amorphous urates; for differentiation:
  • Presence of alkaline pH and a heavy white precipitate post refrigeration.

Calcium Phosphate and Carbonate Crystals

• Phosphate crystals often resemble flat rectangles or thin prisms organized in rosettes; classified as clinically unremarkable.
• Carbonate crystals present as small dumbbell or spherical shapes; generation of gas occurs with acetic acid addition; classified as also being clinically unremarkable.

Ammonium Biurate Crystals

• Introduced as yellow-brown spheres adorned with spicules (commonly referenced as “thorny apples”).
• They are present only in alkaline urine, often encountered in old specimens or those containing urea-splitting bacteria.

Abnormal Crystals

• Cystine Crystals: They appear as hexagonal thin and thick plates; similar to uric acid but vary in polarization properties; presence suggests a disorder named cystinuria, indicating an inability to reabsorb cystine.
• Confirmation of presence can be achieved using cyanide nitroprusside tests.

Cholesterol Crystals

• Found in refrigerated urine, crystalline structures appear as rectangular plates with characteristic notched corners and exhibit high birefringence and indicate nephrotic syndrome alongside fatty casts and oval fat bodies.

Liver Disease Crystals

• Bilirubin Crystals: They appear as clumped needles or granules and exhibit a characteristic yellow color; associated with viral hepatitis that leads to tubular damage and correlate positively with bilirubin reagent strip tests.
• Tyrosine Crystals: Typically display fine yellow needles organized in clumps or rosettes; often appear alongside leucine crystals which appear as yellow-brown spheres marked with concentric circles and radial striations; bilirubin reagent strips will also yield positive results.

Iatrogenic Crystals

• Ampicillin Crystals: Present as colorless needles in bundles, often forming after refrigeration when excessive dosages and inadequate hydration are present.
• Sulfa Crystals: Commonly precipitated and display variable shapes; patients treated for UTIs often demonstrate these crystals adversely affecting testing outcomes.
• Radiographic Dye Crystals: Similar in characteristics to cholesterol crystals and can present diagnostic challenges; a patient history should be taken into account for appropriate assessment reflecting very high specific gravity as discerned with a refractometer.

Artifacts Resembling Casts

• Fibrous materials, including meat and vegetable fibers, as well as hair, can easily be confused with casts in microscopy:
  • Non-casts do not polarize (except for fatty casts); various fibers will show polarization properties.
  • Specific examples include vegetable fibers, hair, and fibers from diapers, which each present unique morphological traits under examination.