Urinalysis and Body Fluids, 5th Edition Lecture Notes

Urinalysis and Body Fluids, 5th Edition

Chapter 6: Microscopic Examination of Urine

Introduction

  • Overview of components identified in urinalysis:
    • Insoluble substances (formed elements)
    • Red blood cells (RBCs)
    • White blood cells (WBCs)
    • Epithelial cells
    • Casts
    • Bacteria
    • Yeast
    • Parasites
    • Mucus
    • Spermatozoa
    • Crystals
    • Artifacts
  • Noted as the least standardized and most time-consuming aspect of urinalysis.

Macroscopic Screening / Chemical Sieving

  • Microscopic examinations are based on physical and chemical results:
    • Parameters include: color, clarity, blood, protein, nitrite, leukocyte esterase, and possibly glucose.
    • Special populations considered for different testing:
    • Pregnant women
    • Pediatric, geriatric, diabetic, immunocompromised, and renal patients.

Clinical and Laboratory Standards Institute (CLSI)

  • Microscopy requests originate from the physician's orders.
  • Focus on:
    • Laboratory-specific populations.
    • Abnormal physical or chemical results must be specified.
    • Laboratory criteria integrated into automated instrumentation.

Sediment Standardization

  • Guidelines for sediment preparation and examination:
    • Volume of sediment examined and methods used for visualization.
    • Reporting of results varies depending on laboratory practices.
  • Commercial systems mentioned:
    • KOVA systems with calibrated centrifuge tubes, special control volume slides, decanting pipettes, and grids for quantitation.

Macroscopic Screening Correlations

  • Screening tests and their significance:
    • Color and Clarity: Identify causes of hematuria vs hemoglobinuria/myoglobinuria; confirm pathologic vs nonpathologic turbidity.
    • Blood: Links to RBCs/RBC casts.
    • Protein: Correlates with casts and cells.
    • Nitrite and Leukocyte esterase: Indicate presence of bacteria and WBCs.
    • Glucose: Associated with yeast infections.

Specimen Preparation

  • Fresh or preserved specimens should be examined.
  • RBCs, WBCs, and casts may lyse in dilute, alkaline urine.
  • Refrigeration may precipitate crystals.
  • Emphasizes the importance of a midstream clean-catch specimen to reduce contamination from epithelial cells.
  • Specimens should be mixed before being placed in the centrifuge tube.

Specimen Volume

  • Recommended centrifuge volume is 10–15 mL of urine (reagent strips commonly require 12 mL).
  • All tubes should be capped after use to avoid evaporation.
  • Discusses implications of insufficient sample volume, which may lead to fewer detected formed elements.
  • Some laboratories may adjust findings based on estimated volume.

Centrifugation

  • Importance of standardizing centrifuge speed and duration:
    • Ideal setting: 5 minutes at relative centrifugal force (RCF) of 400.
    • RCF is essential for accounting for variations in centrifuge head dimensions; RPM values alone are inadequate.
    • Operators should avoid engaging the brake during centrifugation.

Postcentrifuge Sediment

  • Volume to retain after decantation: maintain 0.5–1.0 mL.
  • Concentration Factor: Defined as the volume of urine centrifuged divided by the sediment volume. This factor is crucial for detecting low quantities of formed elements efficiently.
  • Technique recommendation: use aspiration rather than pouring off urine—special pipettes may be utilized for this purpose.
  • Caution to mix sediment gently to avoid disruption of fragile elements.

Volume of Sediment Examined

  • Consistency is critical across specimens; certain commercial systems maintain these controls.
  • Recommended method for examining sediment includes using:
    • 20 μL volume
    • 22 x 22 mm glass cover slip
    • Observations not to overflow the cover slip as heavier elements, such as casts, may flow outside the intended area.

Examination of Sediment

  • Consistency in results is crucial.
  • A minimum of 10 low power fields (lpfs) and 10 high power fields (hpfs) should be reviewed.
  • Low Power: Used for assessing casts and general composition; important to scan edges for casts.
  • High Power: Critical for accurate identification of various elements.
  • Initially focus using low power with reduced light to find epithelial cells, avoiding artifacts at different planes.
  • Continuous fine adjustment is necessary to maintain optimal viewing.

Artifact Interference

  • Artifacts such as large pollen grains may obscure typical sediment elements in view, as their size places them in distinct liquid planes differing from the urine's trapped constituents.

Microscopic Reporting

  • Reports must maintain consistency within the laboratory.
  • Semiquantitative measures include terms such as "rare," "few," "moderate," "many," or a numbering approach (1+, etc.).
  • For casts, report average counts per lpfs, while RBCs and WBCs are reported per hpfs.
  • Other constituents such as epithelial cells and crystals should also be represented in semiquantitative terms.

Sediment Stains

  • Low refractive index elements can be challenging to view using standard bright-field microscopy.
  • Common stains include:
    • Supravital stains like crystal violet and Safranin O, which raise refractive index and accentuate nuclei, cytoplasm, and inclusions.
    • Brand names include Sedi-Stain and KOVA stain.
    • Use of acetic acid can enhance WBC nuclei visualization.

Additional Sediment Stains

  • Specific lipid stains include:
    • Oil Red O and Sudan III highlight triglycerides and neutral fats; cholesterol appears unaffected but can be visualized under polarized light in a Maltese cross pattern.
    • Gram stain assists with identifying bacterial casts.
    • Hansel stain is used for urinary eosinophils.
    • Methylene blue and eosin Y stains outperform the traditional Wright stain for certain applications.
    • Prussian blue is effective for visualizing hemosiderin granules in hemoglobinuria.

Microscopy

  • Bright-field microscopy remains the most prevalent technique in urinalysis.
  • Proper light reduction is crucial for ideal results; standard magnifications utilized include 10x and 40x.
  • The term "par focal" indicates minimal adjustments are required when shifting objectives, emphasizing fine adjustment use for optimal viewing.
  • Rheostat can be used to lower light intensity effectively, and manipulable condenser maintains focused optics.
  • Users must avoid the use of the aperture diaphragm during inspection.

Care of the Microscope

  1. Carry with both hands, supporting the base.
  2. Hold in a vertical orientation.
  3. Clean optical surfaces only with quality lens tissue and commercial cleaner.
  4. Oil should not be used with 10x and 40x objectives.
  5. Always clean the oil immersion lens following use.
  6. Remove slides only with the low-power objective raised.
  7. Store the microscope with the low-power objective positioned and stage centered.

Alternative Microscopy Techniques

  • Phase-contrast: Enhances the refractive index of casts, mucus threads, and trichomonas.
  • Polarizing Microscopy: Useful for analyzing crystals and lipids, allowing light to separate into two beams revealing multicolored crystals; cholesterol creates Maltese cross formations.
  • Interference-contrast Microscopy: Produces three-dimensional representations.

Sediment Constituents

  • Urine samples may manifest a single rare epithelial cell; small quantities of other constituents can be normal or pathogenic, contingent upon clinical evaluation.

Red Blood Cells (RBCs)

  • RBC characteristics:
    • Description: Smooth, non-nucleated, biconcave disks.
    • Appearance changes in varying osmotic environments:
    • Crenated in hypersthenuric urine.
    • Ghost cells manifest in hyposthenuric urine.
  • Identification techniques emphasize high power microscopy.

Identification Dilemmas for RBCs

  • Differential identification among:
    • Yeast (look for buds), oil droplets (obtained using refractibility), air bubbles (by assessing refractibility and liquid plane), starch (which refracts light and polarizes).
  • Correlative results can arise from reagent strip analyses.

Dysmorphic Red Blood Cells

  • Indicate various medical scenarios:
    • Glomerular bleeding
    • Strenuous exercise can alter morphology.
    • Dysmorphic types include acanthocytes and cells exhibiting blebs or fragmentation which aid in diagnosis.

Clinical Significance of RBCs

  • Normal values: Typically 0–3 or 5 per high power field (hpf).
  • Indicative of damage to glomerular membranes or vascular injuries in the genitourinary tract.
  • Correlations between the number of cells and extent of damage:
    • Macroscopic vs microscopic hematuria, noting different presentation indicators (e.g. cloudy versus clear red urine).
    • Other conditions linked include advanced glomerular disease, trauma, acute infections, coagulation disorders, and early malignancies.

White Blood Cells (WBCs)

  • Predominant cell type: Neutrophils, identifiable under high power settings.
  • Features of glitter cells in hypotonic urine result from Brownian movement, causing cells to swell visibly and sparkle with a pale blue hue when stained.

Eosinophils in Urinalysis

  • Indicate drug-induced interstitial nephritis and renal transplant rejection.
  • Staining yields distinct presentation metrics, often reported as a percentage of total WBCs in view.
  • A threshold greater than 1% is considered significant.

Mononuclear Cell Overview

  • Include lymphocytes, monocytes, macrophages, and histiocytes and are generally rare in urine.
  • Distinction is important from renal tubular epithelial (RTE) cells; lymphocytes may mimic RBCs, particularly in early transplant rejection cases.
  • Referral for cytodiagnostic testing may be necessary.

Clinical Significance of WBCs

  • Normal counts are under 5 per hpf, with higher numbers more common in females.
  • Migration through the glomerulus can result from trauma but is also facilitated by amoeboid movement.
  • Increased WBC counts (pyuria) signal various infections and conditions:
    • Cystitis, pyelonephritis, prostatitis, urethritis.
    • Other conditions: Glomerulonephritis, lupus erythematosus, interstitial nephritis, tumors. Presence of bacteria should be reported.

Epithelial Cells

  • Three principal types:
    • Squamous
    • Transitional (urothelial)
    • Renal tubular epithelial (RTE)
  • Classification of epithelial cells:
    • Squamous: Found in vagina, male and female urethra.
    • Transitional: Present in bladder, renal pelvis, calyces, ureters, upper male urethra.
    • RTE: Originating from renal tubules.

Characteristics of Squamous Epithelial Cells

  • Largest cell type in urine, significant for focusing microscopy.
  • Reported as rare, few, moderate, or many.
  • Typical sloughing is normal; contamination could occur if not obtained through a midstream clean catch.

Clue Cells

  • Significance tied to pathologic conditions:
    • Indicative of Gardnerella vaginalis infection, where a coccobacillus envelops much of the squamous cell, demonstrating pathologic relevance.
    • Generally seen in urine, but more prevalent in vaginal wet preparations.

Transitional (Urothelial) Cells

  • Exhibit three forms:
    • Spherical: Swell to become large and round when water is absorbed in the bladder.
    • Caudate: Characterized by a tail-like projection.
    • Polyhedral: Possess multiple sides.
  • Differentiation from RTE is managed via centrally positioned nuclei.
  • Syncytia can appear as clumps, often associated with catheterization or malignancy.

Renal Tubular Epithelial (RTE) Cells

  • Appear with size and shape variations based on specific renal tubular areas:
    • Columnar cells are associated with proximal convoluted tubules (PCT).
    • Round or oval-shaped indicate distal convoluted tubules (DCT).
    • Cuboidal shapes correlate with collecting ducts.
  • Identification of three or more cuboidal cells indicates a renal fragment.

Proximal Convoluted Tubule (PCT) Cells

  • PCT cells are larger in size, characterized as columnar, convoluted shapes with noticeable nuclei and coarse granulation in cytoplasm, potentially resembling casts.

Distal Convoluted Tubule (DCT) Cells

  • DCT cells are generally smaller than PCT cells, identified as round or oval shapes and require observation of eccentrically placed nuclei to differentiate from spherical transitional cells.

Collecting Duct RTEs

  • Identified as cuboidal (never round) with at least one straight edge and an eccentric nucleus.
  • A finding of multiple cells in a clump typically signals renal fragments, signaling more severe conditions; PCTs and DCTs do not appear in such clusters.

Clinical Significance of RTE Cells

  • Most clinically significant among urine epithelial cells, as they indicate tubular necrosis; presence of fragments typically points to severe destruction.
  • Identifying causes includes heavy metals, drug toxicity, myoglobin, viral infections, pyelonephritis, renal transplant rejection, and salicylate poisoning.
  • Single cuboidal cells can suggest salicylate poisoning; capability to absorb substances like bilirubin, hemoglobin, and lipids.
  • Hemosiderin can be identified using Prussian blue staining.

Oval Fat Bodies

  • RTE cells that have absorbed lipids in the filtrate present as oval fat bodies and coexist with free-floating, reflective fat droplets, visible in Maltese cross formation under polarized light.
  • If negatively corroborated, confirmatory testing with Sudan III or Oil Red O stain is needed for clear differentiation.

Lipiduria

  • Conditions such as nephrotic syndrome, acute tubular necrosis, diabetes, and crush syndromes often lead to the emergence of oval fat bodies and lipiduria.

Bacteria in Urine

  • Typically, urine is sterile but can become contaminated during excretion, promoting bacterial growth.
  • WBCs usually accompany bacterial presence in urinary tract infections (UTI).
  • Reporting classifications include descriptions of few, moderate, or many per high power field.
  • Rod-shaped bacteria are the most commonly observed, and nitrite tests aide in confirming the presence of rods over cocci.

Yeast in Urine

  • Yeast appears as single, refractile, budding structures, potentially accompanied by mycelial forms.
  • Reports classify the presence as few, moderate, or many.
  • In diabetic urine, elevated glucose and acidic conditions lead to ideal growth environments for yeast, presenting challenges in differential diagnoses against RBCs due to similar appearances.

Parasites in Urine

  • Most commonly encountered: Trichomonas vaginalis, characterized as a pear-shaped flagellate that swims rapidly across microscopy fields.
  • Other parasites: Schistosoma haematobium and Enterobius vermicularis.

Spermatozoa in Urine

  • Appearance: oval, tapered heads and long tails but are rendered immobile by urine toxicity.
  • Rarely hold significant clinical implications, although they can indicate infertility if expelled into the bladder instead of through the urethra.
  • Results may lead to positive protein tests, as reporting standards vary across laboratories while clinical significance and potential legal ramifications are considered minor.

Mucus in Urine

  • Mucus is a protein derivative from RTE cells and glands in the lower genitourinary tract, presenting threadlike structures with low refractive index.
  • It may be confused with casts; irregular appearances result from Tamm-Horsfall (TH) protein.
  • Generally not significant in female specimens.

Casts in Urine

  • Casts represent elements exclusive to the kidney, forming primarily in the distal convoluted tubule (DCT) and collecting duct.
  • Features: Casts have parallel sides with rounded ends and may include inclusions. Detection requires low power microscopy; identification must be performed at high power.
  • Careful scanning along the edges of the glass coverslip and operating under low light are key to assessing casts accurately.
  • Reporting quantity should reflect counts per low power field.

Composition and Formation of Casts

  • Casts consist primarily of Tamm-Horsfall protein secreted by RTE cells from the DCT and collecting duct, excreted consistently (THP).
  • Production of protein fibrils leads to matrix formation under specific conditions such as stress and increased physical activity.
  • Cast formation is influenced by urine stasis, acid pH levels, as well as sodium and calcium concentration.
  • Notably, TH protein remains undetected using standard reagent strips; increased protein levels generally suggest renal pathology.

Casting Process Overview

  1. Aggregated Tamm-Horsfall fibrils attach to RTEs.
  2. Interweaving forms a loose network to trap elements within.
  3. Enhanced interweaving leads to solid matrix creation.
  4. Elements attach to the matrix forming casts.
  5. Detachment of fibrils occurs from RTE, resulting in cast excretion.
  6. Cylindroids can form, reflecting tapered appearances at either end, carrying similar significance as casts.

Types of Casts

Hyaline Casts
  • Characteristics include low refractive index and colorlessness when unstained.
  • Appear normal or convoluted, wrinkled, and may have adhering cells/granules attached.

Clinical Significance of Hyaline Casts

  • Most commonly observed with normal ranges identified as 0–2 casts.
  • Nonpathologic causes include stress, exercise, fever, heat exposure, and dehydration.
  • Pathologic implications signal glomerulonephritis, pyelonephritis, chronic renal disease, and congestive heart failure.

RBC Casts

  • Notable orange-red color with embedded and adhering cells; fragments may be present.
  • Requirement for positive identification includes confirming free RBCs and reactive test strips for blood presence.
  • Essential to identify both the cast matrix and avoid any misinterpretation of RBC aggregates.

Clinical Significance of RBC Casts

  • Indicative of bleeding within nephron, providing specificity over free RBC determinations in urine.
    - Conditions linked include glomerular damage or nephron capillary injury, such as:
    • Dysmorphic RBCs and elevated protein levels.
    • Often observed post-strenuous exercise.

Cellular Degradation of RBC Casts

  • As urine flow stagnates, RBCs within casts start to disintegrate, particularly under conditions induced by hemoglobin or myoglobin, which can damage tubules.
  • Hemoglobin degenerated to methemoglobin can present as dirty brown casts, necessitating RTE observation to confirm tubular necrosis.

WBC Casts

  • Predominantly contain neutrophils and should demonstrate lobed nuclei and granules, confirmed via staining.
  • Appearance may suggest tightly packed formations; matrix identification helps differentiate casts from mere WBC aggregations.

Infections Related to WBC Casts

  • These casts commonly arise concomitant with infections or inflammatory responses in tubules:
    • Pyelonephritis is indicated by the presence of WBC casts alongside bacteria.
    • Acute interstitial nephritis also presents WBC casts but lacks bacterial presence.
    • WBC casts may occur alongside RBC casts as well.

Bacterial Casts

  • These may contain pure bacterial populations or mixtures with WBCs, resembling granular casts due to their composition. Key to confirm bacterial presence via Gram staining in contexts such as pyelonephritis.

Mixed Cellular Casts

  • Observations for glomerular nephritis may yield RBCs and WBCs; predominant cell types assist in differentiating conditions effectively.

Epithelial (RTE) Casts

  • Formed within the DCT with small round cells where fibrils can draw cells from damaged tubules into the cast matrix.
  • Majority of cells are typically transitively present, necessitating staining to differentiate from WBCs by showcasing a single nucleus.

Clinical Significance of Epithelial Casts

  • These casts point towards tubular damage traceable to various conditions:
    • Heavy metals, drug toxicity, viral infections, graft rejection, pyelonephritis.
  • Bilirubin-stained fragments may appear, with matrix identification helping denote disfortunate tubular segments.

Fatty Casts

  • These present alongside oval fat bodies (OFBs) and fat droplets, known for their high refractility; OFBs may cling to cast matrices.
  • Identified using polarized microscopy or lipid-staining techniques, indicating nephrotic syndrome, diabetes, crush trauma, and tubular necrosis.

Granular Casts

  • Comprised of coarse and finely granular elements originating from:
    • RTE lysosomes that discharge elements during standard metabolism, or excessive quantities especially observed following physical exercise.
    • Disintegration of cellular casts and free cells is noted within various disease states.
  • Detection occurs at low powers, with identification transitioning to high power for accuracy.
  • Longitudinal assessment identifies adherence to solid matrices, guiding differential analysis against debris or crystalline clumps.

Waxy Casts

  • Characterized by brittle, highly refractile nature, often presenting fragmented appearances with jagged ends and notches.
  • Easier to visualize with stains as they emerge from degenerated hyaline and granular casts, often found in extreme cases of urine stagnation and renal failure.

Broad Casts

  • Indicative of severe renal failures, broad casts originate from the destruction and expansion of DCTs.
  • Formation occurs in upper collecting ducts where all forms of casts can appear broad; the most common types encountered are granular and waxy.
  • These casts may present as bilirubin-stained due to causative factors like viral hepatitis.

Urinary Crystals

  • Most crystals found in urinalysis are not clinically significant but should still be reported.
  • Differentiation is essential for identifying potentially abnormal crystals that suggest liver disease, metabolic diseases, or renal tubule damage.
  • Iatrogenic factors include medications or treatments causing abnormal crystallization. Reports specify ranges as rare, few, moderate, or many.

Crystal Formation Mechanism

  • Crystal formation results from solute precipitation of salts, organic compounds, and medications, influenced by urine temperature, concentration, and pH.
  • Observations indicate many crystals may form in refrigerated specimens; however, high specificity requires fresh samples.

General Crystal Identification (ID)

  • Most crystals exhibit unique shapes and colors, with pH being the most valuable identifying factor.
  • Classification divides into normal acidic and alkaline crystals, noting all abnormal crystals present in acid urine.
  • Polarized microscopy aids significantly in crystallization identification.

Solubility Characteristics of Crystals

  • Temperature and pH directly govern both the formation and solubility characteristics:
    • Amorphous urates develop in refrigerated acid urine but dissolve upon heating.
    • Amorphous phosphates arise in alkaline refrigerated urine, dissolving upon exposure to acetic acid; RBCs behave similarly despite their typical stability.

Normal Crystals in Acid Urine

  • Amorphous urates appear as yellow-brown granules under microscopic examination, where sediment exhibits a pink coloration due to uroerythrin adhering to the surface.
  • Typically observed in clumps and may sometimes mimic casts; pH greater than 5.5 is common.

Uric Acid Crystals

  • Characteristics include various forms: rhombic, whetstones, wedges, and rosettes, presenting yellow-brown coloration.
  • Polarization differentiation can substantiate their diagnosis, normally arising from elevated purine levels and nucleic acid turnover, potentially linked to chemotherapy or gout conditions.

Calcium Oxalate Crystals

  • May manifest in both acidic and neutral pH; shapes vary:
    • Dihydrate typically takes an envelope or dual pyramid appearance, representing the most common form.
    • Monohydrate appears oval or dumbbell-shaped, correlating with antifreeze poisoning.
    • Useful for pathology highlighting calcium oxalate's role in renal calculi formation.

Normal Crystals in Alkaline Urine

  • Notable as triple phosphate crystals often described as colorless, prism, or coffin-lid shaped.
  • Generally associated with highly alkaline urine, typically indicative of urinary tract infections without clinical significance.

Amorphous Phosphates

  • May mirror the appearance of amorphous urates; differentiation hinges on pH and heavy white precipitate that forms post-refrigeration.

Calcium Phosphate and Carbonate Crystals

  • Calcium phosphate presents flat rectangles and thin prisms in rosette patterns, while carbonate manifests as small dumbbell and spherical shapes, both lacking clinical significance.
  • Carbonate crystals exhibit gas production upon the addition of acetic acid.

Ammonium Biurate Crystals

  • Characterized as yellow-brown spheres covered with spicules, colloquially termed “thorny apples.”
  • Reflect only urates found in alkaline urine, consequently noted in aged specimens and related to urea-splitting bacteria.

Abnormal Crystals

Cystine Crystals
  • Identified as hexagonal, comprising both thin and thick plates; may resemble uric acid crystals, requiring polarized light for differentiation.
  • Commonly linked to cystinuria, which inhibits cystine reabsorption, confirmable via cyanide nitroprusside tests.
Cholesterol Crystals
  • Observed mainly in refrigerated specimens, presenting as rectangular plates with distinct notched corners; known for high birefringence under polarized light.
  • Commonly seen in nephrotic syndrome alongside fatty casts and oval fat bodies.

Liver Disease Crystals

  • Bilirubin Crystals: Characterized by clumped needles or granules presenting a striking yellow hue, prevalent in situations like viral hepatitis presenting tubular damages corroborated by positive bilirubin reagent strips.
Tyrosine Crystals
  • Comprising fine, yellow needles that may appear either in clumps or rosette formations; often co-occur with leucine crystals, pointing towards inherited amino acid metabolism disorders.

Leucine Crystals

  • Present as yellow-brown spherical structures with concentric circles and radial striations, crucial for diagnostic contexts alongside positive bilirubin reagent strips.

Iatrogenic Crystals

  • Ampicillin Crystals: Manifest as colorless needles forming bundles upon refrigeration—characterized by higher doses combined with patient dehydration.
  • Sulfa Crystals: The most frequent precipitation type presents in diverse shapes and is typically connected to UTI treatment protocols.
  • Radiographic contrast dyes may also induce similar crystal formations, demonstrating significant renal implications in patient histories reflected by high specific gravity noted via refractometry.

Artifacts Resembling Casts

  • Observations may reveal fibers, meat and vegetable particles, and hair, which may mimic casts on microscopic examinations.
  • Unlike casts, non-polarizable materials may easily yield to polarized light, warranting proper procedural confirmation during analyses.