Urinalysis and Body Fluids, 5th Edition

Urinalysis and Body Fluids: Microscopic Examination of Urine

Introduction to Microscopic Examination

  • Identification of Insoluble Substances (Formed Elements):

    • Red blood cells (RBCs)

    • White blood cells (WBCs)

    • Epithelial cells

    • Casts

    • Bacteria

    • Yeast parasites

    • Mucus

    • Spermatozoa

    • Crystals

    • Artifacts

  • Note: This examination is the least standardized and most time-consuming aspect of urinalysis.

Macroscopic Screening / Chemical Sieving

  • Microscopic examination is performed based on physical and chemical results, including:

    • Color

    • Clarity

    • Presence of blood

    • Protein levels

    • Nitrite levels

    • Leukocyte esterase

    • Possible glucose levels

  • Special Populations: Include pregnant women, pediatric, geriatric, diabetic, immunocompromised, and renal patients.

Clinical and Laboratory Standards Institute (CLSI)

  • CLSI guidelines are often requested by physicians and specify:

    • Laboratory-specified population for testing

    • Any abnormal physical or chemical result must be documented

    • Laboratory criteria are programmed into automated instrumentation, enhancing precision and reliability.

Sediment Standardization

  • Important aspects involve:

    • Preparation of sediment

    • Volume of sediment examined

    • Methods of visualization

    • Reporting of results

    • Use of commercial systems, e.g., KOVA, which include calibrated centrifuge tubes, specialized slides for volume control, decanting pipettes, and grids for quantitation.

Macroscopic Screening Correlations

  • Correlations & Screening Test Significance:

    • Color: Indicates potential presence of blood.

    • Clarity: Differentiates hematuria versus hemoglobinuria/myoglobinuria.

    • Blood: Detects RBCs/RBC casts.

    • Protein: Correlates with casts/cells presence.

    • Nitrite: Indicates bacteria/WBCs.

    • Leukocyte esterase: Associated with WBCs/WBC casts/bacteria.

    • Glucose: Suggests possible yeast infection.

Specimen Preparation and Examination

  • Specimen Handling:

    • Specimens should be examined when fresh or preserved.

    • Centrifugation should be performed shortly after collection to minimize contamination and preserve formed elements.

    • Refrigeration can lead to crystalline precipitate formation.

    • Midstream clean-catch specimens minimize contamination with epithelial cells.

    • Specimens should be mixed before decanting to the centrifuge tube.

Specimen Volume

  • Further details on specimen volume include:

    • Centrifuge 10–15 mL of urine (reagent strips fit into 12 mL).

    • Ensure centrifuge tubes are always capped.

    • Low volumes lead to fewer formed elements; some labs correct for volume.

Centrifugation Practice

  • Centrifugation Guidelines:

    • Standardized speed and time for centrifugation is crucial; ideal is 5 minutes at a relative centrifugal force (RCF) of 400. RCF corrects for varying centrifuge head diameters.

    • Do not use braking on the centrifuge as it affects sediment consistency.

Post-Centrifuge Sediment Examination

  • After decantation:

    • Typically, 0.5–1.0 mL of urine should remain for analysis.

    • Calculate the concentration factor as: volume of urine centrifuged/sediment volume, which influences the probability of detecting low quantities of formed elements.

    • Recommended aspiration technique rather than pouring off urine, to minimize disturbance of sediment.

    • Mix sediment gently, avoiding vigorous shaking.

Volume of Sediment Examined

  • Ensure consistency in the amount of sediment examined using commercial systems.

  • The glass slide method typically involves the use of a 20 μL sample and a 22 x 22 mm glass cover slip to ensure accurate results without overflow.

Examination of Sediment

  • Consistent examination methods involve:

    • Minimum examination of 10 low power fields (lpfs) and 10 high power fields (hpfs).

    • Low power for general composition and casts identification; high power for detailed identification.

    • Focus should prioritize epithelial cells over artifacts, utilizing continuous fine adjustment for optimal visibility.

Artifact Interference

  • Awareness of artifacts that may interfere with sediment interpretation:

    • Large pollen grains can obscure usual sediment elements due to differing liquid planes based on size.

Microscopic Reporting

  • Reporting should be systematic within laboratory standards, using qualitative and semiquantitative terms such as:

    • Rare, few, moderate, many, or 1+.

    • Casts quantified as an average per low power field, while RBCs and WBCs are averaged per high power field.

Sediment Stains and Microscopy Basics

  • Stains are often used to improve visibility of low refractive index elements, with common stains including:

    • Supravital Stain: Crystal violet and Safranin O; increases refractive index and enhances cellular visibility.

    • Lipid Stains: Oil Red O and Sudan III detect triglycerides and neutral fats; cholesterol polarizes under polarized light, producing a Maltese cross appearance.

    • Gram Stain: Useful for bacterial casts and identification.

    • Methylene Blue and Eosin Y: Better alternatives to Wright stain for certain cellular components.

  • Microscopy Practices:

    • Bright-field microscopy is predominant; reduced light is essential with magnifications of 10x and 40x.

    • Par focal settings help minimize adjustment between objectives.

Care of the Microscope

  • Proper handling of microscopes is essential:

    1. Carry with two hands, supporting the base.

    2. Keep microscope in a vertical position.

    3. Clean optical surfaces only with quality lens tissue and commercial cleaners.

    4. Avoid oil with non-oil objectives.

    5. Always clean the oil immersion lens post-use.

    6. Remove slides with a low-power objective raised.

    7. Store microscopes with the low-power objective in place and stage centered.

Advanced Microscopy Techniques

  • Phase-Contrast Microscopy: Enhances visualization of casts, mucus threads, and certain microorganisms.

  • Polarizing Microscopy: Useful for differentiating between types of crystals and lipids; provides multicolored views.

  • Interference-Contrast Microscopy: Creates three-dimensional images for detailed examination.

Sediment Constituents Overview

  • Overview of sediment characteristics and clinical correlation:

    • Many urines will contain rare epithelial cells; minor amounts may be normal vs. pathogenic based on clinical context.

    • Some constituents can be easily distorted.

Red Blood Cells (RBCs)

  • RBC Characteristics:

    • Normal RBCs are smooth, non-nucleated, biconcave disks.

    • Types depending on urine osmolality:

    • Crenated cells appear in hypersthenuric urine.

    • Ghost cells are seen in hyposthenuric urine.

    • Identification is best performed under high power.

Identification and Clinical Significance of RBCs

  • Key challenges in identifying RBCs:

    • Potential confusion with yeast (look for buds), oil droplets, air bubbles, and starch due to refractility and size.

    • Reagent strips may help correlate for yeast presence.

  • Dysmorphic RBCs: Indicate glomerular bleeding, strenuous exercise, acanthocytic forms, and can be fragmented and hypochromic, aiding further diagnosis.

  • Clinical Significance: Normal values: 0–3 to 5/hpf.

    • Abnormal counts indicate damage to the glomerular membrane or vascular injury to the genitourinary tract. Conditions include:

    • Macroscopic vs. microscopic hematuria.

    • Cloudy, red urine in advanced glomerular disease or trauma.

    • Clear red urine suggests early glomerular disease, malignancies, or renal calculi.

White Blood Cells (WBCs)

  • Predominant Type: Neutrophils; identified at high power.

  • Glitter Cells: Swollen WBCs in hypotonic urine, sparkling due to Brownian movement. May appear pale blue if stained.

  • Clinical relevance:

    • Normal count generally <5/hpf, higher in females.

    • Increased numbers indicate pyuria, related to infections such as cystitis and pyelonephritis.

Eosinophils

  • Associated with:

    • Drug-induced interstitial nephritis.

    • Renal transplant rejection; identified using Hansel stain with significant percentages over 1% indicating substance.

Mononuclear Cells

  • Comprising lymphocytes, monocytes, macrophages, and histiocytes.

    • Rarely observed but can mimic renal tubular epithelial (RTE) cells.

    • Lymphocytes may resemble RBCs when present in cases of early transplant rejections, potentially requiring cytodiagnostic testing.

Clinical Significance of WBCs

  • An increased number (>5/hpf) indicates possible pyuria, with clinical implications for wen infections.

    • Associated conditions: glomerulonephritis, lupus erythematosus, interstitial nephritis, and certain tumors.

    • Reporting should include presence of bacteria alongside WBC counts.

Epithelial Cells in Urine

  • Three Epithelial Types:

    • Squamous: Found in vagina and urethra; largest cells in urine, indicative of contamination if not midstream clean-catch.

    • Transitional (Urothelial): Found in bladder, upper male urethra; can appear in different shapes. May indicate malignancy when present in syncytia or clumps.

    • Renal Tubular Epithelial (RTE): Also varies in form depending on tubule region; significant in renal pathology.

Clinical Significance of Epithelial Cells

  • RTE cells are particularly concerning, indicating tubular necrosis among other conditions.

  • Influence of certain factors such as heavy metals, drug toxicity, and infections.

Oval Fat Bodies

  • Indicative of nephrotic syndrome; seen with refractile fat droplets demonstrating a Maltese cross under polarized light.

  • Lipiduria can arise from conditions like nephrotic syndrome, and special stains may confirm presence of fats.

Bacteria in Urine

  • Normal urine is sterile, contamination can occur during collection. The presence of WBCs alongside bacteria indicates urinary tract infection (UTI).

  • Reporting structure quantifies as rare, few, moderate, or many.

  • Common bacterial forms include rods and cocci, with rods being the predominant type observed.

Yeast in Urine

  • Characterized by single, refractile, budding forms. Diabetic urine fosters yeast growth due to high glucose and acid levels, and it's crucial to distinguish from RBCs.

Parasites in Urine

  • Most common: Trichomonas vaginalis, identifiable by rapid movement across the field. Others include Schistosoma haematobium and Enterobius vermicularis.

Spermatozoa in Urine

  • Generally of little significance; may appear when expelled into bladder. Reporting should consider laws and clinical relevance but is not routinely significant.

Mucus in Urine

  • Derived from renal tubular epithelium, it appears threadlike and of low refractive index and has clinical relevance when considered with other elements.

Casts: Examination and Clinical Significance

  • Unique to kidneys, formed in distal convoluted tubules and collecting ducts. Identification should occur under low power. Reporting measures the quantity per low power field (lpf).

  • Formed from Tamm-Horsfall protein; consistent excretion is typical in healthy conditions, but proteinuria often indicates abnormality.

Various Cast Types

  • Hyaline Casts: The most common type seen, non-pathologic in small amounts but can signify conditions like glomerulonephritis when present in larger numbers.

  • RBC Casts: Suggest nephron bleeding from glomerular injury; more specific than free RBCs.

  • WBC Casts: Indicative of inflammatory processes, such as pyelonephritis.

  • Bacterial Casts: Combine bacterial components with WBCs or granular casts; evident in infections.

  • Fatty Casts: Associated with nephrotic syndrome and contain oval fat bodies, identifiable under polarized light.

  • Granular Casts: Indicative of cellular disintegration; presence noted often with disease states.

  • Waxy Casts: Reflect advanced renal disease; typically brittle with various characteristics.

  • Broad Casts: Associated specifically with renal failure; may take various forms.

Urinary Crystals Overview and Classification

  • Crystals are often not clinically significant, yet reporting them is standard.

  • Abnormal crystals can indicate underlying pathology such as liver disease or metabolic disorders. Important features include their formation based on conditions like temperature, pH, and concentration.

  • Pathological Crystals: Cystine, cholesterol, bilirubin, tyrosine, and leucine crystals provide diagnostic clues.

Normal Crystals in Urine

  • Acid Urine: Includes amorphous urates and uric acid types, with implications for conditions like gout and metabolic disorders.

  • Alkaline Urine: Contains triple phosphates and invertible salts without adverse clinical significance.

Abnormal Crystals Detailed

  • Notable abnormal crystals such as cystine, bilirubin, and tyrosine have critical diagnostic applications. Confirmatory tests often accompany their identification.

Iatrogenic and Artifact Crystals

  • Iatrogenic crystals are influenced by medications or treatments and may resemble other crystal types, which requires thorough patient history for differentiation.

Conclusion and Considerations

  • Artifacts Resembling Casts: Other materials may resemble casts but lack the essential polarization; identification should consider fiber types and potential contamination sources.

This exhaustive guide encapsulates the full breadth of urinalysis microscopic examination as excerpted from the provided transcript. It encompasses every essential detail necessary for deep understanding and study.