Microscopic Examination of Urine

Introduction to Microscopic Examination of Urine

  • Third part of routine urinalysis: microscopic examination of urinary sediment.
    • Purpose: Detect and identify insoluble materials in urine.
  • Sources of formed elements:
    • Blood
    • Kidney
    • Lower genitourinary tract
    • External contamination
  • Components detected include:
    • Red blood cells (RBCs)
    • White blood cells (WBCs)
    • Epithelial cells
    • Casts
    • Bacteria
    • Yeast
    • Parasites
    • Mucus
    • Spermatozoa
    • Crystals
    • Artifacts
  • Importance of quantification: Some elements are clinically significant, while others are normal unless in increased amounts.
  • Factors affecting microscopic analysis:
    • Methods of sediment preparation
    • Volume of sediment examined
    • Visualization methods and equipment
    • Reporting of results
  • Protocols: Developed to enhance standardization and cost-effectiveness of microscopic urinalysis.

Macroscopic Screening

  • Purpose: Enhance cost-effectiveness of urinalysis through selective microscopic examination.
  • Criteria for microscopic examination: Based on abnormalities in physical and chemical urinalysis results.
    • Parameters considered significant:
    • Color
    • Clarity
    • Presence of blood
    • Protein
    • Nitrite
    • Leukocyte esterase
    • Possibly glucose
  • Automated instruments: Criteria can be programmed into devices for testing.
  • Detection rates of abnormalities vary: Percentages of abnormal specimens undetected differ significantly across studies.
  • Considerations for patient populations:
    • Pregnant women
    • Pediatric patients
    • Geriatric patients
    • Diabetic patients
    • Immunocompromised patients
    • Renal patients
  • CLSI Guidelines: Microscopic examination recommended when:
    • Requested by a physician
    • Laboratory-specified patient population tested
    • Any abnormal results in physical or chemical tests.

Specimen Preparation

  • Examining specimens: Must be fresh or properly preserved to prevent degradation of formed elements.
    • Key elements affected: RBCs, WBCs, and hyaline casts; rapidly disintegrate in dilute alkaline urine.
  • Refrigeration precautions: Can cause precipitation of amorphous urates and phosphates that obscure sediment components.
  • Warm specimens prior to centrifugation: Recommended to 37°C to dissolve certain crystals.
  • Clean-catch midstream specimens: Reduces external contamination for accurate sediment analysis.
  • Dilute random specimens: May produce false negatives; thorough mixing required before decanting.
    • Technical Tip 7-1: Warm refrigerated urine specimens to 37°C before centrifuging to dissolve amorphous urate crystals.

Specimen Volume

  • Standard urine volume for centrifugation: 10 to 15 mL, with 12 mL commonly used.
    • This volume allows for the full engagement of multiparameter reagent strips and is often calibrated in centrifuge tubes.
  • Adjustment for smaller volumes: If less than 12 mL is used (e.g., pediatric cases), report the volume to facilitate corrections.
    • Example: Only using 6 mL, results doubled for reporting accuracy.

Centrifugation

  • Consistent speed and time are critical for effective sedimentation.
    • Recommendations: Centrifuge for 5 minutes at a relative centrifugal force (RCF) of 400.
  • RCF Calculation: Can be calculated using the formula:
    RCF = 1.118 imes 10^{-5} imes ext{radius in cm} imes ext{RPM}^2
  • Calibration Frequency: Calibration of centrifuge should be performed routinely.
  • Avoid Braking Mechanism: Slowing down centrifuge disrupts sediment before decantation.
  • Biohazard Precautions: Must centrifuge all specimens in capped tubes to prevent aerosols.

Sediment Preparation

  • Post-Centrifugation: Maintain a consistent amount of urine and sediment in the tube after decantation, with 0.5 to 1.0 mL commonly retained.
  • Concentration Factor Calculation: Calculated by dividing the volume of urine centrifuged by the sediment volume.
    • Example factors: 24 (if 12 mL urine, 0.5 mL sediment) and 12 (if 12 mL urine, 1.0 mL sediment).
  • Technical Tip 7-2: Utilize commercial systems for decanting that provide a consistent volume for suspension.
  • Methods of aspiration: Preferable to aspirate sediment rather than pour to maintain concentration factor.
  • Thorough Resuspension: Essential to achieve an equal distribution of elements during microscopic examination.

Volume of Sediment Examined

  • Recommended volume to place on slides: 20 μL (0.02 mL), covered by a 22 x 22 mm glass cover slip.
  • Importance of Volume: Flow beyond the cover slip can lead to loss of some heavier elements, like casts.
  • Commercial Systems: Often designed to ensure accurate measurement and volume control for sediment analysis.
    • Slide features: Designed to ensure a monolayer of sediment and calibrated grids for improved quantitation.

Commercial Systems

  • Overview of traditional methods enhanced by commercial slide systems.
    • Examples of systems include:
    • KOVA (KOVA International)
    • Urisystem (Thermo Fisher Scientific)
    • Count-10 System (Myers-Stevens Group)
    • Quick-Prep Urinalysis System (Globe Scientific)
    • CenSlide 2000 (Iris Diagnostics - Beckman Coulter)
    • RS Urine Sediment Workstation (VWR)
  • Features Provided by Commercial Systems:
    • Capped, calibrated centrifuge tubes
    • Decanting pipettes for controlled sediment volume
    • Slides that ensure a consistent layer and provide quantitation grids.
  • Closed systems: Some systems, like the Cen-Slide and RS Workstation, minimize exposure to specimens by permitting direct readings or pumping samples for examination.

Examining the Sediment

  • Consistent techniques during microscopic examination:
    • Minimum of 10 fields must be observed using low (10x) and high (40x) power.
    • Commence examination at low power to detect casts and assess overall sediment composition.
  • Changing settings: Transition to high power when identifying specific elements like casts.
  • Casts location: Often found on the edges of cover slips in the conventional method; may not be an issue with standardized systems.
  • Visual challenges with unstained sediment: Many constituents have similar refractive indices to urine; therefore, illumination adjustments are critical during examination.
  • Initial focusing tips: Can be difficult; epithelial cells may help provide focal reference; avoid focusing artifacts that could distract from examining relevant elements.