Microscopic Examination of Urine Sediment

Introduction to Microscopic Examination of Urinary Sediment

  • The microscopic examination is the third part of routine urinalysis, following physical and chemical analyses.
  • Purpose: To detect and identify insoluble materials in urine.
    • Sources of formed elements:
    • Blood
    • Kidney
    • Lower genitourinary tract
    • External contamination
  • Key components detected:
    • Red blood cells (RBCs)
    • White blood cells (WBCs)
    • Epithelial cells
    • Casts
    • Bacteria
    • Yeast
    • Parasites
    • Mucus
    • Spermatozoa
    • Crystals
    • Artifacts
  • Some components are clinically insignificant, while others are normal unless in increased amounts.
  • Importance of both identification and quantitation of urine elements.

Procedural Variations in Microscopic Analysis

  • Various factors affect microscopic analysis:
    • Methods for preparing the sediment.
    • Volume of sediment actually examined.
    • Methods and equipment for visualization.
    • Results reporting methodology.
  • Developed protocols aim to improve standardization and cost-effectiveness.

Macroscopic Screening

  • Purpose: To enhance cost-effectiveness in urinalysis.
  • Microscopic examination is performed on specimens that meet certain criteria based on physical and chemical abnormalities.
  • Key parameters considered significant (may vary among labs):
    • Color
    • Clarity
    • Presence of:
    • Blood
    • Protein
    • Nitrite
    • Leukocyte esterase
    • Glucose (possibly)
  • Automated systems may be programmed with laboratory-specific criteria.
  • The Clinical and Laboratory Standards Institute (CLSI) recommends microscopic examination under certain conditions:
    • When requested by a physician.
    • When testing a specific patient population.
    • When any abnormal physical or chemical result occurs.

Specimen Preparation

  • Specimens should be examined fresh or preserved adequately.
  • Formed elements like RBCs, WBCs, and hyaline casts disintegrate quickly in dilute alkaline urine.
  • Refrigeration may lead to precipitation of amorphous urates and phosphates, potentially obscuring other elements. Warming urine to 37°C can help dissolve these crystals.
  • Clean-catch midstream specimens are preferable to minimize external contamination.
  • Technical Tip 7-1: Warm refrigerate urine specimens to 37°C before centrifugation.

Specimen Volume

  • Standard volume for centrifugation: 10 to 15 mL (commonly 12 mL).
    • Adequate volume allows for representative sampling of urine elements.
    • Pediatric patients may require documentation of volume discrepancies.
  • Some labs correct results based on the volume used (e.g., multiply results by 2 if only 6 mL is used).

Centrifugation

  • Consistent speed and duration are crucial for optimal sediment collection.
  • Recommended settings: 5 minutes at a relative centrifugal force (RCF) of 400.
  • RCF is preferred over RPM to avoid discrepancies based on centrifuge head diameter.
  • Formula for RCF calculation:
    RCF = 1.118 imes 10^{-5} imes ext{radius in centímetros} imes ext{RPM}^2
  • Routine calibration of centrifuges is necessary.
  • Avoid using the braking mechanism to prevent sediment disruption; use capped tubes to prevent biohazardous aerosols.

Sediment Preparation

  • A uniform volume (0.5 to 1.0 mL) of urine and sediment should remain after decantation.
  • Concentration factor calculated as:
    ext{Concentration Factor} = rac{ ext{Volume of urine centrifuged}}{ ext{Volume of sediment}}
  • Technical Tip 7-2: Commercial systems provide calibrated tubes for decanting and ensure consistent volume for sediment suspension.
  • When aspirating sediment, avoid pouring to reduce sediment disruption. Gentle agitation is essential for resuspension.
    • Aggressive stirring can damage cellular elements.

Volume of Sediment Examined

  • Consistent slide volume is vital for each specimen.
  • Recommended volume for microscopy: 20 μL (0.02 mL) under a 22 x 22 mm cover slip.
  • Ensure sediment does not overflow outside the cover slip to avoid losing heavier elements.

Commercial Systems

  • Enhanced methods via commercial slide systems currently available include:
    • KOVA (KOVA International)
    • Urisystem (Thermo Fisher Scientific)
    • Count-10 System (Myers-Stevens Group)
    • Quick-Prep Urinalysis System (Globe Scientific)
    • CenSlide 2000 Urinalysis System (Iris Diagnostics)
    • RS Urine Sediment Workstation (VWR, Avantor)
  • Features:
    • Capped, calibrated centrifuge tubes.
    • Decanting pipettes for precise sediment volume control.
    • Slides designed for consistent sediment examination and calibrated grids for efficient quantitation.
  • Closed systems like Cen-Slide and RS minimize exposure and contamination risks.

Examining the Sediment

  • Consistent examination method required; minimum of 10 fields observed at both low (10x) and high (40x) power.
  • Start examination with low power to detect casts and assess sediment composition; switch to high power for element identification.
  • Casts typically found at edges of cover slip (in conventional methods), but not in standardized systems.
  • Sediments examined under reduced light due to similar refractive indices with urine.
  • Difficulty focusing initially; use epithelial cells as reference points to ensure accurate plane examination.
    • Avoid focusing on artifacts due to their larger size which can mislead focus.
  • Technical Tip 7-3: Use reduced light for microscopic examination at all magnifications.

Reporting the Microscopic Examination

  • Terminology and methods of reporting will vary, but consistency within laboratories is crucial.
  • Casts reported as average per low-power field (lpf), RBCs and WBCs as average per 10 high-power fields (hpf).
  • Other elements reported semi-quantitatively (e.g., rare, few, moderate, many).
  • Establish laboratory reference values based on sediment concentration factor.
  • Example conversion steps for standardized reporting:
    1. Calculate area of lpf or hpf:
    • ext{Area} = ext{π}r^2 (where diameter of hpf = 0.35 mm)
    • Area of hpf = 0.096 mm².
    1. Determine maximum number of fields in viewing area:
    • Area under cover slip = 484 mm² → rac{484}{0.096} o 5040 ext{ hpf}
    1. Compute number of hpfs per milliliter:
    • ext{hpfs per mL} = rac{5040}{0.02 imes 12} = 21,000 ext{ hpfs/mL}
    1. Calculate formed elements per milliliter:
    • ext{WBCs per mL} = 4 ext{ WBC/hpf} imes 21,000 ext{ hpfs/mL} = 84,000 ext{ WBC/mL}
  • Laboratories should decide on relevance of an additional calculation step in the report.

Correlating Results

  • Microscopic results must correlate with physical and chemical urinalysis findings for accurate reporting.
  • Check specimens with discordant results for technical and clerical errors.
  • Table 7-2 described common correlations in urinalysis, while taking into consideration formed elements, chemical interferences, and specimen age.
  • Technical Tip 7-4: Recheck urine specimens that show discrepancies between physical, chemical, and microscopic results.

Sediment Examination Techniques

  • Various factors can alter urinary sediment appearance:
    • Stage of development and degeneration of cells and casts.
    • Distortion caused by chemical contents of the specimen.
    • Inclusions in cells and casts.
    • Contamination by artifacts.
  • Due to these variables, identification of sediment elements can be challenging even for experienced microscopists.

Historical Note: Addis Count

  • Developed in 1926, standardized quantification of formed elements in urine analysis used a hemocytometer.
  • Normal ranges for the Addis count:
    • RBCs: 0 to 500,000
    • WBCs and epithelial cells: 0 to 1,800,000
    • Hyaline casts: 0 to 5000
  • The Addis count was primarily used to monitor diagnosed renal diseases but has been replaced by modern commercial systems for analyzing non-timed specimens.