Automation and Instrumentation in Clinical Microscopy

• • A goal of the urinaysis laboratory is to maximize productivity and testing quality, while keeping costs and turnaround time at a minimum
  • Studies have shown that the major variable in urinalysis testing is the conscientiousness of the laboratory personnel in their timing and interpretations of the color reactions.
  • Automation brings quality control, precision, faster TAT (turn-around-time), and standardization of results to a method that previously was uncontrolled
• Urine chemistry Semiautomated analyzer

  Daily maintenance is minimal and includes cleaning the reagent strip platform and emptying the reagent strip waste container.
• The semiautomated instrument requires the operator to:

  1. Dip the reagent strip into a urine sample that has been mixed well.
  2. Blot the strip to remove excess urine.
  3. Place the strip onto the reagent strip platform.
  4. Press the analyze/enter button.
• REFLECTANCE PHOTOMETRY 

  • Reflectance photometry uses the principle that light reflection from the test pads decreases in proportion to the intensity of the color produced by the concentration of the test substance
  • A monochromatic light source is directed toward the reagent pads by placing a filter between the light source and the reflective surface of the pad or by using a light-emitting diode (LED) to provide the specific wavelength needed for each test pad color reaction
• REFLECTANCE PHOTOMETRY 

  • The light is reflected to a photodetector, as well as a converter that is either analog or digital. The instruments compare the amount of light reflection with that of known concentrations and then display or print concentration units or transmit data to a laboratory information system (LIS).
  • Reflectance photometry quantifies the intensity of the colored product produced on the reagent strip reaction pads.
• REFLECTANCE PHOTOMETRY 

  • When light strikes a matte or unpolished surface (e.g., a reagent strip), some light is absorbed, and the remaining light is scattered or reflected in all directions. The scattered light is known as diffuse reflectance.
  • Photometers are calibrated using reflectance standards such as magnesium carbonate or barium sulfate that "completely" reflect all incident light
  • The relationship between reflectance and concentration is not linear (inversely proportional)
• Automated Microscopy Analyzers

  • Decrease labor costs and increasing productivity in the urinalysis laboratory
  • Uncentrifuged urine is used, the time spent in handling and preparing concentrated urine sediment for manual microscopy is eliminated
  • Increased standardization of the microscopic examination, which enhances the accuracy and reproducibility (precision) of results
• Types of Automated Microscopy Analyzers

  1. Iris iQ 200
  2. Sysmex UF-Analyzers / Slideless Automated Analyzers
• Iris iQ 200

  • It is an automated system that performs the microscopic examination of urine, as well as cell counts on body fluids
  • Uses patented technologies to capture and automatically classify digital images of urine particles.
  • SOFTWARE USED: AUTO PARTICLE RECOGNITION SOFTWARE or Proprietary neural network software
• Iris iQ 200

  • APR Pre-classifies urine particles in the photographs based on size, shape, texture, and contrast in to 1 to 12 categories. Using the computer monitor, the user can review results, visually assess the particles present, and subclassify them into the 26 or 27 additional categories
• Iris iQ 200

  • The field of view of the microscope is coupled to a digital video camera, and stroboscopic illumination freezes the particles in motion as they stream past, which ensures blur-free imaging. With each sample, the camera captures 500 frames, digitizes them, and sends them to a computer for processing
• iQ200 Auto-classification and Subclassification Categories for Urine Sediment Particles

  • 12 auto-classified sediments are primary parameters measured

  Blood cells

  • Autoclassified by Analyzer: 
  • RBC
  • WBC
  • WBC clump
  • Subclassified by User:
  • RBC clump
• iQ200 Auto-classification and Subclassification Categories for Urine Sediment Particles

  Crystals

  • Autoclassified by Analyzer: unclassified crystals
  • Subclassified by User:
  • Amorphous
  • Calcium carbonate
  • Calcium oxalate
  • Calcium phosphate
  • Triple phosphate
  • Uric acid
  • Cystine
  • Tyrosine
  • Leucine
  • Unclassified crystals
• iQ200 Auto-classification and Subclassification Categories for Urine Sediment Particles

  Casts

  • Autoclassified by Analyzer: 
  • Hyaline 
  • Unclassified casts
  • Subclassified by User:
  • Granular
  • Cellular 
  • Waxy
  • Broad
  • RBCs
  • WBCs
  • Epithelial Cells
  • Fatty 
  • Unclassified casts

  Epithelial cells

  • Autoclassified by Analyzer:
  • Squamous
  • Non squamous
  • Subclassified by User: 
  • Transitional
  • Renal
• iQ200 Auto-classification and Subclassification Categories for Urine Sediment Particles

  Yeast

  • Autoclassified by Analyzer: Budding yeast
  • Subclassified by User: yeast with pseudohyohae

  Others

  • Autoclassified by Analyzer:
  • Bacteria
  • Mucus
  • Sperm
  • Subclassified by User:
  • Trichomonads
  • Fat
  • Oval fat bodies
• Sysmex UF-Analyzers / Slideless Automated Analyzers

  • Principle: Cell flow cytometry (With Electrical impedance for newer models)
  • Urine particles are identified and categorized by fluorescent staining characteristic, light scatter, electrical impedance, and adaptive cluster analysis
  • To perform an automated urine sediment analysis, 1.2 ml of uncentrifuged urine is aspirated into the instrument and divided into two channels: the sediment channel for urine particle analysis and the bacteria channel for bacteria staining and detection
• Sysmex UF-Analyzers / Slideless Automated Analyzers

  • Each channel has a specific stain that targets surface and/or internal components of the cells. The stained urine sample passes through the flow cell, where it is hydrodynamically focused and presented to a red semiconductor laser (635 m)
  • Particles are identified by measuring the height and width of the fluorescent and light scatter signals, which are presented in scattergrams and histograms
• Sysmex UF-Analyzers / Slideless Automated Analyzers

  • In the bacteria channel, the diluent stabilizes the pH and lyses the nonbacterial particles, reducing interference from amorphous crystals. The stain is specific to the ribonucleic acid (RNA) in a bacterial cell, eliminating any nonspecific staining of debris
  • The width of the fluorescent signal measures cellular inclusions, and the width of forward light scatter measures the lenath of cells
• Sysmex UF-Analyzers / Slideless Automated Analyzers

  • A latex particle quality control system monitors performance, and quality control records can be viewed on the instrument screen in a Lew-Jennings graph. The analyzers can store up to 10,000 patient results, including scattergrams, histograms, and specimen characteristics, which can be saved and viewed by the user
• Fluorescent Stain Used in Sysmex UF Analyzers

  • Carbocyanide (blue-green): stains RNA, cell membrane, cytoplasm
  • Phenanthridine (orange): stains DNA, nucleic acid, nucleus, chromosomes
• Light Source Used in Sysmex UF Analyzers

  • Sysmex UF-100 model: Argon laser
  • Sysmex UF-1000i model: red semi-conductor laser
  • For automated particle analysis, the UF-10001 analyzer requires a 4 mL sample volume; however, if the instrument is used in the manual mode, only 1 mL of urine is required Changes in the UF-10001 analyzer include a separate channel for bacterial analysis and the monitoring of lateral or side scatter, which improves detection of bacteria
• Sysmex UF-Analyzers

  • Bacteria is detected specifically by the side light scatter
  • Results are displayed as SCATTERGRAM OR HISTOGRAM
• UF 1000i Particle Detection Categories

  • Particles Enumerated: RBCs, WBCs, Epithelial cells, Hyaline Cast, Bacteria
  • Flagged Particles: Non hyaline (pathologic) cast, Crystals, small round cells (transitional or renal cells), Yeast, Mucus, Sperm
• Additional notes:

  • Determining the specific identity of elements in "flagged" specimens requires a manual microscopic review of the urine by the user.
  • Flagged particles = Particles that cannot be auto-enumerated by the machine and mustbe confirmed by the lab personnel thru manual microscopy
  • The newest Sysmex model, the UN-2000, is an automated, modular, and scalable urinalysis system that combines urine flow cytometry with digital image analysis
• Additional notes:

  • The UF-5000 utilizes fluorescent flow cytometry coupled with a blue semiconductor laser to measure particle length, particle volume, particle internal complexity, and total amount of nucleic acid contained within the particle
• Additional notes:

  • The Sysmex UD-10 is a complementary digital imaging system designed to capture detailed images of urine particles in urine samples that require further review. All captured urine particle images are classified, based on size, into eight different classes. Then the technologist has the opportunity to review the captured images and identify the type(s) of urine particles present
• Additional notes:

  • The UN-2000 uses a single-screen data management system for monitoring analyzer status, result reporting, and reflex rule setting, as well as providing the connection point for interface with the LIS or middleware system
• Full Automated Urinalysis system

  Performing fully automated urinalyses requires combining a urine chemistry analyzer with a microscopy analyzer.