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Hematology

Microscopes

Despite the sophistication of modern automated hematology analyzers, the optical microscope remains the “Gold Standard” for morphological evaluation. When an instrument flags a result for review, or when a manual body fluid analysis is required, the laboratory scientist relies on the microscope to differentiate immature cells, identify inclusions, and diagnose pathologies. The primary instrument used in the clinical laboratory is the Compound Brightfield Microscope, though variations such as Phase Contrast and Polarizing microscopy are essential for specific hematologic and body fluid applications

Principles of Optics

To operate a microscope effectively, the laboratory scientist must understand the physics that govern image quality. The goal of microscopy is not merely magnification (making an image larger), but resolution (making an image clearer)

Magnification

Magnification is the process of enlarging the apparent size of an object. In a compound microscope, this is achieved through a two-stage system: the objective lens and the ocular lens (eyepiece)

  • Total Magnification: Calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens
    • Formula: \(\text{Total Mag} = \text{Objective Power} \times \text{Ocular Power}\)
    • Standard Oculars: Usually 10x magnification
    • 10x Objective (Low Power): Total Mag = 100x. Used for scanning the smear for cell distribution and platelet clumping
    • 40x Objective (High Dry): Total Mag = 400x. Used for estimating WBC counts and selecting an area for the differential
    • 100x Objective (Oil Immersion): Total Mag = 1000x. Used for the identification of cells (WBC differential), RBC morphology, and platelet estimation

Resolution (Resolving Power)

Resolution is defined as the shortest distance between two points at which they can still be distinguished as separate entities. If a microscope has high magnification but poor resolution, the image will be large but blurry (empty magnification)

  • Numerical Aperture (NA): The resolution is determined by the Numerical Aperture of the objective lens. NA is a number that expresses the light-gathering ability of the lens. The higher the NA, the greater the resolution
  • Wavelength: Resolution is also dependent on the wavelength of light used. Shorter wavelengths (blue light) provide better resolution than longer wavelengths (red light). This is why many microscopes have a built-in blue filter

Refractive Index & Immersion Oil

Light travels at different speeds through different media (air vs. glass). When light passes from the glass slide into the air gap between the slide and the objective lens, it bends (refracts). This bending causes light to miss the objective lens, resulting in a loss of resolution

  • The Air Gap: With low-power objectives, the air gap is acceptable. However, at 1000x magnification, the refraction is too great
  • Immersion Oil: Type B immersion oil has the same Refractive Index (\(n = 1.515\)) as glass. By placing oil between the slide and the objective, the air interface is eliminated. Light passes straight from the slide, through the oil, and into the lens without bending. This maximizes the Numerical Aperture and improves resolution

Components of the Microscope

The standard clinical microscope is composed of three systems: the Support System, the Illumination System, and the Optical System

Illumination System

  • Light Source: Typically a Tungsten-Halogen bulb or LED. It is located at the base of the microscope
  • Condenser: Located beneath the stage. Its function is to gather light from the source and concentrate it into a cone of light that illuminates the specimen. The condenser must be centered and focused (moved up or down) to ensure optimal lighting
  • Aperture Diaphragm (Iris): Located within the condenser. This is the most critical adjustment for contrast
    • Opening it: Increases resolution and brightness but decreases contrast
    • Closing it: Increases contrast (making unstained structures visible) and depth of field, but decreases resolution
    • Hematology application: For stained smears (Wright-Giemsa), the iris is generally open to maximize resolution. For wet preps (urine/fluids), the iris is partially closed to increase contrast

Optical System

  • Objectives: The most important lenses, screwed into the revolving nosepiece. They are typically Parfocal, meaning that if the image is in focus at 10x, it will remain roughly in focus when switched to 40x or 100x, requiring only fine adjustment
  • Oculars (Eyepieces): The lenses the laboratory scientist looks through. They can be adjusted for Interpupillary Distance (width between eyes) and Diopter (vision differences between the left and right eye)

Köhler Illumination

Köhler illumination is a method of adjusting the microscope to provide the most uniform, glare-free illumination possible. It ensures that the filament of the light bulb is not visible in the field of view. A microscope that is not adjusted for Köhler illumination will produce artifacts, poor resolution, and eye strain

The Procedure for Setting Köhler Illumination

  1. Focus: Place a specimen on the stage and focus on it using the 10x objective
  2. Close Field Diaphragm: Close the Field Diaphragm (located at the base of the scope) until a small polygon of light is visible
  3. Focus Condenser: Move the condenser up or down until the edges of the polygon are sharp and crisp (magenta/green halo is minimized)
  4. Center: Use the centering screws on the condenser to move the polygon of light to the exact center of the field of view
  5. Open Field Diaphragm: Open the diaphragm until the circle of light just barely clears the field of view. Opening it too wide creates glare
  6. Adjust Aperture: Adjust the Aperture Iris in the condenser to match the Numerical Aperture of the objective (usually by removing an eyepiece and looking down the tube to see that 75-80% of the field is lit)

Specialized Microscopy in Hematology

While Brightfield is used for standard blood smears, other techniques are required for specific body fluids or manual counts

Phase Contrast Microscopy

Phase contrast is used to visualize transparent or low-refractive-index objects that are difficult to see with brightfield microscopy without staining

  • Principle: It converts differences in the “phase” of light waves (which the eye cannot see) into differences in “amplitude” or brightness (which the eye can see). Light passing through a cell is slowed down relative to background light. The phase contrast microscope shifts this background light to create destructive interference, making the object appear dark against a light background
  • Hematology Applications
    • Manual Platelet Counts: Using the ammonium oxalate (Unopette) method, platelets appear as bright, refractile bodies that are easily distinguished from debris
    • Body Fluids: Essential for counting WBCs and RBCs in clear CSF or identifying hyaline casts in urine/fluids

Polarizing Microscopy

Polarizing microscopy is used to identify birefringent (doubly refractive) substances. It utilizes two filters: a polarizer (below the condenser) and an analyzer (between the objective and eyepiece)

  • Principle: When the polarizer and analyzer are crossed, the field is black. If a birefringent object (like a crystal) is placed in the path, it rotates the light, making the object glow against the dark background
  • Hematology Applications
    • Synovial Fluid Analysis: This is the definitive method for distinguishing crystals in joint fluid
    • Monosodium Urate (Gout): Needle-shaped crystals that are Negatively Birefringent (Yellow when parallel to the compensator axis, Blue when perpendicular)
    • CPPD (Pseudogout): Rhomboid crystals that are Positively Birefringent (Blue when parallel, Yellow when perpendicular)

Care & Troubleshooting

  • Cleaning: Objectives should only be cleaned with optical lens paper and approved lens cleaner. Tissues or gauze can scratch the coating. Oil must be removed from the 100x objective immediately after use
  • Oil Error: A common student error is dragging the 40x (High Dry) objective through the oil. The 40x lens is not sealed against oil; if oil penetrates it, the lens is permanently ruined and the image will appear “foggy” indefinitely
  • Micrometer Adjustment: To perform manual counts or measurements, an Ocular Micrometer (a ruler etched into the eyepiece) must be calibrated against a Stage Micrometer (a slide with a known scale) for each objective to determine the physical size of the objects being viewed