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Blood Bank

Microscope

While modern Blood Banks are increasingly automated, the bright-field binocular microscope remains an indispensable tool for manual tube testing. It serves as the final arbiter in distinguishing negative reactions from weak positive reactions and identifying false-positive interferences. Unlike Hematology or Microbiology, where the microscope is used to identify cellular morphology, Blood Bank utilizes the microscope primarily to detect the physical agglutination of red blood cells (RBCs) and to troubleshoot serological discrepancies

Optical Components & Specifications

To ensure accurate interpretation of antigen-antibody reactions, the microscope must be configured correctly. The viewing of unstained, suspended red blood cells requires specific adjustments to contrast that differ from viewing stained smears

Magnification Systems

  • Oculars (Eyepieces): Typically provide 10x magnification. They are binocular (two eyepieces) and often adjustable to accommodate the user’s interpupillary distance and diopter (vision focus) differences between eyes
  • Objectives: The nosepiece usually holds three or four objectives, but Blood Bank primarily utilizes the 10x (Low Power) objective
    • 10x Objective: Combined with 10x oculars, this provides 100x total magnification. This is the standard setting for reading tube reactions. It allows for a wide field of view to see cell dispersion while providing enough magnification to detect small agglutinates (3-5 cells clumping)
    • 40x (High Dry) Objective: Provides 400x total magnification. This is rarely used for routine agglutination reading but may be utilized to investigate “mixed field” reactions or to distinguish non-cellular debris (like glass or fibrin) from cells
    • 100x (Oil Immersion): Generally not used in routine serology. It may be used if Blood Bank performs Gram stains on suspected contaminated units, but this is often referred to Microbiology

Illumination & Contrast

Because red blood cells in saline are translucent and have a refractive index similar to the suspending medium, high light intensity can make them invisible (“washed out”)

  • Condenser: This lens system, located beneath the stage, focuses light onto the specimen. In Blood Bank wet mounts, the condenser is often lowered slightly to increase contrast, though proper Köhler illumination requires it to be at a specific height
  • Aperture Diaphragm (Iris): This is the most critical adjustment for Blood Bank applications. Opening the iris allows more light (resolution) but reduces contrast. Closing the iris reduces light but increases contrast
    • Operational Tip: For viewing wet RBC suspensions, the iris should be partially closed. This creates “shadowing” around the cells, making the edges of the cells and the agglutinates distinct against the bright background

Clinical Applications in Immunohematology

The microscope is used when macroscopic (visual) examination is inconclusive or when protocols mandate a microscopic check to confirm a negative result

Confirmation of Negative Reactions

In tube testing, particularly for antibody detection (IAT) or antigen typing, a reaction that appears negative to the naked eye must often be examined microscopically before recording the result as negative

  • Technique: The cell button is gently resuspended in the tube. A small sample is transferred to a glass slide (or read directly if using an inverted microscope)
  • Interpretation: The laboratory scientist scans for small clumps of cells
    • Negative: Cells are free-floating and evenly distributed
    • Microscopic Positive: Small clusters of 3-5 cells are present against a background of free cells. This is clinically significant in antibody detection
  • Weak D Testing: Microscopic reading is a mandatory step in the Weak D test (after the AHG phase) to confirm the presence or absence of the D antigen

Differentiation of Rouleaux vs. Agglutination

One of the primary troubleshooting roles of the microscope is differentiating true antibody-mediated agglutination from Rouleaux

  • Agglutination: Antibodies form cross-links between RBCs. Under the microscope, this appears as irregular, “grape-like” clusters. The cells are fused in random orientations
  • Rouleaux: Caused by elevated plasma proteins (globulins/fibrinogen) altering the zeta potential. Under the microscope, RBCs appear stacked like distinct “coins” in linear rows
  • Saline Replacement: If Rouleaux is suspected microscopically, the laboratory scientist performs a saline replacement
    • If the clumps disperse after replacing plasma with saline: It was Rouleaux
    • If the clumps remain: It is true agglutination

Identification of Mixed Field Reactions

Macroscopically, a mixed field reaction looks like a cloudy background with small chunks. Microscopically, this presents a very distinct picture

  • Appearance: The field of view contains distinct, solid clumps of agglutinated cells sitting among a sea of completely free, un-agglutinated cells
  • Significance: This indicates two cell populations, commonly seen in:
    • A patient recently transfused with non-ABO identical blood (e.g., Group O cells in a Group A patient)
    • Bone marrow transplant recipients
    • Fetal-maternal hemorrhage

Maintenance & Quality Control

To maintain the optical integrity required for sensitive readings, the microscope requires specific care protocols

Routine Maintenance

  • Cleaning: Optical surfaces (oculars and objectives) must be cleaned with lens paper and approved optical cleaner
    • Caution: Never use Kimwipes, paper towels, or gauze on lenses, as the wood fibers in these products will scratch the optical coating, permanently damaging the focus
  • Protection: When not in use, the microscope should be covered with a dust cover. Dust accumulation in the internal prisms leads to “floaters” in the field of view that can be mistaken for debris
  • Stage Care: Saline spills on the stage are common in Blood Bank. The stage must be wiped dry immediately to prevent corrosion of the mechanical stage clips and knobs

Köhler Illumination

While often set periodically rather than daily, Köhler Illumination is the standard procedure for aligning the light path to ensure the field of view is evenly illuminated without glare

  1. Focus on a specimen
  2. Close the field diaphragm: (at the base) until a small polygon of light appears
  3. Adjust the condenser height: until the edges of that polygon are sharp and crisp
  4. Center the polygon using the condenser centering screws
  5. Open the field diaphragm until the light just fills the field of view

Troubleshooting Common Issues

  • Issue: “The cells look washed out or invisible.”
    • Cause: Too much light or low contrast
    • Fix: Close the aperture diaphragm (iris) or lower the rheostat (light intensity)
  • Issue: “I see a dark shadow rotating when I turn the eyepiece.”
    • Cause: Dirt or mascara on the ocular lens
    • Fix: Clean the oculars with lens paper
  • Issue: “The image is blurry even when I focus.”
    • Cause: Dried saline or oil on the objective lens
    • Fix: Clean the objective. This happens frequently in Blood Bank if a wet tube touches the lens during transfer
  • Issue: “I can focus at 10x, but when I switch to 40x, it hits the slide.”
    • Cause: The microscope is not Parfocal (lenses are not calibrated to maintain focus when switching)
    • Fix: Requires professional service/adjustment