9.8 Hematology Instrumentation: Microscopy, Counters, Analyzers & Centrifuges

Hematology Instrumentation: Microscopy, Counters, Analyzers & Centrifuges

The Laboratory Operations content area names six instrument groups: microscopes, cell counters, differential analyzers (histograms, digital imaging, scatter plots), coagulation analyzers, point-of-care analyzers, and centrifuges. Sections 8.1 and 8.2 covered cell-counting and flow cytometric measurement principles; this section covers the instruments as equipment, including the optics, maintenance, and physical calculations the examination asks about.

---

1. Microscopy

The Brightfield Microscope

Light travels from the illuminator through the condenser, which focuses it on the specimen, then through the objective, which forms the primary magnified image, and finally through the ocular (usually 10x), which magnifies it again. Total magnification = objective power x ocular power, so a 100x oil objective with a 10x ocular gives 1,000x.

• Numerical aperture (NA) describes the light-gathering cone of an objective. Resolution improves as NA increases: $d = \dfrac{0.61\lambda}{NA}$, where a smaller $d$ means finer detail is distinguishable.
• Immersion oil has a refractive index (about 1.515) close to that of glass. It prevents the light rays from bending away as they cross the air gap, preserving the high numerical aperture of the 100x objective. Without oil, the 100x objective cannot resolve the chromatin detail needed for a differential.
• Kohler illumination is the standard alignment procedure: focus the specimen, close the field diaphragm, focus and center it with the condenser height and centering screws, then reopen it just beyond the field of view and set the aperture (iris) diaphragm to roughly 70 to 80 percent of the objective's numerical aperture. Closing the aperture diaphragm too far increases contrast but destroys resolution and creates diffraction halos; the aperture diaphragm is never used to control brightness, which is adjusted with the light intensity control or neutral density filters.

Other Microscopy Modes

Care. Clean oil from objectives after each use with lens paper and an approved lens cleaner, never with xylene on modern cemented optics or with ordinary tissue, which scratches. Carry the microscope with one hand on the arm and one under the base, store with the low-power objective in position and the stage lowered, and cover it when idle.

---

2. Cell Counters and Differential Analyzers

Cell counters operate by electrical impedance, optical light scatter, or a combination, as detailed in Section 8.1. As equipment, their daily requirements are background counts within acceptance limits, aperture and flow-cell cleaning, verification that carryover is within specification, and startup checks before patient testing.

• Coincidence is the simultaneous passage of two cells through the sensing zone, counted as one; hydrodynamic focusing and electronic coincidence correction address it.
• Aperture obstruction by a clot, fibrin, or debris produces erratic low counts with a characteristic pulse-height abnormality and is cleared by the instrument's burn or backflush cycle.
• Carryover is checked with a high-concentration specimen followed by two low-concentration specimens.

Differential analyzers report through three display formats. Histograms are single-parameter frequency distributions (red cell volume, platelet volume, and the impedance three-part leukocyte distribution). Scatter plots (scattergrams) are two-parameter dot plots that create the five-part differential and flag blasts, immature granulocytes, and atypical lymphoid cells. Digital imaging systems photograph the stained monolayer, use a neural network to pre-classify each cell, and present sorted galleries on screen. Digital imaging does not replace the technologist: pre-classification must be verified and reclassified by a qualified operator, and the system must be validated against manual differentials before use. Its advantages are standardization, remote review, and permanent image storage for competency assessment and consultation.

---

3. Coagulation Analyzers

Coagulation analyzers require a validated platelet-poor plasma input, defined as a residual platelet count below 10 x 10^9/L, achieved by centrifugation at approximately 1,500 x g for 15 minutes (a double spin is used for specimens to be frozen for lupus anticoagulant testing).

---

4. Point-of-Care Analyzers

Handheld and benchtop point-of-care devices use the same underlying chemistries in miniature: reflectance photometry for hemoglobin, electrochemical or optical strip endpoints for INR and activated clotting time, and cartridge electrode arrays for blood gases. Their operating requirements are covered in Section 9.4; as equipment, the recurring issues are cartridge and strip storage temperature, lot-to-lot verification, battery and connectivity status, and documented correlation with the central laboratory method.

---

5. Centrifuges

Types

Relative Centrifugal Force

Protocols specify RCF in x g, not revolutions per minute, because the force depends on the rotor radius as well as the speed:

$\text{RCF} = 1.118 \times 10^{-5} \times r \times (\text{rpm})^2$

where $r$ is the rotating radius in centimetres. Two centrifuges running at the same rpm with different rotor radii deliver different forces, which is why a procedure copied between instruments without recalculating rpm produces incorrectly separated plasma.

Safe and Correct Operation

• Balance opposing tube positions by weight, not by appearance; an unbalanced rotor damages bearings and can fail catastrophically. Use a water-filled balance tube of equal weight when an odd number of specimens is spun.
• Never open the lid until the rotor has stopped completely, and use sealed safety cups or a sealed rotor for any specimen that may aerosolize; a tube that breaks in an open rotor generates an aerosol that persists for minutes.
• Spin tubes stoppered to prevent evaporation, aerosol, and pH change.
• Maintenance and verification: check the timer and speed (tachometer or optical tachometer) at defined intervals, inspect the rotor and buckets for corrosion and cracks, clean and disinfect the chamber, and document all of it. Under-centrifugation is a common source of residual platelets in coagulation plasma, which neutralizes heparin and falsely shortens heparin-sensitive assays.