Techniques for WBC differentiation

Laser-based flow cytometry Techniques provides reliable differentiation of the white blood cells

The automated hematology analyzer constitutes a powerful tool that aids physicians in diagnosis and monitoring of disease progression and efficacy of treatment. As such, a hematology analyzer uses sophisticated techniques to provide quantitative analysis results for a variety of parameters.

Complete blood count in health status assessment

Automated hematology analyzers are frequently used in clinical laboratories to assess patients’ health condition. A complete blood count (CBC) is requested by physicians to determine the oxygen-carrying RBCs, the PLTs that help clot the blood, and the WBCs of the immune system. As part of the CBC, a differentiation of the WBCs into neutrophils (NEU), lymphocytes (LYM), monocytes (MONO), eosinophils (EOS), and basophils (BASO) is conducted.

WBC differentiation techniques

Commonly, either a light emitting diode (LED) or laser is used as light source for the WBC differentiation. With LED, light is allowed to pass the cells and transmitted light is detected at zero angle. Unabsorbed (diffused) light passes through the cell, while the absorbed light reflects internal cell structure. A change in the impedance reflects the cell size. Result are displayed in a 2D diagram (Fig 1A).

With laser, cells are exposed to a laser beam and scattered light is detected at three angles. The intensity of the scattered light reflects the cell size and internal structure, while the low-angle signal shows cell size and the middle- and high-angle signals show intracellular (nucleus and cytoplasm) information. With laser scatter, analysis results are displayed in a 3D diagram (Fig 1B).

Compared with polychromatic LED light, laser constitute a more complex monochromatic photometry and the technology is thus costlier. In a laser-based 3D scattergram, however, the cell clusters are well separated, facilitating differentiation of the WBC subpopulations. With a 2D LED-based diagram, close cell clusters might overlap, with the risk of misinterpretation of the results (e.g., over-lysis of NEU could falsely increase LYM).

**Fig 1**. (A) In LED-based WBC differentiation, cell distribution is displayed in a 2D diagram, with cell volume on the x-axis and absorbance on the y-axis. (B) In laser-based WBC differentiation, cell distribution is displayed in a 3D diagram, with the low, mid, and high angle light scatter signals on the axes (scattergram).

Reliability when it counts

Boule’s Swelab Lumi and Medonic M51 hematology analyzers use laser-based flow cytometry for the WBC differentiation. With this technique, cells are forced to flow in a single file through the aperture by a sheath fluid, created by fast-moving diluent that surrounds the slow-moving sample. A laser beam is passed through the sample, and when a cell passes through the sensing zone, the light is scattered and measured by a photoconductor that converts the light into an electrical impulse (Fig 2).

**Fig 2**. Three-angle laser-scatter method, where the low angle signal (about 1° to 5°) represents the cell volume information, the middle angle signal (about 7° to 20°) represents the cell nucleus information, and the high angle signal (about 90°) represents the cell nucleus and cytoplasm information.

Learn more

Read more about the different techniques employed by Boule’s Swelab Lumi and Medonic M51 hematology analyzers.