Automated Instrumentation, Hematology

AbstractHematology analyzers provide information about blood cells and their constituents. The three basic blood cell types are erythrocytes or red blood cells, leukocytes or white blood cells, and thrombocytes or platelets. Hemoglobin is the principal nonaqueous component of red blood cells. Its physiological importance gives it the status of a primary hematological constituent. The aperture impedance principle of blood cell counting and sizing, also called the Coulter principle, exploits the high electrical resistivity of blood cell membranes. In the aperture impedance method, in its simplest form, a d‐c current flows between the electrodes. The resistive cells reduce the current as the cells pass through the aperture, and the current drop is sensed. Aperture impedance counters are flow cytometers, as are all current automated cell counters. Flow cytometers measure cells as the cells flow hydraulically, one at a time, through sensing zones. Aperture impedance counters provide cell volume information as well as cell counts. The light‐scattering principle of cell counting is based on the observation that microscopic particles, such as blood cells, scatter into small (0–15°) angles, most of the visible light incident upon them. This principle is used to count red blood cells, white blood cells, and platelets. The basic single‐angle interval light‐scattering method cannot accurately measure individual red blood cell or platelet volumes, but it can provide mean red cell volume (MCV) and mean platelet volume (MPV). A sphered cell's volume and the hemoglobin concentration can both be accurately determined from Mie Scattering Theory calculations by making simultaneous measurements of light‐scatter intensity over two suitably chosen angle interval. Two suitable angle intervals for red cells are 2–3°, known as low angle scatter (SL), and 5–15°, high angle scatter (SH). Cytochemical techniques can be combined with light‐scattering and absorption measurements to provide five‐part Diffs. Cytochemistry concerns the chemical reactions of cell components. Various method combinations can be used to produce five‐part Diffs including d‐c/r‐f aperture impedance plus light scattering; two‐angle interval light‐scattering plus depolarized light scattering; light scattering plus cytochemistry; and d‐c/r‐f aperture impedance plus cytochemistry. Flow cytometric methods produce five‐part Diffs that are generally more precise and accurate than manual Diffs. However, manual methods can provide more information. Most automated hemoglobinometry methods are derivatives of the manual ICSH reference method, in which the blood sample is diluted in a reagent to lyse the RBC membrane, releasing hemoglobin into solution, ferricyanide present in the diluent diffuses into the hemoglobin molecule yielding methemoglobin, and cyanide diffuses into methemoglobin's interior and reacts with heme to yield cyanmethemoglobin (HiCN), which is measured spectrophotometrically.