The classical way of microscopic view of the peripheral blood smear stained by May-Grünwald-Giemsa method.
The method of automatic differentiation by an automatic counter. The automatic counters differentiate the cells using fixed peripheral stained blood smear.
CONTINUOUS FLOW SYSTEM
Flow system - “three-part” and “five-part” differential. Analyzing the leukocyte volume histogram can also do automated leukocyte differential counting. “Three-part” differential is obtained from a WBC histogram that plots the frequency of the WBC whose size has been altered by treatment of diluted blood with a reagent that lyses erythrocytes and punctures the leukocyte cytoplasmic membrane. The cytoplasm collapses around the shrunken nucleus and granules, and these altered WBCs are then counted and sized by impedance. A computer algorithm classifies particles 30 - 90 fl as lymphocytes, 90 - 160 fl as “mononuclear”, and 160 - 450 fl as granulocytes. Most laboratories that utilize the “three part” differential use laboratory-set action limits (i.e., screening based on other CBC parameters), usually combined with some sort of qualified blood smear review. Instruments such as Coulter STKS, SYSMEX NE-8000, and Cell-Dyn 3000 now provide “Five-part” differential leukocyte counts. The Coulter STKS uses an aliquot of the aspirated blood for making the differential in a separate cell flow. White blood cells are stabilized to maintain native size after red cell lysis. Leukocyte volume, conductivity, and laser light scatter measurements are determined simultaneously on each cell and they are discriminated on the basis of a three-dimensional cluster analysis. Sysmex apparatus employs electrical impedance and radio-frequency conductivity in addition to three specific WBC lysing reagents to distinguish between leukocyte populations. Cell-Dyn 3000 apparatus uses multiangle polarized light scatter to determine leukocyte types.
Flow system - Technicon H-1, H-2, and H-3. Technicon H-1, H-2, and H-3 apparatuses are discrete analyzers that perform complete blood and platelet counts, and leukocyte differential count. The instrument has a tungsten halogen light source and cytometer for leukocyte peroxidase analysis, with the addition of a helium-neon red laser for RBC/platelet and basophil determinations. The peroxidase activity is developed at 750C. Red blood cells are lysed, and fixed leukocytes flow in a stream sheat - a layer of inert liquid of the same refractive index. The stream sheath serves to narrow the sample stream, which prevents clogging and keeps the flow cell clean. Within the cell flow, cells are classified one by one on the basis of size (determined by a dark-field light scatter detector) and cytochemical peroxidase reaction. Measurement of the peroxidase activity is sufficient for the most of the WBC differential classification. Lymphocytes are identified as small, unstained cells. Large atypical lymphocytes, plasma cells, and some blasts are characterized as “large unstained cells” (LUCs). Eosinophils exhibit the strongest peroxidase activity and appear smaller than neutrophils because they absorb some of their own scatter signal. Neutrophils are large and have moderate peroxidase activity. Monocytes have somewhat weaker peroxidase staining and are, therefore, in the area to the left of the neutrophils and to the right of the LUCs. The instrument’s computer automatically performs cluster analysis of the WBC subpopulations. The Technicon systems provide both relative (per cent) and absolute (x 103/µl) cell counts for neutrophils, eosinophils, basophils, monocytes, and LUCs. Red cell morphology for anisocytosis, microcytosis, macrocytosis, variations in hemoglobin concentration (anisochromia), hypochromia, and hyperchromia are based on the RBC volume and hemoglobin concentration histograms.
Automated differential method uses stained fixed blood smears. The analyzer recognizes the cells on the basis of their typical features, such as cell size, density and color intensity of chromatin, color, appearance, and structure of the cytoplasm. The automated counter is linked to a microscopic-televised system, so that each cell can be seen and analyzed on the screen. The system is connected with the computer differentiating cells in the smear. The blood film moves on automatically so that each analyzed cell comes into the center of the field of vision.
VCS system (Coulter) is a system that simultaneously measures leukocyte volume, nucleus conductivity, and scattered laser light.