Automated hematology has totally changed the landscape of the hematology laboratory. Fewer manual techniques are required, as more operations become automated. Work patterns have shifted as hematology professionals are expected to maintain quality and morphologic acuity and adjust to increasingly complex instrumentation. Operators of automated instruments (technologists) are expected to have a variety of interpretive skills. Additionally, most of the white cell differentials that are reviewed are usually abnormal. Accurate and discriminating cell identification skills are essential^
As students are trained in their clinical rotations, they become familiar with the instrumentation provided by their clinical site. Yet few students have the luxury of having been trained on automated instrumentation during the didactic portion of their training. Most universities are only able to offer information rather then actual practice on automated equipment. What is needed for the entry-level practitioner is a way to approach interpreting the visual automated data. This skill is not necessarily practiced at university programs, since owning and operating automated equipment are usually cost prohibitive. Training students on multiparameter instruments is primarily left to clinical rotations. This section will attempt to give students a thoughtful approach to bridging the divide between the classroom and the clinical training ground with respect to automated principles and data interpretation. It will NOT be comprehensive and all inclusive. This presentation will cover basic concepts.
Presently, there is an entire menu of services that automated instrumentation provides including
• Embedded quality control programs
• Flagging systems when data fall out of range
• Preparation, examination, and reporting of white cell differentials
• Automatic maintenance in some instruments
Most hematology instruments operate under several basic principles, and these will be outlined.
The author wishes to acknowledge Joyce Feinberg MT (ASCP) of Beckman-Coulter and Kathy Finnegan MS, MT (ASCP) SH of the MT Program at Stony Brook NY for their assistance with this section.
Using this technology, cells are sized and counted by detecting and measuring changes in electrical resistance when a particle passes through a small aperture. This is called the electrical impedance principle of counting cells. A blood sample is diluted in saline, a good conductor of electrical current, and the cells are pulled through an aperture by creating a vacuum. Two electrodes establish an electrical current. The external electrode is located in the blood cell suspension. The second electrode is the internal electrode and is located in the glass hollow tube, which contains the aperture. Low-frequency electrical current is applied to the external electrode and the internal electrode. DC current is applied between the two electrodes. Electrical resistance or impedance occurs as the cells pass through the aperture causing a change in voltage. This change in voltage generates a pulse (Fig. 20.16). The number of pulses is proportional to the number of cells counted. The size of the voltage pulse is also directly proportional to the volume or size of the cell.1
Radiofrequency (RF) resistance is a high-voltage electromagnetic current flowing between the electrodes to detect the size of cells based on the cellular density. RF is a high-frequency pulsating sine wave. Conductivity or RF measurements provide information about the internal characteristics of the cell. The cell wall acts as a conductor when exposed to high-frequency current. As the current passes through the cell, measurable changes are
Aperture current Vacuum (6" Hg)
Aperture current Vacuum (6" Hg)
detected. The cell interior density or nuclear volume is directly proportional to pulse size or a change in RF resistance. The nuclear to cytoplasmic ratio, nuclear density, and cytoplasmic granulation are determined.1
A sample of blood is diluted with an isotonic diluent and then hydrodynamically focused through a quartz flow cell. Cells pass through a flow cell on which a beam of light is focused. The light source is a laser light that is light amplification by stimulated emission of radiation. Laser light or monochromatic light is emitted as a single wavelength. As the cell passes through the sensing zone, light is scattered in all directions. Photodetectors sense and collect the scattered rays at different angles. These data are then converted to an electric pulse. The number of pulses generated is directly proportional to the number of cells passing through the sensing zone. The patterns of light are measured at various angles: forward light scatter at 180 degrees and right angle scatter at 90 degrees. Cell counts, size, cell structure, shape, and reflectivity are determined by the analysis of the scatter light data. Forward angle light scatter (0 degree) is diffracted light which relates to volume. Forward low-angle light scatter (2 to 3 degrees) relates to cell size or volume. Forward high-angle scatter (5 to 15 degrees) relates to the internal complexity or refractive index of cellular components. Orthogonal light scatter (90 degrees) or side scatter is a combination of reflection and refraction and relates to internal components.1
VCS Technology (Volume, Conductivity, and Scatter)
Low-frequency current measures volume, while high-frequency current measures changes in conductivity, and light from the laser bouncing off white cells characterizes the surface shape and reflectivity of each cell. This technology differentiates white cell characteristics.
This is a technique that narrows the stream of cells to single file, eliminating data above and below the focus points. Hydrodynamic focusing allows greater accuracy and resolution of blood cells. Diluted cells are surrounded by a sheath fluid, which lines up the cells in a single file while passing through the detection aperture. After passing through the aperture, the cells are then directed away from the back of the aperture. This process eliminates the recirculation of cells and the counting of cells twice.
322 PartV • Laboratory Procedures
Flow cells are composed of quartz rather than glass and provide a better atmosphere in which to measure cellular qualities. Light does not bend and UV light can pass through the flow cell. Cell characteristics are then measured. The flow cells measure cell volume, internal content, and cell surface, shape, and reflectivity.
Each cell is analyzed through a flow cytometry cell as it is subjected to a variety of angled light scatter. Five subpopulations of cells are identified.
Basic automated hematology analyzers provide an electronic measured red cell count (RBC), white cell count (WBC), platelet count (Plt), mean platelet volume (MPV), hemoglobin concentration (Hb), and the mean red cell volume (MCV). From these measured quantities, the hematocrit (Hct), mean cell hemoglobin (MCH), mean cell hemoglobin concentration, and the red cell distribution width (RDW) are calculated. The newer analyzers include white cell differential counts, relative or percent and absolute number, and reticulo-cyte analysis. The differential may be a three-part differential that includes granulocytes, lymphocytes, and monocytes or a five-part differential that includes neu-trophils, lymphocytes, monocytes, eosinophils, and basophils. The new generation of analyzers now offers a sixth parameter, which is the enumeration of nucleated RBCs (nRBCs). Hematology instruments also include verification systems. The verification system uses review of past results or delta checks and instrument flagging that includes R flags, population flags, suspect flags, and definitive or quantitative flags.1
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