Till the late 1950s, blood cells were routinely counted manually. In 1956, W. H. Coulter patented a system which used the electrical impedance process, also known as aperture impedance, to count blood cells. This innovation made the blood cell counts production quicker, smoother, and more accessible. The first instruments of hematology conducted only counts of the RBC and the white blood cell (WBC). Later, measurement of hemoglobin was introduced. Instruments are now available that include values for 60 or more parameters, either direct or estimated. Only the RBCs, WBCs, hemoglobin, platelets, and reticulocytes are specifically counted or measured into these instruments. From the results of the RBC count and hemoglobin the hematocrit, red cell indices and other values are determined.
The several measurements given by automated and semi-automated instruments are mainly improvements to the original manual techniques but there are some exciting modern technology applications. Such instruments are reliable and precise, and reproducible measurements. Many manufacturers of instruments employ similar techniques and measurement devices, calculating the same parameter in a slightly different way. Both systems have advantages and drawbacks and it may be claimed that one set of problems can be substituted for another.
Two basic cell-counting technologies
Electrical impedance was the only automated cell-counting method until the 1970s, when light-scatter cell-counting technology was developed. Today’s cell counters are based on one of these two technologies.
Principle of Electrical-Impedance Cell Counting
In an electrolyte solution, which conducts electricity, blood cells counted by electrical impedance are diluted. The electrical current passes through the opening or through the opening of the measuring device in the electrolyte solution from one electrode to another.
Blood cells are bad conductor of electricity. The non-conducting cell creates an impedance or disruption in the electrical circuit when a cell suspended in the electrolyte solution passes through the aperture. The impedance of the electric circuit induces a pulse. Those pulses are known to be cells. The impedance size is proportional to the size of the cell which causes it. The instrument thus not only tracks how many cells move through the aperture but also the size of each cell. The counters of the electrical-impedance cells are based on the concepts of Coulter.
Principle of Light-Scatter Cell Counting
A laser beam or tungsten halogen light beam is guided at a current of blood cells that travel through a narrow channel in the light-scatter cell counters. The channel is incredibly small causing the cells to move in one file. When a cell is struck by the beam of light, the beam is spread at an angle. The intensity and scatter angle of the light beam differ depending on the type of cell. This is known as the cell refractive index. The refraction index is determined by the cell’s form and volume while volume has the greater effect on the scatter. Sensors measure how much light is transmitted, and how much the cell absorbs from the laser.
The laser light is monochromatic, meaning it only has one wavelength, so it flies in one direction. This two features allow it to be more fine tuned than the tungsten halogen light beam and make it more useful in diagnostic hematology for generating scatter patterns. Another drawback is that instruments with laser beams can not be equipped with the same components as most counters. The laser counters can be calibrated only for human blood cells.
A major improvement applied to the light-scatter system is sheath-flow, in which the cells are hydrodynamically oriented. Sheath flow increases efficiency and also helps to prevent the problems of washing and preserving electrical impedance models.
Measurement of Hb is based on the linear relationship between the amount of light absorbed in a given absorption band and the sample absorption source concentration (Beer’s Law) (Skoog DA, West DM, 1965). Many automatic counters use an absorbance reading cyanmethaemoglobin process at wavelengths of 525 or 540 nm. Sysmex applications use sodium lauryl sulphate, with the analyser’s bonus that no cyanide is in the water.
Red cell measurements
Red cells can be counted by aperture impedance, lightscattering techniques – using Laser LED or tungsten or a combination of the two technologies.
Reticulocyte and nucleated RBC measurement
Manufacturers of cell counters also have an evaluation of reticulocytes on their devices. Many devices use RNA fluorescent dyes with laser light to test reticulocyte and related parameters. Some devices use supravital specific dyes that allow reticular material to precipitate and stain, and are then assessed by light absorbance and scatter.
Instrument manufacturers have introduced separable approaches to nucleated RBCs. After the RBC cytoplasm is stripped, nuclei are counted using combinations of light scatter and impedance. The counting is performed on the WBC site, and the complete WBC has been fixed for NuRBC ‘s participation. NuRBC counts are calculated in the Sysmex analyzers through flow cytometry and laser light. The material is combined with an RNA / DNA stain and is aspirated. NucRBC is counted using side fluorescence, providing knowledge about RNA / DNA, and forward scattering, a cell size calculation. The NucRBC count is expressed in all analysts as NucRBC/100 WBC, from the formula:
NucRBC% = (NucRBC ×100)/(NucRBC + WBC)
White cell measurements
White cells can be counted by a variety of technologies and divided into a large number of categories. Both have benefits and drawbacks. The creation of sheath flow-based counting systems , which allow single cells to move through the sensing region, has led to multi-parameter analysis on the same cell by integrating the signals from multiple sensors. The sensing region can be either electrical, conductive, impedant or a mixture of both, depending on the manufacturer, and the sensors can be mounted to look forward to light scatter, side scatter, small- or wide-angle scatter, polarized light scatter, or fluorescence.
The analyzers that generate a 3-part gradient expose the blood cells to a special reagent that shrinks to a certain degree the cytoplasm of each form of WBC, with the lymphocytes shrinking the most. This allows the cells to be divided into three distinct classifications of size: lymphocytes, mononuclear cells and granulocytes. WBCs with a size range of 35-99 fL are grouped as lymphocytes, cells within a range of 100-200 fL are categorized as mononuclear (or “mids”) cells, and cells larger than 200 fL are called granulocytes.
The differential of three parts does not divide the granulocytes into neutrophils, eosinophils and basophils. The machine warns the user to irregular numbers of RBC or WBC, atypical lymphocytes and giant platelets.
Several analyzers for hematology have 5-part differentials. The WBCs are categorized in neutrophils, lymphocytes , monocytes, eosinophils and baseophils in the normal study. Some variant lymphocytes or other immature cells are known as large non-stained cells (LUCs). Instruments conducting a five-part differential apply chemicals or stains like peroxidase and Alcian blue to the sample, subject the sample to a focused light beam and describe the light differential pattern of every cell. The show is called a cytogram or a scattergram.
The impedance, light scatter, a mixture of the two and the flow cytometry with fluorescence can be used to count platelets and thickness. Instruments which use a platelet count impedance from the number of cells in the curve. The accuracy of this count is improved by using a curve fitting software when there are platelets greater than 20 femtoliters (fl). The number of the platelet is expanded to 70 fl above the 35 fl limit.
Some instruments combine the disperse signals for each cell with the high-angle (5–150) and low-angle (2–30). These are translated into an axis volume and refractive index values displayed on the horizontal axis, giving a cytogram of the platelet dispersion. The curved lines reflect a grid with a different refractive index where the platelets fall. The macroplates and red cells can also be distinguished by drawing the refractive index against the platelet volume scale.
- Basic Clinical Laboratory Techniques, 6th Edition, Delmar Cengage Learning
- The science of Laboratory Diagnosis, 2nd Edition, John Wiley & Sons, Ltd