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.
Automated and semi-automated instruments are better than manual techniques because they give more accurate and consistent measurements. These instruments use similar techniques and devices to measure things, but each manufacturer may do it slightly differently. Both methods have pros and cons, so it’s like swapping one set of problems 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 tiny channel in a machine called a light-scatter cell counter guides blood cells in a single line. A laser or light beam shines on them, and when a cell is hit by the light, it scatters in different directions depending on what kind of cell it is. This is because of something called the cell refractive index, which is based on the cell’s shape and size. The machine measures how much light the cell absorbs and scatters to determine the type of cell.
The laser light employed in light-scatter cell counts is unique in that it has just one wavelength and so travels in a straight line. As a result, it is more exact than other forms of light, such as tungsten halogen light beams. The laser beam is very effective in diagnostic hematology because it may produce highly accurate scatter patterns that can be utilized to distinguish various types of blood cells.
There are various disadvantages of employing laser light in cell counting. One drawback is that laser beam instruments cannot be installed in the same components with other types of cell counts. This implies that the laser counters can only be calibrated for use with human blood cells and may require other component to when used to evaluate other types of celluar components.
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.
Hb measurement
The measurement of Hb is based on the linear correlation between the amount of light absorbed in a specific absorption band and the sample absorption source concentration (Beer’s Law) (Skoog DA, West DM, 1965). Several automated counters employ an absorbance reading cyanmethaemoglobin process at wavelengths of 525 or 540 nm. Sysmex applications employ sodium lauryl sulphate, with the added benefit of no cyanide in the solution.
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 also 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
There are several methods for counting white blood cells, each with its own set of advantages and disadvantages. A sheath flow-based approach is one unique method of counting them. Individual cells can travel across a sensing region where many sensors detect distinct aspects of the cell. Depending on the manufacturer, the sensing area can employ various forms of signals such as conductance or impedance, and the sensors can look at various things such as light scatter or fluorescence. This enables a more in-depth examination of each individual cell.
Three-Part Differential
Some machines count white blood cells by using a particular reagent that shrinks the cytoplasm (the fluid inside the cell) of each kind of white blood cell to varying degrees. This results in a three-part gradient where the cells may be categorised based on their size. Lymphocytes are the smallest and decrease the greatest, falling into the 35-99 fL range. Mononuclear cells, often known as “mids,” have a volume of 100-200 fL. Lastly, granulocytes are the biggest cells, with sizes more than 200 fL. It is easier to count and identify the different types of white blood cells in a blood sample when the cells are divided into these three separate size groups.
The 3-part differential system of counting white blood cells does not further differentiate granulocytes into their respective categories, such as neutrophils, eosinophils, and basophils. Instead, the machine simply categorizes them depending on their size, as previously mentioned.
If the machine finds an abnormal number of red or white blood cells, atypical lymphocytes, or large platelets, it will alert the user. These anomalies might be an indication of an underlying health problem that may require further investigation.
Five-Part Differential
Certain hematology analyzers employ a 5-part differential approach to count white blood cells. This approach divides white blood cells into five types: neutrophils, lymphocytes, monocytes, eosinophils, and basophils. This offers a more complete analysis of the different kinds of white blood cells present in the sample.
In addition to the five primary groups, there may be variant lymphocytes or other immature cells known as big non-stained cells (LUCs).
To perform a 5-part differential, the analyzer uses specific reagents or stains on the blood sample, such as peroxidase and Alcian blue. After that, the sample is exposed to a focussed laser beam, and the analyzer evaluates the light differential pattern of each individual cell. This pattern is represented visually as a cytogram or scattergram, which shows the different kinds of cells present in the sample.
Platelet counting
In hematology analyzers, numerous approaches, including impedance, light scatter, a combination of the two, and flow cytometry with fluorescence, can be used to count platelets and measure their size.
Platelet count based on impedance is used by certain equipment to calculate the amount of platelets in a sample. For samples with platelets more than 20 femtoliters (fl), curve fitting software is employed to increase accuracy, extending the platelet count range to 70 fl over the 35 fl limit.
Other equipment combine each cell’s dispersion signals with high-angle (5-150) and low-angle (2-30) data, which are then converted into volume and refractive index measurements. These results are shown on the horizontal axis to form a platelet dispersion cytogram. The graph’s curved lines represent a grid with varied refractive indexes where platelets fall. By graphing refractive index against platelet volume, this approach may distinguish between macroplates, red cells, and platelets.
References:
- Basic Clinical Laboratory Techniques, 6th Edition, Delmar Cengage Learning
- The science of Laboratory Diagnosis, 2nd Edition, John Wiley & Sons, Ltd
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