Cell counts_生物行

Tuesday, March 16, 2004

Description
When one refers to particle counting in the clinical laboratory, he or she is usually referring to the counting of blood cells. Cells are, in fact, particles, and these particles are suspended in a liquid medium such as plasma. The basic principles of particle counting in the laboratory generally fall into two categories:

a. Optical method
b. Coulter method

Procedure
Optical, or Flow-Cell Method

This method is based on the fact that a blood cell suspended in plasma, or a liquid medium, is not as transparent as the surrounding fluid. With this method of blood cell counting, the sample is routed through a small passage known as a flow cell. The diameter of the flow cell is constricted so that very few cells may pass through at a time. In theory, it would be nice if only one cell could get through at a time, but in practice, this does not occur. When two or more cells pass through the flow cell counting area at once, the phenomenon is known as coincidence. As one may discern with some careful thinking, the probability of co-incidence will increase with an increase in the concentration of cells or particles in suspension. Since the concentration of, for example, red cells in blood is on the order of about 5.5 million per cubic millimeter, the probability that more than one cell will pass through at any one given moment will be reduced by making a dilution of the sample. If we reduce the concentration of cells by say, a factor of 400, we also reduce the chances of coincidence by a similar amount. The diluent used is an electrolyte, usually isotonic saline, for reasons which will become apparent later. The flow cell is arranged in such a way that a beam of light is directed to pass through it to a detector (photocell) on the opposite side. When a blood cell passes through the flow cell between the light source and the photocell, the amount of light striking the photocell is diminished, due to the increased opacity of the cell, and the resistance of the detection circuit is increased. This increase in resistance results in a drop in the current flowing through the detector circuit. Since each cell produces only a momentary interruption in the beam of light striking the photocell, there is only a momentary drop in detector current. This is known as a pulse. The amount of the drop in detector current is proportional to the size of the cell, since the amount of light blocked from the photocell increases with the cell size. By including in the detector circuit a device which is sensitive to these fluctuations in current, the pulses may be translated into counts, each pulse representing one cell. There is usually included on the counting instrument either a fixed or adjustable threshold adjustment. Essentially what this does is determine the amount of drop in current that must be registered before the pulse is registered as a count. The higher the threshold adjustment, the larger the cell must be before it is recognized and counted. (discrimination between RBC's, WBC's, Platelets, Dust) We mentioned coincidence before. Most automated counters also include in their circuitry a microprocessor which automatically corrects for coincidence. The correction is simply a matter of solving a probability equation, with coincidence a function of cell concentration, size, flow cell diameter, and rate of flow. One exception to this is the Coulter ABI and F Models which employs a chart used to correct the machine count to the actual count. Most counters today are what is known as decade counters. This means that they register every tenth pulse instead of every pulse. Again we run into the problem of probability. If, for example, there is slight contamination of the diluent, or
suspending fluid, with dust, or some other small particle, we can reduce the probability of counting this contaminant as a cell if we count only every tenth pulse. Contamination of this sort becomes a very important source of error, especially as a cell size being counted become smaller, and the threshold settings decrease. After the required volume of fluid is counted and the pulses registered, the machine calculates the concentration of cells per cu. mm. after correcting for coincidence, dilution, and vol. counted. The results are usually presented on a digital display.

Coulter Method (Impedence)

The Coulter method of cell counting is very similar to the optical method, except that with the Coulter method, cells are made to pass through an aperture, in which there is an electrical current flowing. This is done by suspending electrodes in the diluent, one electrode inside the aperture tube, and one electrode outside the tube in the sample cup. The aperture in the aperture tube is approx. 100 microns in diameter and the diluent containing the suspended particles (cells) is drawn up into the aperture tube through the aperture. Another property of cells and particles, other than their opacity, is their increased resistance to the flow of an electric current, compared to the suspending fluid. Therefore, as each cell passes through the aperture, there is a momentary drop in the current flowing between the two electrodes. This causes a temporary drop in the circuit current and is interrupted in the way previously described. Again, the size of the current drop is proportional to the size of the particle.

Sources of Error:
1) contamination of either glassware or diluent
2) improper dilution of sample
3) improper mixing of sample prior to dilution
4) plugged aperture, or flow cell
5) old or improperly collected sample
6) improper threshold settings

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