Sample cells, light scattering

In principle, the two-angle interval method can produce all CBC parameters within a single measurement channel, uniquely providing ceU-by-ceU hemoglobin concentration. The mean of the concentrations provides an alternative (and direct) measurement of MCHC. The method also provides an alternative HGB measurement, because HGB may be set equal to (RBC x MCV x MCHC)/1000. This method, like the basic light-scattering method, uses the same flow cell to measure platelets and ted cells with the result that the method is capable of providing the CBC parameters RBC, HGB, HCT, MCV, MCHC, MCH, and PLT. The method can also count a sample s white blood cells if the sample s red blood cells have been lysed. 

Scattering of excitation light in the direction of the detector and of the emitted light can influence the precision of the measurement. At very high concentration of analyte, emission may not be seen because most of the exciting light is absorbed near the wall of the sample cell. The fact is that most molecules do not fluoresce and cannot be... 

Figure 10. Optical setup for LDA consisting of a laser beam being split and focused by a lens through a suspension of particles in a sample cell. Scattered light is focused through a pinhole and detected by a photomultiplier tube.

Set up the flow cytometer with fluorescence detectors turned off and the acquisition terminator set for TIME, so that a constant volume from each sample will be analyzed. A time interval (e.g., 1 min) that will be sufficient for the acquisition of approximately 10,000 events in the control samples should be used. Acquire light scatter signals (ESC and SSC) for each sample, vortexing the cell suspensions briefly but vigorously before introducing each sample into the flow cytometer. 

The light scatter assay may be used to determine absolute numbers of viable cells if flow cytometry data from cell suspensions of known concentration are used to construct a standard curve. For that purpose, cell concentrations should be determined in a series of graded, standard cell suspensions with the use of a Coulter counter. A plot of those standard concentrations versus the number of events (Hght scatter signals) acquired during a specified acquisition interval in the flow cytometer may then be used to interpolate cell concentrations for test samples that have been assayed by the light scatter procedure. 

Analyze the samples on the flow cytometer. (See Chapter 36, Fig. 2, for normal light-scatter profiles.) When the fluorescence gain is properly adjusted, the unstimulated control cells (shaded curve. Fig. 2) should be visible on scale with the PMA-stimulated cells (open curves. Fig. 2). PMA (1-100 ng/mL) should stimulate a dose-dependent (graded) response. All normal granulocytes should exhibit an oxidative metabolic response to PMA (see Note 2). 

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