55
ABSOLUTE COUNTS
Determining absolute concentration is difficult because no commercial flow cytometers
presently provide this measurement. The most direct way of adding it would be to build the
flow cytometer with an injector mechanism that either injects a fixed volume or injects the
sample at a known volume flow rate. Although some instruments have such injectors, one
must be careful about how accurate they are. It is a common problem that not all of the
injected sample passes through the measurement point. It is usually necessary to chase the
sample in with an air bubble or a blank sample to be certain that all of the injected sample
was analyzed. In order to use injection rate as the basis for calculating concentration, it is
necessary to insure that the sample is uniformly mixed, or that the average particle flow rate
is used in the calculation. For various technical (and perhaps economic) reasons these
approaches have not yet been implemented.
A method that does not require instrument modification is that of mixing distinctive particles
into the sample at a known concentration. The relative numbers of these particles and the
biological particles of interest can then be used to calculate concentration. This approach has
been used successfully in a number of laboratories. One must to be sure that the added
particles do not aggregate with themselves or with other particles in the sample, that they are
added accurately, that the sample is well mixed, and that the concentration of added particles
is comparable to the concentration of particles of interest. Although this sounds difficult, it
is not hard in practice to find beads that, if added immediately prior to running the sample,
meet these criteria. If the beads are allowed to remain in the sample for a longer period, they
are very likely to interact with the sample in some way that makes the procedure invalid.
ABSOLUTE FLUORESCENCE
A similar approach may be taken in order to obtain calibration of fluorescence in absolute
terms by adding beads to the sample which bear a known amount of antigen on their surface.
The beads are labeled at the same time as the cells during the staining procedure. During
subsequent analysis the level of fluorescence on the beads can be used to calibrate the
fluorescence scale. This procedure also requires beads that are similar to, but distinguishable
from, the biological particles. An example is shown in Figure 3. In this example, beads were
treated with various amounts of antigen (human IgG) which had been trace radiolabeled. After
the IgG had bound to the beads, the mean amount of IgG per bead was calculated using the
radioactive tracer. The beads were then added to preparations of human platelets and the
mixture was labeled with fluorescent antibody to human IgG. During analysis, separate gates
ABSOLUTE COUNTS
Determining absolute concentration is difficult because no commercial flow cytometers
presently provide this measurement. The most direct way of adding it would be to build the
flow cytometer with an injector mechanism that either injects a fixed volume or injects the
sample at a known volume flow rate. Although some instruments have such injectors, one
must be careful about how accurate they are. It is a common problem that not all of the
injected sample passes through the measurement point. It is usually necessary to chase the
sample in with an air bubble or a blank sample to be certain that all of the injected sample
was analyzed. In order to use injection rate as the basis for calculating concentration, it is
necessary to insure that the sample is uniformly mixed, or that the average particle flow rate
is used in the calculation. For various technical (and perhaps economic) reasons these
approaches have not yet been implemented.
A method that does not require instrument modification is that of mixing distinctive particles
into the sample at a known concentration. The relative numbers of these particles and the
biological particles of interest can then be used to calculate concentration. This approach has
been used successfully in a number of laboratories. One must to be sure that the added
particles do not aggregate with themselves or with other particles in the sample, that they are
added accurately, that the sample is well mixed, and that the concentration of added particles
is comparable to the concentration of particles of interest. Although this sounds difficult, it
is not hard in practice to find beads that, if added immediately prior to running the sample,
meet these criteria. If the beads are allowed to remain in the sample for a longer period, they
are very likely to interact with the sample in some way that makes the procedure invalid.
ABSOLUTE FLUORESCENCE
A similar approach may be taken in order to obtain calibration of fluorescence in absolute
terms by adding beads to the sample which bear a known amount of antigen on their surface.
The beads are labeled at the same time as the cells during the staining procedure. During
subsequent analysis the level of fluorescence on the beads can be used to calibrate the
fluorescence scale. This procedure also requires beads that are similar to, but distinguishable
from, the biological particles. An example is shown in Figure 3. In this example, beads were
treated with various amounts of antigen (human IgG) which had been trace radiolabeled. After
the IgG had bound to the beads, the mean amount of IgG per bead was calculated using the
radioactive tracer. The beads were then added to preparations of human platelets and the
mixture was labeled with fluorescent antibody to human IgG. During analysis, separate gates