48
determination of their proportion must be based upon some type of model or on assumptions
about the reason for the lack of resolution. This type of determination is by far the most
common one in medical flow cytometry today and is the basis for such tests as "T4/TS
ratios", phenotyping of leukemias, etc.
Second is the analysis of fluorescence intensity distributions. In this case some, information,
usually the mean or modal fluorescence value for a subpopulation, must be obtained from a
fluorescence distribution. An example is the analysis of a DNA distribution in which the
various populations at different stages of the cell cycle are identified within a continuous
distribution. This type of analysis requires that the response characteristics of the flow
cytometer be known - usually the requirement is that the instrument be linear. It usually
involves measurement of fluorescence in "channels" in arbitrary units and not in any absolute
terms.
Third is the measurement of absolute fluorescence levels. This requires at a minimum that the
flow cytometer be calibrated and that it be stable from one assay to another. Examples include
the measurement of auto-antibodies and cross-matching for organ transplantation. The results
are usually expressed as a channel shift relative to a control or as mean fluorescence, again
in arbitrary units. In those cases where it is desirable to express the measurement in terms of
non-flow cytometric units, such as molecules of fluorochrome, moles of antigen, etc., special
problems are encountered that we will discuss below.
Fourth is the measurement of absolute counts of cells, i.e. cells per unit volume. For reasons
that are somewhat obscure, none of the commercially available flow cytometers offer the easy
determination of absolute cell counts. This has become very important medically in the
determination of the absolute numbers of CD4 lymphocytes in AIDS. The solutions usually
involve obtaining a proportional count in the flow cytometer and then determining the absolute
count with a different instrument. Other solutions include using calibration particles in the
sample at a known concentration, injecting a known volume of sample or injecting the sample
at a known volume rate. Each of these solutions involves special problems that we will
discuss.
The general topic of calibration of a flow cytometer can be broken down into several separate
considerations.
determination of their proportion must be based upon some type of model or on assumptions
about the reason for the lack of resolution. This type of determination is by far the most
common one in medical flow cytometry today and is the basis for such tests as "T4/TS
ratios", phenotyping of leukemias, etc.
Second is the analysis of fluorescence intensity distributions. In this case some, information,
usually the mean or modal fluorescence value for a subpopulation, must be obtained from a
fluorescence distribution. An example is the analysis of a DNA distribution in which the
various populations at different stages of the cell cycle are identified within a continuous
distribution. This type of analysis requires that the response characteristics of the flow
cytometer be known - usually the requirement is that the instrument be linear. It usually
involves measurement of fluorescence in "channels" in arbitrary units and not in any absolute
terms.
Third is the measurement of absolute fluorescence levels. This requires at a minimum that the
flow cytometer be calibrated and that it be stable from one assay to another. Examples include
the measurement of auto-antibodies and cross-matching for organ transplantation. The results
are usually expressed as a channel shift relative to a control or as mean fluorescence, again
in arbitrary units. In those cases where it is desirable to express the measurement in terms of
non-flow cytometric units, such as molecules of fluorochrome, moles of antigen, etc., special
problems are encountered that we will discuss below.
Fourth is the measurement of absolute counts of cells, i.e. cells per unit volume. For reasons
that are somewhat obscure, none of the commercially available flow cytometers offer the easy
determination of absolute cell counts. This has become very important medically in the
determination of the absolute numbers of CD4 lymphocytes in AIDS. The solutions usually
involve obtaining a proportional count in the flow cytometer and then determining the absolute
count with a different instrument. Other solutions include using calibration particles in the
sample at a known concentration, injecting a known volume of sample or injecting the sample
at a known volume rate. Each of these solutions involves special problems that we will
discuss.
The general topic of calibration of a flow cytometer can be broken down into several separate
considerations.