CALIBRATION AND QUANTITATIVE ANALYSIS
K. A. Ault M.D.
Maine Cytometry Research Institute
190 Park Avenue
Portland ME 04102
INTRODUCTION
Flow cytometers have traditionally been used primarily for semi-quantitative measurements.
The earlier flow cytometers were mainly research instruments and were so completely
adjustable that standardization and calibration outside of the boundaries of a single experiment
were both difficult and not really needed. Only recently in the medical applications field has
there been a necessity for quantitation of flow cytometric results, and for standards that can
be applied in many laboratories. The history of attempts to provide standards for quality
control and calibration of flow cytometers in the medical field has been long and difficult.
Clinicians and researchers have long recognized the clear need for standardization but only
in the past few years have there emerged, largely by default rather by careful planning, some
fairly widely recognized standards.
Much of the problem in the field has resulted from confusion about terminology and fuzzy
thinking about what types of controls are needed and what is being controlled for in any given
case. In this overview I will attempt to provide an outline by which it may be possible to
discuss issues of standardization and calibration. I will use some examples drawn from the
medical field in the hope that they may serve as models for applications in marine biology and
oceanography. Unfortunately I must acknowledge that all of the information I will offer here
deals with the use of exogenously applied fluorochromes. To my knowledge the problem of
standardization and calibration of intrinsic fluorescence ("autofluorescence") which is a major
issue in marine biology has not been seriously addressed in the flow cytometric field and this,
I believe, will be a major challenge for the future.
There are four types of quantitative data that can be obtained from a flow cytometer. First is
the determination of proportional populations, i.e. "percent positive". This requires a
minimum of calibration because the measurement is entirely relative and is only meaningful
within the context of a single sample. The only requirement that must be met is that the
population of interest must be resolved from the other cells. If they are not, then the
NATO AS! Series, Vol. G 27
Particle Analysis in Oceanography
Edited by S. Demers
© Springer-Verlag Berlin Heidelberg 1991
K. A. Ault M.D.
Maine Cytometry Research Institute
190 Park Avenue
Portland ME 04102
INTRODUCTION
Flow cytometers have traditionally been used primarily for semi-quantitative measurements.
The earlier flow cytometers were mainly research instruments and were so completely
adjustable that standardization and calibration outside of the boundaries of a single experiment
were both difficult and not really needed. Only recently in the medical applications field has
there been a necessity for quantitation of flow cytometric results, and for standards that can
be applied in many laboratories. The history of attempts to provide standards for quality
control and calibration of flow cytometers in the medical field has been long and difficult.
Clinicians and researchers have long recognized the clear need for standardization but only
in the past few years have there emerged, largely by default rather by careful planning, some
fairly widely recognized standards.
Much of the problem in the field has resulted from confusion about terminology and fuzzy
thinking about what types of controls are needed and what is being controlled for in any given
case. In this overview I will attempt to provide an outline by which it may be possible to
discuss issues of standardization and calibration. I will use some examples drawn from the
medical field in the hope that they may serve as models for applications in marine biology and
oceanography. Unfortunately I must acknowledge that all of the information I will offer here
deals with the use of exogenously applied fluorochromes. To my knowledge the problem of
standardization and calibration of intrinsic fluorescence ("autofluorescence") which is a major
issue in marine biology has not been seriously addressed in the flow cytometric field and this,
I believe, will be a major challenge for the future.
There are four types of quantitative data that can be obtained from a flow cytometer. First is
the determination of proportional populations, i.e. "percent positive". This requires a
minimum of calibration because the measurement is entirely relative and is only meaningful
within the context of a single sample. The only requirement that must be met is that the
population of interest must be resolved from the other cells. If they are not, then the
NATO AS! Series, Vol. G 27
Particle Analysis in Oceanography
Edited by S. Demers
© Springer-Verlag Berlin Heidelberg 1991