Method and materials for calibrating flow cytometers and other analysis instruments

• US 4,704,891 A
• Filed: 08/29/1986
• Issued: 11/10/1987
• Est. Priority Date: 08/29/1986
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Recktenwald, Diether J., Loken, Michael R., Kerndt, Rickie S., Chen, Chia H.
• Primary Examiner(s): Levy, Stewart J.
• Assistant Examiner(s): Raevis, Robert R.
• Application Number:
  US06/901,860
• Time in Patent Office: 438 Days
• Field of Search: 73/1 R, 209/546, 209/576, 209/588, 209/587, 209/DIG. 942, 356/243, 250/252.1, 250/573, 436/10, 424/7.1, 424/2, 424/3, 377/11
• US Class Current: 250/252.1
• CPC Class Codes: G01N 15/1012 Calibrating particle analysers; References therefor
• CPC Class Codes: G01N 15/1456 without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1014 Constitution of reference particles
• CPC Class Codes: G01N 2015/1477 Multiparameters

Abstract

A method for calibrating an instrument for using that instrument to obtain at least one light-related signal from particles under analysis comprises directing an incident beam of light at calibration particles having one or more known characteristics related to the particles expected to be analyzed. Both a light signal and a noise signal from the calibration particles are detected. A measurement is made of the ratio of the detected light signal to the detected noise signal, and that measurement is reported. The measured ratio is then compared to a predetermined ratio which represents a threshold for minimum instrument performance. This method further includes adjusting, if the predetermined ratio has not been attained, the operation of the instrument, while the calibration particles are within the incident beam of light, until the measured ratio reaches the predetermined ratio whereby the instrument is calibrated for subsequently obtaining the light signal from particles to be analyzed. A kit of reagents for use in calibrating an instrument for performing two-color analysis of particles and particle standards for calibrating fluorescence analysis instruments are also part of the instant invention.

Claims

1. A method for calibrating a flow cytometry instrument for using said instrument to obtain at least one light-related signal from particles passing through the instrument for analysis comprising:

passing calibration particles, having characteristics of like nature to particles expected to be tested, in a liquid flow stream so that each calibration particle passes, substantially one at a time, through an incident beam of light;

detecting a light signal from the calibration particles passing through said beam of light;

detecting a noise signal from the calibration particles passing through said beam of light;

determining the ratio of the level of detected light signal to the level of detected noise signal;

reporting said ratio as a measured separation value between the light signal and the noise signal;

comparing said measured separation value to a predetermined separation value which represents a threshold for minimum instrument performance; and

adjusting, if the predetermined separation value has not been attained, the operation of the instrument, while the calibration particles are passing therethrough, until the measured separation value reaches said predetermined separation value whereby the instrument is calibrated for subsequently analyzing particles.

2. The method of claim 1 wherein said calibration particles are substantially spherical beads.

3. The method of claim 2 wherein said beads have a diameter between 0.5 and 20 microns.

4. The method of claim 3 wherein the diameter of said beads is between 2 and 8 microns.

5. The method of claim 2 wherein said beads are made of plastic material.

6. The method of claim 5 wherein said plastic material is selected from the group of plastics consisting of styrene, acrylamide, propylene and vinylchloride.

7. The method of claim 1 wherein said calibration particles are chicken red blood cells.

8. The method of claim 1 wherein said calibration particles are capable of emitting fluorescence at a known wavelength and the light-related signal for which the instrument is to be calibrated is said known wavelength of fluorescence.

9. The method of claim 8 wherein said calibration particles are fluorescently-labeled osmiumtetraoxide-fixed chicken red blood cells.

10. The method of claim 1 wherein said calibration particles are capable of emitting fluorescence at a plurality of known wavelengths and the light-related signal for which the instrument is to be calibrated is each of the plurality of known wavelengths of fluorescence.

11. The method of claim 1 wherein said calibration particles are unlabeled so as to be substantially incapable of emitting fluorescence.

12. The method of claim 1 wherein said calibration particles are included in a mixture wherein two particle species are labeled with different fluorescence agents and another particle species is unlabeled so that two fluorescence signals are calibrated, said unlabeled particles serving as the medium to introduce a noise signal which is detected to establish the signal to noise ratio for each fluorescence signal.

13. The method of claim 12 wherein the adjusting step includes compensating each fluorescent signal by substantially eliminating spectral overlap.

14. The method of claim 13 wherein said compensating step includes electrically subtracting a proportion of the first fluorescent light signal from the second fluorescent light signal and electrically subtracting a proportion of the second fluorescent light signal from the first fluorescent light signal.

15. The method of claim 14 further including displaying the uncompensated fluorescence signals in a dot-population display wherein a first group of dots represents the first fluorescent light signal and a second group of dots represents the second fluorescent light signal.

16. The method of claim 15 wherein said dot population display includes a digital readout of compensation values for said first and said second fluorescent light signals, said adjusting step including the adjustment of the compensation values until a predetermined compensation value is reached for each fluorescent light signal whereby each fluorescent light signal is compensated.

17. The method of claim 12 wherein the fluorescently-labeled calibration particles and the unlabeled calibration particles are provided in substantially equal amounts in a carrier liquid which is used to form said liquid flow stream containing the calibration particles.

18. The method of claim 1 wherein said reporting step includes displaying said ratio in a form visible to an operator performing the calibration method.

19. The method of claim 18 wherein said reporting step includes displaying a histogram for the light signal being detected, said histogram showing the light signal mean channel and the noise signal mean channel.

20. The method of claim 19 wherein said reporting step includes displaying, in digital readout form, the value of the light signal mean channel, the value of the noise signal mean channel, the separation value between the light signal mean channel and the noise signal mean channel representing said signal to noise ratio, and said predetermined separation value representing said threshold for minimum instrument performance.

21. The method of claim 20 wherein said reporting step includes displaying a plurality of histograms, each for a different light-related signal being detected, and further displaying said digital readout values associated with each different histogram of light-related signals.

22. The method of claim 21 wherein said reporting step includes displaying three histograms and associated digital readout values for detecting three different light-related signals, two of said light signals being fluorescence at different wavelengths, the third signal being a light scatter signal.

23. The method of claim 1 wherein said reporting step expresses the ratio as a linearized difference between the light signal and the noise signal.

24. The method of claim 1 wherein the adjusting step includes adjusting detectors to positions which improve receipt of the respective signals which are detected.

25. A method for calibrating an instrument for using said instrument to obtain at least one light-related signal from particles under analysis comprising:

directing an incident beam of light at calibration particles having one or more known characteristics related to particles expected to be analyzed;

detecting a light signal from the calibration particles;

detecting a noise signal from the calibration particles;

measuring the ratio of the level of detected light signal to the level of detected noise signal and reporting same;

comparing said measured ratio to a predetermined ratio which represents a threshold for minimum instrument performance; and

adjusting, if the predetermined ratio has not been attained, the operation of said instrument, while the calibration particles are within the incident beam of light, until the measured ratio reaches said predetermined ratio whereby the instrument is calibrated for subsequently analyzing particles.

26. The method of claim 25 wherein said calibration particles include labeled particles and unlabeled particles, and wherein the detected light signal is associated with said labeled particles and the detected noise signal is associated with said unlabeled particles.

27. The method of claim 25 wherein said ratio is reported as a linearized separation value between the light signal and the noise signal.

28. A method for calibrating a flow cytometry instrument for using said instrument to obtain at least two different fluorescence signals from particles passing through the instrument for analysis comprising:

passing substantially spherical calibration beads in a liquid flow stream so that each bead passes, substantially one at a time, through an incident beam of light, said beads being labeled with two different fluorochromes and provided in substantially equal amounts in a carrier liquid which is used to form said flow stream;

detecting each of the two fluorescence signals from the beads passing through said beam of light;

detecting noise signals associated with each of the two fluorescence signals, the unlabeled beads providing the noise signal for each of the two fluorescence signals;

determining the ratio of the level of detected fluorescence signal to the level of detected noise signal for each of the two fluorescence signals;

displaying said ratio for each of the two fluorescence signals as a measured separation value between each respective light signal and the noise signal, said displaying step utilizing a log scale relationship in which the ratio of signal to noise is expressed by the mean channel linear difference between the light signal and the noise signal;

comparing said measured separation value for each of the two fluorescence signals to predetermined separation values each of which represents a threshold for minimum instrument performance; and

adjusting, if the predetermined separation values for either of the two fluorescence signals has not been attained, the operation of the instrument while the calibration beads are passing therethrough until each measured separation value reaches the predetermined separation value, whereby the instrument is calibrated for subsequently obtaining said two fluorescence signals from particles to be analyzed.