Calibration method for flow cytometry using fluorescent microbeads and synthesis thereof

• US 4,767,206 A
• Filed: 12/24/1984
• Issued: 08/30/1988
• Est. Priority Date: 12/24/1984
• Status: Expired due to Term

• Current Assignee: Bangs Laboratories Incorporated (Polysciences Incorporated)
• Original Assignee: Flow Cytometry Standards Corporation
• Inventors: Schwartz, Abraham
• Primary Examiner(s): Rosenberger, R. A.
• Application Number:
  US06/685,464
• Time in Patent Office: 1,345 Days
• Field of Search: 356/39, 356/73, 356/243, 356/72, 436/10, 436/172, 422/55, 422/68, 523/201, 523/223, 250/252.1, 250/459.1, 250/461.2, 250/458.1, 377/10-12, 377/29, 364/555, 364/571, 209/3.1, 209/3.3
• US Class Current: 356/73
• CPC Class Codes: G01N 15/1012 Calibrating particle analysers; References therefor
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1014 Constitution of reference particles
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]

Abstract

A method of calibrating a flow cytometer is based on a set of highly uniform microbeads associated with a fluorescent dye in such a way that the microbeads have the same excitation and emission spectral properties as the samples which are to be measured. The calibration values of the microbeads are plotted against the relative fluorescence intensity peak channel for each microbead in the set. From this calibration plot, the relative fluorescence intensity peak channel of the sample is translated into equivalent soluble fluorescent dye molecules per sample particle. The calibration values of the standard microbeads are determined against solutions of the dyes. In cases where the background scatter of the bulk microbeads suspensions is too high for a direct determination against the solutions, a different set of microbeads with low background scatter is calibrated against the dye solutions and used to make an initial calibration of the flow cytometer, which in turn, is used to calibrate the uniform microbead standards. A novel method of making the microbead standards is also disclosed.

Claims

1. The method of establishing a standard reference for calibrating a flow cytometer, comprising the steps of:

(a) making up a first batch of microbeads to serve as standard microbeads, each said standard microbead comprising;

(i) a highly-uniform sized body substantially within the range of 3 to 15 microns in size, formed of hydrophobic polymeric material; and

(ii) fluorescent dye material covalently bonded to the surface of said body material via chemical functional groups and in such manner that the fluorescent spectra thereof remains unaltered;

(b) making up a second batch of microbeads to serve as primary microbeads, each said primary microbead comprising;

(i) a hydrophilic polymeric material; and

(ii) a fluorescent dye copolymerized within the microbead and made up of a material allowing free access of aqueous media throughout the microbead, and having a fluorescent spectra that remains unaltered after said copolymerization;

(c) determining that the excitation-emission spectra of the microbeads of both batches are substantially identical to each other, substantially identical to the same free dye when in the same suspending solution and also substantially identical to a particle sample such as cells having the same known dye incorporated therein;

(d) based on the identities in step (c) being established, calibrating said second batch of microbeads against a sample of said free dye to determine the equivalent soluble fluorescent molecules per microbead;

(e) developing a calibration curve for said second batch of microbeads representing equivalent soluble fluorescent molecules per relative fluorescent channel of a flow cytometer; and

(f) measuring the fluorescent intensities of the highly uniform microbeads of said first batch with a flow cytometer to determine the relative fluorescent channel thereof and thereafter determining from step (e) the number of equivalent soluble fluorescent molecules per microbead in said first batch, thereby establishing said first batch of microbeads as a standard reference.

2. A method for calibrating a flow cytometer for measuring cell and other particle samples, comprising the steps of:

(a) preparing microbeads with a fluorescent dye associated therewith in a manner such that the microbeads have the same excitation and emission spectra as the samples being measured, said microbeads comprising a first batch of hydrophobic microbeads having a fluorescent dye material covalently bonded to their surfaces, and a second batch of low background scatter hydrophilic microbeads having a fluorescent dye copolymerized within the microbeads and made up of a material allowing free access of aqueous media throughout the hydrophilic microbeads;

(b) calibrating said low background scatter hydrophilic microbeads against solutions of the free fluorescent dye, by the steps comprising;

(i) determining that the hydrophilic microbeads in the same suspending solution have an excitation-emission spectra substantially identical to solutions of the free dye;

(ii) determining the fluorescent intensity of a suspension of said hydrophilic microbeads with a fluorometer as compared to solutions of the free fluorescent dye, to determine the number of equivalent soluble dye molecules per unit volume of the hydrophilic microbead suspension;

(iii) determining the number of microbeads per unit volume of the hydrophilic microbead suspension; and

(iv) dividing the equivalent soluble dye molecules per unit volume of the hydrophilic microbeads suspension by the number of microbeads per unit volume of the hydrophilic microbeads suspension to yield the number of equivalent soluble fluorescent dye molecules per hydrophilic microbead;

(c) calibrating the hydrophobic microbeads in terms of equivalent numbers of soluble fluorescent dye molecules per microbead, by measuring the fluorescent intensities of the highly uniform hydrophilic microbeads to determine the relative fluorescent channel thereof and thereafter determining from step (b) (iv) the number of equivalent soluble fluorescent molecules per microbead in said hydrophilic microbeads, thereby establishing said hydrophilic microbeads as a standard reference; and

(d) calibrating the flow cytometer in terms of the number of equivalent soluble fluorescent dye molecules per fluorescent intensity channel of the cytometer by the use of said hydrophobic microbeads.

3. A method for calibrating a flow cytometer for measuring cell particle samples, comprising the steps of:

(a) preparing uniform microbeads with a fluorescent dye associated therewith in a manner such that the microbeads have the same excitation and emission spectra as the samples being measured;

(b) determining the number of equivalent soluble fluorescent dye molecules necessary to give rise to the same level of fluorescence intensity as said microbeads, by the steps of;

(i) determining the fluorescence intensity of a suspension of said microbeads with a fluorometer as compared to solutions of the free fluorescent dye, to determine the number of equivalent soluble dye molecules per unit volume of said suspension of microbeads;

(ii) determining the number of microbeads per unit volume in said suspension thereof; and

(iii) dividing the number of equivalent soluble dye molecules per unit volume of said suspension of microbeads by the number of microbeads per unit volume of the suspension to yield the number of equivalent soluble fluorescent dye molecules per microbead;

(c) for microbeads of known number of equivalent soluble fluorescent dye molecules per microbead as determined from step (b)(iii), determining the peak fluorescence intensity channel of the flow cytometer for said microbeads;

(d) constructing a calibration plot of the number of equivalent soluble fluorescent dye molecules as a function of the peak of fluorescence intensity channel of the flow cytometer for said microbeads whose peak fluorescence intensity channels wee determined in step (c);

whereby at the same instrument parameters as employed to establish said calibration plot, the peak fluorescence intensity channel of said cell or particle sample is measured on the flow cytometer, and from such peak fluorescence intensity channel value, the number of equivalent soluble fluorescence dye molecules in said sample is determined from said calibration plot.