Fluorescent calibration microbeads simulating stained cells

• US 4,714,682 A
• Filed: 04/03/1987
• Issued: 12/22/1987
• Est. Priority Date: 12/11/1985
• Status: Expired due to Term

• Current Assignee: Bangs Laboratories Incorporated (Polysciences Incorporated)
• Original Assignee: Flow Cytometry Standards Corporation
• Inventors: Schwartz, Abraham
• Primary Examiner(s): Kyle, Deborah L.
• Assistant Examiner(s): Wallen, T. J.
• Application Number:
  US07/033,625
• Time in Patent Office: 263 Days
• Field of Search: 436/8-18, 436/19, 424/3, 264/4-4.7, 427/213.3-213.36
• US Class Current: 436/10
• CPC Class Codes: G01N 15/1012 Calibrating particle analysers; References therefor
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/1014 Constitution of reference particles
• CPC Class Codes: G01N 2015/1486 Counting the 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 33/585 with a particulate label, e.g. coloured latex
• CPC Class Codes: Y10T 436/10 Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]
• CPC Class Codes: Y10T 436/109163 Inorganic standards or controls

Abstract

A method of calibrating a flow cytometer or fluorescent microscope 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 a flow cytometer or fluorescent microscope, 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 making a calibration microbead standard to minimize aggregation and maximize yield comprising the steps:

(a) homogenizing a highly insoluble (<

10^-3 g/l) compound of molecular weight <

1000 in a surfactant solution to obtain a first homogenate;

(b) swelling small seed microbeads, 0.1-3u in diameter, with said first homogenate;

(c) homogenizing an aqueous surfactant solution containing a large stabilizing alkaline halide salt with a mixture of oil soluble polymerizable monomers within which is dissolved 0.5-5% by weight an oil soluble initiator to obtain a second homogenate;

(d) swelling the seed microbeads a second time with said second homogenate; and

(e) placing said twice swollen microbeads under conditions appropriate to cause said initiator to polymerize the monomers in the swollen microbeads.

2. The method of claim 6 wherein said highly water soluble compound is 1-chlorododecane.

3. The method of claim 6 wherein said stabilizing alkaline halide salt is potassium chloride, said oil soluble monomers methyl methacrylate and glycidyl methacrylate, and said oil soluble initiator is benzoyl peroxide.