Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – Polymers from only ethylenic monomers or processes of...
Reexamination Certificate
1999-11-02
2003-02-18
Henderson, Christopher (Department: 1713)
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
Polymers from only ethylenic monomers or processes of...
C526S243000, C526S247000
Reexamination Certificate
active
06521729
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to products and methods used in flow cytometry instrumentation for the analysis of biological particles or cells. More particularly, the invention relates to synthetic polymer materials and methods for standardizing or calibrating flow cytometry instruments prior to using the instruments for analyzing such particles, for example, the cellular components of whole blood.
BACKGROUND OF THE INVENTION
The analysis of particles, particularly biological particles, and cells is routinely performed using a variety of commercially-available instruments which determine the characteristics of such particles based on one or more light-related signals which pass through the instrument. Flow cytometers allow the determination of the characteristics of particles using techniques in which the particles are moving in a liquid stream or carried in a suspension. Typically, in flow cytometry instruments, cells or other biological particles flow in a liquid stream so that each particle, virtually one cell at a time, passes through a sensing region that is capable of measuring the physical or chemical characteristics of the particles.
A variety of signals associated with different characteristics of the particles under analysis may be detected. Such signals include electrical, acoustical, optical and radioactive. Flow cytometers generally rely on optical signals for the analysis of particles which pass through the instrument. Whether or not an instrument analyzes particles in a static or a dynamic state, those skilled in the art will appreciate that calibration and standardization are required prior to performing particle analyses. Under normal circumstances, calibration occurs as one or more preliminary presteps in preparing instruments for proper use and measurement and to ensure accurate and reliable assay results. This is especially important since cells, or other biological particles, are extremely small and the signals to be detected, in relation to the size of the cells or particles, are often at a low magnitude.
Flow cytometers and other biological particle and cell analysis instruments are commonly calibrated with particles which simulate or approximate the types of particles or cells that are intended to undergo analysis. Thus, calibration particles should be selected or designed so that they have characteristics and parameters that are quite similar to those of the particles or cells to be tested in the instruments. Exemplary characteristics and parameters include similarities in size, volume, surface characteristics, granularity properties, and, if necessary, color features, such as stains, dyes, immunofluorescent tags, and the like. Accordingly, it is to be understood that the particles to be analyzed after calibration or standardization of an analyzer need not be restricted biological cells. For example, particles to be assayed may be found in oil-in-water suspensions, milk, or another nonbiological medium.
Hematology analyzers represent but one specific type of instrumentation designed and employed for the determination and measurement of the characteristics of cells and biological particles in whole blood. As a particular, yet nonlimiting, example, hematology systems commercially available from Bayer Corporation optically analyze, determine the characteristics of and measure erythrocytes (red blood cells), reticulocytes (immature red blood cells), leukocytes (white blood cells) and platelets in whole blood samples.
Past and present calibration procedures for flow cytometry instruments, including hematology analyzers, involve the use of fixed and/or sphered red blood cells, for example, human and chicken red blood cells, for the calibration steps and for standardizing optical signals such as light scattering. See, for example, U.S. Pat. No. 4,489,162 to Hawkins et al. and U.S. Pat. No. 4,777,139 to S.-C. Wong et al. While the use of red blood cells procured from mammals and humans may be reliable in some aspects of calibration procedures, there are drawbacks to the use of these types of “natural” calibrator cells. For example, the preparation of sphered and fixed red blood cells entails the blood-drawing process and other blood manipulations which involve contact with potentially biohazardous material. Further, because the stability of red cells as optical standards is limited, i.e., about six months, the cell standards must be reproduced periodically, typically, two or more times a year.
In addition to biological samples for calibration and standardization purposes, microspheres or microbeads have also been used for calibrating cellular analysis instruments. For example, U.S. Pat. No. 4,331,862 to W. L. Ryan describes beads composed of latex material for calibrating a particle counting instrument. Plastic microbeads are disclosed for calibrating flow cytometers and cell analysis instruments in U.S. Pat. No. 4,704,891 to D. J. Recktenwald et al. Calibration beads for calibrating flow cytometry instruments are commercially available from the Flow Cytometry Standards Corporation, Research Triangle Park, N.C. In general, such microsphere beads are not produced with a consideration for the proper refractive index values of actual cells and biological particles.
Nonbiological bead polymers having an average particle diameter of from 0.5 to 10 &mgr;m and containing 1% to 60% of chemically bound fluorine are disclosed in U.S. Pat. No. 5,093,445 to W. Podszun et al. for use as matting agents and spacers in photographic recording materials. This patent does not relate to particle calibrators for flow cytometry and does not recognize refractive index as a critical parameter for such use. Indeed, the present inventors have determined that the polymer beads exemplified in this patent have refractive indices which are significantly higher than 1.45, which is considered to be at or near the upper limit of refractive index, particularly as it relates to blood cells, as described further hereinbelow.
Other types of flow cytometry instruments, such as fluorescence flow cytometers, use polymer beads made of materials such as polystyrene to standardize the optical signals. The signals typically include forward scatter (0-2 degrees), side scatter (90 degrees) and fluorescence intensity. The polymer bead materials, although safe and stable for about five years or more, are particularly unsatisfactory for light scattering flow cytometry instruments. More particularly, for particles of a given volume, currently-available polymer beads produce scattering signals that are different from those of red blood cells of the same volume, even if the red blood cells are sphered. This is because the refractive index (r.i.) of a polymeric material such as polystyrene is quite different from that of red blood cells. While the r.i. of polystyrene is high, i.e., about 1.59, the r.i. of a red blood cell is about 1.40-1.42. It is well known from the Mie Scattering Theory that the light scattering properties of small spheres are significantly influenced by their r.i. values. Thus, the scattering patterns of polymer beads currently used in flow cytometry instruments are not truly representative of the scattering patterns of actual red blood cells. Consequently, the forward and side scatter signals do not provide important information, such as cell size and refractive index, which are related to cell density and activation state.
In addition, platelet light scattering signals are currently standardized using either sphered and fixed red blood cells in the case of some types of analyzers, for example, commercial hematology analyzers, or using appropriately-sized polymer beads in the case of fluorescence flow cytometers. However, the scattering signals of platelets are typically much smaller than those of red blood cell (i.e., by more than a factor of 10). Also, the refractive index of platelets is only about 1.35 to about 1.40. Accordingly, the present invention advantageously provides a material with the light scattering properties associated with actual platelets, as well
DeClercq Ronny
Kuckert Eberhard
Louwet Frank
Moskalski William
Podszun Wolfgang
Bayer Corporation
Henderson Christopher
Morgan & Finnegan , LLP
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