US 6,228,652 B1Granted

Method and apparatus for analyzing cells in a whole blood sample

AssigneeCoulter International Corp.

InventorsCarlos M. Rodriguez, Jose M. Cano, Barbara Carrillo, Kristie M. Gordon, Allan F. Horton, Ronald D. Paul, Mark A. Wells, James L. Wyatt

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Abstract

A blood analyzing instrument includes a single transducer for simultaneously measuring the DC volume, RF conductivity, light scattering and fluorescence characteristics of blood cells passing through a cell-interrogation zone. Preferably, the transducer includes an electro-optical flow cell which defines a cell-interrogation zone having a square transverse cross-section measuring approximately 50×50 microns, and having a length, measured in the direction of cell flow, of approximately 65 microns.

Claims24

Dependent claims

Apparatus for differentiating and enumerating various types of blood cells in a whole blood sample, said apparatus comprising:

(a) a single flow cell comprising an optically transparent element in the form of a rectangular prism defining a passageway through which formed bodies in said sample can be made to pass one at a time, said passageway defining a cell-interrogation zone having a substantially square transverse cross-section, determined perpendicular to the direction of blood cell flow, of approximately 50 microns on each side and a length of approximately 65 microns, measured in the direction of blood cell flow;
(b) means for aspirating a sample of whole blood from a container;
(c) means for producing at least a first aliquot of the aspirated whole blood sample;
(d) means for subjecting the first aliquot of whole blood to a lysing reagent to provide a lysed sample containing predominantly white blood cells and nucleated red blood cells;
(e) means for subjecting one or more subsets of cells contained in the lysed sample to a fluorescent material, thereby causing said subsets of cells to become labeled with such fluorescent material;
(f) metering means for producing a metered volume of the lysed sample;
(g) means for causing said metered volume of said lysed sample to flow through the cell-interrogation zone of the flow cell so that blood cells in said metered volume flow through said cell-interrogation zone seriatim;
(h) circuit means for (a) simultaneously establishing DC and RF current flows through said cell-interrogation zone while blood cells are flowing therethrough, each of said blood cells being effective to (i) modulate the DC current in said zone as a function of the cell's volume, and (ii) modulate the RF current flow in said zone as a function of the cell's internal conductivity;
and (b) detecting modulations in said DC and RF currents;
(i) optical means for irradiating individual blood cells flowing through said cell-interrogation zone with a beam of radiation propagating along an axis perpendicular to the direction of sample flow, said radiation being adapted to cause the fluorescent material to fluoresce;
(j) radiation scatter-detecting means for detecting radiation scattered from an irradiated blood cell in said zone;
(k) fluorescence-detecting means for detecting radiation fluorescing from a fluorescent material-labeled subset of cells as a result of being irradiated by said optical means;
and
(l) analyzing means, operatively coupled to said circuit means and to the respective radiation scatter- and fluorescence-detecting means, for differentiating and determining the respective concentrations of different subsets of white cells and fluorescent material-labeled cells based upon their respective DC volume (V), RF conductivity (C), light scatter (S) and fluorescence (F) properties.

11 more elements

Apparatus for differentiating and enumerating various types of blood cells in a whole blood sample, said apparatus comprising:

(a) a single flow cell comprising an optically transparent element in the form of a rectangular prism defining a passageway through which formed bodies in said sample can be made to pass one at a time, said passageway defining a cell-interrogation zone having a substantially square transverse cross-section, determined perpendicular to the direction of formed body flow, of approximately 50 microns on each side and a length of approximately 65 microns, measured in the direction of formed body flow;
(b) means for aspirating a sample of whole blood from a container;
(c) means for producing at least a first aliquot of the aspirated whole blood sample;
(d) means for subjecting the first aliquot of whole blood to a diluent to provide a diluted sample containing predominantly red blood cells and platelets;
(e) means for subjecting one or more subsets of cells contained in the diluted sample to a fluorescent material, thereby causing said subsets of red blood cells or platelets to become labeled with such fluorescent material;
(f) metering means for producing a metered volume of the diluted sample;
(g) means for causing said metered volume of said diluted sample to flow through the cell-interrogation zone of the flow cell so that blood cells in said metered volume flow through said cell-interrogation zone seriatim;
(h) circuit means for (a) simultaneously establishing DC and RF current flows through said cell-interrogation zone while blood cells are flowing therethrough, each of said blood cells being effective to (i) modulate the DC current in said zone as a function of the cell's volume, and (ii) modulate the RF current flow in said zone as a function of the cell's internal conductivity;
and (b) detecting modulations in said DC and RF currents;
(i) optical means for irradiating individual blood cells flowing through said cell-interrogation zone with a beam of radiation propagating along an axis perpendicular to the direction of sample flow, said radiation being adapted to cause the fluorescent material to fluoresce;
(j) radiation scatter-detecting means for detecting radiation scattered from an irradiated blood cell in said zone, said radiation scatter detecting means being adapted to detect scattered radiation within a plurality of discrete angular ranges measured with respect to said axis;
(k) fluorescence-detecting means for detecting radiation fluorescing from a fluorescent material-labeled subset of cells as a result of being irradiated by said optical means;
and
(l) analyzing means, operatively coupled to said circuit means and to the respective radiation scatter- and fluorescence-detecting means, for differentiating and determining the respective concentrations of different subsets of red blood cells and platelets based on their respective DC volume, RF conductivity, light scatter and fluorescence properties.

11 more elements

An automated method for determining the respective concentrations of different cell types and certain respective subsets thereof in a whole blood sample comprising the steps of:

(a) providing a flow cell adapted to simultaneously measure the respective DC volume (V), RF conductivity (C), light scattering (S) and fluorescent (F) properties of blood cells passing seriatim through an interrogation zone within said flow cell, said flow cell comprising an optically transparent element in the form of a rectangular prism defining a passageway through which blood cells in said sample can be made to pass one at a time, said passageway defining a cell-interrogation zone having a substantially square transverse cross-section, determined perpendicular to the direction of blood cell flow, of approximately 50 microns on each side and a length of approximately 65 microns, measured in the direction of blood cell flow;
(b) aspirating a sample of whole blood from a container;
(c) producing first and second aliquots of the aspirated whole blood sample;
(d) subjecting said first aliquot of whole blood to a lysing reagent to provide a lysed sample of predominantly white blood cells;
(e) subjecting said second aliquot of whole blood to a diluent to provide a diluted sample;
(f) subjecting one or more subsets of cells contained in the diluted or lysed samples to a fluorescent material, thereby causing said subsets of cells to become labeled with such fluorescent material, said fluorescent material being adapted to fluoresce when exposed to radiation of predetermined wavelength;
(g) dispensing metered volumes of said lysed and diluted samples and causing the respective metered volumes of lysed and diluted samples to flow through said interrogation zone sequentially and so that the respective blood cells in said metered volumes flow through said interrogation zone seriatim;
(h) irradiating individual blood cells flowing through said interrogation zone with a beam of radiation of said predetermined wavelength propagating along an axis while establishing DC and RF current flows through said interrogation zone, each of said blood cells being effective, while passing through said interrogation zone, to (i) modulate the DC current in said interrogation zone as a function of the cell's volume, and (ii) modulate the RF current flow in said passageway as a function of the cell's internal conductivity;
(i) detecting modulations in said DC and RF currents while detecting radiation scattered by and fluorescence emitted from an irradiated blood cell in said interrogation zone;
(j) differentiating and counting normal and mature white cells in said lysed sample based upon each cell's DC volume (V), RF conductivity(C) and light scattering (S) properties;
and
(k) differentiating and counting different types of red blood cells and platelets in said diluted sample based upon each cell's DC volume (V), RF conductivity (C), fluorescent label (F) and light scattering (S) properties.

9 more elements

An automated method for determining the respective concentrations of different cell types and certain respective subsets thereof in a whole blood sample comprising the steps of:

(a) providing a flow cell adapted to simultaneously measure the respective DC volume (V), RF conductivity (C), light scattering (S) and fluoresent (F) properties of blood cells passing seriatim through an interrogation zone within said flow cell, said flow cell comprising an optically transparent element in the form of a rectangular prism defining a passageway through which blood cells in said sample can be made to pass one at a time, said passageway defining a cell-interrogation zone having a substantially square transverse cross-section, determined perpendicular to the direction of blood cell flow, of approximately 50 microns on each side and a length of approximately 65 microns, measured in the direction of bold cell flow;
(b) aspirating a sample of whole blood from a container;
(c) producing an aliquot of the aspirated whole blood sample;
(d) subjecting said aliquot of whole blood to a lysing reagent to provide a lysed sample of prediminantly white blood cells;
(e) subjecting one or more subsets of cells contained in said lysed sample to a fluorescent material, thereby causing said subsets of cells to become labeled with such fluorescent material, said fluorescent material being adapted to fluoresce when exposed to radiation of predetermined wavelength;
(f) dispensing a metered volume of said lysed sample and causing the metered volume of said lysed sample to flow through said interrogation zone sequentially and so that the respective blood cells in said metered volume flow through said interrogation zone seriatim;
(g) irradiating individual blood cells flowing through said interrogation zone with a beam of radiation of said predetermined wavelength propagating along an axis while establishing DC and RF current flows through said interrogation zone, each of said blood cells beiing effective, while passing through said interrogation zone, to (i) modulate the DC current in said interrogation zone as a function of the cell's volume, and (ii) modulate the RF current flow in said passageway as a function of the cell's internal conductivity;
(h) detecting modulations in said DC and RF currents while detecting radiation scattered by and fluorescence emitted from an irradiated blood cell in said interrogation zone;
(i) differentiating and counting different subsets of mature white cells in said lysed sample based upon each cell's DC volume (V), RF conductivity (C) and light scattering (S) properties;
and
(j) differentiating and counting different subsets of white cells labeled with fluoresent material based upon each cell's DC volume (V), RF conductivity (C), fluorescent label (F) and light scattering (S) properties.

8 more elements

An automated method for determining the respective concentrations of different cell types and certain respective subsets thereof in a whole blood sample comprising the steps of:

(a) providing a flow cell adapted to simultaneously measure the respective DC volume (V), RF conductivity (C), light scattering (S) and fluorescent (F) properties of blood cells passing seriatim through an interrogation zone within said flow cell, said flow cell comprising an optically transparent element in the form of a rectangular prism defining a passageway through which blood cells in said sample can be made to pass one at a time, said passageway defining a cell-interrogation zone having a substantially square transverse cross-section, determined perpendicular to the direction of blood cell flow, of approximately 50 microns on each side and a length of approximately 65 microns, measured in the direction of blood cell flow;
(b) aspirating a sample of whole blood from a container;
(c) producing an aliquot of the aspirated whole blood sample;
(d) subjecting said aliquot of whole blood to a diluent to provide a diluted sample of predominantly red blood cells, white blood cells and platelets;
(e) subjecting one or more subsets of cells contained in said diluted sample to a fluorescent material, thereby causing said subsets of cells to become labeled with such fluorescent material, said fluorescent material being adapted to fluoresce when exposed to radiation of predetermined wavelength;
(f) dispensing a metered volume of said diluted sample and causing the metered volume of said diluted sample to flow through said interrogation zone so that the respective blood cells in said metered volume flow through said interrogation zone seriatim;
(g) irradiating individual blood cells flowing through said interrogation zone with a beam of radiation of said predetermined wavelength propagating along an axis while establishing DC and RF current flows through said interrogation zone, each of said blood cells being effective, while passing through said interrogation zone, to (i) modulate the DC current in said interrogation zone as a function of the cell's volume, and (ii) modulate the RF current flow in said passageway as a function of the cell's internal conductivity;
(h) detecting modulations in said DC and RF currents while detecting radiation scattered by and fluorescence emitted from an irradiated blood cell in said interrogation zone;
(i) differentiating and counting red blood cells, white blood cells and platelets in said diluted sample based upon each cell's DC volume (V), RF conductivity (C) and light scatter (S);
and
(j) differentiating and counting different subsets of red cells, white cells and platelets labeled with fluorescent material based upon each cell's DC volume (V), RF conductivity (C), fluorescent label (F) and light scattering (S) properties.

8 more elements

Dependencies are inferred from the claim text.

Facts

Application: 09/251,019Filed 16 Feb 1999
Granted: 8 May 2001
Term ends: Not estimatedThe source records don’t support an estimate.

Classification11 codes

G01N 15/14
Investigating individual particles: optical investigation techniques, e.g. flow cytometry
G01N 2015/1019
Investigating individual particles: associating Coulter-counter and optical flow cytometer [OFC]
G01N 2015/139
By observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle:…
Y10S 435/808
Chemistry: molecular biology and microbiology: optical sensing apparatus