PHOTO-DAMAGE APPARATUS AND METHODS FOR SORTING PARTICLES

• US 20110217722A1
• Filed: 05/12/2011
• Published: 09/08/2011
• Est. Priority Date: 03/28/2003
• Status: Active Grant

• Current Assignee: Inguran LLC
• Original Assignee: Inguran LLC
• Inventors: HATCHER, JEREMY T., WESTFALL, LON A., WALLACE, JEFFREY D., DURACK, GARY, VANDRE, GARY P., NAYAK, NIRAJ V.
• Granted Patent: US 8,206,987 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: A01N 1/0284 Temperature processes, i.e. using a designated change in temperature over time
• CPC Class Codes: C12N 5/06 Animal cells or tissues; Human cells or tissues preservation of living cells or tissues A01N1/02
• CPC Class Codes: C12N 5/061 Sperm cells, spermatogonia
• CPC Class Codes: C12N 5/0612 sorting of gametes, e.g. according to sex or motility
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: C12Q 3/00 Condition responsive control processes apparatus therefor C12M1/36; controlling or regulating in general G05
• CPC Class Codes: G01N 15/01 specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/1468 with spatial resolution of the texture or inner structure of the particle
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1406 Control of droplet point
• CPC Class Codes: G01N 2015/1415 Control of particle position
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/63 optically excited
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/48 Biological material, e.g. blood, urine G01N33/02, G01N33/26, G01N33/44, G01N33/46 take precedence; Haemocytometers counting blood corpuscules distributed over a surface by scanning the surface G06M11/02
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• 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 436/115831 : Condition or time responsive
• CPC Class Codes: Y10T 436/12 : Condition responsive control
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/2525 : Stabilizing or preserving
• CPC Class Codes: Y10T 436/25375 : Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 : including use of a solid sorbent, semipermeable membrane, or liquid extraction
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A system and method for sorting a mixture of stained particles in a fluid flow path, including stained particles from two distinguishable groups. The system and method can include an electromagnetic radiation source for exciting fluorescence emissions from the stained particles, a photodetector for detecting the fluorescence emissions from the stained particles, a processor for classifying the stained particles; and a photo-damaging laser for damaging selected particles in the flow path.

Claims

1-80. -80. (canceled)

81. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, into at least one population, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles in the flow path according to their fluorescence emissions as either stained particles having characteristic A or stained particles having characteristic B;

e. a control in communication with the detector for selecting stained particles to damage; and

f. a photo-damaging laser in communication with the control for ablating selected particles in the flow path.

82. A system for sorting a mixture of stained particles according to claim 81 further comprising a sort strategy.

83. A system for sorting a mixture of stained particles according to claim 82, wherein the control is responsive to vary the sort strategy as a function of at least one of the following:

a. the purity of the at least one population with respect to either characteristic A particles or characteristic B particles; and

b. the quantity of undamaged characteristic A particles or undamaged characteristic B particles in the at least one population relative to the total quantity of characteristic A particles and characteristic B particles in the flow path.

84. A system for sorting a mixture of stained particles according to claim 81, wherein the control is responsive to information from the processor to vary the fluid delivery rate as a function of at least one of the following:

a. the purity of the at least one population with respect to either characteristic A particles or characteristic B particles; and

b. the quantity of undamaged characteristic A particles or undamaged characteristic B particles in the at least one population relative to the total quantity of characteristic A particles and characteristic B particles in the flow path.

85. A system for sorting a mixture of stained particles according to claim 84, wherein the control increases the rate of fluid delivery when the purity of the said at least one population is greater than a desired purity and decreases the rate of fluid delivery when the purity is less than the desired purity.

86. A system for sorting a mixture of stained particles according to claim 84, wherein the control determines the purity of the said at least one population, the system further including an operator input to the control for indicating a desired purity, and wherein the control varies the rate of fluid delivery to obtain the desired purity.

87. A system for sorting a mixture of stained particles according to claim 81, wherein the stained particles are stained sperm cells, and wherein characteristic A is indicative of a live X-chromosome bearing sperm cell (X) and wherein characteristic B is indicative of something other than a live X-chromosome bearing sperm cell (˜

X).

88. A system for sorting a mixture of stained particles according to claim 81, wherein the stained particles are stained sperm cells, and wherein characteristic A is indicative of a live Y-chromosome bearing sperm cell (Y) and wherein characteristic B is indicative of something other than a live Y-chromosome bearing sperm cell (˜

Y).

89. A system for sorting a mixture of stained particles according to claim 81 wherein the stained particles are sperm cells stained with a DNA selective fluorescent dye.

90. A system for sorting a mixture of stained particles according to claim 89 wherein the processor classifies sperm cells having characteristic A or characteristic B based upon a comparison of the fluorescence emissions of the sperm cells in the flow path.

91. A system for sorting a mixture of stained particles according to claim 81, wherein a stream is formed within the flow path containing particles following generally one after another in a series which includes sequential sets of particles, including:

a. first particle sets each including one or more particles having characteristic A;

b. second particle sets each including one or more particles having characteristic B; and

c. third particle sets each including two or more closely spaced particles at least one of which has characteristic A and at least one of which has characteristic B.

92. A system for sorting a mixture of stained particles according to claim 91, wherein the laser damages either the second particle sets or the second and third particles sets.

93. A system for sorting a mixture of stained particles according to claim 81, wherein the control increases the rate of fluid delivery either:

a. when the quantity of undamaged characteristic A particles in the said at least one population is greater than an acceptable quantity of undamaged characteristic A particles in the at least one population, and wherein the control decreases the rate of fluid delivery when the quantity of undamaged characteristic A particles in the said at least one population is less than the acceptable quantity;

or

b. when the quantity of characteristic A particles in the said at least one population is greater than an acceptable quantity of characteristic A particles in the said at least one population, and wherein the control decreases the rate of fluid delivery when the quantity of characteristic A particles in the at least one population is less than an acceptable quantity of characteristic A particles in the at least one population.

94. A system for sorting a mixture of stained particles according to claim 81, wherein the control determines the quantity of undamaged characteristic A particles in the said at least one population based on output signals from the flow cytometry apparatus, the system further including an operator input to the control for indicating an acceptable quantity of undamaged characteristic A particles in the said at least one population, and wherein the control varies the fluid delivery rate to obtain the acceptable quantity of undamaged characteristic A particles in the said at least one population.

95. A system for sorting a mixture of stained particles according to claim 81, wherein the control determines the total number of characteristic A particles in the said at least one population based on output signals from the flow cytometry apparatus, the system further including an operator input to the control for indicating an acceptable quantity of characteristic A particles in the at least one population, and wherein the control varies the rate so that the quantity of characteristic A particles in the at least one population to obtain the acceptable quantity or percentage.

96. A method of sorting a mixture of stained particles including stained particles having a characteristic A and stained particles having a characteristic B into at least one population, the method comprising the steps of:

a. flowing a fluid containing stained particles through a flow path;

b. exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B flowing in the flow path;

c. detecting the fluorescence emissions from the excited particles;

d. classifying the stained particles as either having characteristic A or particles having characteristic B based upon the fluorescence emissions;

c. selecting stained particles in the flow path based on their classification;

f. photo-damaging selected particles.

97. A method of sorting a mixture of stained particles according to claim 96 wherein the step of selecting stained particles in the flow path to photo-damage further comprises the step of photo-damaging particles based upon a sort strategy.

98. A method of sorting a mixture of stained particles according to claim 97 further comprising the step of varying the sorting strategy as a function of at least one of the following:

a. the purity of the said at least one population with respect to either characteristic A particles or characteristic B particles; and

b. the quantity of undamaged characteristic A particles or undamaged characteristic B particles in the said at least one population relative to the total quantity of characteristic A particles or characteristic B particles in the stream.

99. A method of sorting a mixture of stained particles according to claim 97 further comprising the step of varying the fluid delivery rate as a function of at least one of the following:

a. the purity of the said at least one population with respect to either characteristic A particles or characteristic B particles; and

b. the quantity of undamaged characteristic A particles or undamged characteristic B particles in the said at least one population relative to the total quantity of characteristic A particles or characteristic B particles in the stream.

100. A method of sorting a mixture of stained particles according to claim 96, wherein the method further includes the step of forming the fluid into a stream containing particles following generally one after another in a series which includes sequential sets of particles, including:

a. first particle sets each including one or more particles having characteristic A;

b. second particle sets each including one or more particles having characteristic B; and

c. third particle sets each including two or more closely spaced particles at least one of which has characteristic A and at least one of which has characteristic B.

101. A method of sorting a mixture of stained particles according to claim 100, the method further includes the step of either photo-damaging the second particles sets and not the first and third particle sets or photo-damaging the second and third particle sets and not the first particle sets.

102. A method of sorting a mixture of stained particles according to claim 96, wherein the stained particles are stained sperm cells, and wherein characteristic A is indicative of a live X-chromosome bearing sperm cell (X) and wherein characteristic B is indicative of something other than a live X-chromosome bearing sperm cell (˜

X).

103. A method of sorting a mixture of stained particles according to claim 96, wherein the stained particles are stained sperm cells, and wherein characteristic A is indicative of a live Y-chromosome bearing sperm cell (Y) and wherein characteristic B is indicative of something other than a live Y-chromosome bearing sperm cell (˜

Y).

104. A method of sorting a mixture of stained particles according to claim 96, further comprising the step of increasing the rate of fluid delivery when the purity of the said at least one population is greater than a desired purity and decreasing the rate of fluid delivery when the purity is less than the desired purity.

105. A method of sorting a mixture of stained particles according to claim 96 further comprising the step of increasing the rate of fluid delivery when the quantity of undamaged characteristic A particles in the said at least one population is greater than an acceptable quantity of undamaged characteristic A particles in the at least one population, and decreasing the rate of fluid delivery when the quantity of undamaged characteristic A particles in the said at least one population is less than the acceptable quantity.

106. An apparatus for sorting particles contained in a fluid stream according to one more characteristics of the particles, the apparatus comprising:

a. a fluid delivery system for delivering a fluid stream containing the particles to a first location;

b. an analyzer for classifying the particles according to said characteristics, and sorting the particles according to said classification into at least one population containing desired particles; and

c. a laser for photo-damaging particles in the stream, the analyzer comprising an epi-illumination optics system including a focusing lens, the optics system being operable to direct a laser beam through the focusing lens in a forward direction along a beam axis intersecting the fluid stream at the first location so that the cells pass through the beam, resulting in emissions of electromagnetic radiation from the cells directed along the beam axis in a rearward direction.

107. The apparatus according to claim 106, wherein the particles are sperm cells.

108. The apparatus according to claim 106, wherein the analyzer comprises a flow cytometer with an orienting nozzle having an interior surface configured to exert a force on the particles tending to bring them into a desired orientation prior to passing through the beam.

109. The apparatus according to claim 106, wherein the one more characteristics of the particles are indicative of a live X-chromosome bearing sperm cell (X) or indicative of other than a live X-chromosome bearing sperm cell (˜

X).

110. The apparatus according to claim 106, wherein the one more characteristics of the particles are indicative of a live Y-chromosome bearing sperm cell (Y) or indicative of other than a live Y-chromosome bearing sperm cell (˜

Y).