Methods, compositions, and automated systems for separating rare cells from fluid samples

• US 20040142463A1
• Filed: 11/04/2003
• Published: 07/22/2004
• Est. Priority Date: 10/11/2001
• Status: Active Grant

• Current Assignee: AVIVA Biosciences Corporation
• Original Assignee: AVIVA Biosciences Corporation
• Inventors: Wu, Lei, Xu, Junquan, Huang, Mingxian, Tao, Guoliang, Hujsak, Paul, Walker, George, Yap, Charina, Snyder, Sara, Yamanishi, Douglas
• Granted Patent: US 7,166,443 B2
• Time in Patent Office: Days
• US Class Current: 435/325
• CPC Class Codes: A61M 1/36 Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
• CPC Class Codes: A61M 1/3616 Batch-type treatment
• CPC Class Codes: A61M 1/3618 Magnetic separation
• CPC Class Codes: A61M 1/362 changing physical properties of target cells by binding them to added particles to facilitate their subsequent separation from other cells, e.g. immunoaffinity
• CPC Class Codes: A61M 1/3679 by absorption A61M1/3675 takes precedence
• CPC Class Codes: B01D 2325/0214 Tapered pores
• CPC Class Codes: B01D 2325/04 Characteristic thickness
• CPC Class Codes: B01D 57/02 by electrophoresis treatment of water, waste water, sewage or sludge by electrophoresis C02F1/469; electrophoretic production of compounds or non-metals C25B7/00; investigating or analysing materials by using electrophoresis G01N27/26
• CPC Class Codes: B01D 61/18 Apparatus therefor
• CPC Class Codes: B01D 67/0034 by micromachining techniques, e.g. using masking and etching steps, photolithography
• CPC Class Codes: B01D 67/0062 by micromachining techniques, e.g. using masking and etching steps, photolithography
• CPC Class Codes: B01D 71/0213 Silicon
• CPC Class Codes: B01D 71/027 Silicium oxide
• CPC Class Codes: B01D 71/04 Glass
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0457 passive flow or gravitation
• CPC Class Codes: B01L 2400/0478 pistons
• CPC Class Codes: B01L 2400/0644 : rotary valves
• CPC Class Codes: B01L 2400/065 : sliding valves
• CPC Class Codes: B01L 3/502 : with fluid transport, e.g. in multi-compartment structures centrifugal-type cuvettes G01N21/07; analysis by separation into components G01N30/00; automatic analysers G01N35/00
• CPC Class Codes: B01L 3/502753 : characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/502761 : specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: G01N 1/40 : Concentrating samples
• CPC Class Codes: G01N 15/0272 : with screening; with classification by filtering B01D takes precedence
• CPC Class Codes: G01N 15/05 : in blood
• CPC Class Codes: G01N 2001/4016 : being a selective membrane, e.g. dialysis or osmosis
• CPC Class Codes: G01N 2015/012 : Red blood cells
• CPC Class Codes: G01N 2015/0288 : Sorting the particles
• CPC Class Codes: G01N 2015/0294 : Particle shape
• CPC Class Codes: G01N 2015/0681 : Purposely modifying particles, e.g. humidifying for growing

Abstract

The present invention recognizes that diagnosis and prognosis of many conditions can depend on the enrichment of rare cells from a complex fluid sample. In particular, the enrichment of fetal cells from maternal samples, such as maternal blood samples, can greatly aid in the detection of fetal abnormalities or a variety of genetic conditions. In addition, the present invention recognizes that the enrichment of rare malignant cells from patient samples, can aid in diagnosis, prognosis, and development of therapeutic modalities for patients. The invention includes microfabricated filters for filtering fluid samples and methods of enriching rare cells of fluid samples using microfabricated filters of the present invention. The invention also includes solutions for the selective sedimentation of red blood cells (RBCs) from a blood sample and methods of using selective RBC sedimentation solutions for enriching rare cells of a fluid sample. Yet another aspect of the invention is an automated system for processing a fluid sample that includes: at least one filtration chamber that includes a microfabricated filter; automated means for directing fluid flow through at least one filtration chamber of the automated system, and means for collecting enriched rare cells. The present invention also includes methods of using automated systems for separating rare cells from fluid samples. Preferred fluid samples are blood, effusion, or urine samples, and rare cells that can be enriched from such sample include nucleated red blood cells and cancer cells.

Claims

1. A microfabricated filter comprising at least one tapered pore.

2. The microfabricated filter of claim 1, comprising silicon, silicon dioxide, glass, metal, ceramics, or a polymer.

3. The microfabricated filter of claim 2, comprising silicon dioxide.

4. The microfabricated filter of claim 1, wherein said filter is between 0.01 mm² and 0.1 m².

5. The microfabricated filter of claim 4, wherein said filtration area is between 0.25 mm² and 100 cm².

6. The microfabricated filter of claim 5, wherein said filtration area is between 1 mm² and 9 cm².

7. The microfabricated filter of claim 1, wherein the thickness of said filter in said filtration area is between about 10 and 500 microns.

8. The microfabricated filter of claim 7, wherein the thickness of said filter in said filtration area is between about 40 and 100 microns.

9. The microfabricated filter of claim 1, comprising two or more tapered pores.

10. The microfabricated filter of claim 9, wherein the variation in size of said two or more tapered pores is within 20%.

11. The microfabricated filter of claim 10, wherein the variation in size of said two or more tapered pores is less than 10%.

12. The microfabricated filter of claim 11, wherein the variation in size of said two or more tapered pores is less than 5%.

13. The microfabricated filter of claim 12, wherein said two or more tapered pores are two or more tapered slots.

14. The microfabricated filter of claim 13, wherein said two or more tapered pores have tapering angles between about 0 degree and about 90 degrees.

15. The microfabricated filter of claim 14, wherein said two or more tapered pores have tapering angles between 0.1 degrees and 45 degrees.

16. The microfabricated filter of claim 15, wherein said two or more tapered pores have tapering angles between about 0.5 degrees and 10 degrees.

17. The microfabricated filter of claim 16, wherein said filter comprises from four to 1,000,000 tapered slots.

18. The microfabricated filter of claim 17, wherein said filter comprises from 100 to 250,000 tapered slots.

19. The microfabricated filter of claim 18, wherein said slots are from about 0.1 microns to about 1000 microns in length.

20. The microfabricated filter of claim 19, wherein said slots are from about 20 to about 200 microns in length.

21. The microfabricated filter of claim 18, wherein said slots are from about 0.1 micron to about 100 microns in width.

22. The microfabricated filter of claim 21, wherein said slots are from about 1 micron to about 10 microns in width.

23. The microfabricated filter of claim 22, wherein said slots are from about 2.5 microns to about 6 microns in width.

24. The microfabricated filter of claim 23, wherein the variation in slot width is less than 2 microns.

25. The microfabricated filter of claim 24, wherein the variation in slot width is less than 1 micron.

26. The microfabricated filter of claim 25, wherein the variation in slot width is less than 0.5 micron.

27. The microfabricated filter of claim 1, comprising two or more electrodes.

28. A filtration chamber comprising or engaging one or more filters of claim 1.

29. The filtration chamber of claim 28, wherein said filtration chamber has a volume of from about 0.01 ml to about 2 liters.

30. The filtration chamber of claim 29, wherein said filtration chamber has a volume of from about 0.2 ml to about 20 ml.

31. The filtration chamber of claim 30, wherein said filtration chamber comprises glass, silicon, silicon dioxide, metal, ceramics, at least one plastic, or at least one polymer.

32. The filtration chamber of claim 31, wherein said filtration chamber comprises or engages two or more electrodes.

33. The filtration chamber of claim 31, wherein said filtration chamber comprises or engages at least one acoustic element.

34. A cartridge comprising the filtration chamber of claim 31.

35. An automated system comprising the chamber of claim 31.

70. A method for enriching rare cells of a fluid sample, comprising:

a. dispensing a fluid sample into a filtration chamber of claim 28;

and

b. providing fluid flow of said fluid sample through said filtration chamber, wherein components of the fluid sample flow through or are retained by said at least one microfabricated filter based on the size, shape, or deformability of said components; and

c. collecting enriched rare cells from the filtration chamber.

71. The method of claim 70, wherein in said at least one filtration step one or more desirable components of said sample are retained by said at least one filter and one or more undesirable components of said sample flow through said at least one filter.

72. The method of claim 70, wherein in said at least one filtration step rare cells of interest of said sample are retained by said at least one filter.

73. The method of claim 70, wherein said filter comprises silicon, silicon dioxide, glass, metal, ceramics, a hard plastic, or a polymer.

74. The method of claim 73, wherein said filter comprises silicon dioxide.

75. The method of claim 74, wherein said filter is between 0.01 mm² and 0.1 m².

76. The method of claim 75, wherein the filtration area of said filter is between 0.25 mm² and 100 cm².

77. The method of claim 76, wherein the filtration area of said filter is between 1 mm² and 9 cm².

78. The method of claim 77, wherein the thickness of said filter in the filtration area is between about 10 and 500 microns.

79. The method of claim 78, wherein the thickness of said filter in the filtration area is between about 40 and 100 microns.

80. The method of claim 79, wherein said filter comprises two or more tapered pores.

81. The method of claim 80, wherein the variation in size of said two or more tapered pores is less than about 20%.

82. The method of claim 81, wherein the variation in size of said two or more tapered pores is less than about 10%.

83. The method of claim 82, wherein the variation in size of said two or more tapered pores is less than 5%

84. The method of claim 80, wherein said two or more tapered pores are two or more tapered slots.

85. The method of claim 184, wherein said filter comprises from four to 1,000,000 tapered slots.

86. The method of claim 85, wherein said filter comprises from 100 to 250,000 tapered slots.

87. The method of claim 86, wherein said slots are from about 0.1 microns to about 1000 microns in length.

88. The method of claim 87, wherein said slots are from about 20 to about 200 microns in length.

89. The method of claim 88, wherein said slots are from about 0.1 micron to about 100 microns in width.

90. The method of claim 89, wherein said slots are from about one micron to about ten microns in width.

91. The method of claim 90, wherein said slots are from about 2.5 microns to about 6 microns in width.

92. The method of claim 91, wherein the variation in slot width is less than about 2 microns.

93. The method of claim 92, wherein the variation in slot width is less than about 1 micron.

94. The method of claim 70, wherein said filtration step occurs in an automated system.

95. The method of claim 70, wherein said sample is blood, an effusion, urine, a bone marrow sample, ascities fluid, pelvic wash fluid, or pleural fluid, spinal fluid, lymph, serum, mucus, sputum, saliva, urine, semen, occular fluid, extracts of nasal, throat or genital swabs, cell suspension from digested tissue, or extracts of fecal material.

96. The method of claim 95, wherein said sample is a urine sample.

97. The method of claim 95, wherein said sample is a blood sample and said filtering removes red blood cells.

98. The method of claim 97, wherein said at least one filter comprises at least two tapered slots, wherein said at least two tapered slots are from about 20 to about 200 microns in length, and wherein said slots are from about 2.5 microns to about 6 microns in width, further wherein the variation in slot width is less than 1 micron.

99. The method of claim 198, further comprising a selective lysis step.

100. The method of claim 99, wherein said selective lysis step utilizes a buffer that selectively lyses red blood cells.

101. The method of claim 100, wherein said selective lysis step occurs prior at least one of said at least one filtration steps.

102. The method of claim 70, further comprising selectively removing one or more undesirable components from said sample.

103. The method of claim 102, wherein said selectively removing uses at least one specific binding member that specifically binds said one or more undesirable components.

104. The method of claim 103, wherein said selectively removing comprises capturing said one or more undesirable components to a solid support.

105. The method of claim 104, wherein said solid support comprises magnetic beads.

106. The method of claim 105, wherein said removing one or more undesirable components occurs in a tube or separation column.

107. The method of claim 106, wherein said tube or separation column comprises or engages one or more permanent magnets.

108. The method of claim 105, wherein said at least one specific binding member directly binds said magnetic beads.

109. The method of claim 105, wherein said at least one specific binding member indirectly binds said magnetic beads.

110. The method of claim 109, wherein said at least one specific binding member indirectly binds said magnetic beads through a biotin -streptavidin linkage.

111. The method of claim 103, wherein said sample is a blood sample and said one or more undesirable components are white blood cells.

112. The method of claim 111, wherein said at least one specific binding member is a lectin.

113. The method of claim 111, wherein said at least one specific binding member is an antibody.

114. The method of claim 113, wherein said antibody is a CD3 antibody, a CD11b antibody, a CD14 antibody, a CD17 antibody, a CD31 antibody, a CD45 antibody, a CD50 antibody, a CD53 antibody, a CD63 antibody, a CD69 antibody, a CD81 antibody, or a CD84 antibody.

115. The method of claim 114, further comprising debulking said blood sample with a solution that selectively sediments red blood cells.

116. The method of claim 115, wherein said debulking and said selectively removing undesirable components are performed simultaneously.

117. The method of claim 116, wherein said at least one specific binding member directly or indirectly binds magnetic beads, and further wherein said debulking and said selectively removing are performed in a tube that engages at least one magnet.

118. The method of claim 103, wherein said at least one filtration step is more than one filtration step.

119. The method of claim 118, wherein at least one of said more than one filtration steps is performed after said selectively removing one or more undesirable components from said sample.

120. The method of claim 70, further comprising separating one or more rare cells from said sample.

121. The method of claim 12, wherein said separating uses at least one specific binding member that specifically binds said one or more rare cells.

122. The method of claim 121, wherein said separating comprises capturing said one or more rare cells to a solid support.

123. The method of claim 122, wherein said solid support comprises magnetic beads.

124. The method of claim 123, wherein said specific binding member is directly bound to said magnetic beads.

125. The method of claim 123, wherein said specific binding member can indirectly bind said magnetic beads.

126. The method of claim 125, wherein said specific binding member can indirectly bind said magnetic beads through a biotin-streptavidin linkage.

127. The method of claim 123, wherein said separating occurs in a separation column.

128. The method of claim 123, wherein said separating occurs on an electromagnetic chip.

129. The method of claim 120, wherein said separating is by dielectrophoretic retention of said desirable components.

130. The method of claim 129, wherein said separating occurs on a chip that comprises at least two electrodes.

131. The method of claim 130, wherein said at least two electrodes are arranged in a castellated pattern.

132. The method of claim 130, wherein said at least two electrodes are arranged in a spiral pattern.

133. The method of claim 120, wherein said fluid sample is blood, urine, an effusion, a bone marrow sample, ascities fluid, pelvic wash fluid, or pleural fluid, spinal fluid, lymph, serum, mucus, sputum, saliva, urine, semen, occular fluid, extracts of nasal, throat or genital swabs, cell suspension from digested tissue, or extracts of fecal material.

134. The method of claim 133, wherein said fluid sample is a blood sample blood sample and said one or more rare cells are non-hematopoietic cells, subpopulations of blood cells, fetal red blood cells, stem cells, or cancerous cells.

135. The method of claim 133, wherein said fluid sample is an effusion or a urine sample and said one or more rare cells are cancerous cells or nonhematopoietic cells.

136. The method of claim 120, further comprising selectively removing undesirable components from said fluid sample.

137. The method of claim 136, comprising more than one filtration step.

138. The method of claim 137, wherein at least one of said more than one filtration steps is performed after said selectively removing one or more undesirable components from said sample.

139. The method of claim 120, further comprising at least one debulking step.

140. The method of claim 139, wherein said at least one debulking step is a gradient centrifugation step.

141. The method of claim 139, wherein said at least one debulking step is a selective sedimentation step.

142. The method of claim 141, wherein said fluid sample is a blood sample and said selective sedimentation step utilizes a solution that selectively sediments red blood cells.

143. The method of claim 136, wherein said sample is a blood sample and said undesirable components are white blood cells.

144. The method of claim 143, wherein said selectively removing undesirable components comprises capturing said white blood cells to a solid support using a specific binding member that selectively binds said white blood cells.

145. The method of claim 144, wherein said desirable components are nucleated red blood cells.

146. The method of claim 144, wherein said desirable components are cancer cells or nonhematopoietic cells.

167. An automated system for separating rare cells from a fluid sample, comprising:

a) at least one filtration chamber of claim 28;

b) at least one power supply or signal source or control circuit for automated control and powering fluid flow through said at least one filtration chamber;

c) means for collecting enriched rare cells.

168. The automated system of claim 167, wherein said filtration chamber comprises at least one microfabricated filter that comprises at least two tapered pores, wherein the variation in the size of said at least two tapered pores is less than 20%.

169. The automated system of claim 168, wherein said at least one microfabricated filter comprises a polymer, glass, ceramics, metal, silicon, or silicon dioxide.

170. The filtration chamber of claim 168, wherein said filtration chamber comprises glass, silicon, silicon dioxide, metal, ceramics, at least one plastic, or at least one polymer.

171. The automated system of claim 170, wherein said filtration chamber has a volume of from about 0.01 ml to about 2 liters.

172. The automated system of claim 171, wherein said filtration chamber has a volume of from about 0.2 ml to about 80 ml.

173. The automated system of claim 168, wherein said at least one filtration chamber is one filtration chamber.

174. The automated system of claim 173, wherein said filtration chamber comprises one filter.

175. The automated system of claim 174, wherein said automated system comprises at least one valve that can control the flow of said fluid sample into or out of said filtration chamber.

176. The automated system of claim 175, wherein said at least one valve can be automatically opened and closed.

177. The automated system of claim 176, wherein said at least one valve is at least two valves.

178. The automated system of claim 177, wherein said automated means for producing fluid flow through said at least one filtration chamber comprises an electrically powered pump or negative pressure system.

179. The automated system of claim 178, further comprising automated means for transferring sample into said filtration chamber.

180. The automated system of claim 179, further comprising automated means for adding at least one solution or reagent to said fluid sample.

181. The automated system of claim 180, further comprising automated means for transferring enriched cells into a collection container.

182. The automated system of claim 181, further comprising automated means for sensing the volume of a sample or a portion thereof.

183. The automated system of claim 182, further comprising means for securing and rocking fluid sample tubes.

184. The automated system of claim 183, comprising at least one tray for loading one or more sample tubes.

185. The automated system of claim 184, further comprising at least one magnet.

186. The automated system of claim 168, wherein said filtration chamber comprises two filters.

187. The automated system of claim 186, comprising at least one inlet for introducing a fluid sample into said automated system.

188. The automated system of claim 187, comprising conduits that can direct fluid through said automated system.

189. The automated system of claim 188, wherein said automated means for producing fluid flow through said at least one filtration chamber comprises an electrically powered pump or negative pressure system.

190. The automated system of claim 189, further comprising one or more active chips.

191. The automated system of claim 190, wherein at least one of said one or more active chips comprises one or more acoustic elements.

192. The automated system of claim 191, wherein at least one of said one or more active chips is a chip comprising at least two electrodes.

193. The automated system of claim 192, further comprising at least one separation chamber.

195. The automated system of claim 190, comprising at least one cartridge.

196. The automated system of claim 195, wherein said at least one cartridge is disposable.

197. The automated system of claim 196, wherein at least one of said at least one cartridges comprises at least one separation column.

198. A method of enriching rare cells from a fluid sample, comprising:

a) introducing a fluid sample into the automated system of claim 168;

b) filtering said fluid sample using at least one filtration chamber of the automated system; and

c) collecting enriched rare cells from at least one vessel or at least one outlet of the automated system.

199. The method of claim 198, wherein said fluid sample is a biological sample.

200. The method of claim 199, wherein said sample from a human.

201. The method of claim 200, wherein said sample is a blood sample, an effusion, a urine sample, semen, fecal matter, bone marrow aspirate, spinal fluid, cell suspension from tissue, mucus, sputum, or saliva.

202. The method of claim 201, wherein said rare cells are non-hematopoietic cells or cancer cells.

203. The method of claim 201, wherein said sample is a blood sample.

204. The method of claim 203, wherein said rare cells are nucleated red blood cells or a subpopulation of blood cells.

36. A solution for enriching rare cells of a blood sample, comprising a chemical RBC aggregation inducing agent and at least one specific binding member that specifically binds red blood cells, wherein said solution promotes the sedimentation of red blood cells.

37. The solution of claim 36, wherein said chemical RBC aggregation inducing agent is a polymer.

38. The solution of claim 37, wherein said polymer is dextran.

39. The solution of claim 38, wherein said dextran has a molecular weight of between 68 and 500 kilodaltons.

40. The solution of claim 38, wherein concentration of said dextran is between about 0.2% and 10%.

41. The solution of claim 40, wherein said concentration is between about 0.5% and about 6%.

42. The solution of claim 36, wherein said specific binding member that specifically binds red blood cells is a lectin.

43. The solution of claim 42, wherein said at least one lectin is concanavalin A, Dolichos biforus agglutinin, Datura Stramonium lectin, Sambucus Nigra lectin, Erythrina Cristagalli lectin, Griffonia Simplicifolia lectin I, Griffonia Simplicifolia lectin II, Lens culinaris agglutinin, Artocarpus integrifolia agglutinin, Lycopersicon esculentum lectin, Maackia amurensis lectin , phaseolus vulgaris lectin, phaseolus vulgaris agglutinin leucoagglutinin, phaseolus vulgaris agglutinin erythroagglutinin, peanut agglutinin, Pisum Sativum Agglutinin, Ricinus Communis Agglutinin I, Soybean Agglutinin, Sophora Japonica Agglutinin, Solanum Tuberosum lectin, Succinylated wheat germ agglutinin, Ulex europaeous agglutinin I, or wheat germ agglutinin.

44. The solution of claim 43, wherein said at least one lectin is Maackia amurensis lectin, phaseolus vulgaris lectin, phaseolus vulgaris agglutinin leucoagglutinin, phaseolus vulgaris agglutinin erythroagglutinin, peanut agglutinin, Ricinus Communis Agglutinin I, Soybean Agglutinin, or Ulex europaeous agglutinin I.

45. The solution of claim 42, wherein said lectin is present at a concentration of between 0.01 microgram per ml. and 500 milligram per ml.

46. The solution of claim 36, wherein said at least one specific binding member is an antibody.

47. The solution of claim 46, wherein said antibody is present at a concentration of between about 0.01 microgram per milliliter and about 1 milligram per milliliter.

48. The solution of claim 47, wherein said at least one antibody is an antibody to a red blood cell surface epitope.

49. The solution of claim 48, wherein said at least one antibody is an antibody to glycophorin A.

50. The solution of claim 49, wherein said at least one antibody to glycophorin A is a multivalent antibody.

51. The solution of claim 36, further comprising at least one additional specific binding member that can specifically bind undesirable components of the sample other than RBCs.

52. The solution of claim 51, wherein said at least one additional specific binding member that can specifically bind undesirable components of the sample other than RBCs is a receptor ligand, a lectin, or an antibody.

53. The solution of claim 52, wherein said at least one additional specific binding member that can specifically bind undesirable components of the sample other than RBCs is a lectin.

54. The solution of claim 52, wherein said at least one additional specific binding member that can specifically bind undesirable components of the sample other than RBCs is an antibody.

55. The solution of claim 52, wherein said at least one additional specific binding member that can specifically bind undesirable components of the sample other than RBCs is bound to magnetic beads.

56. The solution of claim 52, wherein said at least one additional specific binding member that can specifically bind undesirable components of the sample other than RBCs can indirectly bind magnetic beads.

57. The solution of claim 52, wherein said at least one additional specific binding member that can specifically bind undesirable components of the sample other than RBCs is a specific binding member that selectively binds white blood cells.

58. The solution of claim 57, wherein said at least one additional specific binding member that selectively binds white blood cells is an antibody that binds white blood cells.

59. The solution of claim 58, wherein said antibody that binds white blood cells is an antibody to CD3, CD11b, CD14, CD17, CD31, CD45, CD50, CD53, CD63, CD69, CD81, CD84, CD102, or CD166.

60. The solution of claim 59, wherein said antibody that specifically binds white blood cells is bound to magnetic beads.

61. The solution of claim 60, wherein said magnetic beads have a diameter of from about 0.5 to 5 microns.

62. The solution of claim 61, further comprising at least one salt.

63. The solution of claim 62, wherein said at least one salt is supplied as a saline solution.

64. The solution of claim 63, wherein said saline solution is PBS or Hanks balanced saline solution.

65. The solution of claim 62, further comprising a chelator of bivalent metals.

66. The solution of claim 65, wherein said chelator of bivalent metals is EDTA.

67. The solution of claim 66, wherein said concentration of EDTA is between about 0.1 mM and about 50 mM.

68. The solution of claim 62, further comprising heparin.

69. The solution of claim 68, wherein said heparin concentration is from about 1 to 100 units per ml.

147. A method of enriching rare cells from a blood sample, comprising

a) adding the solution of claim 36 to a blood sample;

b) mixing said blood sample and said solution of claim 36;

c) allowing red blood cells to sediment; and

d) removing a supernatant from said blood sample that comprises enriched rare cells.

148. The method of claim 147, wherein said solution of claim 36 is added to said sample at a solution to blood sample volume ratio of about 0.01 to 100.

149. The method of claim 148, wherein said solution of claim 36 is added to said sample at a solution to blood sample volume ratio of about 0.25 to 5.

150. The method of claim 149, wherein said allowing red blood cells to sediment comprises an incubation period of from five minutes to twenty-four hours.

151. The method of claim 150, wherein said allowing red blood cells to sediment comprises an incubation period of from ten minutes to two hours.

152. The method of claim 151, wherein said allowing red blood cells to sediment comprises an incubation period of from fifteen minutes to one hour.

153. The method of claim 147, further comprising selectively removing undesirable components from said sample.

154. The method of claim 153, wherein said undesirable components are white blood cells and said selectively removing undesirable components from said sample comprises adding a specific binding member that specifically binds white blood cells to said sample.

155. The method of claim 154, wherein said specific binding member that specifically binds white blood cells is bound to magnetic beads and selectively removing undesirable components from said sample is performed by magnetic capture of said white blood cells.

156. The method of claim 154, wherein said specific binding member that specifically binds white blood cells can indirectly bind magnetic beads and said selectively removing undesirable components is performed by magnetic capture of said white blood cells.

157. A method of enriching rare cells from a blood sample, comprising

a) adding the solution of claim 51 to a blood sample in a tube;

b) mixing said blood sample and said solution of claim 51;

c) allowing red blood cells to sediment;

d) allowing undesirable components to bind a solid support; and

e) collecting enriched rare cells.

158. A method of enriching rare cells from a blood sample, comprising

a) adding the solution of claim 60 to a blood sample in a tube;

b) mixing said blood sample and said solution of claim 60;

c) allowing red blood cells to sediment;

d) allowing rare cells to bind a solid support; and

e) collecting enriched rare cells.

159. The method of claim 158, further comprising a filtration step.

160. The method of claim 159, wherein said filtration step comprises filtering said sample through a chamber that comprises or engages at least one microfabricated filter that comprises at least one tapered pore.

161. The method of claim 160, wherein filtration step occurs after said sedimenting.

162. The method of claim 161, performed in an automated system.

163. The method of claim 158, further comprising a selective lysis step.

164. The method of claim 163, wherein said selective lysis step uses a buffer that selectively lyses red blood cells.

165. The method of claim 158, further comprising separating desirable components from said sample.

166. The method of claim 165, wherein said separating desirable components comprises at least one of:

capture of said desirable components using specific binding members, magnetic separation, filtration, flow cytometry, or dielectrophoretic separation.

194. The automated system of 193, wherein at least one of said at least one separation chamber comprises a chip that comprises one or more electromagnetic units.

205. An automated system for enriching rare cells of a fluid sample, comprising:

a) automated fluid volume sensing means for sensing the volume of at least one sample or a portion thereof provided in at least one tube or vessel, said automated fluid volume sensing means comprising a light transmission-light sensing system;

b) at least one filtration chamber that comprises or engages at least one microfabricated filter comprising one or more tapered pores;

c) at least one power supply or signal source or control circuit for automated control and powering of fluid flow through said at least one filtration chamber; and

d) means for collecting enriched rare cells.

206. The automated system of claim 205, wherein said automated fluid volume sensing means can direct an automated fluid uptake system to remove a portion or essentially all of said at least one sample.

207. The automated system of claim 205, wherein said automated fluid volume sensing means comprises at least one collated light source and at least one light-sensor.

208. The automated system of claim 207, wherein said at least one collated light source emits light at a wavelength that can be absorbed by at least a portion of said sample.

209. The automated system of claim 208, wherein said at least one collated light source comprises at least one laser.

210. The automated system of claim 208, wherein said at least one laser emits light of a wavelength from between about 700 to about 900 nanometers.

211. The automated system of claim 210, wherein said at least one laser emits light of a wavelength from between about 750 to about 850 nanometers.

212. The automated system of claim 207, wherein said at least one collated light source can move to scan said at least one sample or portion thereof in said at least one tube or vessel.

213. The automated system of claim 212, wherein said at least one collated light source and said at least one light sensor can move to scan at least one sample in said at least one tube or at least one vessel.

214. The automated system of claim 213, wherein at least one of said at least one collated light source can move coordinately with at least one of said at least one light sensor to scan said at least one sample in said at least one tube or vessel.

215. The automated system of claim 207, further comprising at least one rack that can hold said at least one tube or vessel.

216. The automated system of claim 215, wherein said wherein said at least one collated light source can move to scan a sample in said at least one tube or vessel held in said rack.

217. The automated system of claim 216, wherein at least one of said at least one collated light source can move coordinately with at least one of said at least one light sensor to scan said at least one sample in said at least one sample tube or vessel held in said rack.

218. The automated system of claim 217, wherein said at least one sample tube is at least two sample tubes.

219. The automated system of claim 218, wherein said automated fluid volume sensing means comprises least two collated light sources and at least two light-sensors.

220. The automated system of claim 219, wherein at least one of said at least two collated light sources can move coordinately with at least one of said at least two light sensors to scan one of said at least two samples in one of said at least two tubes or vessels.

221. The automated system of claim 220, wherein each of said at least two samples in said at least two sample tubes or vessels can be scanned by one of said at least two collated light sources that moves coordinately with one of said at least two light sensors.

222. The automated system of claim 217, wherein said automated fluid volume sensing means can detect an interface or boundary between a sample supernatant and a sample precipitate.

223. The automated system of claim 222, wherein said automated fluid volume sensing means can direct an automated fluid uptake system to remove a portion or essentially all of said sample supernatant.

224. The automated system of claim 215, wherein said at least one rack can move in an automated fashion to rock, shake, or invert said at least one sample tube or vessel held in said rack.

225. The automated system of claim 224, further comprising at least one magnet that can be automatically positioned in proximity to said rack.

226. The automated system of claim 205, wherein said at least one filtration chamber comprises an antechamber and a post-filtration subchamber separated by a microfabricated filter.

227. The automated system of claim 226, wherein:

the thickness of said microfabricated filter in the filtration area is from about 40 to about 100 microns;

further wherein;

said microfabricated filter comprises from about 100 to 250,000 tapered slots,

wherein said tapered slots are from approximately 20 microns to 200 microns in length and from about 2 microns to about 6 microns in width, and the tapering of said slots if from about 0 degrees to about 10 degrees, and

wherein the variation in slot size of said tapered slot is less than about 10%.

250. A method of using the automated system of claim 205, comprising,

a) providing a fluid sample in a tube or vessel;

b) using said automated fluid volume sensing means to determine the volume of said sample, or a portion thereof, in said tube or vessel;

c) using said determination in part b) to automatically remove a portion of said sample, and dispensing said sample portion in said filtration chamber;

d) filtering said sample in said filtration chamber; and

e) collecting enriched rare cells from said automated system.

228. A filtration unit, comprising:

a filtration chamber, wherein said filtration chamber comprises an antechamber and a post-filtration subchamber separated by one microfabricated filter comprising one or more tapered pores;

wherein said antechamber comprises an inlet and wherein said post-filtration subchamber comprises an outlet and at least one side port;

a frame comprising;

a loading reservoir;

at least one sample loading valve that can provide fluid communication between said loading reservoir and said filtration chamber;

at least one waste port that can reversibly engage said post-filtration subchamber via said outlet; and

at least one port for collecting enriched rare cells that can reversibly engage said antechamber via said inlet.

229. The filtration unit of claim 228, wherein said inlet of said antechamber and said outlet of said post-filtration subchamber are not on the same axis.

230. The filtration unit of claim 229, wherein said filtration chamber can rotate within said filtration unit.

231. The filtration unit of claim 230, wherein said side port is along the axis of filtration chamber rotation.

232. The filtration unit of claim 228, wherein said filtration chamber can rotate within said filtration unit between 0 and approximately 360 degrees.

233. The automated system of claim 232, wherein said filtration chamber can rotate within said filtration unit approximately 180 degrees or more.

234. The filtration unit of claim 233, wherein, when said filtration unit is in the filtering position, said antechamber is above said post-filtration subchamber, said inlet of said antechamber engages said at least one sample loading valve, and said outlet of said post-filtration subchamber engages said waste port;

and further wherein, when said filtration unit is in the collection position, said post-filtration subchamber is above said antechamber, said inlet of said antechamber engages said collection port, and said outlet of said post-filtration subchamber is blocked.

235. An automated filtration unit for enriching rare cells of a fluid sample, comprising:

a) the filtration unit of claim 234;

b) at least one power supply or signal source or control circuit for automated control and powering of fluid flow through said at least one filtration chamber; and

c) means for collecting enriched rare cells.

236. A method of filtering a sample through the filtration unit of claim 234, comprising the following automated steps:

a) with said filtration unit in said filtering position with said waste port closed, and said side port and said sample loading valve open, adding buffer to said filtration chamber from said side port until said buffer reaches said sample loading valve;

b) closing said side port;

c) adding a sample to said loading reservoir;

d) opening said waste port and pumping said sample through said filtration chamber, such that filtered sample exits said filtration chamber via said waste port;

e) adding buffer to said loading reservoir;

f) pumping said buffer through said filtration chamber;

g) closing said sample loading valve, opening said side port, and adding buffer to said post-filtration subchamber via said side port to wash said post-filtration subchamber;

h) rotating said filtration chamber approximately 180 degrees within the filtration unit, so that said filtration unit is in said collection position, wherein said antechamber is below said post-filtration subchamber, wherein said inlet of said antechamber engages said collection port;

i) pumping buffer into said filtration chamber through said side port so that enriched rare cells exit said antechamber through said collection port; and

j) collecting said enriched rare cells.

237. The method of claim 236, optionally comprising pushing air through said side port after steps g) and i).

238. The method of claim 236, wherein said filtering is performed at a rate of approximately 10-60 milliliters per hour.

239. An automated system for enriching rare cells of a fluid sample, comprising:

a) automated fluid volume sensing means for sensing the volume of at least one sample or a portion thereof provided in at least one tube or vessel, said automated fluid volume sensing means comprising a light transmission-light sensing system;

b) at least one filtration unit of claim 234;

d) at least one power supply or signal source or control circuit for automated control and powering of fluid flow through said at least one filtration chamber; and

e) means for collecting enriched rare cells.

240. The automated system of claim 239, wherein said filtration chamber can rotate within said filtration unit between 0 and approximately 360 degrees.

241. The automated system of claim 240, wherein said filtration chamber can rotate within said filtration unit approximately 180 degrees or more.

242. The filtration unit of claim 241, wherein, when said filtration unit is in the filtering position, said antechamber is above said post-filtration subchamber, said inlet of said antechamber engages said at least one sample loading valve, and said outlet of said post-filtration subchamber engages said waste port;

and further wherein, when said filtration unit is in the collection position, said post-filtration subchamber is above said antechamber, said inlet of said antechamber engages said collection port, and said outlet of said post-filtration subchamber is blocked.

243. The automated system of claim 242, further comprising at least one rack that can hold at least one tube or vessel.

244. The automated system of claim 243, further wherein said automated fluid volume sensing means can direct an automated fluid uptake system to remove a portion or essentially all of said at least one sample.

245. The automated system of claim 244, wherein said automated fluid volume sensing means comprises at least one collated light source and at least one light-sensor, wherein said at least one collated light source emits light at a wavelength that can be absorbed by at least a portion of said sample.

246. The automated system of claim 245, wherein said at least one collated light source and said at least one light sensor can move to scan at least one sample in said at least one tube or at least one vessel.

247. The automated system of claim 246, wherein at least one of said at least one collated light source can move coordinately with at least one of said at least one light sensor to scan said at least one sample in said at least one sample tube or vessel held in said rack.

248. The automated system of claim 243, wherein said at least one rack can move in an automated fashion to rock, shake, or invert said at least one sample tube or vessel held in said rack.

249. The automated system of claim 248, further comprising at least one magnet that can be automatically positioned in proximity to said rack.

251. A method of using the automated system of claim 248, comprising,

a) providing a fluid sample in a tube or vessel;

b) adding to said sample at least one reagent that can cause at least one sample component to sediment;

c) positioning said tube or vessel in said rack;

d) automatically rocking or inverting said tube or vessel positioned in said rack;

e) allowing at least one sample component to sediment, resulting in a sample supernatant and a sample precipitate;

d) determining the volume of said sample supernatant or said sample precipitate in said tube or vessel using said automated fluid volume sensing means;

e) using the determination in part d) to automatically remove a volume of said sample supernatant, and dispensing said volume of said sample supernatant in said filtration chamber;

f) filtering said sample or portion thereof in said filtration chamber; and

g) collecting enriched rare cells from said automated system.

252. The method of claim 251, wherein said sample is a blood sample.

253. The method of claim 252, wherein said enriched rare cells are nucleated red blood cells or cancer cells.

254. A method of using the automated system of claim 249, comprising,

a) providing a fluid sample in a tube or vessel;

b) adding to said sample at least one reagent that promotes the sedimentation of at least one undesirable component of said sample;

c) adding to said sample a preparation of magnetic beads directly or indirectly linked to at least one specific binding member that can bind at least one other undesirable sample component;

d) positioning said tube or vessel in said rack;

e) automatically rocking or inverting said tube or vessel positioned in said rack to mix said reagent that promotes the sedimentation of at least one sample component of said sample and said preparation of magnetic beads with said sample, allowing said at least one other undesirable sample component to bind said magnetic beads;

f) while maintaining said tube in a stationary position, positioning at least one magnet adjacent to said tube;

g) allowing said at least one sample component to sediment and said at least one other undesirable sample component bound to magnetic beads to adhere to the side of the tube adjacent to said magnet, resulting in a separation of said sample into a sample supernatant, a sample precipitate, and magnetic beads adhering to one side of said tube;

h) using said automated fluid volume sensing means to determine the volume of said sample supernatant in said tube or vessel;

i) using the determination in part h) to automatically remove a volume of said sample supernatant, and dispensing said volume of said sample supernatant in said loading reservoir of said filtration chamber;

j) filtering said sample or portion thereof in said filtration chamber; and

k) collecting enriched rare cells from said automated system.

255. The method of claim 254, wherein said sample is a blood sample.

256. The method of claim 255, wherein said at least one undesirable component is red blood cells.

257. The method of claim 256, wherein said enriched rare cells are nucleated red blood cells or cancer cells.

258. The method of claim 257, wherein said at least one other undesirable component is white blood cells, and said at least one specific binding member that can bind at least one undesirable sample component is a specific binding member that binds white blood cells.

259. The method of claim 258, wherein said reagent that promotes the sedimentation of at least one sample component of said sample is provided in a solution that selectively sediments red blood cells.

260. The method of claim 259, wherein said reagent that promotes the sedimentation of at least one undesirable sample component and said preparation of magnetic beads are both added in a combined solution for sedimenting red blood cells and selectively removing undesirable sample components of a blood sample.

261. An automated fluid volume sensing system for determining the level of a sample or a portion of a sample, comprising:

at least one bar in an essentially horizontal orientation, that is positioned or can be positioned proximal to a rack or sample holder, wherein said bar comprises;

a light source on one end of said bar and a light sensor on another end of said bar, wherein the vertical level of the bar corresponds to the vertical level of the light source;

wherein when a sample is in said rack or sample holder, said light source is positioned on one side of said sample, and said light sensor is positioned on the opposite side of said sample;

further wherein said bar can move in a vertical direction, to allow said light source and said light sensor to scan a sample held in said rack or sample holder.

262. The fluid volume system of claim 261, wherein said light source is a collated light source.

263. The fluid volume system of claim 261, wherein said collated light source is a laser.

264. An automated system for enriching rare cells from a fluid sample comprising the automated fluid volume sensing system of claim 261.

265. An automated fluid volume sensing/ fluid volume uptake system for determining a volume of a sample supernatant in a sample tube or vessel and removing said volume of a sample supernatant from said sample tube or vessel, comprising:

at least one bar in an essentially horizontal orientation, that is positioned or can be positioned proximal to a rack or sample holder, wherein said bar comprises;

a light source on one end of said bar and a light sensor on another end of said bar, wherein the vertical level of the bar corresponds to the vertical level of the light source;

wherein when a sample is in said rack or sample holder, said light source is positioned on one side of said sample, and said light sensor is positioned on the opposite side of said sample;

further wherein said bar can move in a vertical direction, to allow said light source and said light sensor to scan a sample held in said rack or sample holder;

a collection tip, wherein said collection tip is comprises a conduit for the uptake and dispensing of fluid, further wherein said collection tip can move in a horizontal plane, to position itself over a tube in said rack or holder, and can move in a vertical plane, to reach the level of the bar or a level within said sample tube.

266. The fluid volume system of claim 265, wherein said light source is a collated light source.

267. The fluid volume system of claim 266, wherein said collated light source is a laser.

268. The automated fluid volume sensing/ fluid volume uptake system of claim 265, wherein said rack or sample holder can hold two or more samples;

wherein said at least one bar is at least two bars; and

wherein said at least one collection tip is at least two collection tips.

269. An automated system for enriching rare cells from a fluid sample comprising the automated fluid volume sensing/ fluid volume uptake system of claim 265.

270. An automated system for enriching rare cells from a fluid sample comprising the automated fluid volume sensing/ fluid volume uptake system of claim 267.

271. A method of determining the level of a sample supernatant or upper phase, and removing the sample supernatant or upper phase of a fluid sample using the fluid volume sensing/fluid volume uptake system of claim 267, comprising the following automated steps:

providing a sample in a tube or vessel in said rack;

optionally positioning said bar proximal to said tube; and

allowing said fluid uptake system to automatically calculate the volume of and remove a portion of said fluid sample, comprising the steps;

a) said bar moves vertically along the length of said tube, and during said movement said light source emits light that can be detected by said light sensor;

b) when said light sensor detector registers a critical value above threshold, said bar stops moving such that the level of said bar is at the interface between said sample supernatant and a sample precipitate, or between said sample upper phase and a sample lower phase;

c) after said bar stops moving, said collection tip moves downward over said bar until said collection tip contacts said bar;

d) said collection tip moves upward to a level above the level of the tube, positions itself over the tube, and moves downward into the tube;

e) when said collection tip sense the fluid sample supernatant or upper phase, said collection tip takes up said fluid sample supernatant or upper phase while moving downward into the tube;

f) when said collection tip reaches the level of the bar, said collection tip stops moving downward and stops taking up fluid;

g) said collection tip moves upward; and

h) said collection tip moves away from said tube.