Methods and compositions for prenatal testing

Abstract

This disclosure provides a method and system for isolating circulating fetal cells (CFCs) from maternal biological samples of pregnancy-related conditions. A method for identifying a genetic disorder in a target fetus(s) may include assaying a biological sample derived from the target to isolate CFCs, characterizing biomarkers such as the sequence of genetic material or the abundance of gene expression substances derived from the CFCs, and determining the presence or absence of pregnancy-related conditions such as maternal or fetal genetic order or existing health risks.

Claims

1. A composition comprising an ex vivo cell population derived from a maternal blood sample, wherein the ex vivo cell population comprises a maternal cell subpopulation and a fetal cell subpopulation, and the fetal cell subpopulation constitutes at least about 75% of the cells in the ex vivo cell population.

2. The composition according to claim 1, wherein the embryonic cell subpopulation comprises at least about 80% of the cells in the ex vivo cell population.

3. The composition according to claim 1 or claim 2, wherein the embryonic cell subpopulation comprises at least about 90% of the cells in the ex vivo cell population.

4. The composition according to any one of claims 1 to 3, wherein the maternal cell subpopulation accounts for about 30% or less of the cells in the ex vivo cell population.

5. The composition according to any one of claims 1 to 4, wherein the maternal cell subpopulation accounts for about 20% or less of the cells in the ex vivo cell population.

6. The composition according to any one of claims 1 to 5, wherein the maternal cell subpopulation accounts for about 10% or less of the cells in the ex vivo cell population.

7. The composition according to any one of claims 1 to 6, wherein the embryonic cell subpopulation includes trophoblasts.

8. The composition according to any one of claims 1 to 7, further comprising a second subpopulation of fetal cells, wherein the second subpopulation of fetal cells has a different cell type from the subpopulation of fetal cells.

9. The composition according to claim 8, wherein the second subpopulation of fetal cells includes erythroblasts.

10. The composition according to claim 8, wherein the second subpopulation of fetal cells includes megakaryocytes.

11. The composition according to claim 8, wherein the second subpopulation of fetal cells includes vascular endothelial cells.

12. The composition according to claim 8, wherein the second subpopulation of fetal cells includes fetal stromal cells.

13. The composition according to claim 8, wherein the first subpopulation of fetal cells includes fetal cells in the early stages of gestation, and the second subpopulation of fetal cells includes fetal cells in the relatively later stages of gestation.

14. The composition according to any one of claims 1 to 13, wherein the maternal cell subpopulation and the fetal cell subpopulation in the ex vivo cell population are quantified by flow cytometry.

15. A method comprising sequencing a nucleic acid sequence, wherein the nucleic acid sequence is derived from an ex vivo cell population derived from a maternal blood sample, the ex vivo cell population comprises a maternal cell subpopulation and a fetal cell subpopulation, and the fetal cell subpopulation comprises at least about 70% of the cells in the ex vivo cell population.

16. The method according to claim 15, wherein the embryonic cell subpopulation comprises at least about 80% of the cells in the ex vivo cell population.

17. The method according to claim 15 or claim 16, wherein the embryonic cell subpopulation comprises at least about 90% of the cells in the ex vivo cell population.

18. The method according to any one of claims 15 to 17, wherein the maternal cell subpopulation accounts for about 30% or less of the cells in the ex vivo cell population.

19. The method according to any one of claims 15 to 18, wherein the maternal cell subpopulation is about 20% or less of the cells in the ex vivo cell population.

20. The method according to any one of claims 15 to 19, wherein the maternal cell subpopulation is about 10% or less of the cells in the ex vivo cell population.

21. The method according to any one of claims 15 to 20, further comprising extracting the maternal cell subpopulation and the fetal cell subpopulation from the maternal blood sample to prepare an extract prior to sequencing the nucleic acid sequence, wherein the extract comprises the maternal cell subpopulation and the fetal cell subpopulation derived from the maternal blood sample.

22. The method according to claim 21, further comprising enriching the extract with the fetal cell subpopulation to prepare a sample in which fetal cells are enriched and maternal cells are depleted.

23. The method according to claim 21, further comprising preparing a sample enriched with fetal cells and depleted with maternal cells by sorting the fetal cell subpopulation in the extract from the maternal cell subpopulation in the extract.

24. The method according to any one of claims 15 to 23, wherein, prior to the sequencing of the nucleic acid sequence, an extract is prepared by extracting the maternal cell subpopulation and the fetal cell subpopulation from the maternal blood sample, and the extract comprises the maternal cell subpopulation and the fetal cell subpopulation derived from the maternal blood sample.

25. The method according to claim 24, wherein the fetal cell subpopulation in the extract is enriched from the maternal cell subpopulation in the extract, thereby preparing a sample in which fetal cells are enriched and maternal cells are depleted.

26. The method according to claim 24, wherein the fetal cell subpopulation in the extract is sorted from the maternal cell subpopulation in the extract, thereby preparing a sample in which fetal cells are enriched and maternal cells are depleted.

27. The method according to any one of claims 15 to 26, wherein the embryonic cell subpopulation includes trophoblasts.

28. The method according to any one of claims 15 to 27, wherein the ex vivo cell population further comprises a second embryonic cell subpopulation, and the second embryonic cell subpopulation has a different cell type from the first embryonic cell subpopulation.

29. The method according to claim 28, wherein the second subpopulation of fetal cells includes erythroblasts.

30. The method according to claim 28, wherein the second subpopulation of fetal cells includes megakaryocytes.

31. The method according to claim 28, wherein the second subpopulation of fetal cells includes vascular endothelial cells.

32. The method according to claim 28, wherein the second subpopulation of fetal cells includes stromal cells.

33. The method according to claim 28, wherein the subpopulation of fetal cells includes fetal cells in the early stages of gestation, and the second subpopulation of fetal cells includes fetal cells in the relatively later stages of gestation.

34. The method according to any one of claims 15 to 33, further comprising contacting the maternal blood sample with a first isolation agent prior to sequencing the nucleic acid sequence, and coupling the first isolation agent to a first biomarker in the fetal cell subpopulation to isolate the fetal cell subpopulation from the maternal blood sample.

35. The method according to claim 34, further comprising incubating the maternal blood sample after the contact.

36. The method according to claim 35, wherein the first isolated agent is a nucleic acid probe.

37. The method according to claim 35, wherein the first isolated drug is a ligand.

38. The method according to claim 35, wherein the first isolated agent is coupled to an intracellular biomarker.

39. The method according to claim 35, wherein the first isolated agent is coupled to a cell surface biomarker.

40. The method according to claim 35, wherein the first isolated agent is coupled to a soluble cell surface biomarker.

41. The method according to claim 35, wherein the first isolated agent is coupled to a nucleic acid biomarker.

42. The method according to any one of claims 15 to 41, wherein, prior to sequencing of the nucleic acid sequence, the fetal cell subpopulation is isolated from the maternal blood sample, the maternal blood sample is brought into contact with a first isolation agent, and the first isolation agent is coupled to a first biomarker in the fetal cell subpopulation.

43. The method according to claim 42, wherein the maternal blood sample is incubated after the contact.

44. The method according to claim 42, wherein the first isolated agent is a nucleic acid probe.

45. The method according to claim 42, wherein the first isolated drug is a ligand.

46. The method according to claim 42, wherein the first isolated agent is coupled to a cell surface biomarker.

47. The method according to claim 42, wherein the first isolated agent is coupled to a soluble cell surface biomarker.

48. The method according to claim 42, wherein the first isolated agent is coupled to an intracellular biomarker.

49. The method according to claim 42, wherein the first isolated agent is coupled to a nucleic acid biomarker.

50. The method according to any one of claims 15 to 49, wherein, prior to sequencing of the nucleic acid sequence, the maternal cell subpopulation is isolated from the maternal blood sample, the maternal blood sample is brought into contact with a second isolation agent, and the second isolation agent is coupled to a second biomarker in the maternal cell subpopulation.

51. The method according to claim 50, wherein the maternal blood sample is incubated after the contact.

52. The method according to claim 50, wherein the second isolated drug is a nucleic acid probe.

53. The method according to claim 50, wherein the second isolated drug is a ligand.

54. The method according to claim 50, wherein the second isolated agent is coupled to a cell surface biomarker.

55. The method according to claim 50, wherein the second isolated agent is coupled to a soluble cell surface biomarker.

56. The method according to claim 50, wherein the second isolated drug is coupled to an intracellular biomarker.

57. The method according to claim 50, wherein the second isolated agent is coupled to a nucleic acid biomarker.

58. The method according to any one of claims 15 to 57, further comprising contacting the maternal blood sample with a second isolation agent prior to sequencing of the nucleic acid sequence, and coupling the second isolation agent to a second biomarker in the maternal cell subpopulation to isolate the maternal cell subpopulation from the maternal blood sample.

59. The method according to claim 58, further comprising incubating the maternal blood sample after the contact.

60. The method according to claim 58, wherein the second isolated agent is a nucleic acid probe.

61. The method according to claim 58, wherein the second isolated drug is a ligand.

62. The method according to claim 58, wherein the second isolated agent is coupled to a cell surface biomarker.

63. The method according to claim 58, wherein the second isolated agent is coupled to a soluble cell surface biomarker.

64. The method according to claim 58, wherein the second isolated drug is coupled to an intracellular biomarker.

65. The method according to claim 58, wherein the second isolated agent is coupled to a nucleic acid biomarker.

66. The method according to any one of claims 15 to 65, wherein the sequencing is performed on a next-generation sequencing platform.

67. The method according to any one of claims 15 to 66, wherein the nucleic acid sequence is derived from the embryonic cell subpopulation.

68. The method according to claim 67, wherein the nucleic acid sequence is a DNA sequence.

69. The method according to claim 67, wherein the nucleic acid sequence is an RNA sequence.

70. The method according to any one of claims 15 to 69, wherein the maternal cell subpopulation and the fetal cell subpopulation in the ex vivo cell population are quantified by flow cytometry.

71. A method comprising performing an assay on an ex vivo cell population derived from a maternal blood sample, wherein the ex vivo cell population comprises a maternal cell subpopulation and a fetal cell subpopulation, the fetal cell subpopulation comprising at least about 70% of the cells in the ex vivo cell population.

72. The method according to claim 71, wherein the assay is a cell morphology assay.

73. The method according to claim 71, wherein the assay is a cell count assay.

74. The method according to claim 71, wherein the assay is a cell viability determination assay.

75. The method according to claim 71, wherein the assay is a cell proliferation assay.

76. The method according to claim 71, wherein the assay is a cytotoxicity assay.

77. The method according to claim 71, wherein the assay shows the likelihood that a fetus associated with the fetal cell subpopulation is susceptible to a hereditary disease.

78. The method according to claim 77, wherein the hereditary condition is Down syndrome.

79. The method according to claim 77, wherein the hereditary pathological condition is cystic fibrosis.

80. The method according to claim 77, wherein the hereditary pathological condition is muscular dystrophy.

81. The method according to claim 77, wherein the hereditary condition is sickle cell disease.

82. The method according to claim 77, wherein the hereditary disease is Tay-Sachs disease.

83. The method according to any one of claims 71 to 82, wherein the maternal cell subpopulation and the fetal cell subpopulation in the ex vivo cell population are quantified by flow cytometry.

84. The method according to any one of claims 71 to 83, wherein the maternal cell subpopulation and the fetal cell subpopulation in the ex vivo cell population are quantified by microscopy.

85. (a) Sequencing a first nucleic acid sequence obtained from a first type of fetal cell derived from an ex vivo fetal cell population. (b) Sequencing a second nucleic acid sequence obtained from a second type of fetal cell derived from the ex vivo fetal cell population. (c) Sequencing a third nucleic acid sequence obtained from a third type of fetal cell derived from the ex vivo fetal cell population. (d) Sequencing a fourth nucleic acid sequence obtained from a fourth type of fetal cell derived from the ex vivo fetal cell population. (e) Sequencing a fifth nucleic acid sequence obtained from a fifth type of fetal cell derived from the ex vivo fetal cell population, and (f) Analyze at least one of the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, the fourth nucleic acid sequence, or the fifth nucleic acid sequence to determine the likelihood that a fetus associated with an ex vivo fetal cell population is susceptible to a hereditary disease. Methods that include...

86. The method according to claim 85, wherein at least one of the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, the fourth nucleic acid sequence, or the fifth nucleic acid sequence includes a DNA sequence.

87. The method according to claim 85, wherein at least one of the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, the fourth nucleic acid sequence, or the fifth nucleic acid sequence includes an RNA sequence.

88. The method according to any one of claims 85 to 87, wherein the ex vivo fetal cell population is in a solution containing artiodactyl serum.

89. The method according to any one of claims 85 to 88, further comprising extracting the ex vivo maternal cell population and the ex vivo fetal cell population from a maternal blood sample to prepare an extract, wherein the extract comprises the ex vivo maternal cell population and the ex vivo fetal cell population.

90. The method according to claim 89, wherein the maternal blood sample is derived from a pregnant subject.

91. The method according to claim 89, wherein the maternal blood sample is plasma.

92. The method according to claim 89, wherein the maternal blood sample is serum.

93. The method according to claim 89, wherein the maternal blood sample is a buffy coat.

94. The method according to claim 89, wherein the maternal blood sample includes peripheral blood mononuclear leukocytes.

95. The method according to claim 89, wherein the maternal blood sample contains nucleated leukocytes.

96. The method according to claim 89, wherein the maternal blood sample is whole blood.

97. The method according to claim 89, further comprising enriching the ex vivo fetal cell population in the extract with the ex vivo maternal cell population in the extract to prepare a sample in which fetal cells are enriched and maternal cells are depleted.

98. The method according to claim 89, further comprising preparing a sample enriched with fetal cells and depleted with maternal cells by sorting the ex vivo fetal cell population in the extract from the ex vivo maternal cell population in the extract.

99. The method according to any one of claims 85 to 98, wherein, prior to (a) to (f), an extract is prepared by extracting the ex vivo fetal cell population and the ex vivo maternal cell population from a maternal blood sample, and the extract comprises the ex vivo fetal cell population and the ex vivo maternal cell population.

100. The method according to claim 99, wherein the maternal blood sample is derived from a pregnant subject.

101. The method according to claim 99, wherein the maternal blood sample is plasma.

102. The method according to claim 99, wherein the maternal blood sample is serum.

103. The method according to claim 99, wherein the maternal blood sample is a buffy coat.

104. The method according to claim 99, wherein the maternal blood sample includes peripheral blood mononuclear leukocytes.

105. The method according to claim 99, wherein the maternal blood sample contains nucleated leukocytes.

106. The method according to claim 99, wherein the maternal blood sample is whole blood.

107. The method according to claim 99, wherein the ex vivo fetal cell population in the extract is enriched with the ex vivo maternal cell population in the extract, thereby preparing a sample in which fetal cells are enriched and maternal cells are depleted.

108. The method according to claim 99, wherein the ex vivo fetal cell population in the extract is sorted from the ex vivo maternal cell population in the extract, thereby preparing a sample in which fetal cells are enriched and maternal cells are depleted.

109. The method according to any one of claims 85 to 108, further comprising incubating a blood sample containing the ex vivo fetal cell population and the ex vivo maternal cell population with a first isolation agent, and coupling the first isolation agent to a first biomarker in the ex vivo fetal cell population, prior to (a) to (f).

110. The method according to claim 109, further comprising sorting the ex vivo fetal cell population based on the presence of the first isolated agent that couples to the first biomarker in the ex vivo fetal cell population.

111. The method according to claim 109, further comprising sorting the ex vivo fetal cell population based on the absence of a second isolated agent coupled to a second biomarker in the ex vivo maternal cell population.

112. The method according to claim 109, wherein the first isolated drug is a nucleic acid probe.

113. The method according to claim 109, wherein the first isolated drug is a ligand.

114. The method according to claim 109, wherein the first isolated agent is coupled to a cell surface biomarker.

115. The method according to claim 109, wherein the first isolated agent is coupled to a soluble cell surface biomarker.

116. The method according to claim 109, wherein the first isolated drug is coupled to an intracellular biomarker.

117. The method according to claim 109, wherein the first isolated agent is coupled to a nucleic acid biomarker.

118. The method according to any one of claims 85 to 117, wherein, prior to (a) to (f), a blood sample containing the ex vivo fetal cell population and the ex vivo maternal cell population is incubated with a first isolation agent that couples to a first biomarker in the ex vivo fetal cell population.

119. The method according to claim 118, wherein the ex vivo fetal cell population is sorted based on the presence of the first isolation agent that couples to the first biomarker in the ex vivo fetal cell population.

120. The method according to claim 118, wherein the ex vivo fetal cell population is sorted based on the absence of a second isolation agent that couples to a second biomarker in the ex vivo maternal cell population.

121. The method according to claim 118, wherein the first isolated agent is a nucleic acid probe.

122. The method according to claim 118, wherein the first isolated drug is a ligand.

123. The method according to claim 118, wherein the first isolated agent is coupled to a cell surface biomarker.

124. The method according to claim 118, wherein the first isolated agent is coupled to a soluble cell surface biomarker.

125. The method according to claim 118, wherein the first isolated agent is coupled to an intracellular biomarker.

126. The method according to claim 118, wherein the first isolated agent is coupled to a nucleic acid biomarker.

127. The method according to any one of claims 85 to 126, further comprising incubating a blood sample containing the ex vivo fetal cell population and the ex vivo maternal cell population with a second isolation agent before (a) to (f), and coupling the second isolation agent to a second biomarker in the ex vivo maternal cell population.

128. The method according to claim 127, further comprising sorting the ex vivo maternal cell population based on the presence of the second isolated agent that couples to the second biomarker in the ex vivo maternal cell population.

129. The method according to claim 127, further comprising sorting the ex vivo maternal cell population based on the absence of a first isolation agent coupled to a first biomarker in the ex vivo fetal cell population.

130. The method according to claim 127, wherein the second isolated agent is a nucleic acid probe.

131. The method according to claim 127, wherein the second isolated drug is a ligand.

132. The method according to claim 127, wherein the second isolated agent is coupled to a cell surface biomarker.

133. The method according to claim 127, wherein the second isolated agent is coupled to a soluble cell surface biomarker.

134. The method according to claim 127, wherein the second isolated drug is coupled to an intracellular biomarker.

135. The method according to claim 127, wherein the second isolated agent is coupled to a nucleic acid biomarker.

136. The method according to any one of claims 85 to 135, wherein, prior to (a) to (f), a blood sample containing the ex vivo fetal cell population and the ex vivo maternal cell population is incubated with a second isolation agent that couples to a second biomarker in the ex vivo maternal cell population.

137. The method according to claim 136, wherein the ex vivo maternal cell population is sorted based on the presence of the second isolation agent that couples to the second biomarker in the ex vivo maternal cell population.

138. The method according to claim 136, wherein the ex vivo maternal cell population is sorted based on the absence of a first isolation agent that couples to a first biomarker in the ex vivo fetal cell population.

139. The method according to claim 136, wherein the second isolated agent is a nucleic acid probe.

140. The method according to claim 136, wherein the second isolated drug is a ligand.

141. The method according to claim 136, wherein the second isolated agent is coupled to a cell surface biomarker.

142. The method according to claim 136, wherein the second isolated agent is coupled to a soluble cell surface biomarker.

143. The method according to claim 136, wherein the second isolated agent is coupled to an intracellular biomarker.

144. The method according to claim 136, wherein the second isolated agent is coupled to a nucleic acid biomarker.

145. The method according to any one of claims 85 to 144, wherein the ex vivo fetal cell population includes trophoblasts in the early gestational period.

146. The method according to any one of claims 85 to 145, wherein the ex vivo fetal cell population includes trophoblasts from a relatively late gestational stage.

147. The method according to any one of claims 85 to 146, wherein the ex vivo fetal cell population includes erythroblasts.

148. The method according to any one of claims 85 to 147, wherein the ex vivo fetal cell population includes megakaryocytes.

149. The method according to any one of claims 85 to 148, wherein the ex vivo fetal cell population includes stromal cells.

150. The method according to any one of claims 85 to 149, wherein the ex vivo fetal cell population includes endothelial cells.

151. The method according to any one of claims 85 to 150, wherein the ex vivo fetal cell population includes lymphocytes.

152. The method according to any one of claims 85 to 151, wherein the ex vivo fetal cell population includes nucleated red blood cells.

153. The method according to any one of claims 85 to 152, wherein the ex vivo fetal cell population includes hematopoietic progenitor cells.

154. The method according to any one of claims 85 to 153, wherein the ex vivo fetal cell population includes mesenchymal progenitor cells.

155. A method comprising performing a single enrichment of a fetal cell population from a maternal cell population in a cell sample, wherein the single enrichment enriches the fetal cell population from the cell sample to a level that is sufficient to obtain an amount of fetal nucleic acid sufficient for characterization as fetal nucleic acid rather than maternal nucleic acid.

156. The method according to claim 155, wherein the single enrichment provides an enriched sample, and the level at which the amount of fetal nucleic acid sufficient for characterization as fetal nucleic acid rather than maternal nucleic acid is obtained is at least about 50% of the fetal cells in the enriched sample.

157. The method according to claim 156, comprising performing the enrichment once and less than once.

158. The method according to any one of claims 155 to 157, wherein the cell sample is a blood sample.

159. The method according to claim 158, wherein the blood sample is plasma.

160. The method according to claim 158, wherein the blood sample is serum.

161. The method according to claim 158, wherein the blood sample is a buffy coat.

162. The method according to claim 158, wherein the maternal blood sample includes peripheral blood mononuclear leukocytes.

163. The method according to claim 158, wherein the maternal blood sample contains nucleated leukocytes.

164. The method according to claim 158, wherein the blood sample is whole blood.

165. The method according to any one of claims 155 to 164, wherein the cell sample is derived from a human subject.

166. The method according to any one of claims 155 to 165, further comprising extracting the maternal cell population and the fetal cell population from the cell sample to prepare an extract before performing the above, wherein the extract comprises the maternal cell population and the fetal cell population.

167. The method according to claim 166, further comprising preparing a sample enriched with fetal cells and depleted with maternal cells by sorting the fetal cell population in the extract from the maternal cell population in the extract.

168. The method according to any one of claims 155 to 167, wherein, prior to the above-mentioned execution, an extract is prepared by extracting the fetal cell population and the maternal cell population from the cell sample, and the extract comprises the maternal cell population and the fetal cell population.

169. The method according to claim 168, wherein the fetal cell population in the extract is sorted from the maternal cell population in the extract, thereby preparing a sample in which the fetal cells are enriched and the maternal cells are depleted.

170. The method according to any one of claims 155 to 169, wherein, prior to performing the above, the cell sample is incubated with a first isolation agent specific to a first biomarker in the embryonic cell population.

171. The method according to claim 170, wherein the first isolated drug is a nucleic acid probe.

172. The method according to claim 170, wherein the first isolated drug is a ligand.

173. The method according to claim 170, wherein the first biomarker includes a cell surface marker.

174. The method according to claim 170, wherein the first biomarker includes a soluble cell surface marker.

175. The method according to claim 170, wherein the first biomarker includes an intracellular biomarker.

176. The method according to claim 170, wherein the first biomarker includes a nucleic acid marker.

177. The method according to any one of claims 155 to 176, further comprising incubating the cell sample with a first isolation agent specific to a first biomarker in the embryonic cell population before performing the above.

178. The method according to claim 177, wherein the first isolated drug is a nucleic acid probe.

179. The method according to claim 177, wherein the first isolated drug is a ligand.

180. The method according to claim 177, wherein the first biomarker includes a cell surface marker.

181. The method according to claim 177, wherein the first biomarker includes a soluble cell surface marker.

182. The method according to claim 177, wherein the first biomarker includes an intracellular biomarker.

183. The method according to claim 177, wherein the first biomarker includes a nucleic acid marker.

184. The method according to any one of claims 155 to 183, wherein, prior to performing the above, the cell sample is incubated with a second isolation agent specific to a second biomarker in the maternal cell population.

185. The method according to claim 184, wherein the second isolated agent is a nucleic acid probe.

186. The method according to claim 184, wherein the second isolated drug is a ligand.

187. The method according to claim 184, wherein the second biomarker includes a cell surface marker.

188. The method according to claim 184, wherein the second biomarker includes a soluble cell surface marker.

189. The method according to claim 184, wherein the second biomarker includes an intracellular biomarker.

190. The method according to claim 184, wherein the second biomarker includes a nucleic acid marker.

191. The method according to any one of claims 155 to 190, further comprising incubating the cell sample with a second isolation agent specific to a second biomarker in the maternal cell population before performing the aforementioned procedure.

192. The method according to claim 191, wherein the second isolated agent is a nucleic acid probe.

193. The method according to claim 191, wherein the second isolated drug is a ligand.

194. The method according to claim 191, wherein the second biomarker includes a cell surface marker.

195. The method according to claim 191, wherein the second biomarker includes a soluble cell surface marker.

196. The method according to claim 191, wherein the second biomarker includes an intracellular biomarker.

197. The method according to claim 191, wherein the second biomarker includes a nucleic acid marker.

198. The method according to any one of claims 155 to 197, further comprising sequencing nucleic acid sequences derived from the fetal cell population.

199. a) Extracting ex vivo maternal cell subpopulations and ex vivo fetal cell subpopulations from maternal blood samples. b) After the extraction, enrich the ex vivo fetal cell subpopulation from the ex vivo maternal cell subpopulation. c) After enriching, prepare a sample in which fetal cells are enriched and maternal cells are depleted by sorting the ex vivo fetal cell subpopulation from the ex vivo maternal cell subpopulation, and d) After sorting, sequencing of nucleic acids derived from the ex vivo fetal cell subpopulation, Methods that include...

200. a) Contacting a maternal blood sample containing an ex vivo fetal cell subpopulation and an ex vivo maternal cell subpopulation with an isolation agent that couples to a biomarker in the ex vivo fetal cell subpopulation. b) After contact, incubate the isolated drug together with the maternal blood sample. c) After incubation, isolating the ex vivo fetal cell subpopulation in the maternal blood sample from the ex vivo maternal cell subpopulation in the maternal blood sample, and d) After isolation, sequencing of nucleic acids derived from the ex vivo fetal cell subpopulation, Methods that include...

201. a) Contacting a maternal blood sample containing an ex vivo fetal cell subpopulation and an ex vivo maternal cell subpopulation with an isolation agent that couples to a biomarker in the ex vivo fetal cell subpopulation. b) After contact, incubate the isolated drug together with the maternal blood sample. c) After incubation, isolate the ex vivo fetal cell subpopulation in the maternal blood sample from the ex vivo maternal cell subpopulation in the maternal blood sample. d) After isolation, sorting the ex vivo fetal cell subpopulation from the ex vivo maternal cell subpopulation, and e) After sorting, sequencing of nucleic acids derived from the ex vivo fetal cell subpopulation, Methods that include...

202. A method comprising incubating a cell population derived from a target sample with a first isolation agent, a second isolation agent, a third isolation agent, a fourth isolation agent, a fifth isolation agent, and a sixth isolation agent, wherein the cell population includes a subpopulation of fetal cells and a subpopulation of maternal cells, the first isolation agent is specific to a first biomarker, the second isolation agent is specific to a second biomarker, the third isolation agent is specific to a third biomarker, the fourth isolation agent is specific to a fourth biomarker, the fifth isolation agent is specific to a fifth biomarker in the fetal cell subpopulation, and the sixth isolation agent is specific to a sixth biomarker in the maternal cell subpopulation.

203. The method according to claim 202, wherein the subject is a human subject.

204. The method according to claim 202 or claim 203, wherein the sample is a blood sample.

205. The method according to claim 204, wherein the blood sample is plasma.

206. The method according to claim 204, wherein the blood sample is serum.

207. The method according to claim 204, wherein the blood sample is a buffy coat.

208. The method according to claim 204, wherein the maternal blood sample includes peripheral blood mononuclear leukocytes.

209. The method according to claim 204, wherein the maternal blood sample contains nucleated leukocytes.

210. The method according to claim 204, wherein the blood sample is whole blood.

211. The method according to any one of claims 202 to 210, wherein one of the first, second, third, fourth, fifth, and sixth isolated agents is a nucleic acid probe.

212. The method according to any one of claims 202 to 211, wherein one of the first isolated agent, the second isolated agent, the third isolated agent, the fourth isolated agent, the fifth isolated agent, and the sixth isolated agent is a ligand.

213. The method according to any one of claims 202 to 212, wherein one of the first, second, third, fourth, fifth, and sixth isolation agents is specific to a cell surface marker.

214. The method according to any one of claims 202 to 213, wherein one of the first, second, third, fourth, fifth, and sixth isolation agents is specific to a soluble cell surface marker.

215. The method according to any one of claims 202 to 214, wherein one of the first, second, third, fourth, fifth, and sixth isolated agents is specific to an intracellular biomarker.

216. The method according to any one of claims 202 to 215, wherein one of the first, second, third, fourth, fifth, and sixth isolated agents is specific to a nucleic acid marker.

217. The method according to any one of claims 202 to 216, further comprising enriching the fetal cell subpopulation from the maternal cell subpopulation after incubation.

218. The method according to any one of claims 202 to 217, wherein, after incubation, the fetal cell subpopulation is enriched from the maternal cell subpopulation.

219. A method comprising incubating a cell population with a first isolation agent, a second isolation agent, a third isolation agent, a fourth isolation agent, a fifth isolation agent, and a sixth isolation agent, wherein the cell population comprises a subpopulation of fetal cells and a subpopulation of maternal cells derived from a sample of interest, the first isolation agent being specific to a first biomarker in the fetal cell subpopulation, the second isolation agent being specific to a second biomarker in the fetal cell subpopulation, the third isolation agent being specific to a third biomarker in the fetal cell subpopulation, the fourth isolation agent being specific to a fourth biomarker in the maternal cell subpopulation, the fifth isolation agent being specific to a fifth biomarker in the maternal cell subpopulation, and the sixth isolation agent being specific to a sixth biomarker in the maternal cell subpopulation.

220. The method according to claim 219, wherein the subject is a human subject.

221. The method according to claim 219 or claim 220, wherein the sample is a blood sample.

222. The method according to claim 221, wherein the blood sample is plasma.

223. The method according to claim 221, wherein the blood sample is serum.

224. The method according to claim 221, wherein the blood sample is a buffy coat.

225. The method according to claim 221, wherein the maternal blood sample includes peripheral blood mononuclear leukocytes.

226. The method according to claim 221, wherein the maternal blood sample contains nucleated leukocytes.

227. The method according to claim 221, wherein the blood sample is whole blood.

228. The method according to any one of claims 219 to 227, wherein one of the first, second, third, fourth, fifth, and sixth isolated agents is a nucleic acid probe.

229. The method according to any one of claims 219 to 228, wherein one of the first isolated agent, the second isolated agent, the third isolated agent, the fourth isolated agent, the fifth isolated agent, and the sixth isolated agent is a ligand.

230. The method according to any one of claims 219 to 229, wherein one of the first, second, third, fourth, fifth, and sixth isolation agents is specific to a cell surface marker.

231. The method according to any one of claims 219 to 230, wherein one of the first, second, third, fourth, fifth, and sixth isolation agents is specific to a soluble cell surface marker.

232. The method according to any one of claims 219 to 231, wherein one of the first, second, third, fourth, fifth, and sixth isolated agents is specific to an intracellular biomarker.

233. The method according to any one of claims 219 to 232, wherein one of the first, second, third, fourth, fifth, and sixth isolation agents is specific to a nucleic acid marker.

234. The method according to any one of claims 219 to 233, further comprising enriching the fetal cell subpopulation from the maternal cell subpopulation after incubation.

235. The method according to any one of claims 219 to 234, wherein, after incubation, the fetal cell subpopulation is enriched from the maternal cell subpopulation.

236. A method comprising incubating a cell population with a first isolation agent, a second isolation agent, a third isolation agent, a fourth isolation agent, a fifth isolation agent, a sixth isolation agent, and a seventh isolation agent, wherein the cell population includes a subpopulation of fetal cells and a subpopulation of maternal cells derived from a sample of interest. The method wherein the first isolated agent is specific to the first biomarker, the second isolated agent is specific to the second biomarker, the third isolated agent is specific to the third biomarker, the fourth isolated agent is specific to the fourth biomarker, the fifth isolated agent is specific to the fifth biomarker, the sixth isolated agent is specific to the sixth biomarker in the maternal cell subpopulation, and the seventh isolated agent is specific to the seventh biomarker in the fetal cell subpopulation.

237. The method according to claim 236, wherein the subject is a human subject.

238. The method according to claim 236 or claim 237, wherein the sample is a blood sample.

239. The method according to claim 238, wherein the blood sample is plasma.

240. The method according to claim 238, wherein the blood sample is serum.

241. The method according to claim 238, wherein the blood sample is a buffy coat.

242. The method according to claim 238, wherein the maternal blood sample includes peripheral blood mononuclear leukocytes.

243. The method according to claim 238, wherein the maternal blood sample contains nucleated leukocytes.

244. The method according to claim 238, wherein the blood sample is whole blood.

245. The method according to any one of claims 236 to 244, wherein one of the first, second, third, fourth, fifth, sixth, and seventh isolation agents comprises a nucleic acid probe.

246. The method according to any one of claims 236 to 245, wherein one of the first isolated agent, the second isolated agent, the third isolated agent, the fourth isolated agent, the fifth isolated agent, the sixth isolated agent, and the seventh isolated agent comprises a ligand.

247. The method according to any one of claims 236 to 246, wherein one of the first, second, third, fourth, fifth, sixth, and seventh isolation agents is specific to a cell surface marker.

248. The method according to any one of claims 236 to 247, wherein one of the first, second, third, fourth, fifth, sixth, and seventh isolation agents is specific to a soluble cell surface marker.

249. The method according to any one of claims 236 to 248, wherein one of the first, second, third, fourth, fifth, sixth, and seventh isolated agents is specific to an intracellular biomarker.

250. The method according to any one of claims 236 to 249, wherein one of the first, second, third, fourth, fifth, sixth, and seventh isolation agents is specific to a nucleic acid marker.

251. The method according to any one of claims 236 to 250, further comprising enriching the fetal cell subpopulation from the maternal cell subpopulation after incubation.

252. The method according to any one of claims 236 to 251, wherein, after incubation, the fetal cell subpopulation is enriched from the maternal cell subpopulation.

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