阅读说明：本技术 冷冻干燥的抗体组 (Lyophilized antibody panel ) 是由 斯蒂芬·K·H·利 丹尼尔·马约尼斯 弗拉迪米尔·巴拉诺夫 奥尔加·奥尔纳茨基 贝迪卢·阿洛 于 2020-03-27 设计创作，主要内容包括：公开了用于使用元素分析进行研究的冷冻干燥的抗体组。所述抗体组包含分别用一种或多种同位素进行元素标签化或元素标记的多种抗体,从而每种不同的抗体与其他抗体是同位素可区分的。每种元素标签可以包含一种或多种独特的元素或独特的同位素组合。可以将元素标签化的抗体组混合冷冻干燥。因此,在通过元素分析仪,如质谱仪研究前,可以容易且有效地将冷冻干燥的元素标签化的抗体组再混悬并与样品混合。这种冷冻干燥的元素标签化的抗体组可以提供元素标签化测定的益处,同时还易于使用并长期保持稳定。(A freeze-dried antibody panel for studies using elemental analysis is disclosed. The antibody set comprises a plurality of antibodies that are each element-tagged or element-labeled with one or more isotopes, such that each different antibody is isotopically distinguishable from the other antibodies. Each elemental tag may comprise one or more unique elements or unique isotopic combinations. The element-tagged antibody panel can be mixed and lyophilized. Thus, a freeze-dried element-tagged antibody set can be easily and efficiently resuspended and mixed with a sample prior to study by an element analyzer, such as a mass spectrometer. Such a freeze-dried element-tagged antibody panel can provide the benefits of an element-tagged assay, while also being easy to use and stable over long periods of time.)

1. A set for elemental analysis, comprising:

a plurality of conjugated antibodies, wherein each of the plurality of conjugated antibodies is tagged with a different elemental tag, wherein each different elemental tag is distinguishable based on its isotopic composition, and wherein the plurality of conjugated antibodies are in a lyophilized mixture.

2. The panel of claim 1, wherein the plurality of conjugated antibodies comprises two or more antibodies from the list comprising: cd45, Cd45RA, Cd45RO, Cd123, Cd4, Cd8a, Cd11C, Cd57, CXCR3, Cd185, Cd38, Cd56, Cd3, Cd20, Cd66b, HLA-DR, IgD, Cd27, Cd28, Cd127, Cd19, Cd16, Cd161, Cd194, Cd25, Cd294, Cd197, Cd14, CCR6, and TCR δ γ.

3. The panel of claim 1, wherein a majority of the conjugated antibodies are specific for a cell type in human peripheral blood.

4. The panel of claim 1, wherein a majority of the conjugated antibodies are specific for cell surface markers.

5. The panel of claim 1, wherein the plurality of conjugated antibodies comprises ten or more conjugated antibodies in the lyophilized mixture.

6. The set of claim 1, wherein each different element tag comprises a plurality of element atoms of an isotope.

7. The panel of claim 1, wherein at least two conjugated antibodies of the plurality of conjugated antibodies are tagged with element tags having different isotopes of a single element.

8. The panel of claim 1, further comprising a biomolecule coupled to other element tags, wherein the biomolecule is not an antibody, and wherein the other element tags are distinguishable from each different element tag based on their isotopic composition.

9. The set of claim 1, further comprising a non-antibody metal-containing moiety comprising a metal isotope distinguishable from each different elemental tag based on its isotopic composition.

10. The set of claim 1, wherein each different element tag comprises a metallic element having an atomic weight greater than 80 amu.

11. The panel of claim 1, wherein each different element tag comprises a chelated metal.

12. The panel of claim 1, wherein each different element tag comprises elements that are not endogenous to human peripheral blood.

13. The group of claim 1, wherein the group has a moisture content at or less than 5% by weight.

14. The group of claim 1, wherein the group has a moisture content at or less than 3% by weight.

15. The group of claim 1, wherein the group has a moisture content at or less than 1% by weight.

16. The group of claim 1, wherein the group has a moisture content at or between 0.05 to 1% by weight.

17. The set of claim 1, further comprising a freeze-dried intercalating agent, wherein the freeze-dried intercalating agent is contained in the freeze-dried mixture.

18. The set of claim 1, further comprising a freeze-dried calibration material, wherein the freeze-dried calibration material comprises a known amount of one or more known isotopes, and wherein the freeze-dried calibration material is contained in the freeze-dried mixture.

19. The set of claim 1, further comprising a supplemental reagent, wherein the supplemental reagent is useful for conducting an assay using the plurality of conjugated antibodies, wherein the supplemental reagent is lyophilized, and wherein the supplemental reagent is contained in the lyophilized mixture.

20. An assay kit for use with elemental analysis comprising:

A closed container; and

the panel of claim 1, wherein the set of freeze-dried mixtures is stored within the closed container.

21. The assay kit of claim 20, wherein the closed container comprises an internal atmosphere of inert gas or dry air.

22. The assay kit of claim 20, wherein the plurality of conjugated antibodies comprises a first antibody specific for a cell surface marker and a second antibody specific for an intracellular target.

23. The assay kit of claim 20, further comprising:

other closed containers; and

a set of other antibodies comprising at least one other conjugated antibody tagged with a different element tag distinguishable from each different element tag based on its isotopic composition, wherein the other conjugated antibody is freeze-dried and stored within a further closed container.

24. The assay kit of claim 23, wherein the plurality of conjugated antibodies comprises antibodies specific for one or more cell surface markers, and wherein the other conjugated antibodies are specific for intracellular targets.

25. The assay kit of claim 20, further comprising an intercalating agent comprising additional different elemental tags distinguishable from each different elemental tag based on their isotopic composition.

26. The assay kit of claim 20, further comprising a plurality of sample barcoding reagents for labeling a plurality of samples, wherein each of the plurality of sample barcoding reagents comprises a different combination of isotopes.

27. The assay kit of claim 26, further comprising a plurality of containers, wherein each of a plurality of sample barcoding reagents is contained within a different container of the plurality of containers.

28. The assay kit of claim 26, wherein each of the plurality of sample barcoding reagents binds to a majority of cells in the sample.

29. The assay kit of claim 26, wherein each of the plurality of sample barcoding reagents comprises an elemental tag functionalized to covalently bind on or within a cell of the sample.

30. The assay kit of claim 26, wherein each of the plurality of sample barcoding reagents comprises a sample barcoded antibody that specifically binds a target present on a majority of cells in the sample or multiple targets that together bind a majority of cells in the sample.

31. The assay kit of claim 30, wherein each of the sample barcoded antibodies specifically binds one or more of CD45, CD298, and b2 m.

32. The assay kit of claim 30, wherein the element tag of the sample barcoded antibody provides a weaker signal than the element tag of a majority of the other antibodies in the set when analyzed using an element analyzer.

33. The assay kit of claim 26, wherein the different combinations of isotopes comprise cadmium.

34. The assay kit of claim 26, wherein the different combinations of isotopes comprise platinum in cisplatin.

35. The assay kit of claim 26, wherein each of the plurality of sample barcoding reagents comprises a set of sample barcoded antibodies, wherein each sample barcoded antibody comprises all isotopes of a different combination of the isotopes.

36. The assay kit of claim 26, wherein each of the plurality of sample barcoding reagents is capable of barcoding a living cell, and wherein each of the plurality of sample barcoding reagents is non-toxic to the living cell.

37. The assay kit of claim 20, further comprising assay barcoding reagents comprising additional antibodies for detecting different analytes, wherein each assay barcoding reagent comprises a different combination of isotopes.

38. The assay kit of claim 37, wherein each assay barcoded reagent is an assay barcoded bead comprising a different combination of the isotopes.

39. The assay kit of claim 38, wherein the assay barcoding reagent is contained in the set of lyophilized mixtures.

40. The assay kit of claim 38, wherein each assay barcoded bead comprises a unique combination of isotopes present within the interior of the assay barcoded bead.

41. The assay kit of claim 37, wherein the assay barcoding reagents comprise at least ten assay barcoding reagents for barcoding at least ten different analytes, and wherein the assay barcoding reagents are provided in a mixture.

42. The assay kit of claim 37, wherein the different analyte is a free analyte in human peripheral blood.

43. The assay kit of claim 37, further comprising a combination of reporter antibodies that specifically bind the different analytes, wherein each reporter antibody comprises an element tag detectable by elemental analysis.

44. The assay kit of claim 43, wherein each of the combined element tags of the reporter antibodies comprises an isotopically identical element detectable by elemental analysis.

45. The assay kit of claim 37, wherein each assay barcoding reagent is functionalized to attach to a sample barcode of a sample barcoded composition comprising an isotope.

46. The assay kit of claim 45, further comprising a sample barcode comprising a sample barcoded composition of isotopes.

47. The assay kit of claim 45, wherein the sample barcode can bind to cells of the sample stained with the freeze-dried population.

48. The assay kit of claim 20, further comprising barcoding reagents, wherein each barcoding reagent comprises an isotopically determined barcoded composition and an isotopically sampled barcoded composition, wherein each differently isotopically determined barcoded composition is associated with a different analyte, and wherein each differently isotopically sampled barcoded composition may be associated with a different sample.

49. The assay kit of claim 48, wherein each barcoded reagent is a bead, and wherein the sample barcoded composition of isotopes is located inside the bead.

50. The assay kit of claim 48, wherein each barcoded reagent is a bead, and wherein the sample barcoded composition of isotopes is located on the surface of the bead.

51. The assay kit of claim 20, further comprising an anticoagulant.

52. The assay kit of claim 20, further comprising a calibration material, wherein the calibration material comprises a known amount of a known isotope.

53. An assay kit for use with elemental analysis comprising:

a plurality of closed containers; and

the set of claim 1, wherein the freeze-dried mixture of the set is dispensed in the plurality of closed containers.

54. The assay kit of claim 53, further comprising a plurality of sample barcoding reagents for labeling a plurality of samples, wherein each of the plurality of sample barcoding reagents comprises a different combination of isotopes, and wherein each of the plurality of sample barcoding reagents is contained within a different container of the plurality of closed containers.

55. A barcoding system, comprising:

a barcode reagent comprising an assay barcode, wherein the assay barcode comprises an isotopic composition associated with a target analyte, wherein the isotopic composition are distinguishable by elemental analysis, wherein the barcode reagent comprises one of a plurality of sample barcodes or is functionalized to bind to at least one of the plurality of sample barcodes, wherein each sample barcode of the plurality of sample barcodes comprises a unique other composition of isotopes distinguishable from the assay barcode composition of isotopes by elemental analysis, wherein each sample barcode of the plurality of sample barcodes can be associated with a different sample.

56. The system of claim 55 wherein the barcoded reagent is a bead, and wherein the sample barcode is present on the interior of the bead.

57. The system of claim 55, wherein the barcoded reagents are beads, and wherein surfaces of the beads are functionalized to bind to the plurality of sample barcodes.

58. The system of claim 55 wherein the barcoded reagent is a bead, and wherein the surface of the bead comprises one of the plurality of sample barcodes.

59. The system of claim 55 wherein the barcoding reagent is functionalized to bind to the plurality of sample barcodes, and wherein the system further comprises each sample barcode of the plurality of sample barcodes in a separate container.

60. The system of claim 55 further comprising a sample barcoding reagent comprising at least one of the plurality of sample barcodes, wherein the sample barcoding reagent can bind both the barcoding reagent and cells of the sample.

61. The system of claim 55, wherein the barcoded reagent is a bead, and wherein the assay barcode is present in the interior of the bead, and wherein the interior of the bead comprises a solid metal core, a metal chelating polymer interior, a nanocomposite interior, or a hybrid material interior.

62. The system of claim 61, wherein the bead has a solid metal core and a polymeric surface.

63. The system of claim 62, wherein the polymer surface is bound to an antibody that binds to a target analyte.

64. The system of claim 63, wherein the target analyte is a free analyte present in blood.

65. The system of claim 55, further comprising a reporter antibody that specifically binds to a target analyte and comprises an element tag or a combination of a high intensity element tag and a low intensity element tag.

66. The system of claim 55, wherein the assay barcoding reagents comprise at least ten assay barcoding reagents for barcoding at least ten different analytes, and wherein individual mixtures of a plurality of the assay barcoding reagents each comprise an isotopically distinguishable sample barcode.

67. The system of claim 66, further comprising reporter biomolecules that specifically bind to the target analytes of the assay barcoded reagents, wherein the reporter biomolecules comprise affinity reagents or oligonucleotides, respectively, and wherein each reporter biomolecule comprises an element tag or a combination of high and low signal element tags.

68. The system of claim 67, wherein at least some of the reporter biomolecules that specifically bind different target analytes comprise the same elemental signature.

69. A method, comprising:

providing a plurality of antibodies;

conjugating each of the plurality of antibodies with a different element tag, wherein each different element tag is distinguishable based on its isotopic composition, and wherein each of the plurality of antibodies is distinguishable by its different element tag;

mixing the plurality of conjugated antibodies together into a mixture; and is

Freeze drying the mixture.

70. The method of claim 69, further comprising spin filtering the plurality of conjugated antibodies.

71. The method of claim 69, further comprising:

selecting a study protocol for studying the sample; and is

The plurality of antibodies is selected based on the selected study protocol.

72. The method of claim 69, wherein providing the plurality of antibodies comprises providing two or more antibodies from the list comprising Cd45, CD45RA, CD45RO, Cd123, CD4, CD8a, CD11C, CD57, CXCR3, CD185, CD38, CD56, CD3, CD20, CD66b, HLA-DR, IgD, CD27, CD28, CD127, CD19, CD16, CD161, CD194, CD25, CD294, CD197, CD14, CCR6, and TCR δ γ.

73. The method of claim 69, wherein each of the plurality of antibodies is specific for a cell type in human peripheral blood.

74. The method of claim 69, wherein each of the plurality of antibodies is specific for a cell surface marker.

75. The method of claim 69, wherein mixing the plurality of conjugated antibodies together comprises mixing ten or more antibodies together.

76. The method of claim 69, wherein each different element tag comprises a plurality of element atoms of an isotope.

77. The method of claim 69, wherein at least two of the different element tags have different isotopes of a single element.

78. The method of claim 69, further comprising providing a biomolecule comprising other element tags, wherein the other element tags are distinguishable from each different element tag based on its isotopic composition, wherein the biomolecule is not an antibody, and wherein mixing the plurality of conjugated antibodies together further comprises mixing the biomolecule with the plurality of conjugated antibodies.

79. The method of claim 69, wherein each different elemental tag comprises a metallic element having an atomic weight greater than 80 amu.

80. The method of claim 69, wherein each different element tag comprises a chelated metal.

81. The method of claim 69, wherein each different element tag comprises elements that are not endogenous to human peripheral blood.

82. The method of claim 69, wherein freeze-drying the mixture comprises reducing the water content to 5% by weight or less.

83. The method of claim 69, further comprising mixing an intercalating agent into the mixture prior to freeze-drying the mixture, wherein the intercalating agent comprises additional elemental tags distinguishable from each different elemental tag based on their isotopic composition.

84. The method of claim 69, further comprising mixing a calibration material into the mixture prior to freeze-drying the mixture, wherein the calibration material comprises a known amount of a known isotope.

85. The method of claim 69, further comprising mixing a supplemental reagent into the mixture prior to freeze-drying the mixture, wherein the supplemental reagent is available to aid in conducting an assay using a plurality of conjugated antibodies.

86. The method of claim 69, wherein freeze-drying the mixture further comprises storing the freeze-dried mixture in a closed container having an internal atmosphere of inert gas or dry air.

87. The method of claim 69, wherein the plurality of antibodies comprises a first antibody specific for a cell surface marker and a second antibody specific for an intracellular target.

88. The method of claim 69, further comprising:

providing at least one additional antibody;

conjugating the at least one other antibody with at least one other distinct elemental tag distinguishable from each distinct elemental tag based on its isotopic composition;

freeze-drying the at least one additional antibody; and is

Storing the freeze-dried at least one other antibody independently of the freeze-dried mixture.

89. The method of claim 88, wherein the plurality of antibodies comprises antibodies specific for one or more cell surface markers, and wherein the other antibodies are specific for an intracellular target.

90. The method of claim 69, further comprising providing a plurality of sample barcoding reagents for labeling a plurality of samples, wherein each of the plurality of sample barcoding reagents comprises a different combination of isotopes.

91. The method of claim 90, further comprising:

providing a plurality of containers; and is

Storing each of the plurality of sample barcoding reagents in a different container of the plurality of containers.

92. The method of claim 90, wherein each of the plurality of sample barcoding reagents binds to a majority of cells in the sample.

93. The method of claim 90, wherein each of the plurality of sample barcoding reagents comprises an elemental tag functionalized to covalently or otherwise permanently bind on or within cells of the sample.

94. The method of claim 90, wherein each of the plurality of sample barcoding reagents comprises a sample barcoded antibody that specifically binds a target present in a majority of cells in the sample.

95. The method of claim 94 wherein each of the sample barcoded antibodies specifically binds one or more of CD45, CD298, and b2 m.

96. The method of claim 94 wherein each of the sample barcoded antibodies specifically binds to a target present in the sample selected such that the mass signal of a single sample barcoded antibody is weaker than the mass signal of the majority of the plurality of conjugated antibodies.

97. The method of claim 90, wherein the different combinations of isotopes comprise cadmium.

98. The method of claim 90, wherein the different combinations of isotopes comprise platinum in cisplatin.

99. The method of claim 90, wherein each of the plurality of sample barcoding reagents comprises a set of sample barcoded antibodies, wherein each sample barcoded antibody comprises all isotopes of a different combination of the isotopes.

100. The method of claim 90 wherein each of the plurality of sample barcoding reagents is capable of barcoding a living cell, and wherein each of the plurality of sample barcoding reagents is non-toxic to the living cell.

101. The method of claim 69, further comprising:

assay barcoding reagents comprising other antibodies for detecting different analytes are provided, wherein each assay barcoding reagent comprises a different combination of isotopes.

102. The method of claim 101 wherein each assay barcoded reagent is an assay barcoded bead containing a different combination of isotopes.

103. The method of claim 102, wherein each assay barcoded bead comprises a unique combination of isotopes present within the interior of the assay barcoded bead.

104. The method of claim 101, wherein the assay barcoding reagents comprise at least ten assay barcoding reagents for barcoding at least ten different analytes, and wherein the assay barcoding reagents are provided in a mixture.

105. The method of claim 101, wherein the different analyte is a free analyte in human peripheral blood.

106. The method of claim 101, further comprising providing a combination of reporter antibodies that specifically bind the different analytes, wherein each reporter antibody comprises an element tag detectable by elemental analysis.

107. The method of claim 106, wherein each of the elemental tags of the combination of reporter antibodies comprises an isotopically identical element detectable by elemental analysis.

108. The method of claim 101 further comprising functionalizing each assay barcoding reagent to attach to a sample barcode of a sample barcoding composition comprising an isotope.

109. The method of claim 108 wherein the sample barcode binds cells of the sample determined by the freeze-dried mixture.

110. The method of claim 108 further comprising providing the sample barcode, the sample barcode comprising a sample barcoded composition of isotopes.

111. The method of claim 69 further comprising providing barcoding reagents, wherein each barcoding reagent comprises an isotopically determined barcoding composition and an isotopically sampled barcoding composition, wherein each differently isotopically determined barcoding composition is associated with a different analyte, and wherein each differently isotopically sampled barcoding composition is associated with a different sample.

112. The method of claim 111 wherein each barcoded reagent is a bead, and wherein a sample barcoded composition of the isotope is located inside the bead.

113. The method of claim 111 wherein each barcoded reagent is a bead, and wherein a sample barcoded composition of the isotope is located on a surface of the bead.

114. The method of claim 69, further comprising providing an anticoagulant.

115. The method of claim 69, further comprising titrating and diluting each of the plurality of conjugated antibodies to a predetermined concentration prior to mixing the plurality of conjugated antibodies.

116. The method of claim 69, further comprising mixing the plurality of conjugated antibodies with an excipient prior to lyophilizing the mixture.

117. The method of claim 116, wherein the excipients comprise a sugar and bovine serum albumin.

118. The method of claim 116, further comprising mixing the plurality of conjugated antibodies with a viability stain prior to freeze-drying the mixture.

119. The method of claim 118, wherein the viability stain is a rhodium intercalator.

120. A method, comprising:

preparing a sample;

providing a set of lyophilized antibodies comprising a plurality of conjugated antibodies, wherein each of the plurality of conjugated antibodies is tagged with a different elemental tag, and wherein each different elemental tag is distinguishable based on its isotopic composition;

performing surface staining of cells of the sample using the freeze-dried antibody panel; and is

The sample is studied using elemental analysis to detect the presence of the different elemental tags.

121. The method of claim 120, wherein studying the sample using elemental analysis comprises processing the sample on an inductively coupled plasma mass spectrometer to detect the presence of different elemental tags of the set of freeze-dried antibodies.

122. The method of claim 120, further comprising staining the sample of cells with a viability stain.

123. The method of claim 120, wherein the viability stain is provided as part of the freeze-dried antibody panel.

124. The method of claim 123, wherein the viability stain is rhodium.

125. The method of claim 120, further comprising performing FcR blocking of the sample of cells.

126. The method of claim 120, further comprising fixing the sample after performing the surface staining.

127. The method of claim 120, further comprising staining the sample with an intercalating agent, wherein the intercalating agent comprises other distinct elemental tags distinguishable from each distinct elemental tag based on their isotopic composition.

128. The method of claim 127, wherein staining of the sample occurs by the intercalating agent after permeabilizing the sample.

129. The method of claim 120, further comprising permeabilizing the sample and performing intracellular staining of the sample of cells with at least one other antibody, wherein the at least one other antibody is labeled with a further different element label that is distinguishable from each different element label based on its isotopic composition.

130. The method of claim 120, wherein preparing the sample comprises collecting whole blood.

131. The method of claim 130, wherein preparing the sample comprises isolating peripheral blood mononuclear cells from the whole blood.

132. The method of claim 120 further comprising labeling the sample with a sample barcoding reagent, wherein the sample barcoding reagent comprises different combinations of isotopes available for distinguishing the sample barcoding reagent from other sample barcoding reagents.

133. The method of claim 132 wherein investigating the sample comprises acquiring data by elemental analysis, identifying the sample barcoding reagent by different combinations of the isotopes in the acquired data and correlating the acquired data with the sample.

134. The method of claim 133, wherein studying the sample further comprises mixing the sample with other samples prior to acquiring data by elemental analysis.

135. The method of claim 120, wherein performing surface staining comprises:

adding a suspension of cells of the sample to the freeze-dried antibody panel or a re-suspension of the freeze-dried antibody panel; and is

Unbound antibody is removed.

136. The method of claim 120, further comprising:

providing assay barcoding reagents comprising other antibodies for detecting different analytes, wherein each assay barcoding reagent comprises a different combination of isotopes; and is

Prior to studying the sample, the assay barcoding reagent is mixed with the sample and unbound antibody is removed.

137. The method of claim 136, wherein the sample comprises plasma, and wherein the different analyte is a free analyte within the plasma.

138. The method of claim 136 wherein providing the assay barcoded reagent and providing the freeze-dried set of antibodies occur by providing a mixture of the freeze-dried set of antibodies and the assay barcoded reagent.

139. The method of claim 136, wherein preparing the sample comprises collecting whole blood.

140. The method of claim 139, wherein preparing the sample further comprises separating peripheral blood mononuclear cells and plasma, wherein performing the surface staining comprises mixing the freeze-dried antibody panel with the peripheral blood mononuclear cells and removing unbound antibodies; and wherein mixing of the assay barcoded reagent with the sample comprises mixing of the assay barcoded reagent with the plasma and removing unbound antibody.

141. The method of claim 120, wherein studying the sample further comprises automatically identifying cell viability.

142. The method of claim 120, wherein studying the sample further comprises automatically identifying a population of cells.

143. The method of claim 142, wherein studying the sample further comprises identifying characteristics of an automatically identified cell population.

144. The method of claim 143, wherein studying the sample further comprises comparing the identified features for the entire identified cell population or comparing the identified features associated with one of the identified cell populations to other identified characteristics associated with the same one of the cell populations identified from other samples.

145. The method of claim 143, wherein identifying a characteristic of the automatically identified cell population comprises determining an abundance of one or more targets on or in cells of the identified cell population.

146. The method of claim 143, wherein the identified characteristic comprises a percentage of cells in the population of cells.

147. The method of claim 120, wherein studying the sample further comprises generating at least one of a histogram, a 2D dot plot, and a tSNE plot based on known targets of the set of lyophilized antibodies.

148. The method of claim 147, further comprising automatically accessing a stored map of known targets of the set of lyophilized antibodies, wherein the stored map will move to a target associated with an associated mass channel.

149. The method of claim 136, further comprising:

labeling the sample with a sample barcoding reagent, wherein the sample barcoding reagent comprises different combinations of isotopes that distinguish the sample barcoding reagent from other sample barcoding reagents;

providing a further sample;

labeling the other sample with an other sample barcoding reagent;

performing additional surface staining of additional cells of the additional sample using the freeze-dried antibody panel;

mixing the assay barcoding reagent with the further sample and removing unbound antibody; and is

Mixing the sample with the other sample prior to studying the sample, wherein studying the sample comprises studying a mixture of the sample and the other sample.

150. The method of claim 149, wherein mixing the sample with the other sample occurs before the surface staining is performed.

151. The method of claim 149 wherein functionalizing the assay barcode reagent to bind to the sample barcode reagent, wherein labeling the sample with the sample barcode reagent comprises binding the sample barcode reagent to a first portion of the assay barcode reagent such that a second portion of the assay barcode reagent is free of the sample barcode reagent, wherein mixing the assay barcode reagent with the other sample comprises mixing the second portion of the assay barcode reagent with the other sample, and wherein mixing the sample with the other sample occurs after mixing the assay barcode reagent with the other sample.

152. The method of claim 120, wherein investigating the sample comprises obtaining data relating to the sample using an elemental analysis device, wherein the method further comprises automatically analyzing the data.

153. The method of claim 152, wherein automatically analyzing the data comprises applying a cleansing model to the data, wherein applying the cleansing model comprises accessing gaussian-type measurements produced by an elemental analysis device related to ionization of the sample.

154. The method of claim 152, wherein automatically analyzing the data comprises:

accessing an element tag specification model, wherein the element tag specification model comprises information associating each of the different element tags of the set of freeze-dried antibodies with a cell type;

identifying presence information of different elemental tags of the set of freeze-dried antibodies; and is

For each cell of the sample, determining the cell type using the identified presence information for the different element tag and the element tag designation model.

155. A barcoded kit for elemental analysis, comprising:

a plurality of sample barcodes for labeling a plurality of samples, wherein each of the sample barcodes comprises a different combination of isotopes distinguishable by elemental analysis, and wherein each of the sample barcodes is stored in a different container; and

A set of biomolecules capable of binding to the plurality of samples, wherein the set of biomolecules comprises or is functionalized to bind to the plurality of sample barcodes.

156. The barcoded kit of claim 155, wherein each of the set of biomolecules comprises a unique sample barcode of the plurality of sample barcodes.

157. The barcoding kit of claim 155, wherein each of the set of biomolecules is functionalized to bind to the plurality of sample barcodes, and wherein the set of biomolecules is stored separately from the plurality of sample barcodes.

158. The barcoded kit of claim 155, wherein said set of biomolecules comprises a plurality of beads.

159. The barcoding kit of claim 158, wherein each bead comprises an outer surface functionalized to bind to the plurality of sample barcodes.

160. The barcoded kit of claim 159, wherein each bead comprises an assay barcode located inside the bead, wherein each assay barcode comprises a combination of other isotopes distinguishable from a different combination of isotopes of the sample barcode by elemental analysis.

161. A method, comprising:

providing a plurality of samples comprising a first sample and a second sample;

providing a plurality of sample barcodes comprising a first sample barcode and a second sample barcode, wherein each of the sample barcodes comprises a different combination of isotopes distinguishable by elemental analysis;

providing a plurality of biomolecules capable of binding to the plurality of samples, wherein each biomolecule comprises one of the plurality of sample barcodes or is functionalized to bind to the plurality of sample barcodes, and wherein the plurality of biomolecules comprises a first biomolecule and a second biomolecule;

mixing the first biomolecule with the first sample;

mixing the second biomolecule with the second sample;

removing any unbound biomolecules;

studying the plurality of samples using elemental analysis to obtain elemental data;

detecting the presence of each different combination of isotopes in the elemental data; and is

Correlating the elemental data with one of the plurality of samples using the presence of each different combination of isotopes detected.

162. The method of claim 161, further comprising:

Mixing the first sample barcode with a mixture comprising the first biomolecule and the first sample; and is

Mixing the second sample barcode with a mixture comprising the second biomolecule and the second sample.

163. The method of claim 161, further comprising:

mixing the first sample barcode with the first biomolecule prior to mixing the first biomolecule with the first sample; and is

Mixing the second sample barcode with the second biomolecule prior to mixing the second biomolecule with the second sample.

164. The method of claim 161, further comprising mixing the first sample and the second sample prior to studying the plurality of samples.

165. The method of claim 161, wherein the plurality of biomolecules comprises a plurality of beads.

166. The method of claim 165 wherein each bead comprises an outer surface functionalized to bind to the plurality of sample barcodes.

167. The method of claim 166 wherein each bead comprises an assay barcode located inside the bead, wherein each assay barcode comprises a combination of other isotopes distinguishable from a different combination of isotopes of the sample barcode by elemental analysis.

168. A kit for determining barcoding, comprising:

a plurality of reporter molecules for detecting the presence of a target analyte; and

a plurality of individual assay beads comprising:

a capture biomolecule that specifically binds to a target and binds to the bead surface; and

an assay barcode comprising a composition of isotopes or elements associated with the captured biomolecules, wherein the assay barcode is distinguishable from an assay barcode of assay beads having captured biomolecules bound to different targets by mass spectrometry.

169. The kit of claim 168, wherein the plurality of individual assay beads are provided in a mixture.

170. The kit for assay barcoding of claim 168 or 169, wherein the reporter molecule comprises an antibody or oligonucleotide conjugated to a nanoparticle mass tag.

171. The kit for assay barcoding of any one of claims 168 to 170, the reporter molecule comprising a reporter oligonucleotide that hybridizes directly to the target analyte.

172. The kit for assay barcoding of any one of claims 168 to 170, wherein the reporter molecule comprises a mass-tagged oligonucleotide hybridized to an intermediate reporter oligonucleotide that is directly or indirectly hybridized to the target analyte.

173. The assay barcoded kit of any one of claims 168-172, wherein the target is a target analyte.

174. The kit for assay barcoding of any one of claims 168 to 172, wherein the target comprises an intermediate capture oligonucleotide comprising a complementary sequence of an oligonucleotide of the capture biomolecule, and wherein the target further comprises an assay biomolecule that binds to a target analyte of a sample.

175. The kit for assay barcoding of claim 175, wherein the intermediate capture biomolecule further comprises a mass tag that provides a sample barcode.

176. The kit for assay barcoding of any one of claims 168 to 175, wherein a reporter molecule that binds to the target analyte comprises an oligonucleotide sequence that hybridizes directly or indirectly to a mass-tagged oligonucleotide.

177. The assay barcoded kit of any one of claims 168-176, wherein the reporter molecules for different target analytes comprise the same mass tag.

178. The assay barcoded kit of any one of claims 168-177, comprising a mixture of reporter molecules for different target analytes.

179. The kit for assay barcoding of any one of claims 168 to 178, the assay barcoded bead comprising one of a plurality of sample barcodes, or being functionalized to bind to the plurality of sample barcodes, wherein each sample barcode of the plurality of sample barcodes comprises a unique other composition of isotopes distinguishable from the isotopic composition by elemental analysis, wherein each sample barcode of the plurality of sample barcodes is associated with a different sample.

180. The assay barcoded kit of any one of claims 168-178, wherein the reporter molecule comprises a system for signal amplification.

181. The kit for detecting barcoding of claim 180, wherein said signal amplification is by a hybridization protocol.

182. A method of analyzing a target analyte using the assay barcoded beads of any of the kits of claims 168-181, comprising:

incubating the assay barcoded beads with a target analyte in a sample, thereby binding the target analyte to the assay barcoded beads;

incubating the assay barcoded beads with the plurality of reporter molecules;

Detecting the assay barcode on each bead and the mass label of the reporter by mass spectrometry.

183. An elemental analysis method, comprising:

dividing the sample cells into a plurality of fractions;

staining cells with a common mass-tagged antibody panel;

staining cells of each fraction with a mass-tagged biomolecule of a difference group, wherein each difference group comprises biomolecules that are not in the other difference groups, but are tagged with mass tags that are present in the other difference groups; and

the sample is studied using elemental analysis to detect the presence of different mass tags on individual cells.

184. The method of claim 183, wherein the consensus group is conserved between two or more moieties.

185. The method of claim 183 or 184, wherein the consensus group detects positively expressed surface targets that distinguish parent populations that together cover a majority of the immune cells.

186. The method of any one of claims 183-185, wherein one or more individual sets of differences detect positively expressed surface targets that distinguish between subpopulations within one of the parental populations, but do not distinguish between subpopulations of a majority of immune cells, respectively.

187. The method of any one of claims 183 to 185, further comprising labeling the divided cells with a set barcode that identifies the set of differences.

188. The method of any one of claims 183-186, further comprising classifying cells of a single study into a cell population based on the consensus group and its variance group.

189. The method of claim 188, wherein classification is automated by software trained on said common and difference groups.

190. The method of claim, further comprising integrating the population of cells under study identified based on the different difference groups into the same dataset based on the consensus group.

191. A kit for performing any one of claims 183 to 190.

192. A kit for elemental analysis, comprising:

a consensus group comprising a plurality of antibodies respectively conjugated to different mass labels, wherein each different mass label is distinguishable based on its isotopic composition, wherein the plurality of conjugated antibodies are in a mixture; and

a plurality of difference sets comprising mass tagged biomolecules, wherein the difference sets comprise biomolecules that are different from each other, but comprise overlapping mass tags.

193. A kit for cell segmentation, comprising:

a membrane stain comprising a plurality of antibodies to different cell surface targets, wherein the antibodies are conjugated to the same mass tag;

wherein the membrane stain does not comprise antibodies that stain a target within a compartment other than the plasma membrane.

194. The kit of claim 194, wherein the membrane stain comprises an anti-connexin antibody and an anti-non-connexin antibody.

195. A method of staining cells in a tissue slice sample using the kit of claim 193 or 194.

196. The method of claim 195, wherein the membrane stain is applied after other antibody stains.

197. The method of claim 195 or 196, further comprising studying the sample by imaging mass cytometry.

198. The method of claim 197, further comprising segmenting the cell based at least in part on the membrane stain.

199. The method of claim 198, further comprising identifying a population of segmented cells based on detection of antibodies against cell surface markers, wherein the antibodies are conjugated to different mass labels.

200. The method of claim 198 or 199, wherein cell segmentation is performed automatically by software.

Technical Field

The present disclosure relates generally to biological assays and, more particularly, to biological assays studied by elemental analysis.

Background

The biometric may be used to determine information about the sample. For example, certain biological assays may be performed to determine the presence of different cell types in a blood sample. The biometric results can be used in research and medicine.

In some current assays, multiple antibodies are labeled with different fluorescent substances. Each antibody may be specific for a particular protein (e.g., an antigen) that may or may not be present in the sample. When these fluorescence-tagged antibodies are mixed with the sample, antibodies specific for proteins in the sample can bind to those proteins. Then, after washing the sample, the resulting sample can be optically studied to identify the presence of fluorescent substances.

However, these fluorescence assays are limited by the number of channels that can be distinguished and the time required to perform the assay. For example, the multiplexing of conventional fluorescence microscopy is limited by the overlap of the emission spectra of the fluorophores. Recent developments in imaging mass spectrometry of samples stained with element-labeled antibodies have enabled more targets to be imaged simultaneously, as each target can be bound to a unique isotope through an antibody intermediate. However, conventional techniques for generating and storing assays for elemental signatures can be sensitive to damage and other error-causing effects, which can lead to inaccurate results.

Drawings

The present specification makes reference to the following drawings, wherein the use of like reference numerals in different drawings is intended to indicate similar or analogous components.

FIG. 1 is a schematic diagram of an element-tagged section in accordance with certain aspects of the present disclosure.

Fig. 2A is a schematic illustration of barcoding reagents and a separate sample barcode according to certain aspects of the present disclosure.

Fig. 2B is a schematic illustration of the barcoding reagent shown in fig. 2A after attachment of the sample barcode, in accordance with certain aspects of the present disclosure.

Fig. 3 is a schematic diagram of a barcoding reagent 308 and an integrated sample barcode 312 in accordance with certain aspects of the present disclosure.

FIG. 4 is a schematic diagram of a system for assaying one or more samples using an elemental analyzer and a freeze-dried antibody panel, according to certain aspects of the present disclosure.

FIG. 5 is a schematic diagram illustrating analysis and processing of unknown particles according to certain aspects of the present disclosure.

FIG. 6 is a schematic diagram showing 3 example element tags and a diagram showing their respective element data, according to certain aspects of the present disclosure.

FIG. 7 is a schematic illustration of a sample cell tagged with an element-tagged portion, a sample barcode, and a barcoding reagent, according to certain aspects of the present disclosure.

Fig. 8 is a schematic diagram showing sample cells barcoded with multiple isotope samples, in accordance with certain aspects of the present disclosure.

Fig. 9 is a schematic diagram showing sample cells partially labeled with a dispensed sample barcode, according to certain aspects of the present disclosure.

Fig. 10 is a schematic illustration of a barcoding reagent and a reporter antibody according to certain aspects of the present disclosure.

Fig. 11 is a schematic diagram showing preparation of a freeze-dried antibody panel, according to certain aspects of the present disclosure.

Fig. 12 is a schematic illustration of a method for preparing barcoding reagents, according to certain aspects of the present disclosure.

Fig. 13 is a flow chart showing a method for staining and analyzing a blood sample, according to certain aspects of the present disclosure.

FIG. 14 is a schematic diagram illustrating an example gating strategy for automatically analyzing element data, in accordance with certain aspects of the present disclosure.

Fig. 15 is a schematic diagram showing a technique for sample labeling of barcoded reagents and analysis of a set of samples, according to certain aspects of the present disclosure.

FIG. 16 is a schematic diagram showing a technique for sample labeling and analysis of a set of samples, according to certain aspects of the present disclosure.

Fig. 17 is a schematic diagram showing preparation of a set of preconfigured sample barcode labeled barcoding reagents, according to certain aspects of the present disclosure.

Detailed Description

Certain aspects and features of the present disclosure relate to a freeze-dried antibody panel for studies using elemental analysis. The antibody may be a fragment thereof, such as a nanobody or a Fab fragment. Furthermore, in the embodiments discussed herein, conventional affinity reagents (including not only antibodies, but also other affinity reagents such as lectins, aptamers, and non-antibody protein-ligand pairs, such as streptavidin-biotin) may be used in place of antibodies.

The set of antibodies may include a plurality of different antibodies that are labeled or element-labeled, respectively, with one or more isotopic elements, such that each different antibody is isotopically distinguishable from the other antibodies. Each elemental tag may include one or more unique elements, isotopes, or unique combinations of isotopes. The element-tagged antibody panel can be mixed and lyophilized. Thus, a freeze-dried element-tagged antibody set can be easily and efficiently resuspended and mixed with a sample prior to study by an element analyzer, such as a mass spectrometer. Such a freeze-dried element-tagged antibody panel can provide the benefits of an element-tagged assay, while also being easy to use and stable over long periods of time. The element tag of any subject method or kit can be a mass tag, such as when the detection method is mass spectrometry and/or when the element tag comprises an enriched isotope. Identical mass labels or overlapping mass labels refer to mass labels that have the same isotopic (or isotopic mass) label atom and are difficult to distinguish by mass spectrometry.

Certain aspects and features of the present disclosure also relate to techniques for uniquely barcoding different samples, thereby allowing the samples to be combined prior to study by an elemental analyzer. Different sample barcoding reagents may each include a unique combination of isotopes that can be used to distinguish the sample barcoding reagent from other sample barcoding reagents. Multiple samples can be individually mixed with different sample barcoding reagents, allowing the sample barcoding reagents to bind to targets in the samples (e.g., assay barcoded beads and/or cells), thus labeling each sample with its own unique barcode. Even after the samples are mixed together, data for each sample can be extracted based on the presence of unique isotope combinations in the data collected from the elemental analyzer. In some cases, the samples may even be mixed together prior to performing an assay, such as an assay using a freeze-dried set of element-tagged antibodies.

Elemental analysis is a method in which the elemental and sometimes isotopic composition of a sample is analyzed. Elemental analysis can be achieved by methods including: optical atomic spectroscopy, such as flame atomic absorption, graphite furnace atomic absorption, and inductively coupled plasma atomic emission, which probes the external electronic structure of the atoms; mass spectrometry atomic spectroscopy, such as inductively coupled mass spectrometry, which detects atomic mass; x-ray fluorescence, particle-induced X-ray emission, X-ray photoelectron spectroscopy, and auger electron spectroscopy, which probe the internal electronic structure of an atom. Other elemental analysis techniques may be used.

The mass spectrometer used in the present invention may be selected based on the needs of the operator or the particular application. Example types of mass spectrometers include quadrupole, time-of-flight, magnetic sector, high resolution, single or multiple collector mass spectrometers. Typically, time-of-flight or magnetic sector mass spectrometers are used to record fast, transient events where transmission durations (transitions) are expected from particles (e.g., beads, cells, or laser ablated plumes). In certain aspects, a time-of-flight mass spectrometer can have a high pass filter, such as having a mass cutoff of 80amu or more. In certain aspects, an atomization and ionization source, such as an Inductively Coupled Plasma (ICP), may be located upstream of a time-of-flight or fan-shaped magnetic field mass detector.

Certain aspects of the present disclosure are particularly suited for use with a class of elemental analysis, referred to as mass analysis, in which the mass of an isotope or element is determined. The mass analysis may include atomic mass analysis, such as mass cytometry. Mass analysis may be particularly suitable for identifying isotopes of an element, and thus distinguishing one isotope from another different isotope or distinguishing one combination of isotopes from another different combination of isotopes. Thus, where appropriate, the mass analysis may be in the form desired for elemental analysis for all aspects of the disclosure disclosed herein.

An elemental analyzer is an instrument for quantifying the atomic composition of a sample. The elemental analyzer may use one of the elemental analysis techniques described herein. The elemental analyzer may have a set number of channels for detecting or distinguishing isotopes. For example, a mass spectrometer may have a number of mass channels that can be used to detect different isotopes. The number of channels available to a particular elemental analyzer may be limited. Thus, for a particular purpose (e.g., labeling the antibodies of a freeze-dried antibody panel or barcoding a sample), the use of any subset of channels may make the remaining channels available for other purposes. For example, empty channels may be used to perform other assays or other barcoding.

In some cases, elemental analysis may be performed on a single particle basis, which is referred to as particle elemental analysis. Particle elemental analysis includes determining the elemental composition of individual particles (e.g., cell-by-cell), such as using a mass spectrometer-based flow cytometer. Certain aspects of the present disclosure utilize particle element analysis on a cell-by-cell basis, which may be referred to as cytometric element analysis. In some cases, elemental analysis may be performed on an overall basis, which is referred to as overall elemental analysis or solution elemental analysis. Bulk elemental analysis involves determining the elemental composition of the entire sample volume.

Elemental analysis can be used to study samples, such as biological samples. If the sample is labeled with a known elemental tag, detection of the elemental tag during elemental analysis can indicate a sample characteristic related to the elemental tag.

As referred to herein, mass cytometry is any method of detecting elemental tags (mass tags) in a biological sample, such as simultaneously detecting multiple distinguishable mass tags at single cell resolution. Mass cytometry can include analysis of mass-tagged beads independently of or in addition to cells. Any of the subject kits and methods can include or be adapted for mass cytometry. Mass cytometry included suspension mass cytometry and Imaging Mass Cytometry (IMC).

Suspension mass spectrometry flow cytometry involves analysis of suspended, elemental tagged cells and/or beads by mass spectrometry (e.g., by atomic weight spectroscopy) and is described in U.S. patent publications, including US20050218319, US20150183895, US20150122991, all of which are incorporated herein by reference.

Imaging mass cytometry includes any imaging mass spectrometry (e.g., imaging atomic mass spectroscopy) of an element-tagged biological sample, such as a tissue slice or cell smear. The IMC may atomize and ionize the mass labels of the cell sample by one or more of laser radiation, ion beam radiation, electron beam radiation, and/or Inductively Coupled Plasma (ICP). Mass cytometry can detect different mass labels from a single cell simultaneously, such as by time-of-flight (TOF) or magnetic sector Mass Spectrometry (MS). Examples of mass cytometry include suspension mass cytometry, where cells flow into ICP-MS, and imaging mass cytometry, where a cell sample (e.g., a tissue slice) is sampled, for example, by laser ablation (LA-ICP-MS) or by a primary ion beam (e.g., for SIMS). Laser-based IMCs are described in U.S. patent publications US20160131635, US20170148619, US20180306695, and US 20180306306695, all of which are incorporated herein by reference. In certain aspects, when the sample is a cell smear for analysis by IMC, the cells can be processed, as described herein, such as by staining with a freeze-dried group, sample barcoding, and/or assay barcoding. Similarly, assay beads described herein can be analyzed by IMC, alone or in admixture with cells.

The mass labels can be sampled, atomized and ionized prior to elemental analysis. For example, mass labels in biological samples can be sampled, atomized and/or ionized by radiation, such as a laser beam, an ion beam or an electron beam. Alternatively or additionally, the mass labels may be atomized and ionized by a plasma, such as Inductively Coupled Plasma (ICP). In suspension mass cytometry, whole cells including mass tags can be flowed into an ICP-MS, such as ICP-TOF-MS. In imaging mass cytometry, a form of radiation can remove (and optionally ionize and atomize) a solid biological sample, such as a portion of a tissue sample (e.g., a pixel, a region of interest), that includes a mass tag. Examples of IMC include LA-ICP-MS and SIMS-MS of mass-tagged samples. In certain aspects, ion optics may eliminate ions other than isotopes of the mass label. For example, ion optics can remove lighter ions (e.g., C, N, O), organic molecular ions. In ICP applications, ion optics can remove gases, such as Ar and/or Xe, such as by a high-pass quadrupole filter. In certain aspects, the IMC may provide an image of the mass label (e.g., the target to which the mass label binds) at cellular or sub-cellular resolution.

Similar to fluorescence immunohistochemistry methods, the workflow of mass cytometry (including imaging mass cytometry) can include cell (e.g., tissue) fixation and/or permeabilization prior to staining with antibodies and/or other specific binding partners. In contrast to fluorescent methods, in mass cytometry, a mass tag (e.g., comprising a non-cellular endogenous heavy metal) is bound to a target analyte by a specific binding partner, such as an antibody. Imaging mass cytometry, such as fluorescence microscopy, can include an antigen retrieval step in which a sample is exposed to conditions, such as heat, to expose a target analyte for binding by a biomolecule. Unbound biomolecules are typically washed away before the mass label is detected by mass spectrometry. Notably, other detection methods, such as elemental analysis (e.g., emission spectroscopy or X-ray dispersion spectroscopy) are also within the scope of the subject patent application.

Notably, antigen retrieval conditions can be particularly important for IMC over other imaging methods, as element tags (e.g., mass tags) can create more steric hindrance in tissue than other tags, such as fluorophores. However, high stringency repair conditions (e.g., prolonged exposure of tissue sections to high temperatures) can denature the sample, thereby destroying the epitope to be detected. In certain aspects, a metal heating block, such as an aluminum heating block, may be provided in the kit, or may be used to heat tissue sections for antigen retrieval. The inventors have found that such a block provides a uniform heat distribution and allows for a suitably rapid temperature transition for controlled antigen retrieval. As such, the methods or kits of the subject patent application, e.g., for any imaging mass cytometry application or another imaging application, e.g., light microscopy.

Other reagents for mass cytometry include metal-containing biosensors (e.g., biosensors deposited or bound under conditions such as hypoxia, protein synthesis, cell cycle, and/or cell death) and/or metal-containing histochemical compounds that bind to structures (e.g., DNA, cell membranes, layers) based on chemical properties. Additionally, mass tags (e.g., the mass tags of the subject patent application or other mass tags) can be combined to provide a unique barcode to label a particular sample or experimental condition before mixing with other samples or experimental conditions.

In IMC, the tissue sample may be a slice, for example, a slice having a thickness in the range of 1-10 μm, such as between 2-6 μm, may be used. In some cases, ultrathin sections less than 500nm, 400nm, 200nm, 100nm, or 50nm thick may be used, such as samples cut from resin-embedded tissue pieces. Techniques for preparing such sections are well known in the IHC art, e.g., using microtomes, including dehydration steps, fixation, embedding, permeabilization, sectioning, and the like. Thus, the tissue can be chemically fixed and then sections can be prepared at the desired plane. Cryosectioning or laser capture microtomy can also be used to prepare tissue samples. The sample may be permeabilized, for example, to allow the agent to label the intracellular target. The proximity of the biomolecule to the analyte may be sterically hindered even after antigen retrieval (e.g., by heating). As such, smaller biomolecules and certain mass tags may best bring the biomolecule close to its target analyte.

Cell segmentation in imaging mass cytometry may allow for the use of automated cell sorting as well as other aspects of the main patent application. However, different tissues and cell types have different surface markers, which makes single universal membrane staining difficult.

A kit for cell segmentation (cell segmentation) may include a membrane stain comprising a plurality of antibodies to different cell surface targets, wherein the antibodies are conjugated to the same mass tag. In certain aspects, the membrane stain comprises an antibody against a connexin and an antibody against a non-connexin.

The membrane stain does not contain antibodies that stain targets within the compartment other than the plasma membrane. The membrane stain may bind to membranes of more cell types than any single antibody of the membrane stain. The plurality of antibodies may be in a mixture.

The kit may also include a nuclear stain and/or a cytosolic stain, for example, to better enable the individual to recognize the cells and guide cell segmentation along the membrane. The kit also comprises a set of antibodies against the cell surface target, wherein the antibodies of the set are conjugated to different mass labels and as described herein, for identifying a population of cells (e.g., by automated classification of the population).

In certain aspects, the membrane stain can be applied before, simultaneously with, or after the other antibody groups. Cell segmentation can be performed on images obtained by imaging mass cytometry. Segmentation methods are known in the art and are described, for example, by Wang et al in "Cell Segmentation for Image Cytometry: Advances, Insufficients, and Challenges," Cytometry. part A (2019): 708-. In general, cell segmentation benefits from clear labeling of the cell membrane and the interior of the cell (e.g., the nucleus of the cell visualized by nuclear stains).

Information on Protein localization has focused on Human Protein maps (Human Protein Atlas) that are searchable to identify Protein targets that specifically localize to the plasma membrane (e.g., cell junctions) between different tissues and cell types. A complementary set of protein targets can thus be identified that recognize cell membranes within some tissues and between some tissues, and the proteins of those targets can be tagged with the same element tags to provide the membrane stain of the subject patent application. Membrane stains can include 1, 2, 3, 4, or more antibodies that specifically bind to synapsin 4, solute carrier family 16 member 1, erythrocyte membrane protein band 4.1-like protein 3, linker-associated protein complex 2mu 1 subunit, G protein subunit β 2, moesin, EZR, CTNNB1, atpase Na +/K + transporter subunit β 3, phosphatidylethanolamine-binding protein 1, catenin β 1, solute carrier family 1 member 5, Ezrin (Ezrin), S100, calcium-binding protein a4, and ankyrin 3, which are also reported to express membrane proteins that are conserved between different cell lines. Alternatively or additionally, the membrane stain may include 1, 2, 3, 4, or more antibodies that specifically bind to CDH17, CTNNA1, DNAJC18, GJB6, TJP3, and C4of19, which are also reported to express cell-linking proteins that are conserved between different cell lines and/or tissues.

For detection of RNA, cells in a biological sample as discussed herein can be prepared for analysis of RNA and protein content using the methods and devices described herein. In certain aspects, the cells are fixed and permeabilized prior to the hybridization step. The cells may be provided as fixed and/or permeabilized. Cells can be fixed by cross-linking fixatives such as formaldehyde, glutaraldehyde. Alternatively or additionally, the cells may be fixed using a precipitation fixative, such as ethanol, methanol, or acetone. Cells can be permeabilized by detergents such as polyethylene glycol (e.g., Triton X-100), polyethylene oxide (20) sorbitan monolaurate (tween-20), saponins (a group of amphiphilic glycosides), or chemicals such as methanol or acetone. In some cases, the same reagent or set of reagents may be used for immobilization and permeabilization. Immobilization and Permeabilization techniques are discussed by Jamur et al in "Permeabilization of Cell Membranes" (Methods mol. biol., 2010).

The biological sample may include any sample having a biological property that requires analysis. For example, the sample may include biomolecules, tissues, fluids and cells of animals, plants, fungi or bacteria. They may also include molecules of viral origin. Typical samples include, but are not limited to, sputum, blood cells (e.g., leukocytes), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells derived therefrom. Biological samples may also include tissue sections, such as frozen sections taken for histological purposes. Another typical source of biological samples are viruses and cell cultures of animals, plants, bacteria, fungi, where the expression state of genes can be manipulated to explore relationships between genes. In some cases, other samples, such as artificial samples, may be studied. Certain aspects of the present disclosure are particularly useful when studying samples of human origin, and are particularly useful when studying samples of human peripheral blood.

Samples can be tagged or labeled with elemental tags to aid in the determination of useful information by elemental analysis studies. An elemental tag is a detectable isotope (e.g., an element or an isotope of an element) that can be detected by elemental analysis. In some cases, the elemental tag may comprise only the detectable isotope itself, although this need not always be the case. In some cases, the elemental tag may include a substrate to which one or more isotopes are coupled or a substrate in which one or more isotopes are additionally contained. In this manner, to produce an element-tagged moiety, a detectable isotope can be coupled to a substrate independently (e.g., before, simultaneously with, or after) the substrate coupled to the moiety. For example, the element tag can include a polymer chain comprising a plurality of pendant groups (e.g., metal-containing chelating groups) comprising a detectable isotope, the polymer chain capable of binding to a moiety to tag the moiety with an element. In another example, the element tag may comprise a bead or nanoparticle that may contain one or more detectable isotopes therein or on the surface thereof, the bead or nanoparticle being capable of binding to a moiety to tag the moiety with an element. As used herein, an isotope or isotopic composition located on the surface of a bead or nanoparticle includes an isotope or isotopic composition embedded or bound (e.g., covalently bound or otherwise) to the surface by a polymer on the surface. In some cases, the beads or nanoparticles may comprise a solid metal core, optionally encapsulated by a silica shell, or a polymer core and polymer surface embedded and/or chelated with metal (e.g., using poly-L-lysine, PEG (polyethylene glycol), PEG MEA (methyl ether acrylate), PVMS (methyl vinyl polysiloxane), polydopamine, polystyrene, and/or other suitable polymers). In certain aspects, the polymer (e.g., and/or monomeric precursors thereof) can be hydrophobic, e.g., can include acrylic acids, amides and imides, carbonates, dienes, esters, ethers, fluorocarbons, olefins, and/or aryl groups, e.g., phenolic groups, such as catechol. The polymer may also provide reactive groups, such as amine and/or thiol reactive groups. For example, the reactive group can be a reactive functional group (e.g., a thiol, amine, thiol-reactive, amine-reactive, or click chemistry functional group), such as a benzoquinone formed by polymerization. The polymer may be formed into a film, such as a monolayer film.

The beads of the subject patent application may be element-encoded particles suitable for biomolecule attachment, thereby enabling large-scale multiplexed bioanalytical methods. For example, polymeric beads can be prepared according to one or more aspects of U.S. patent publication 20100144056, which is incorporated by reference and summarized below.

In one method, polymer particles containing embedded metal ions or atoms are synthesized. The polymeric matrix of the particles can be used to encapsulate metal ions, but at the same time provide colloidal stability in aqueous vehicles. The polymeric matrix of the particles minimizes direct contact of metal ions with the aqueous phase and the functional groups on the surface of the particles are available for attaching antibodies or other biomolecules to the particles. These functional groups may also be used to attach linker arm or spacer arm groups to which biomolecules may be attached. The polymer particles of interest have diameters in the range of about several nm to about 20 μm, with those of most interest having diameters of about 50nm to about 500 nm.

Polymer particles containing embedded metal ions or atoms can be synthesized. The polymeric matrix of the particles can be used to encapsulate metal ions, but at the same time provide colloidal stability in aqueous vehicles. In certain aspects, chelating lanthanide (or other metal) ions may be used.

The polymeric matrix of the particles minimizes direct contact of the metal ions with the aqueous phase, and functional groups on the surface of the particles may be available for attaching antibodies or other biomolecules to the particles. These functional groups may also be used to attach linker arm or spacer arm groups to which biomolecules may be attached. Alternatively, polymer membranes with different structures can be synthesized on the bead surface and allow for biomolecule attachment, as discussed herein. In certain aspects, the beads are assay barcoded beads as described herein. The interior of the beads may contain an assay barcode such as a distinguishable combination of metal isotopes. The interior of the bead may be any of a variety of suitable structures, such as a solid metal core, a metal chelating polymer interior, a nanocomposite interior, or a hybrid (hybrid) interior. The solid metal core may be formed by subjecting a mixture (e.g., a solution) of one or more metallic elements and/or isotopes to high heat and/or pressure. The nanocomposite structure can comprise a combination of nanoparticles/nanostructures (e.g., a matrix) (e.g., each comprising different physical properties and facilitating one or more assay barcode elements/isotopes and/or providing a scaffold for other nanoparticles comprising assay barcode elements/isotopes). The interior of the bead may comprise a polymer (e.g., via a pendant group, such as DOTA, DTPA, or derivatives thereof) that entraps and/or sequesters the assay barcode metal. Suitable polymer backbones can be branched (e.g., hyperbranched) or form a matrix. In some aspects, the polymer may be formed in an emulsion. The polymer may comprise subunits of styrene, acrylates, any derivatives thereof, or other polymers known in the art. In certain aspects, an inert surface (e.g., such as a solid metal surface) may be present inside the assay bead that requires functionalization (e.g., by polymerization reactions over the entire surface) prior to attachment to the assay biomolecule (e.g., oligonucleotide or antibody). Element tags have been provided to mass cytometry for conjugation or pre-conjugation to biomolecules, e.g., affinity reagents, such as antibodies (e.g., antibodies or derivatives thereof, such as antibody Fab fragments), that specifically bind to a target analyte. The elemental tag used for conjugation may comprise a metal-preloaded polymer, or a separate metal solution for loading onto the polymer (e.g., prior to conjugation to the biomolecule). The polymer can comprise a backbone and a plurality of pendant metal binding groups, such as pendant groups comprising a chelating agent (e.g., DTPA, DOTA, or derivatives thereof). Elemental tags conjugated to biomolecules, such as antibodies, may be preloaded with a metal. In certain aspects, the metal is an enriched isotope, such as a lanthanide isotope. The lanthanides are chemically similar, and lanthanide tags (including those conjugated to antibodies) have been found to be stable in solution to a degree suitable for mass cytometry. However, non-lanthanide metals may not have similar stability. In certain aspects, the metal element or isotope thereof can be outside of the lanthanide family, such as a non-lanthanide transition metal or a post-transition metal. Suitable late transition metals for mass cytometry include elements having atomic numbers 48-50 (e.g., cadmium, indium, and/or tin) and 80-84. The stability of post-transition metal (post-transition metal) loaded polymers can be improved by freeze-drying. The subject methods and kits include these non-lanthanide tags lyophilized as described herein, provided that they are separate from or mixed with other element tags or biomolecules tagged with other elements. Medium weight elements (e.g., or isotopes thereof) can provide weaker signals by mass flow cytometry, such as when the element is near (e.g., within 30, 20, or 10amu from) a high pass filter that removes argon dimers from ICP and/or organic molecular elements. These medium heavy elements or isotopes may have an atomic number between 39 and 52. In addition, non-lanthanide elements, such as certain transition metals and post-transition metals, may not be chelated, as is readily achieved for pendant groups on the polymer. As such, the non-lanthanide metals described herein (e.g., isotopes thereof) can be useful for cell barcoding, e.g., live cell barcoding with element-tagged CD 45. For example, a live cell barcode may include freeze-dried cadmium isotope labeled CD 45.

The assay bead surface may comprise a polymer, a linker to space the assay biomolecule from the surface and/or to increase colloidal stability (e.g., a PEG linker), a functional group for attaching (or attaching) to the assay biomolecule and/or the sample barcode.

Multiple different isotopes or isotopes may be usedSpecific isotopic combinations produce elemental tags. Unique elemental tags may have different (e.g., distinguishable by elemental analysis) elements, isotopes, or combinations of isotopes. For example, in a set of element tags, an element tag may be unique if distinguishable from other element tags of the set by mass (e.g., by the composition of one or more isotopes thereof). For purposes of clarity, reference is made throughout this specification to an element tag or a unique element tag, however it will be understood that certain aspects of the present disclosure utilize multiple copies of a unique element tag. The elemental tag may include one or more isotopes, such as one or more isotopes of a single element (e.g.,¹⁴²nd and¹⁴³nd) or one or more isotopes of various elements (e.g.,¹⁴²nd and¹⁴¹pr). In certain aspects, the elemental tag can include a metal atom having an atomic weight above 80 amu. The metal may be selected from noble metals, lanthanides, transition metals and/or post-transition metals. The element tag may comprise a polymer. For example, the polymer can chelate a metal (e.g., lanthanide series) to a metal-binding pendant group (such as DOTA or DTPA) on the polymer backbone, or can incorporate a metal (e.g., tellurium) into the carbon backbone of the polymer itself, or can be used to surround (entrap) the metal. The element tag may comprise a metal nanoparticle, such as a metal nanocrystal (e.g., functionalized on its surface to bind affinity reagents). The metal of the elemental tag may be isotopically pure. The element tag can be bound and functionalized to bind biomolecules (e.g., affinity reagents, such as antibodies).

Each element tag may include one or more discernible isotopes detectable by elemental analysis to determine the presence of the element tag. Since different element tags may have unique isotopes or isotope combinations, elemental analysis may be used to identify the element tags based on their unique isotopic characteristics (signatures). The discernible isotope may be selected to facilitate detection using elemental analysis. For example, the discernible isotope may be selected to be an isotope outside the expected range of endogenous isotopes in the sample or an isotope introduced as a result of a particular elemental analysis technique. For example, biological samples will generally not inherently include greater than 80amu of metal, and thus elemental tags for use with these samples may include greater than 80amu of metal. In another example, certain types of inductively coupled plasma mass spectrometry (ICP-MS) utilize argon, and thus the elemental tags used for analysis by ICP-MS may utilize elements with masses greater than argon, such as metals greater than 80 amu. Filtering techniques can be used to exclude elements outside of those selected as discernible elements in the element tag being used, thus automatically excluding elements endogenous to the sample and elements introduced during elemental analysis, if any. For example, in ICP-MS, a quadrupole filter may be applied to reject ions with a mass of less than 80amu, thus leaving the detector uncovered (overhelm) by ions that are not of interest (e.g., ions that are unlikely to be detectable isotopes).

When referring to a combination of isotopes of a given element tag, the combination can exist at each instance of the element tag or at different instances of the element tag mixed together.

An element tagged moiety is a chemical moiety that includes an element tag. Any suitable moiety may be used, although in certain aspects of the present disclosure, a moiety having a targeting function is used. A targeting function is the ability to bind to a target (e.g., a target protein or molecule). As used herein, reference to a moiety that binds to a target will be understood to be a moiety that is capable of binding to the target, whether or not actual binding has occurred. Targeting functions can occur through specific binding (e.g., as in an antibody specific for a particular antigen), covalent bonding, hybridization (e.g., of nucleic acids), or other suitable bonding mechanisms. The metal-containing moiety can be a moiety tagged with a metal-tagged element. Examples of suitable metal-containing moieties include metal-containing small molecules or drugs (e.g., cisplatin), histochemical staining (e.g., ruthenium red, trichrome staining, or osmium tetroxide), metal-tagged oligonucleotides, and metal-tagged antibodies. In some cases, the element-tagged portion may be a biomolecule, although this need not always be the case. The biomolecule may be any biomolecule, such as an affinity agent (e.g., an antibody, an aptamer), a nucleic acid (e.g., a nucleic acid that hybridizes to a target), a lectin, a sugar, or other such molecule.

As used herein, the term affinity agent may refer to a biological molecule (e.g., an antibody, aptamer, lectin, or sequence-specific binding peptide) that is known to form highly specific non-covalent bonds with individual target molecules (e.g., peptides, antigens, or small molecules). An affinity reagent labelled with a unique element tag is an affinity reagent labelled with an element tag that is unique and distinguishable from a plurality of other element tags in the same sample. When conjugated to an element tag, the affinity reagent may be considered to be an element tagged moiety. Although some embodiments and examples herein list antibodies, affinity reagents (e.g., antibodies or non-antibody affinity reagents) may be used in place of antibodies in any of these embodiments.

The element tagged portion may include or be bound to an element tag. In some cases, the element-tagged moiety may comprise an element tag, such as in the case of cisplatin which contains platinum as an element tag. In some cases, the element-tagged moiety can be bound to an element tag, such as an antibody bound to an element tag, which comprises a polymer scaffold having a metal-chelating group to which a distinguishable isotope (e.g., metal) is bound. The use of a polymer scaffold may allow multiple isotopes to be bound to a single element tagged moiety. Thus, the polymer scaffold can provide a stronger elemental signal by including more copies of the distinguishable isotope. In some cases, the polymer scaffold can be used to help provide distinguishable isotope combinations by having different isotopes contained at different locations along the polymer scaffold. Such a polymer scaffold may contain some metal-chelating ligands attached to more than one subunit of the polymer. The number of metal-chelating groups capable of binding at least one metal atom in the polymer can be between about 1 and 10,000, such as 5-100, 10-250, 250-5,000, 500-2,500, or 500-1,000. At least one metal atom may be bound to at least one metal-chelating group. The polymer may have a degree of polymerization of between about 1 and 10,000, such as 5-100, 10-250, 250-5,000, 500-2,500, or 500-1,000. Thus, the polymer-based element tag may comprise about 1 to 10,000, such as 5-100, 10-250, 250-5,000, 500-2,500, or 500-1,000 tag atoms.

In some cases, the elemental tag may use a polymer selected from the group consisting of: linear polymers, copolymers, branched polymers, graft copolymers, block polymers, star polymers, and hyperbranched polymers. The backbone of the polymer may be derived from substituted polyacrylamides, polymethacrylates, or polymethacrylamides and may be substituted derivatives of acrylamide, methacrylamide, acrylates, methacrylates, homopolymers or copolymers of acrylic or methacrylic acid. Polymers can be synthesized from reversible addition fragmentation polymerization (RAFT), Atom Transfer Radical Polymerization (ATRP), and anionic polymerization. The step of providing a polymer may comprise synthesis of the polymer from a compound selected from the group consisting of: n-alkylacrylamides, N-dialkylacrylamides, N-arylacrylamides, N-alkylmethacrylamides, N-dialkylmethacrylamides, N-arylmethacrylamides, methacrylates, acrylates and functional equivalents thereof. The polymer may be water soluble. The moiety is not limited to chemical content. However, if the backbone has relatively repeatable dimensions (e.g., length, number of tag atoms, repeatable dendrimer properties, etc.), it simplifies the analysis. The requirements for stability, solubility and non-toxicity are also considered. Thus, the preparation and characterization of functional water-soluble polymers is performed by a synthetic strategy that places multiple functional groups plus different reactive groups (linking groups) along the backbone, which can be used to attach the polymer to a molecule (e.g., affinity reagents) through linkers and optionally spacer arms. By controlling the polymerization reaction, the size of the polymer is controllable. Typically, the size of the polymer will be chosen such that the cyclotron radiation of the polymer is as small as possible, such as between 2 and 11 nanometers. The length of the moiety bound to the element tag may be on the order of about 10 nanometers, and thus an excessively large polymer tag may sterically hinder the targeting function of the moiety relative to the size of the moiety to which the element tag is bound.

In some cases, the metal-chelating group capable of binding at least one metal atom may comprise at least 4 acetate groups. For example, the metal chelating group can be a diethylenetriaminepentaacetic acid (DTPA) group; a 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA) group; or a DTPA or DOTA derivative group. Alternative groups include ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis (β -aminoethylether) -N, N' -tetraacetic acid (EGTA). The metal-chelating group may be attached to the polymer scaffold by an ester or by an amide. Examples of suitable metal chelating polymers include X8 and DM3 polymers from Fluidigm Canada, inc.

The element-tagged portions may be labeled with element tags that are discernable by elemental analysis. The element-tagged moiety can be selected to bind or couple to any suitable structure (e.g., target) desired to be labeled by an element label and then detected by elemental analysis. For example, suitable element-tagged moieties may include oligonucleotide probes for hybridization, cell status probes (e.g., cisplatin as a viability marker or organic tellurium as a hypoxia or synthesis marker), or other suitable molecules.

In certain aspects of the present disclosure, element-tagged antibodies are used as element-tagged moieties. The antibody may be specific for a particular cell type. A cell type (e.g., a cell population) can represent a cell population or a particular subset of a cell population. An antibody specific for a cell type can have an antigen binding site (e.g., paratope) that is capable of binding to a molecule or antigen (e.g., an epitope) present in or on that particular cell type. In some cases, the antibody may be specific for a target on or within peripheral blood, such as a surface target or an intracellular target of Peripheral Blood Mononuclear Cells (PBMCs).

Certain aspects of the present disclosure describe the components as being in a mixture (mix or an mix). As used herein, the term mixture (mix or additure) includes components that are joined together (e.g., physically located together in the same container or housing). The container or housing comprises any suitable volume of space that is separable from another volume of space. The container or housing may be sealed, e.g. airtight. Examples of suitable containers or housings include tubes (e.g., test tubes), cassettes, pouches, wells of multi-well plates, or other such containers.

In some cases, aspects of the present disclosure may utilize calibration materials to aid in calibrating elemental analyzers. The calibration material may be any suitable metal or metal-containing material useful for providing calibration to an elemental analyzer. In some cases, the calibration material may be metal-containing beads having a mixture of different isotopes from different elements within a certain elemental mass range. The calibration material may contain a known amount of one or more known isotopes.

Certain aspects of the present disclosure utilize element-tagged antibodies to enable highly multiplexed single cell analysis of biological samples using elemental analysis, such as mass cytometry. These techniques may enable the separation of multiple markers in a single set without compensation. In some cases, more than 30 markers in a single set can be isolated.

The large number of detection channels (e.g., greater than 20 but less than 60) enabled by mass cytometry provides the opportunity to utilize combinatorial assays. For example, the number of targets detected, which is typically equal to the number of channels, can be increased by correlating groups of antibodies with each other through a common subset of biomolecules (e.g., antibodies) and using the same mass tag for antibodies that differ between the groups. For example, the number of targets detected in a given cell sample (e.g., dataset) can be at least 1.2, 1.5, or 2 times the detection channel used.

Cells can be stained with a consensus set so that cell populations identified in different sets can be correlated with each other, but with the proviso that mass signature channels remain for use in different sets of individual aliquots. Since panels use some mass labels for different antibodies (to detect different targets), the sample barcode can be applied to the cells in the aliquot as a "panel barcode" that identifies which mass labels are associated with which antibodies. Alternatively, the "master" consensus group can stain a portion (aliquot) of cells of a sample and be used to correlate (and be necessary for such correlation) populations identified by other groups with each other. The main consensus group may have different or only partially overlapping antibody subgroups common to each of a plurality of distinct groups staining different cell aliquots, respectively. There may be 2 or more, 3 or more, 4 or more, 5 or more or 10 or more distinct groups of antibodies shared with the common group. A variance group may have more mass signatures common to another variance group than to common (in common) antibodies.

The workflow will involve dividing a single sample (e.g., human PBMC) into different aliquots and staining each aliquot with different cell type-specific groups. On a panel basis, the same subset of mass channels will be used for different cell type-specific markers (e.g., a T cell panel may not have any or some of the B cell markers in the B cell panel, and vice versa). Amazon-type software solutions will identify cells stained by different groups and can combine them into the same dataset.

Barcoding of cells by groups may allow software to automatically identify groups stained for each cell. Aspects of the subject patent application include this software, including software that also implements other automated analysis described in the present patent application.

A common subset of markers that identify all basic cell types may be its own set or a subset of all sets. This can be used to identify the relative abundance of each of the major cell types, which can then be used in combination with the cell type specific set to identify the relative abundance of the specific cell type (population) in the entire set.

The software solution may exclude cellular events in the set that do not have a cell type of interest, thereby staining each cellular event for a target of interest. For example, any B cell event in the T cell group can be eliminated. As described in the focus above, this can be done after calculating the relative abundance of the major cell types. One or more of the difference groups may have a study marker. Some or all of these may be shared throughout the group. There may be one or more different channels for a user to detect their own targets of interest.

In certain aspects, an elemental analysis method comprises: dividing the sample cells into a plurality of fractions; staining cells with a consensus set of mass-tagged antibodies; staining cells of each fraction with a mass-tagged biomolecule of a difference group, wherein each difference group comprises biomolecules that are not in the other difference groups, but are tagged with mass tags that are present in the other difference groups; and/or studying the sample using elemental analysis to detect the presence of different mass labels on individual cells. The consensus group may be conserved in two or more portions, such as 3 or more, 4 or more, or 5 or more portions. The consensus group can detect surface targets that differentiate positive expression of parent populations (parent populations) that together cover a large fraction of immune cells. One or more (e.g., 2 or more, 3 or more, or 4 or more) individual differential sets detect a positive marker that distinguishes a subpopulation within one of the parental populations, but does not distinguish a subpopulation of a majority of immune cells, respectively.

A consensus group may not stain all portions (e.g., may only stain one portion), and the consensus group comprises different subsets of antibodies that are identical to antibodies of two or more distinct groups.

Each difference group may be a subpopulation within a parent population identified by the providing of the consensus group, wherein the parent population is selected from the group consisting of: t cells, CD 4T cells, regulatory T cells, CD 8T cells, NK cells, B cells, dendritic cells, and/or monocytes/macrophages. Alternatively or additionally, the panel of differences may identify cellular function, for example, when the panel of differences detects targets involved in intracellular signal transduction, cytokine production, and/or the cell cycle.

In certain aspects, an oligonucleotide is used in addition to or in place of an antibody in the difference set, e.g., when the oligonucleotide specifically hybridizes to a target RNA, either directly or indirectly. For example, the oligonucleotide may hybridize to RNA encoding a cytokine as described herein.

Wherein staining with the difference set comprises signal amplification by staining each moiety with an oligonucleotide-tagged antibody and hybridizing mass-tagged oligonucleotides directly or indirectly to the antibody-tagged oligonucleotides.

One or more of the consensus and difference groups may be freeze-dried as described herein for the groups.

The cells are stained with the consensus group prior to being divided into fractions, or the cells are divided prior to being stained with the consensus group (e.g., where the consensus group is mixed with each of the variance groups). The method may further comprise labeling the compartmentalized cell with a group barcode that identifies the set of differences (and thus the target associated with the mass label of the group), e.g., wherein the group barcode is provided mixed with its set of differences prior to addition to the compartmentalized cell. The sections were combined after labeling with the group barcode and before study. The gang barcode may be applied in a similar manner to the sample barcode shown in fig. 13, or may be applied in addition to the sample barcode.

The above method can be performed on one or more other samples (which are themselves divided into aliquots for separate stains) prior to investigation, with the samples being barcoded with different samples and the barcoded samples being combined. The barcoded samples may be combined prior to separation into portions.

The method may further comprise classifying the cells of the individual study into cell populations based on the consensus group and the variance group thereof. For example, a consensus group may identify a parent population of T cells, but a different T cell group for one of the portions may identify a subpopulation within the parent population. Other populations and groups suitable for these methods are discussed in the present application.

Classification can be by automated software trained on consensus and variance groups (e.g., on similar samples, such as samples including PBMCs). Classification can be done by gating, by a trained clustering algorithm running in a high dimensional space (where the dimensions are related to the number of surface markers used for classification), or by a neural network.

The difference group can be used to identify a subpopulation of the parent population identified by the consensus group. The method may further comprise integrating the population of cells under study identified based on different distinct groups (differential differentiation panels) into the same dataset based on the consensus group. The integrating may include: discarding data for cells of the parental population identified as the consensus group unable to identify the subpopulation thereof; identifying a proportion of cells in the original sample in the subpopulations identified by the different sets of the respective fractions; and/or displaying the expression of targets detected by different groups for the same parental population detected by the consensus group.

The method may further comprise stimulating cells of different samples or sample portions under different conditions prior to staining.

Kits may include a common group and a differential group packaged for use in any of the above methods. The consensus and differential groups may be packaged in a kit as described in the methods above.

In certain aspects, kits for elemental analysis (e.g., mass spectrometry) can include a common set comprising a plurality of conjugated antibodies. A consensus group may include a plurality of antibodies conjugated to different mass labels, where each different mass label is distinguishable based on its isotopic composition. The plurality of antibodies may be in a mixture. The plurality of difference sets may comprise mass-tagged biomolecules (e.g., antibodies and/or oligonucleotides), wherein each difference set comprises biomolecules that are not present in one or more other sets, but are tagged with mass tags present in the one or more other sets.

Certain aspects of the present disclosure enable freeze-drying of antibody panels to achieve panels with high stability. The stability of the freeze-dried antibody panel can be described in terms of a comparison of the ion counts of the elemental tag isotopes of the samples determined using the freeze-dried antibody panel relative to the same samples determined using a non-freeze-dried antibody panel. The higher stability of the freeze-dried antibody panel relative to the non-freeze-dried antibody panel can be viewed as a higher ion count of the element tags from the freeze-dried antibody panel than from the non-freeze-dried antibody panel. In addition, stability can be described as a function of antibody destruction (e.g., destruction affecting antibody binding activity) and/or a function of metal retention of metal atoms of the element tag and/or the distinguishable isotope on the element-tagged portion. For example, a stable set will show little to no disruption of antibody binding activity and/or little to no loss of metal atoms from the element tag and/or element-tagged moiety. The freeze-dried group described herein showed better stability than the non-freeze-dried group with the same composition.

Element tags have been provided for mass cytometry flow for conjugation or pre-conjugation to antibody biomolecules. The elemental tag used for conjugation may comprise a metal-preloaded polymer, or a separate metal solution for loading onto the polymer (e.g., prior to conjugation to the biomolecule). The polymer can comprise a backbone and a plurality of pendant metal binding groups, such as pendant groups comprising a chelating agent (e.g., DTPA, DOTA, or derivatives thereof). Elemental tags conjugated to biomolecules, such as antibodies, may be preloaded with a metal. In certain aspects, the metal is an enriched isotope, such as a lanthanide isotope. The lanthanides are chemically similar, and lanthanide tags (including those conjugated to antibodies) have been found to be stable in solution to a degree suitable for mass cytometry. However, non-lanthanide metals may not have similar stability. In certain aspects, the metal element or isotope thereof can be outside of the lanthanide family, such as a non-lanthanide transition metal or a post-transition metal. Suitable late transition metals for mass cytometry include elements having atomic numbers 48-50 (e.g., cadmium, indium, and/or tin) and 80-84. The stability of the late transition metal loaded polymer can be improved by freeze-drying. The subject methods and kits include these non-lanthanide tags lyophilized as described herein, provided that they are separate from or mixed with other element tags or biomolecules tagged with other elements.

Medium weight elements (e.g., or isotopes thereof) can provide weaker signals by mass flow cytometry, such as when the element is near (e.g., within 30, 20, or 10amu from) a high pass filter that removes argon dimers from ICP and/or organic molecular elements. These medium heavy elements or isotopes may have an atomic number between 39 and 52. In addition, non-lanthanide elements, such as certain transition metals and post-transition metals, may not be chelated, as is readily achieved for pendant groups on the polymer. As such, the non-lanthanide metals described herein (e.g., isotopes thereof) can be useful for cell barcoding, e.g., live cell barcoding with element-tagged CD 45. For example, a live cell barcode may include freeze-dried cadmium isotope labeled CD 45.

In general, generating and obtaining large multiplex groups may include optimization of antibody titers, complete group validation, sample preparation and analysis. Preparing a staining mixture from a plurality of individual tubes (e.g., 30 different tubes) is cumbersome and may be prone to error. In addition, especially when based on metal-tagged antibodies, the pre-prepared staining mixture may tend to break down over time and may show poor stability. For example, a non-freeze-dried metal-tagged antibody panel containing a plurality of antibodies can remain stable for about 1 week or less, whereas certain aspects of the present disclosure relate to a similar freeze-dried antibody panel that can remain stable for about 1 year or more. Thus, the freeze-dried antibody panel can facilitate large-scale production and distribution of the antibody panel while allowing long-term storage of the antibody panel, thereby improving consistency in subsequent assays utilizing the same panel.

Sample preparation according to the subject methods can be performed, at least in part, by an automated sample preparation system. The system can process the sample prior to staining, can contact the sample with one or more antibody sets, assay beads, and/or barcodes, can perform centrifugation and washing steps, and/or can deliver the sample to a mass spectrometry flow cytometry system.

Accordingly, certain aspects of the present disclosure relate to freeze-dried multi-way (e.g., 30-way) immunophenotyping groups contained in a single housing (e.g., a single tube) that can be used with efficient workflow and automated software solutions for human whole blood analysis using mass cytometry. In some cases, the panel may be focused on T cell lineages while also capturing other relevant immune populations. Blood can be added directly to the freeze-dried antibody tube, followed by Red Blood Cell (RBC) lysis, washing and fixation steps, and finally data collection of the stained sample on an elemental analyzer (e.g., ICP-MS system).

The software tool can receive data from the elemental analyzer and automatically generate reports on various cell count information, such as viable cell number, percentage of specific cell population, staining intensity, histogram, 2D dot plot, and t-distribution random neighborhood embedding (tSNE) plot. The software can automatically calculate the population frequency equivalent to manually gating. The automated software can eliminate user bias that may occur during manual analysis (e.g., manual gating) and significantly reduce the amount of time required to analyze each data file. The set and software tools may enable researchers to streamline immunophenotyping of whole blood while accurately and reproducibly monitoring changes in immune cell subtypes in patient samples. In some cases, appropriate element tags may be automatically loaded into the software tool to enable rapid decoding of the element analyzer data into useful results.

Antibodies and other element-tagged moieties can be used to recognize the presence of a cellular target in a sample. The antibody or element-tagged moiety may have a targeting function for a cellular target and thus remain with the sample after washing the sample. Thus, any detection of a different isotope or combination of isotopes associated with a particular element tag on an antibody or element-tagged moiety is indicative of the presence of that cellular target. The cellular target may be a surface target or an intracellular target. Examples of intracellular targets may include cell cycle and proliferation targets, intracellular signaling targets, and intracellular cytokine targets. Since permeabilizing a cell can sometimes destroy cell surface markers, the sample can be stained with an antibody or element-tagged portion associated with the surface target prior to permeabilization. However, since permeabilization of the cell is sometimes necessary for proper access to the intracellular target, the sample is stained with an antibody or element-tagged portion associated with the intracellular target after permeabilization.

In some cases, an intercalator (intercalator) may be used. In some cases, the intercalator may be an element tagged moiety. The intercalator may comprise or be coupled to an element of an element tag. For example, iridium may be used as an intercalator and may simultaneously function as its own elemental tag. Thus, detection of iridium during the study indicates the presence of the intercalator. Other suitable intercalating agents include rhodium and cisplatin. The intercalating agent may be included in or provided separately from the freeze-dried component. If the intercalator is mixed with the sample cell prior to permeabilization, the intercalator may be a cell viability stain, which indicates whether the cell is a living cell. In these cases, the presence of the intercalating agent detected during the study is indicative of dead cells. However, if the intercalator is mixed with the sample cell after permeabilization, the intercalator may function as a cell identification stain or a cell presence stain, which may indicate the presence of a cell, as the intercalator should enter all permeabilized cells. In these cases, the presence of the intercalating agent detected during the study is indicative of the cell being analyzed by the elemental analyzer. In some cases, an intercalating agent, such as rhodium, can be included with or mixed with the lyophilized antibody panel.

The antibodies selected for use in the freeze-dried group may be selected to perform certain assays. In some cases, a freeze-dried set or subset of freeze-dried sets can be generated for use in a variety of assays, thus facilitating the desired assay by simply selecting the appropriate set or combination of subsets of sets. Various groups or subgroups of groups are described herein for use with certain aspects of the present disclosure. Each group or subset of groups described with respect to a list of possible antibodies can include two or more antibodies from the list or any number of antibodies from the list, up to and including all antibodies from the list. As used herein, a subset of a panel can include any combination of antibodies from the panel described herein. In some cases, a subset of a set can be combined with a different set or subset of sets to produce one or more of the sets described herein.

In some cases, the freeze-dried population may comprise two or more antibodies from the list comprising: cd45, Cd45RA, Cd45RO, Cd123, Cd4, Cd8a, Cd11C, Cd57, CXCR3, Cd185, Cd38, Cd56, Cd3, Cd20, Cd66b, HLA-DR, IgD, Cd27, Cd28, Cd127, Cd19, Cd16, Cd161, Cd194, Cd25, Cd294, Cd197, Cd14, CCR6, and TCR δ γ. In some cases, the freeze-dried group can include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 antibodies from the list. As disclosed herein, certain aspects of the present disclosure are useful for incorporating multiple different element-tagged antibodies into a single freeze-dried panel. In some cases, such a set may be particularly useful for immunophenotyping of human peripheral blood. In some cases, such a set can be a common set in a kit or method that includes a differential set of labeled portions (e.g., aliquots) of cells from the same sample, as further described herein. These sets of differences may include one or more of the following sets.

In some cases, the freeze-dried group may include a leukemic group and/or a lymphoma group that includes antibodies that are particularly suited for determining information about the cell count of leukemic cells and/or lymphoma cells.

Various example freeze-dried groups are described herein. For purposes of description, groups may be described with respect to antibodies and targets, where targets are indicated in parentheses. In some cases, a different antibody may be substituted for the different antibody with any given antibody, as long as the different antibody is specific for the target of the substituted antibody. In certain embodiments, multiple freeze-dried sets may be provided (e.g., in a kit) such that they may be combined based on the sample and/or application.

In some cases, a freeze-dried panel for human Acute Myeloid Leukemia (AML) typing may include antibodies (and targets) from the list including HIB19(CD19), 104D2(CD117), ICRF44(CD11B), 10.1(CD64), CD7-6B7(CD7), 6H6(CD123), HI30(CD45), WM53(CD33), clone (target) (clone (target)), W6D3(CD15), 581(CD34), UCHT1(CD3), IM7(CD44), HIT2(CD38), L243(HLA-DR), and 12G5(CXCR 4). In some cases, the set may be particularly useful for AML typing. AML is the most common type of acute leukemia in adults, and is the generation of malignant tumors in the bone marrow due to the destruction of normal hematopoiesis. AML is produced within precursors of myeloid, erythroid, megakaryocytic, and monocytic cell lineages due to the generation of chromosomal rearrangements and multigenic mutations. The immunophenotype of AML is highly heterogeneous; markers often expressed by AML include CD15, CD33, CD34, and CD 64.

In some cases, a freeze-dried panel for human B cell typing may include antibodies (and targets) from the list including HIB19(CD19), 1a6-2(IgD), 2H7(CD20), polyclonal (IgA), BL13(CD21), L128(CD27), HIB22(CD22), CB3-1(CD79B), HIT2(CD38), ML5(CD24), MHM-88(IgM), and L243 (HLA-DR). In some cases, the panel can facilitate identification and typing of human B cells, including initial, memory, transitional, and plasma B cell populations.

In some cases, a freeze-dried panel used to identify cell cycle information may include antibodies (and targets) from the list including N/a (S-phase (IdU)), J112-906(pRb [ Ser807/811]), GNS-1 (cyclin B1(CyclinB1)), B56(Ki67), and HTA28(pHistone H3[ Ser28 ]). In some cases, the panel may help to assess cell cycle status: proliferation, G0 (senescence), G1, S-phase, G2 and M-phase (mitosis). This group in combination with other groups may be particularly useful for identifying cell cycle status and other cell count information.

In some cases, a freeze-dried group for human helper T cell typing may include antibodies (and targets) from the list including G034E3(CCR6), NP-6G4(CCR5), RPA-T8(CD8), C398.4A (ICOS), HI100(CD45RA), UCHT1(CD38), G025H7(CXCR3), 205410(CCR4), GP-3G10(CD161), UCHL1(CD45RO), 2A3(CD25), RF8B2(CXCR5), SK3(CD4), EH12.2H7(PD-1), and a019D5(CD 127). In some cases, this The panel can be used for identification and typing of human CD4+ helper T cell subtypes, including helper T1 cells (T)_H1)、T_H2、T_H17、T_H22. Follicular helper T cells (T)_FH) And T regulatory cells (T)_REG). Differentiation of CD4+ T cells into functionally distinct helper T cell subtypes may be important for normal immune regulation. These subtypes are illustrated by external and internal lines, and the resulting cell population acquires a stable phenotype defined by the expression of characteristic cytokines, "master regulators" transcription factors and characteristic cell surface phenotypes.

In some cases, a freeze-dried group for substantially human peripheral blood typing may include antibodies (and targets) from the list including 2H7(CD20), HI30(CD45), M5E2(CD14), 3G8(CD16), SK1(CD8), UCHT1(CD3), and SK3(CD 4). The set may be useful for assaying fresh or frozen human whole blood or PBMCs. This panel can be used to identify CD4+ T, CD8+ T, B cells, monocytes, NK and granulocytes.

In some cases, other freeze-dried groups for primary human peripheral blood typing may include antibodies (and targets) from the list including RPA-T4(CD4), RPA-T8(CD8a), 2H7(CD20), 3G8(CD16), HI30(CD45), M5E2(CD14), and UCHT1(CD 3). The set may be useful for assaying fresh or frozen human whole blood or PBMCs. This panel can be used to identify CD4+ T, CD8+ T, B cells, monocytes, NK and granulocytes.

In some cases, a freeze-dried group for human peripheral blood typing may include antibodies (and targets) from the list including UCHT1(CD3), RPA-T4(CD4), RPA-T8(CD8a), Bu15(CD11c), M5E2(CD14), 3G8(CD16), HIB19(CD19), 2H7(CD20), O323(CD27), HIT2(CD38), HI30(CD45), HI100(CD45RA), VI-PL2(CD61), CD66a-B1.1(CD66), 6H6(CD123), HIR2(CD235a/B), and L (HLA-DR). The set may be useful for assaying fresh or frozen human whole blood or PBMCs. The group can be useful for the identification of major peripheral blood cell subtypes, including granulocytes, basophils, plasmacytoid dendritic cells, natural killer cells, effector T killer cells, naive T killer cells, activated T killer cells, memory T killer cells, effector T helper cells, naive T helper cells, activated T helper cells, memory B cells, naive B cells, plasma B cells, spinal cord dendritic cells, atypical monocytes, canonical monocytes, and platelets.

In some cases, a freeze-dried group for human T cell typing may include antibodies (and targets) from the list including HI111(CD11a), RPA-T4(CD4), RPA-T8(CD8a), 3G8(CD16), 2A3(CD25), HI30(CD45), G043H7(CCR7), FN50(CD69), UCHL1(CD45RO), BJ18(CD44), O323(CD27), HI100(CD45RA), UCHT1(CD3), HCD57(CD57), L243(HLA-DR), and a019D5(CD 127). The set may be useful for assaying fresh or frozen human whole blood or PBMCs. This group may be useful for the identification of major T cell subtypes, including naive, central memory, effector and effector memory CD4+ and CD8+ cells, and may also classify the activation and homing status of these subtypes.

In some cases, a lyophilized panel of human T cell-types for expansion may include antibodies (and targets) from the list including TS1/8(CD2), UCHT2(CD5), CD7-6B7(CD7), SN 4C 3-3a2(CD9), CD28.2(CD28), 9F10(CD49d), HP-3G10(CD161), 205410(CCR4), NP-6G4(CCR5), and G025H7(CXCR 3). The set may be useful for assaying fresh or frozen human whole blood or PBMCs. This group may be particularly useful when combined with human T cell typing, in which case the combined group may be used to identify all major T cell subtypes, including naive, central memory, effector and effector memory CD4+ and CD8+ cells, naive and memory tregs, TH1 and TH2 cells, and to classify the activation and homing status of these subtypes.

In some cases, a freeze-dried panel for human Embryonic Stem (ES) cell or Induced Pluripotent Stem (iPS) cell typing may include antibodies (and targets) from the list including TRA-1-60(TRA-1-60), O3O-678(Sox2), 40/Oct-3(Oct-3/4), N31-355(Nanog), IM7(CD44), and 9E10 (c-Myc).

In some cases, a freeze-dried panel for human hematopoietic stem and progenitor cell typing may include antibodies (and targets) from the list including HI10a (CD10), WM15(CD13), 581(CD34), G0H3(CD49f), 104D2(CD117), DL-101(CD138), and 12G5(CXCR 4). This panel can be used for the identification and typing of hematopoietic progenitor cell populations, including Hematopoietic Stem Cells (HSCs), in human bone marrow and cord blood. This panel can be usefully combined with human peripheral blood typing so that lineage positive cells can be excluded from the gating strategy.

In some cases, a lyophilized group for human intracellular cytokine I assay may include antibodies (and targets) from the list including B27(IFN γ), MQ1-17H12(IL-2), MP4-25D2(IL-4), TRFK5(IL-5), MQ2-13a5(IL-6), N49-653(IL-17A), SHLR17(IL-17F), B (granzyme), B-D48 (perforin), D21-1351(MIP1 β), and Mab11(TNF α). This set can be used for the assay of fresh or frozen human whole blood, PBMCs or cell lines. This panel can help measure 11 major cytokines as well as lytic proteins, granzyme B and perforin. This panel can be combined with human peripheral blood typing to allow for broad immunophenotyping of cytokine-expressing cells.

In some cases, a freeze-dried group for human regulatory T cell typing may include antibodies (and targets) from the list including 9F10(CD49D), RPA-T4(CD4), 205410(CCR4), HI100(CD45RA), UCHT1(CD3), a1(CD39), PCH101(Foxp3), DX2(CD95), UCHL1(CD45RO), 2A3(CD25), 14D3(CD152), L243(HLA-DR), and a019D5(CD 127). This panel can be used to identify regulatory T cells. Regulatory T cells (tregs) are an inhibitory subtype of CD4+ helper T (th) cells important for the regulation of immune responses. Tregs are defined by the expression of the transcription factor Foxp 3. Other Treg markers include constitutive expression of high-avidity IL-2 ra chain (CD25) and cytotoxic T lymphocyte-associated antigen 4(CTLA-4) and low expression of IL-7 ra chain (CD 127). CD4+ CD25+ Foxp3+ tregs can be divided into two main types: tregs (ttregs) of thymic origin and tregs (ptregs) of peripheral origin.

In some cases, the freeze-dried panel for human monocyte/macrophage typing may include antibodies (and targets) from the list including HIB19(CD19), ICRF44(CD11B), CD7-6B7(CD7), CD66a-B1.1(CD66), 5-271(CD36), GHI/61(CD163), HI30(CD45), Bu15(CD11c), M5E2(CD14), 3G8(CD16), HIT2(CD38), 15-2(CD206), WM53(CD33), UCHT1(CD3), and L243 (HLA-DR). This panel can be used to identify and type monocytes and macrophages. Monocytes circulate in the blood, bone marrow and spleen and account for approximately 2-12% of total human leukocytes. Monocytes have been considered to be a systemic reservoir of spinal cord precursors for tissue phagocytosis and dendritic cell renewal, despite the presence of DC and macrophage subpopulations that are produced independently of monocytes. The recruited monocytes are innate effectors of the immune response to microorganisms, and they kill pathogens through phagocytosis, the production of Reactive Oxygen Species (ROS), Nitric Oxide (NO), myeloperoxidase, and inflammatory cytokines. Monocytes can be classified as "classical" (CD14+ CD16-), intermediate (CD14+ CD16+), and non-classical (CD14loCD16+) based on the expression of CD14 and CD 16.

In some cases, a lyophilized set for signaling assays may include antibodies (and targets) from the list including 47(pSTAT5), 58D6(pSTAT1), D3F9(P38), 4/P-Stat3(pSTAT3), L35a5(I κ β α), D13.14.4E (pERK1/2), and N7-548(pS 6). This panel can be used to quantify basal and induced phosphorylation of multiple key signaling pathways: JAK/STAT, NF κ B, and MAPK. This panel can be combined with other panels to measure cell signaling in heterogeneous samples (e.g., blood or spleen cells). Alternatively, when a homogeneous sample, such as a cell line, is measured, it can be used as a separate group.

In some cases, a freeze-dried group for spleen/lymph node typing of a basal mouse may include antibodies (and targets) from the list including 30-F11(CD45), M1/70(CD11B (MAC1)), 145-2C11(CD3e), 53-6.7(CD8a), RM4-5(CD4), and RA3-6B2 (B220). The set may be useful for identifying CD4+ T, CD8+ T, B cells, macrophages and monocytes in fresh or frozen isolated mouse splenocytes and thymocytes.

In some cases, a freeze-dried group for spleen/lymph node typing in mice may include antibodies (and targets) from the list including RB6-8C5(Ly6G/C (Gr1)), N418(CD11C), H1.2F3(CD69), 30-F11(CD45), M1/70(CD11B (MAC1)), 6D5(CD19), 3C7(CD25), 145-2C11(CD3e), TER119(TER-119), MEL-14(CD62L), 53-6.7(CD8a), H57-597(TCR β), PK136(NK1.1), IM7(CD44), 4-5(CD4), and RA3-6B2 (B220). The panel can be useful for identifying the major mouse spleen/lymphocyte subsets among fresh or frozen isolated mouse splenocytes and thymocytes, including effector CD4+ T, effector memory CD4+ T, central memory CD4+ T, activated CD4+ T, effector CD8+ T, effector memory CD8+ T, central memory CD8+ T, Treg, plasmacytoid DCs, spinal DCs, erythrocytes, macrophages, monocytes, NK cells, and granulocytes.

In some cases, a freeze-dried group for mouse intracellular cytokine I assay may include antibodies (and targets) from the list including XMG1.2(IFNy), JES6-5H4(IL-2), 11B11(IL-4), TRFK5(IL-5), MP5-20F3(IL-6), JES5-16E3(IL-10), TC11-18H10.1(IL-17A), and MP6-XT22 (TNFa). This panel can be used to determine the major mouse cytokines in fresh or frozen mouse-derived leukocytes, including splenocytes, thymocytes, bone marrow and lymph node cells or cell lines. This group can be used with a mouse spleen/lymph node typing group to allow for broad immunotyping of cytokine expressing cells.

In some cases, other groups or subgroups of groups may include pairs of regulatory T cell surface markers (e.g., PD-1, CTLA-4, GITR, CXCR3, IL-12R, IL-4R, CRTH2, IL-17Rb, IL-23R, CCR6, IL-1Rb, OX40L, CD40L, SLAM, IL-21R, ICOS, CXCR5, TIM3, 1B11, LAG3, and/or BTLA); intracellular regulatory T cell transcription factor markers (e.g., FoxP3, RORgT, T-beta, Bcl6, and/or GATA 3); intracellular cytokine and chemokine markers (e.g., IFNg, IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, IL-17A, IL-22, IL-23, TGFb, TNFa, perforin and/or granzyme b); druggable target markers (e.g., BTLA, GITR, 4-1BB, OX40, TIGIT, Helios, and/or ICOS); and/or spinal cord derived inhibitory cell markers (e.g., CD11b, CD15, and/or CD 33).

The set of CD 4T cells may include at least two of the following: CCR6(G034E3), CD45RA (HI100), CD4(RPA-T4), LAG3 (polyclonal; R & D Systems), CCR4(205410), CD62L (DREG-56; BioLegend), CD49b (AK-7; BD Biosciences), CXCR3(G025H7), CD161(HP-3G10), TIGIT (MBSA 43; eBioscience), ICOS (DX 29; BD Biosciences), CD226(11A 8; BioLegend), CD8 a (SK1), CD25(2A3), CTLA-4(14D3), CXCR5(51505), CD3(UCHT 1; BioLegend), PD-1(EH12.2H7), optionally in the form of a clone listed in parentheses. In some cases, the freeze-dried group can include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from the list. The set may be provided in a freeze-dried form or as a solution. The group may be a diversity group as described herein.

The NK group may include at least two of the following: CD27, CD4, CD8, CD57, TRAIL, KIRDL2/L3/S2, CD16, CD117, KIR2Ds4, LILRB1, NKp46, NKG2D, NKG2C, 2B4, NKp30, CD122, KIR3DL1, CD94, CCR7, KIRDL3, NKG2A, HLA-DR, KIR2DL4, CD56, CD45, KIR2DL5 and CD 25. In some cases, the freeze-dried group can include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from the list. The set may be provided in a freeze-dried form or as a solution. The group may be a diversity group as described herein.

The set of CD 8T cells may include at least two of the following: CD3, CCR7, CD11a, CD7, CD8, CD27, CD28, CD29, CD43, CD45RA, CD45RO, CD49d, CD57, CD62L, KLRG1 and HLA-DR. In some cases, the freeze-dried group can include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from the list. The set may be provided in a freeze-dried form or as a solution. The group may be a diversity group as described herein.

The Treg group may include at least two of the following: CCR 6G 034E3, LAP TW4-2F8, CD45RA HI100, CD103 Ber-ACT8, CD31 WM59, CD8 RPA-T8, CD147 HIM6, GITR 621, CCR 4205410, CD28 CD28.2, CD49D 9F10, CD62L DREG-56, CD3 UCHT1, CXCR 3G 025H7, CD73 AD 73, CD161 HP-3G 73, CD 73A 73, ICOS 73, OX 73 Ber-ACT 73, CCR 106588-5, CD 1374B 73-1, CD 73L 128, GARP 7B 36243, CD 252A 73, CD 73M-KT 251, CD 414D 73, CD73 SK 73, CD 73T 73, CD 73-HIL 73, CD KL 80, CD 73-RG 7B 73, HLA KL 73/RG 73 and HLA KL 73/RG 73. In some cases, the freeze-dried group can include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from the list. The set may be provided in a freeze-dried form or as a solution. The group may be a diversity group as described herein.

The B cell group may include at least two of: CD10, CD117, CD11c, CD127, CD16, CD179a, CD179b, CD19, CD20, CD21, CD22, CD23, CD235, CD24, CD27, CD33, CD34, CD38, CD40, CD43, CD45, CD45RA, CD49d, CD5, CD61, CD62L, CD66b, CD7, CD72, CD79b, CXCR4, HLADR, IgD, mi/igh (igh), κ, λ, Pax5, PreBCR, RAG1 and TdT. In some cases, the freeze-dried group can include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from the list. The set may be provided in a freeze-dried form or as a solution. The group may be a diversity group as described herein.

The monocyte and MDSC group may include at least two of the following: CD14, CD16, HLA-DR, CD163, CD206, CD33, CD36, CD32, CD64, CD13, CD11b, CD11c, CD86, CD274, CCR2, CD163, CD13, CD123, and CD 206. In some cases, the freeze-dried group can include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from the list. The set may be provided in a freeze-dried form or as a solution. The group may be a diversity group as described herein.

The DC group may include at least two of: EpCAM/CD326, CD117, CD11, BDCA/CD 303, CD 127/IL-7/IL 3, CD66, CD163, CD, CCR, CD11, BDCA/CD 141, CD335/NKp, BDCA-1/CD1, CD 172/b/SIRPa, HLADR, CD115/CSF1, CX3 CR/CX 3CR, CD116/GMSFR, CD275/ICOSL, TLR, CD274/PDL, CLEC 9/DNGR, CD135/FLT, Dectin-1/CLEC7, CD206/MMR, CD, Langerin (Langerin)/CD207, CD45, CD (UC), CD XCR-PE, anti-APC antibody, Siglec-6/CD PE, CD100-APC, AxCDM/SynCAM, CD205/DEC205, BDCA/DEC 123/CD 395, CD 650/CD 304, CD 650/BCA/BCB, CD304, CD 650/BCB, CD304, CD III, B V, CD III, B III, B V, B III, C, CD III, CD2, C, CD III, C, III, B III, B III, B III, B III, B III, B III, CD19 PerCP/Cy5.5, CD20 PerCP/Cy5.5, CD1a Pacific Blue, CD2 APC/Cy7, CD206/MMR APC (cline 15-2), CD3 PerCP/Cy5.5, CD335 PerCP/Cy5.5, CD4 BV785, CD45 BV785, CD5 BV737, CD66b PerCP/Cy5.5, CD8a APC/Cy7, CD81 PerCP/Cy5.5, CLEC9A/DNGR1 APC, CD209/DC-SIGN APC, Dectin1/CLEC7A PE, HLADR BV605, Langerin (Langerin)/CD207 PE, TCF4/E2-2, Anti-fibroblast (Angellas-fibrest) (TE7), CD a, DARC 3-desmoplastin 3-3). In some cases, the freeze-dried group can include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from the list. The set may be provided in a freeze-dried form or as a solution. The group may be a diversity group as described herein. In certain aspects, a panel can include element-tagged oligonucleotide probes that hybridize (directly or via a hybridization protocol) to a target RNA, such as an RNA encoding a cytokine. For example, a panel may include two or more element-tagged oligonucleotide probes encoding RNAs: IFNg, IL-2, TNF, CXCL8, IL8, CCl4, IL-1B, IL-6, CCL2, ILRN and Il1 a. In some cases, the freeze-dried group can include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from the list. The set may be provided in a freeze-dried form or as a solution. The group may be a diversity group as described herein.

Although various example freeze-dried panels are described herein, other combinations of antibodies or other element-tagged portions can be used to help perform the desired assay. In some aspects, one or more of the groups described above may be provided in a non-lyophilized form, such as in a solution. Two or more groups may be provided in a mixture (in a combined group).

Each of the freeze-dried groups disclosed herein may be associated with a specific gating strategy for identifying certain characteristics based on the antibodies in the group. To facilitate rapid analysis, gating strategies associated with a particular group may be preloaded into the software used to analyze the elemental analyzer data. Once selected (e.g., manually or automatically), the software can use the gating strategy to generate results from the elemental analyzer data. For example, using a human peripheral blood typing kit, a pre-loaded gating strategy can distinguish between different major peripheral blood cell subtypes based on the presence of multiple antibodies in the sample (e.g., the presence of an elemental tag bound to the antibodies). In some cases, multiple groups or combinations of subgroups of groups, such as those identified herein, may enable the use of new or other gating strategies. For example, when combining a human peripheral blood typing group with a human hematopoietic stem cell and progenitor cell typing group, then a new gating strategy can be used to help exclude lineage positive cells from the results.

Within each group, each antibody may be tagged with a unique elemental tag, such that a unique isotope or combination of isotopes is associated with each antibody in the group. In some cases, particularly for groups designed to be used in conjunction with another group, the unique element tags from one group are different from the unique element tags of another group. The location of each antibody for each isotope or combination of isotopes can be stored as an elemental tag location map (mapping). The element tag mapping map may be used by the software disclosed herein to interpret data received from the element analyzer.

In some cases, the freeze-dried antibody panel can include one or more supplemental reagents. These supplemental reagents may include any suitable freeze-dryable reagent useful in performing assays using antibodies of the freeze-dried antibody panel. Examples of suitable supplemental reagents include red blood cell lysates, wash buffers, fixing reagents, permeabilizing reagents, and the like.

The method of preparing a freeze-dried group may include obtaining antibodies, conjugating the antibodies to their respective elemental tags, titrating the conjugated antibodies in a liquid form group for quality control, diluting the liquid antibodies in a stabilizer based on the titration results, combining the diluted antibodies with an excipient, combining the antibody and excipient mixtures together to form a single mixture, freeze-drying the mixture, then filling back the freeze-dried mixture and sealing it in a container. In some cases, the individual antibody and excipient mixtures may be freeze-dried prior to combining them together in the mixture, although multiple antibody and excipient mixtures are typically combined prior to freeze-drying. Any suitable excipient may be used, such as sugars (e.g., trehalose, sucralose, and mannitol). The use of trehalose and/or sucrose may help inhibit protein unfolding while providing a glass matrix. The use of mannitol may act as a bulking agent. In some cases, the excipient may include a sugar, either alone or in combination with Bovine Serum Albumin (BSA). In some cases, the excipient can be a mixture of about 5% -20% (e.g., 10%) sugar in PBS and 5% -20% (e.g., 10%) BSA in PBS.

The freeze-drying itself may occur in a number of stages, including a heating stage, an evacuation stage, a drying stage, and a holding stage. During each stage, specific settings of temperature, warm-up time, hold time, and/or vacuum (e.g., freeze-drying settings) may be used to control the freeze-drying apparatus. The freeze-drying device may be any device suitable for controlling the temperature and vacuum of the mixture to be freeze-dried. In some cases, each stage may include a plurality of different sub-stages, each sub-stage having a different freeze-drying setting. During the heating phase, the temperature may be maintained at a temperature between-60 and 0 ℃ for a duration of time while the vacuum is maintained in a range between 100 and 500 torr. During the evacuation phase, the vacuum may be reduced to a range between 100 and 500 mTorr. During the drying stage, the temperature can be manipulated in the range between-50 to 30 ℃ while maintaining the vacuum in the range of 10-150 mtorr. During the hold phase, the temperature may be maintained at a temperature between 10 and 30 ℃ while the vacuum is raised to a range between 100 and 500 mTorr. After the hold period, the freeze-dried mixture may be returned, which may include increasing the pressure in the container, such as in a range of up to 200 torr to 760 torr, although in some cases, the return occurs at a pressure below ambient pressure. In some cases, the temperature of the mixture does not rise above the glass transition temperature of the mixture during all stages of freeze-drying.

During the freeze-drying process, the water content of the mixture decreases. In some cases, the freeze-dried group may have a moisture content at or less than 5% by weight, although in some cases, the moisture content may be at or less than 0.5% by weight. In some cases, the freeze-dried group may have a moisture content at or less than: 5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1%, 3%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%,. 95%, 0.9%, 0.85%, 0.8%, 0.75%, 0.7%, 0.65%, 0.6%, 0.55%, 0.5%, 0.49%, 0.48%, 0.47%, 0.46%, 0.45%, 0.44%, 0.8%, 0.75%, 0.7%, 0.65%, 0.6%, 0.55%, 0.5%, 0.35%, 22.35%, 22.0.19%, 22.0%, 19%, 30.0.0.0%, 19%, 30.0.0.0.0.0.25%, 30%, 19%, 30%, 0.0.0.0.0.25%, 25%, 28%, 25%, 0.0.0.0.0% or more, 0.14%, 0.13%, 0.12%, 0.11% and/or 0.1%. In some cases, the freeze-dried group may have a moisture content at or greater than 0.05% by weight, such as between 0.05 and 1% by weight.

The freeze-dried group can be stored in any suitable container, such as a tube, pouch, or well of a well plate. In some cases, a single group may storeIn a single container. In some cases, a single group may be uniformly distributed among multiple receptacles, such as multiple wells of a well plate. In some cases, the freeze-dried group can be stored in an inert atmosphere (e.g., N)₂) Or in air (e.g., dry air). The freeze-dried group can be stored in a closed container.

The freeze-dried group can be used similarly to the other antibody groups. The freeze-dried groups can be resuspended in solution before or after addition of the sample to be analyzed. In some cases, the human peripheral blood sample may be mixed with heparin prior to mixing with the freeze-dried group. In some cases, the blood sample may be mixed directly into the container containing the freeze-dried group, although this need not always be the case. After mixing the sample with the freeze-dried group, the mixed sample can be incubated for a period of time, washed, and then studied using an elemental analyzer. In some cases, further staining, such as intracellular target staining, may occur prior to the study, which may require permeabilization and fixation of the sample.

In certain aspects, the kit includes an elemental standard (e.g., provided in the kit itself or with other reagents described herein, such as a freeze-dried set and/or barcoded beads). Elemental standards may include microbeads containing known amounts of a variety of different metal isotopes. In certain aspects, elemental standards may comprise microbeads having varying amounts of one or more metal isotopes. For example, the elemental standard microbeads may comprise populations of 2 or more, 3 or more, 4 or more, or 5 or more microbeads, wherein each population comprises a similar amount of each of the plurality of metal isotopes, but the populations differ in the amount of at least one metal isotope. A similar amount of a given isotope within a population can represent a standard deviation of the amount of the given isotope (the number of atoms of that isotope in the beads of the population) can be less than 10%, less than 25%, less than 50%, less than 100%, less than 200%, or less than 300% of the average amount of the given isotope for the beads in the population. A population of beads may have at least one isotope present in a significantly different amount than each other population in the kit (e.g., a significantly different isotope as compared to a first other population may be different from an isotope of a second other population). For example, the difference between the average amounts of isotopes between populations may be greater than 2-fold, greater than 3-fold, greater than 4-fold, or greater than 5-fold of the standard deviation within one or both of the populations.

In certain aspects, the elemental standard microbeads may comprise at least 2, 3, 4, 5, 6, 7, 8, or 9 variable isotopes that differ in magnitude between at least some of the population of microbeads. Isotopes (e.g., variable isotopes) of elemental standard microbeads may cover mass ranges greater than 10, greater than 20, greater than 30, greater than 40, or greater than 50 amu. The at least one isotope may remain constant within all or some of the bead populations, such that the ratio of constant isotope to variable isotope may be used to represent the bead population (e.g., to identify the expected amount of variable isotope in the bead).

The microbeads may have a size of greater than 100nm to less than 1000um, such as between 500nm to 100 um. As such, a microbead can have greater than one thousand, ten thousand, one hundred thousand, or one million atoms per bead. The microbeads can be prepared according to any of the methods described herein for other beads, such as methods of preparing assay and/or sample barcoded beads (e.g., without a step of binding biomolecules, such as binding of SBPs to a surface) or any suitable method.

Elemental standards can be used for calibration of mass cytometry and/or normalization of the signals obtained for the entire sample run. For example, elemental standards can be used to quantify the amount of antibody bound, such as the amount of antibody bound per cell. Elemental standard microbeads can be mixed with cells and run on a mass cytometer (e.g., so that microbead events and cellular events can be detected separately). As such, the mass of at least one isotope in the microbead may be different from any mass tag used for cells.

In certain aspects, the methods comprise calibrating a mass cytometer (at the beginning and/or during a sample run) based on an elemental standard described herein (e.g., a kit comprising an elemental standard). Alternatively or additionally, the method can include normalizing the mass cytometry data based on an elemental standard described herein (e.g., a kit including an elemental standard). For example, isotope signals from cells (i.e., from a particular mass channel) can be normalized to the intensity of isotopes from microbeads having similar masses and/or amounts collected at similar times. For example, the signal from a metal isotope-labeled antibody bound to a specific analyte of a cell can be normalized to the intensity of the isotope signal of most similar mass and intensity obtained over the time window of the cellular event. A standard curve can be generated for different masses and/or intensities to normalize the signal from the cellular event based on the elemental standard microbeads detected within the time window of the cellular event. The time window may be less than 1000 seconds, less than 100 seconds, or less than 10 seconds. Cellular events for elemental standard microbeads of the population for which no acquisition was made within the time window may be discarded from the data set. The cellular events may be normalized based on the most recent microbead events (e.g., based on the signal normalization provided by the most recent microbead events for each microbead population). In certain aspects, the same calibration curve may be used to calibrate all cellular events within a time window.

In certain aspects, the amount (e.g., average number of atoms and optionally deviation) of each of the plurality of isotopes in an element standard microbead (e.g., a population of element standard microbeads) can be known, and the amount of atoms (e.g., average number of atoms and optionally deviation) of a given isotope attached to an antibody bound to a cellular analyte can be known and used together to calculate the antibody bound (e.g., bound to the analyte) for each cell. For example, fractionation and analysis of labeled antibodies can be performed by FPLC (e.g., size exclusion FPLC and/or anion exchange FPLC), and the overall amount of protein can be used to back calculate the amount of antibody, and the entire metal (bulk metal) can be determined by mass spectrometry. Elemental standard microbeads can be examined (e.g., by electron microscopy) to determine size and uniformity. Calibration of the instrument (e.g., across sample runs and/or between sample runs) may further enable quantification of bound antibody.

In certain aspects, the kits or methods with elemental standard microbeads may be combined with other aspects described herein, including freeze-dried mixtures of metal-tagged antibodies and/or assay barcoded beads. For example, elemental standard microbeads (e.g., mixed with lyophilized antibodies) may be provided in the same kit or even container. Alternatively, cells stained with the freeze-dried antibodies described herein can be subsequently combined with elemental standard microbeads prior to analysis by mass cytometry. Normalization (e.g., quantification of antibody bound by each cell) can improve the ability to gate a population of cells, e.g., by the automated gating software described herein.

The following example procedure lists the steps of staining a human peripheral blood sample. Heparin sodium salt was added to blood aliquots requiring staining (10uL 10KU/mL to 1mL blood) at a final concentration of 100U/mL. The mixture was vortexed for 2 seconds and incubated at room temperature for at least 20 min. Antibody mixtures (e.g., lyophilized antibody panels) were prepared in 5ml tubes. A volume of heparin-blocked blood was added directly to the 5mL tube so that the final volume (blood + antibody mixture) was 300 μ L, and the mixture was vortexed for 2 seconds to ensure resuspension of the lyophilized product. The mixture was incubated at room temperature for 30 minutes. Immediately after staining was complete, 250. mu.L of Cal-Lyse lysis solution was added to each tube. Incubate at room temperature in the dark for 10 minutes. To each tube was added 3mL of water. The tube was vortexed for 2 seconds and incubated at room temperature for 10 minutes. The cell suspension will initially be opaque and become translucent after 10 minutes of incubation. If the cell suspension is not completely translucent after 10 minutes, the sample is vortexed again and incubated at room temperature for an additional 5 minutes. The tube was centrifuged and the supernatant poured into a 50mL waste tube. To each tube was added 3mL of cell staining buffer. The tube was centrifuged and the supernatant was poured off. The washing step was repeated 2 times for a total of 3 washes. After each wash, the cell pellet and cell supernatant were visually inspected. If the cell pellet or supernatant is red, the washing is repeated until the supernatant is clear and the cell pellet is white. Then, the cells are fixed and the DNA is inserted. With phosphorus Acid salt buffered saline (PBS) a fresh dilution of 16% formaldehyde was prepared as a 1.6% working solution. To each tube was added 1mL of 1.6% formaldehyde. The tube was vortexed for 2 seconds and incubated at room temperature for 10 minutes. Centrifuge at 800 XG for 5min at room temperature and pour the supernatant. The intercalator was diluted to a final concentration of 125nM in the fixation and permeabilization buffer. To each tube was added 1mL of the intercalator + buffer mixture. The tube was vortexed for 2 seconds and incubated overnight at 4 ℃. The cells can be maintained in formaldehyde at 4 ℃ for up to 48 hours. If desired or available, the immobilized cells can be seeded onto the prepared slide. The cells are then washed prior to sample collection. To each tube was added 2mL of cell staining buffer. The tube was vortexed for 2 seconds and centrifuged at 800 × G for 5min at room temperature. Once centrifugation was complete, the supernatant was decanted. The washing step was repeated 1 time for a total of 2 washes. To each tube was added 2mL of cell harvest solution. The tube was roughly swirled. Cells were counted to determine the final volume of resuspension during harvest. Centrifuge at 800 XG for 5min at room temperature. Once centrifugation is complete, the supernatant is aspirated or pipetted off. The cell pellet was retained and stored at 4 ℃ until ready for harvest. A mixture of 90% cell harvest solution and 10% of 4 elemental calibration beads by volume was generated in a 15mL tube. Once ready for collection, cells were plated in a mixture of cell collection solution +4 elemental calibration beads at 0.5X 10 ⁶One cell/mL to 1.5X 10⁶Concentrations between cells/mL were resuspended. The cell filter cover was removed from the 5mL polystyrene round bottom tube with cell-filter cover (cell-purifier cap) and placed on the 5mL polypropylene round bottom tube. The resuspended cells were filtered through a cell filter cover. Samples were collected on a pre-conditioned ICP-MS instrument. Samples were collected at no more than 600 samples/s. At least 400,000 events should be collected.

As used herein, fixation and permeabilization refers to the creation of pores in the cell membrane by chemical cross-linking of cellular components by reagents such as glutaraldehyde, formaldehyde, formalin, ethanol, methanol, and the like, and detergent. Suitable detergents may be readily selected from non-ionic detergents. Desirably, these detergents are used at concentrations between about 0.001% to about 0.1%. One detergent that may be used is Triton X-100(Sigma T9284). Examples of other suitable detergents include Igepal and Nonidet P-40. Other suitable detergents can be readily selected by those skilled in the art.

Certain aspects of the present disclosure are useful for analyzing whole blood, such as human peripheral blood. In some cases, whole blood may be separated into PBMCs and separated plasma prior to staining with the freeze-dried group. The freeze-dried group can stain PBMCs. In some cases, the sample or PBMCs from the sample, whether before or after staining of the panel by freeze-drying, may be tagged with a sample barcode as disclosed herein. When labeled, the samples can be mixed together prior to investigation by an elemental analyzer. Stained PBMCs can be studied using an elemental analyzer, such as a mass spectrometer.

The data from the elemental analyzer may include data indicating the presence of multiple isotopes in the sample. In particular, the data may include the presence of a detectable isotope of the elemental tag that remains bound to the sample after the sample is washed. The elemental analyzer may study the sample cell-by-cell or particle-by-particle, thus generating data cell-by-cell or particle-by-particle. For example, data from an elemental analyzer may indicate the presence of multiple isotopes for each cell or each sample particle.

During the automated analysis process, the software can decode the elemental analyzer data into useful and readable results. The software may be incorporated into the elemental analyzer or provided separately (e.g., on a separate computing device). Decoding the element analyzer data may result in identifying a variety of element tags detected by the element analyzer, which may be referred to as element tag data. Each elemental tag may be associated with a specific marker, such as an antibody, a viable cell marker, or a sample barcode. These dependencies may be stored as a software accessible element tag location map. The software can then apply the element tag locator map to the element tag data to produce useful and readable results. In some cases, the software may select (e.g., manually or automatically) a specific gating scheme (gating scheme) to use in the interpretation of the element tag data. For example, for a particular freeze-dried antibody panel, a particular gating scheme may be used to help interpret the element tag data, such that the presence or absence of multiple antibodies from the freeze-dried antibody panel may help identify a particular cell type (e.g., activated T helper cells, memory T helper cells, or memory B cells). The software may then output the appropriate results for display or storage in a file.

In some cases, the software may automatically identify the cell type of the sample. The automatic identification of cell types may be based on a predetermined gating of similarly expressed cell populations of a common surface marker subset. Alternatively or additionally, the identification of cell types may also be guided by a clustering algorithm.

In some cases, the software may output the cell type results. Cell type results can include relative quantification of various cell types (e.g., total cell%, parental cell (population of maternal cells%), progenitor cell%, etc.), such as CD4 α β T cells (e.g., total CD4, initial, central memory, effector memory and regulatory); CD8 α β T cells (e.g., total CD8, initial, central memory, effector memory); delta gamma T cells; b cells (e.g., total B cells, initial, memory, resting memory, transitional); an NK cell; (ii) a monocyte; and/or dendritic cells.

In some cases, the software may output marker intensities. Marker intensity may include the intensity (e.g., median intensity) of each cell type's marker and/or any other marker at the discretion of the user. For example, a visual output may be generated that shows the intensity "color" of each cell type in a phenotypic tree. In another example, each result is compared to a user-generated data set containing reportable results (reportable) of frequency, intensity, and cell count, which can be displayed by visual display (e.g., color can be deemed "hot" if the median value is 1.5 sigma greater than normal and color can be deemed "cold" if the median value is 1.5 sigma less than normal).

In some cases, the software may output other results, such as a dot plot of any two markers and/or a histogram of any marker. In some cases, the software can automatically flag any marker having more than one pattern of distribution (e.g., a bimodal distribution). In some cases, the software may output a report text file with the desired reportable results (e.g., frequency, intensity, and cell count). In some cases, the software may maintain a database of reportable results (e.g., frequency, intensity, and cell count). In some cases, the software may output a report in a print quality format with user-selected drawings and tables.

Certain aspects of the present disclosure relate to barcoding samples with elemental tags on a per sample and optionally per assay basis. As disclosed herein, elemental tags can be unique and distinguishable based on their isotopic composition. For example, an element tag (or combination of element tags) used in a sample or assay barcode may have a unique isotope or unique combination of isotopes that distinguishes it from other element tags. Thus, when an assay barcode is identified during elemental analysis of a sample, the following inference can be made: the particular cells or particles under study have undergone assays related to assaying barcodes (e.g., treatment/stimulation conditions, staining with specifically labeled SBP panels, etc.). Notably, the assay and/or sample barcodes can be used to identify particle doublets (and exclude data from) comprising two or more assay barcodes or two or more sample barcodes.

Assay barcoding may be accomplished by binding analytes in a sample to an assay barcode associated with a particular target analyte being detected. The assay barcode may comprise a distinguishable combination of a solid support (e.g., an assay bead), a capture biomolecule (e.g., an assay biomolecule that specifically binds to a target analyte in a sample and to the surface of the assay bead), and an isotope associated with the target analyte (assay barcode). The solid support can be a planar surface/slide (e.g., containing an assay barcoded array) or an assay barcoded bead. The assay biomolecule may be an oligonucleotide (e.g., which specifically hybridizes to a target RNA or DNA), an affinity agent (e.g., an antibody, aptamer, or lectin), or a substrate (e.g., a peptide comprising an element tag that is cleaved in the presence of a target enzyme, such as a target protease). The assay biomolecules may be bound to the assay beads such that different assay biomolecules (e.g., assay biomolecules bound to different target analytes) are bound to beads having different assay barcodes. As described herein, a reporter molecule (reporter) can provide for the detection of an assay biomolecule. The reporter molecule may comprise a reporter biomolecule (e.g., an oligonucleotide or an antibody) that binds (directly or indirectly) to the target analyte. The reporter molecule can also include an elemental tag for detecting the presence of a target analyte bound to the assay bead (e.g., by binding to an assay biomolecule that is itself bound to the bead).

In general, the assay biomolecule and reporter molecule may provide improved specificity, as both need to bind to the target analyte to provide a signal. Furthermore, by determining that the biomolecule-bound analyte will be present on the surface of the bead (or spaced apart from the surface), the reporter molecule with the large element tag can still bind to the target. As such, the signal amplification methods described herein may be used to detect a target analyte.

As used herein in the context of mass cytometry, signal amplification is the binding of greater than 30, greater than 50, greater than 100, greater than 200, or greater than 500 labeled atoms (e.g., enriched isotopes) to a target analyte (i.e., a single instance of a target analyte bound by a specific binding partner). In certain aspects, the tagging atom may be a heavy metal, such as a lanthanide or transition metal. In certain aspects, signal amplification may be performed for greater than 2, 5, 10, or 20 target analytes. In certain aspects, signal amplification may include the use of branched conjugation of mass tags to biomolecules, high sensitivity polymers, large mass tag particles, mass tag nanoparticles, hybridization schemes where multiple element-tagged oligonucleotides (e.g., including multiple instances of the same element tag) bind to a target, and/or enzymatic deposition of multiple element tags. In certain aspects, signal amplification uses a mass tag polymer as described herein.

The mass-tagged oligonucleotide may be directly or indirectly hybridized to the target oligonucleotide. For example, one or more intermediate oligonucleotides may provide a scaffold on which a plurality of mass-tagged oligonucleotides can be hybridized, thereby amplifying the signal. Accordingly, aspects of the subject patent application include oligonucleotides for hybridization-based signal amplification.

The target oligonucleotide may be a DNA or RNA molecule (e.g., coding RNA, small interfering RNA, or microrna) endogenous to the cell. The target oligonucleotide may be single stranded. The target oligonucleotide may have a known specific sequence (or homology to a known specific sequence). In certain aspects, biomolecules, such as antibodies or derivatives thereof, may be conjugated to target oligonucleotides, thereby being conjugated to synthetic single-stranded DNA oligonucleotides comprising known sequences. In these cases, both the antibody and the oligonucleotide may be referred to as biomolecules.

After binding of the biomolecule to the analyte in the sample, the plurality of mass-tagged oligonucleotides may be directly or indirectly hybridized to the first oligonucleotide. The hybridization may be branched or linear. In certain aspects, the polymerase can extend the first oligonucleotide along the template to provide additional sites for element-tagged attachment (e.g., additional hybridization sites for element-tagged oligonucleotides). The mass-tagged oligonucleotide may comprise a single labeling atom, or may comprise a polymer comprising multiple labeling atoms. The mass-tagged oligonucleotide may comprise a labeling atom, such as a heavy metal atom, in the chemical structure of the oligonucleotide itself.

Signal amplification may be uniquely beneficial for bead-based assays, where the same reporter tag (labeled metal element or isotope) may be amplified and used in different beads and their target analytes. In certain aspects, mass-tagged oligonucleotides can be mass-tagged with the high-sensitivity polymers or nanoparticles described herein, in addition to use in signal-amplification hybridization protocols.

In certain aspects, the assay beads may be customizable such that a user may assay a biomolecule of interest for each assay barcoded bead. Such attachment may be through a chemical bond, or may be through specific assay barcoded beads that localize the assay biomolecule to the same mixture. Such localization may be carried out by providing assay barcoded beads which provide oligonucleotide sequences unique to each assay barcode, and a user may attach different localizing oligonucleotides to different biomolecules to attach to the bead surface, wherein the localizing oligonucleotides specifically hybridize to one of the unique oligonucleotide sequences.

In the case where the assay biomolecule is an oligonucleotide, the element-tagged reporter oligonucleotide may hybridize to another portion of the target RNA or DNA, thereby providing a signal when the target RNA or DNA is bound to the bead. In the case where the assay biomolecule is an affinity reagent, such as an antibody that binds the analyte at a first epitope, the element-tagged reporter affinity reagent (e.g., reporter antibody) may bind to another epitope on the analyte, thereby providing a signal when the target analyte binds to the bead. The analyte may also be bound by a reporter molecule, such as an element-tagged reporter antibody or oligonucleotide. The reporter molecule can include a highly sensitive (e.g., intensity) elemental tag that provides a highly abundant isotope (e.g., greater than 50, 100, 200, 500, 1000 copies of a single isotope), thereby enabling detection of a smaller number of target analytes bound to the assay beads. Such high sensitivity element tags may include nanoparticles (e.g., metal nanocrystal surfaces comprising functionalization to bind biomolecules, such as antibodies or oligonucleotides) or hyperbranched polymers. For example, multiple reporter biomolecules containing the same gold nanoparticle element tag will provide high signals and take advantage of the fact that: individual reporter biomolecules (e.g., beads that provide their specific analytes) comprising the same elemental tag can be distinguished by measuring the barcode. In certain aspects, nanoparticle labels (e.g., gold nanoparticles) can be bound to the reporter probes via biotin-avidin (e.g., biotin-streptavidin) interactions. For example, nanoparticles (e.g., gold nanoparticles) can be conjugated to streptavidin. The reporter molecule may also include a low sensitivity elemental tag that provides a low abundance isotope (e.g., less than 100, 50, 30, 20, 10, or 5 copies of the isotope) that is different from the high abundance isotope, thereby allowing detection/quantification of the amount of analyte that is too high in abundance, so that the high abundance isotope will saturate the detector. In certain aspects, the high and low abundance isotopes have a mass difference (e.g., greater than 5, 10, 20, 30, 40, or 50amu) such that saturation of the detector by the high abundance isotope does not affect detection of the low abundance isotope. The reporter molecules of different analytes (e.g., antibodies comprising target analytes bound to different assay beads) may comprise the same isotope or combination of isotopes, as the analytes will be distinguished by the unique assay barcodes of the beads.

In certain aspects, the reporter molecule can include a reporter system that provides signal amplification through the binding of multiple instances of an element tag to a single instance of a target analyte. Signal amplification can be by enzymatic deposition, hybridization (e.g., branched chain hybridization, strand hybridization, and/or hybridization of multiple reporter oligonucleotides to a single long intermediate oligonucleotide), extension (e.g., single extension, rolling circle extension), and/or a series of branched chain conjugation. In certain aspects, multiple (e.g., all) analytes detected by the assay beads can be detected by the same reporter system. In certain aspects, the signal amplification reporting system may have a high sensitivity element tag.

For example, an elemental tag comprising an enzyme substrate moiety can be deposited from solution onto a bead (or a molecule attached to a bead) by an enzyme attached to a reporter biomolecule. This reaction may be by covalent binding of a tyramine element tag via the action of horseradish peroxidase bound to a reporter biomolecule.

Aspects include hybridization protocols whereby multiple element-tagged oligonucleotides hybridize indirectly (via one or more oligonucleotide intermediates) to a single oligonucleotide target. For example, the oligonucleotide target may be a target RNA or DNA (e.g., gDNA or cDNA) sequence, or may be an oligonucleotide present on a reporter antibody.

As described herein, mass cytometry enables sufficient detection channels (mass channels) to detect both the sample in the bead and the assay barcode, while allowing other channels for reporter molecules (e.g., for detecting assay targets). As such, the bead assays described herein can be samples for mass cytometry and/or assay barcodes. For example, a variety of different conditions (e.g., drug candidates, such as an enzyme or an agonist or antagonist of one or more enzymes) can be applied to a biological sample, and its effect on a variety of targets can be detected by enzymatic assay beads. Each condition can be identified by a sample barcode common to different assay beads exposed to the same condition. Assay barcoded beads can be incorporated prior to analysis, e.g., prior to exposure to conditions. The sample barcoded beads may be combined prior to analysis.

In certain aspects, the enzyme may be a protease, a kinase, a phosphatase, or a DNA modifying protein, such as a DNA methyltransferase. The target may be a substrate acted upon by an enzyme, and the reporter molecule (e.g., a reporter biomolecule as described herein) may only bind to the target before or after its action by the enzyme. For example, a phosphorylation-specific antibody detects a phosphorylated form of a protein target, the abundance of which can be increased when acted upon by a kinase or decreased when acted upon by a phosphatase. When the enzyme is a protease, the reporter molecule can bind to the end of the peptide substrate and be removed from binding to the bead when the substrate is cleaved (so that a decrease in the reporter label indicates an increase in protease activity).

The sample barcode may be used to indicate which of several enzymes (or agonists/antagonists thereof) are tested in a particular assay. For example, candidate enzymes, agonists, antagonists may be added to a biological fluid, such as a cell lysate, and the sample then contacted with an assay barcoded bead to detect the activity of the enzyme. Alternatively, the candidate may be administered to a cell (e.g., directly or by genetic engineering) or organism, such as a patient or mammalian test subject, and a sample taken from that source may be contacted with the assay beads. Sample barcoding allows for simultaneous screening of multiple candidates. In either case, a sample barcode may be added to identify candidates as described herein for beads. For example, greater than 10, greater than 20, greater than 50, greater than 100, greater than 500, or greater than 1000 different samples can be barcoded. For example, a unique combination of 6 of 12 different isotopes provides 924 different combinations (e.g., for barcoding of up to 924 samples). Another 12 different isotopes can be used for barcoding nearly 1000 assays. As such, greater than 10, greater than 20, greater than 50, greater than 100, greater than 500, or greater than 1000 different assay bead formats can be coded (e.g., beads that detect the amount of different substrates acting through a candidate). As described herein, at least one channel will be retained for detection of a substrate by a reporter molecule. This may allow unprecedented screening using direct reading by mass cytometry.

Post-translational modification of proteins is performed by enzymes within living cells. Known post-translational modifications include protein phosphorylation and dephosphorylation, as well as methylation, prenylation, sulfation, and ubiquitination. The presence or absence of phosphate groups on proteins, particularly enzymes, is known to play a regulatory role in a variety of biochemical and signal transduction pathways.

Bead-based kinase assays for mass cytometry are discussed in U.S. patent publication US20070190588, which is incorporated by reference and summarized below. However, such bead-based assays have not been proposed for both sample and assay barcoding, which provides a screening advantage and is uniquely achievable by high multiplexing of mass cytometry.

The function of kinases is to transfer phosphate groups (phosphorylation) from high energy donor molecules, such as ATP, to specific target molecules (substrates). Enzymes that remove phosphate groups from targets are called phosphatases. The largest group of kinases are protein kinases, which act on specific proteins and alter their activity. A number of other kinases that act on small molecules (lipids, carbohydrates, amino acids, nucleotides, etc.) are commonly named for their substrates and include: adenylate kinase, creatine kinase, pyruvate kinase, hexokinase, nucleoside diphosphate kinase, thymidine kinase.

Protein kinases catalyze the transfer of phosphate esters from Adenosine Triphosphate (ATP) to serine, threonine, or tyrosine residues of targeted peptide or protein substrates. Protein kinases are distinguished by their ability to phosphorylate substrates on various sequences. Commercially available kinases can be in an active form (phosphorylated by the supplier) or in an inactive form and require phosphorylation by another kinase.

Protein phosphatases hydrolyze phosphomonoesters at phosphoserine, phosphothreonine, or phosphotyrosine residues to phosphate ions and protein or peptide molecules having free hydroxyl groups. This effect is directly opposite to protein kinases. Examples include: protein tyrosine phosphatases, alkaline phosphatases, serine/threonine phosphatases and inositol monophosphatase enzymes that hydrolyze phospho-tyrosine residues.

Another aspect is to provide a method for phosphatase determination comprising: incubating a plurality of element-labeled supports having metal ion coordination complexes attached thereto in a plurality of solutions, each solution comprising a different free phosphorylated substrate labeled with an element tag (which may optionally be the same element tag for all substrates), in such a way that each type of phosphorylated substrate labeled with an element tag (which may optionally be the same element tag for all substrates) is attached to a single type of element-labeled support; separating free phosphorylated substrate from bound substrate attached to metal ion coordination complex (metal ion coordination complex attached to macroelement labeled support) in a plurality of separate solutions; incubating a plurality of element-labelled carriers having attached thereto a plurality of phosphorylated substrates labelled with an element tag (which may optionally be the same element tag for all substrates) by attachment to a metal ion coordination complex attached to the carriers in a single solution with ADP and at least one phosphatase under conditions such that the phosphatases are capable of dephosphorylating the phosphorylated substrates; separating free non-phosphorylated substrate from bound phosphorylated substrate labeled with an element tag (the element tag may optionally be the same element tag for all substrates) attached to a metal ion coordination complex attached to a macroelement labeled support; and performing a particulate elemental analysis on the bound phosphorylated substrate labeled with an element tag (the element tag may optionally be the same element tag for all substrates), the element tag being attached to a metal ion coordination complex, the metal ion coordination complex being attached to a macroelement-labeled support.

Another aspect of the applicant's teachings of the present invention is to provide a kit for detecting and measuring an element in a sample, wherein the element being measured comprises an element tag attached to a phosphorylated substrate, a metal ion coordination complex element, and a uniquely labeled carrier element, comprising: an element tag for direct tagging of a phosphorylated substrate; a plurality of phosphorylated substrates; a uniquely labeled vector; a metal ion coordination complex; and optionally, a phosphatase buffer, and ADP.

Another aspect of applicants' teachings of the present invention is to provide a method for a kinase assay comprising: incubating ATP, at least one kinase, a free metal ion coordination complex, and a plurality of non-phosphorylated substrates immobilized on an element-labeled support under conditions that enable the kinase to phosphorylate the substrates in a manner that the single type of non-phosphorylated substrate is attached to the single type of element-labeled support; separating the plurality of phosphorylated substrates immobilized on the element-labeled support with the attached metal ion coordination complex from the free metal ion coordination complex and the plurality of immobilized non-phosphorylated substrates; and measuring a quantity of phosphorylated substrate immobilized on an element-labeled support having an attached metal ion coordination complex by elemental analysis.

Another aspect of the applicants' teachings is to provide a kit for detecting and measuring an element in a sample, wherein the element being measured comprises an element tag attached to a non-phosphorylated substrate and a metal ion coordination complex comprising: an element tag for direct tagging of a non-phosphorylated substrate; a non-phosphorylated substrate; a solid support; a metal ion coordination complex; and optionally, a kinase; a kinase buffer; and ATP.

As discussed herein, the carrier may be encoded beads that are striped as a sample.

Due to their role in maintaining homeostasis in animals, enzyme assays and pharmacological regulation have become key elements in the identification of potential therapeutic agents. Proteases are a subset of proteolytic enzymes that have recently been shown to play an important role in signal transduction pathways, and dysregulation of which can lead to cancer, cardiovascular disease, and neurological disorders. Of about 400 known human proteases, tens are being investigated as potential drug candidates. Small-molecule inhibitors of proteases are currently considered to be valuable therapeutic leads for the treatment of degenerative diseases, for cancer treatment and as antibacterial, antiviral and antifungal agents.

Bead-based protease assays for mass cytometry are discussed in U.S. patent publication US20170023583, which is incorporated by reference and summarized below. However, such bead-based assays have not been proposed for both sample and assay barcoding, which provides a screening advantage and is uniquely achievable by high multiplexing of mass cytometry.

There remains a need for robust, sensitive and quantitative enzyme assays that allow simultaneous measurement of multiple enzymatic reactions. The assay may allow retention of valuable biological samples and reagents, achieve high throughput and reduce assay time, and reduce the overall cost of the enzymatic assay.

One aspect of the invention is a method for detecting protease activity in a biological fluid. The method may comprise attaching the encoded bead to a first amino acid of a peptide substrate to form an immobilized peptide substrate comprising the first amino acid and a last amino acid and being a substrate for a protease: attaching an element tag to the last amino acid of the peptide substrate to form a tagged peptide substrate: incubating the immobilized, tagged peptide substrate with a biological fluid: and detecting the elemental tag and the encoded bead in the biological fluid by elemental analysis. As discussed herein, the encoded beads can be assay and sample barcoded.

The protease assay kit may include an assay-encoding bead of a first amino acid attached to a peptide substrate (immobilized peptide substrate) that may include the first amino acid and a last amino acid and may be a substrate for a protease. The element tag may be attached at or near the last amino acid of the peptide substrate to form a tagged peptide substrate. As discussed herein, the encoded beads can be assay and sample barcoded.

The mixture of assay beads may together target at least 5, 10, 20, 50, 100, 200, 500, 1000 or more analytes. In certain aspects, the sample barcode can distinguish assay barcoded beads and/or cells from at least 5, 10, 20, 50, or 100 or more different samples.

In certain aspects, the number and/or size of barcoded beads can be determined by the change in the analyte for which they are specific. For example, an assay barcoded bead that specifically binds a high abundance target analyte may have fewer, or a greater number of, antibodies on its surface than an assay barcoded bead that specifically binds a low abundance target analyte in the same sample. For example, in a mixture of assay barcoded beads, the abundance of assay barcoded beads specific for a first target analyte may be 2, 5, 10, 20, 50, or 100 times that of assay barcoded beads specific for a second target analyte. When the abundance of the first analyte is greater than the second analyte, it will be diluted in the large number of assay barcoded beads to which it is bound. The assay barcode kit may comprise one or more assay barcoded beads and/or reporter molecules as described herein.

Barcoding of each sample can be achieved by barcoding the sample with the sample associated with the particular sample. Thus, when a sample barcode is identified during elemental analysis, it can be inferred that the particular cell or particle under study is part of a particular sample. Thus, multiple samples can be mixed together at any time between application of the sample barcode and the use of elemental analysis studies, without losing the ability to correlate the cells or particles of interest with their respective samples. By allowing the samples to mix together, the elemental analyzer can be run at higher throughput and can simplify the handling and storage of the samples.

The sample barcode may include an element tag coupled to an element-tagged moiety (e.g., an assay barcoding reagent or a sample barcoding reagent) that has a targeting function for all, a majority, or a majority of or all of the cells or particles of the sample. As used herein, the term substantial portion may include at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells or particles of the sample.

In some cases, the barcoding reagent may be a bead capable of containing or being coupled to an element tag. In some cases, the unique assay barcode may be located within a bead and the surface of the bead may contain, be coupled to, or functionalized to couple to the unique sample barcode. Any bead suitable for containing or coupling to an element tag, such as a polymer-assisted surface functionalized bead, may be used. Such a polymer may be poly-L-lysine, PEG (polyethylene glycol), PEG MEA (methyl methacrylate), PVMS (methyl vinyl polysiloxane), polydopamine, polystyrene and/or another polymer or derivative known to those skilled in the art. In some cases, the bead may be functionalized with a first functional group for binding to a moiety with targeting functionality (e.g., an antibody) and a second functional group for binding to a sample barcode. However, in some cases, the beads may be functionalized with only a single functional group capable of binding both the moiety with targeting function and the sample barcode. In some cases, a blocking agent (e.g., albumin) can be used to control the attachment of the moieties and sample barcodes to the beads. In some cases, reactive functional groups (e.g., thiol, amine, thiol-reactive, amine-reactive or click chemistry functional groups, or even highly reactive functional groups such as isothiocyanates) can be used to help label beads blocked with blocking agents, provided that the functional groups react with the cell surface or the cells are permeabilized prematurely. These functional groups may be located on the sample barcode to aid in the labeling of the barcoding reagent or the sample itself. Alternatively, free metal (e.g., a set of sample barcode isotopes) may be provided in solution or may be bound by chelating groups on the bead surface. In certain aspects, the beads may be functionalized to bind to (or be bound by) the same sample barcoding reagent as used to barcode cells in the assay. In certain aspects, the combination of isotopes in the sample barcode may be the same for beads and cells from the same sample. In some cases, the assay beads may be blocked (e.g., using a blocking reagent, such as BSA). In these cases, the sample barcode may be bound to the blocking reagent.

Sample barcoding reagents for cells can include element-tagged antibodies (that bind to multiple cell types or multiple cells in a sample), element tags functionalized to non-specifically bind to cells (e.g., by covalent interactions), and/or metals in solution. The sample barcoding reagents for the cells may also include reagents for barcoding the sample into the cells (e.g., DMSO, cell permeabilizing reagents such as detergents or ethanol, etc.). Sample barcoding reagents for assaying barcoded beads may be present within the beads, on the surface of the beads or may be coated onto the beads. If used for application to beads, the sample barcoding reagent may comprise a functional group as described herein to bind to the surface of the bead (e.g., to a functional group provided by the bead or a blocking reagent present on the bead surface). The sample barcoding reagent for a given sample may include a unique combination of isotopes specific for that sample. In certain aspects, cells from the same sample (e.g., a single blood sample) can be labeled with the same assay barcode and the assay barcoded beads. The same assay barcode used for labeling of cells and beads may contain the same combination of isotopes and/or the same means of attachment (e.g., functional groups).

The sample barcoding reagent may be provided in a mixture or with a lyophilized antibody set. The sample barcoded reagents may be provided in a mixture or with assay barcoded beads. Assay barcoded beads may be provided in a mixture or with a freeze-dried antibody panel. The assay barcoded beads and sample barcoded reagents may be provided in a mixture or with a set of lyophilized antibodies (e.g., when the sample barcoded reagents bind both the assay barcoded beads and the cells in the sample). In certain embodiments above, the sample barcoding reagents may be provided in, on or with the assay barcoded beads.

In some cases, highly reactive functional groups (e.g., thiol, amine, thiol-reactive, amine-reactive, or click chemistry functional groups) can be used to facilitate cell labeling using the sample barcode. These functional groups may be located on the sample barcode to help label the barcoding reagent or the sample itself. Alternatively, free metal (e.g., a set of sample barcode isotopes) may be provided in solution, which may be administered to cells (e.g., in the presence of reagents such as alcohols, detergents, and/or DMSO to allow the metal to enter the cells).

In some cases, the element tags used to determine barcodes and sample barcodes may include elements and/or isotopes that are not commonly used by element-tagged freeze-dried groups or are outside the lanthanide family. For example, the assay barcode and/or the sample barcode may be barcoded using Pd or Te. In some cases, the element tag used to determine the barcode and/or the sample barcode can be a cadmium element tag containing a cadmium isotope chelated to a polymer (e.g., a DOTA group chelated to a polymer) or bound to a cadmium binding protein.

In some cases, kits may be provided that contain barcoded reagents, such as beads, in a single container and in separate containers that each contain a unique sample barcode. The barcoding reagent may or may not include an assay barcode. The user can mix separate volumes of these barcoded reagents with different samples to be tested. Each sample may then be mixed with a unique sample barcode, allowing the sample barcode to bind to barcoded reagents already in or bound to the sample and/or to cells or particles of the sample itself. After washing, such sample-barcode-labeled samples will include barcoded reagents all comprising the same sample barcode, but with or without the assay barcode. Thus, such sample barcodes may be used to identify the cells or particles of such particular sample. The same process can be carried out with other samples and other sample barcodes, thus resulting in a batch of multiple sample-barcode-tagged samples, wherein each sample is tagged with a unique and distinguishable barcode. Alternatively, in some cases, the sample barcode may be used without a separate barcoding reagent, as the sample barcode itself may directly target cells or particles of the sample.

In some cases, the relationship between a particular sample and its sample barcode may be stored, such as in a location map. Such sample barcode information can be accessed by research or analysis software to automatically attribute elemental analyzer data or other results to the appropriate sample based on the detected presence of the sample barcode.

In some cases, a reporter reagent may be provided along with an assay reagent and/or for detecting the presence of an analyte bound to the assay reagent. The reporter reagent may bind directly (specifically or non-specifically) to the analyte in the sample before or after binding of the analyte by the assay beads. The reporter agent may comprise a biomolecule such as an oligonucleotide (e.g., an oligonucleotide that hybridizes to target RNA or DNA of an assay barcoded bead) or an affinity agent (e.g., an antibody, lectin, or aptamer). Such reporter biomolecules can be used to help identify the presence of barcoded reagents (e.g., beads) and/or target particles during the study. The reporter reagent may include an elemental tag that is common to all reporter reagents of the same subject (e.g., all reporter reagents of a bead or all reporter reagents of a particular target analyte), but distinguishable from other elemental tags (e.g., a sample barcoded elemental tag). Each reporter agent can be associated with a particular object, such as a barcoded reagent (e.g., a bead) or a target analyte. The reporter reagents may include antibodies or other moieties that have targeting functions for their associated subjects. Thus, when a reporter reagent is mixed with other reagents (e.g., barcoded reagents) or samples, the reporter reagent can tag their associated object with the elemental tag of the reporter reagent. When the elemental signature is detected during a study by elemental analysis, it can be inferred that the particle under study is of a type associated with a particular reporter reagent. For example, if an element tag associated with a reporter reagent of a barcoded bead is detected, it can be inferred that the object being detected at that time, and thus the element detected within a particular window before and after that time, is associated with the barcoded bead (e.g., is an isotope associated with the element tag within or on the bead). As used herein, a cell-type reporter reagent can be an antibody form of a lyophilized antibody panel.

The reporter biomolecule may comprise an elemental tag (e.g., a series of one or more isotopes on a polymer chain or embedded within or on a bead). In some cases, a reporter reagent (e.g., a reporter antibody) can be specific for a free analyte associated with a particular assay reagent (e.g., bound by an assay reagent, such as an assay antibody present on the surface of a bead). The reporter antibody and the assay antibody (e.g., an antibody coupled to an assay bead) can be the same type of antibody or can be different types of antibodies. In some cases, if the reporter antibody is different from the assay antibody (e.g., if a polyclonal antibody is used), a reporter antibody can be selected that does not interfere with the binding of the assay antibody to the target analyte. In some cases, the reporter reagent and the assay reagent may be generated from two different antibodies (e.g., monoclonal antibodies), although this need not always be the case.

When multiple different assay reagents are used for multiple target analytes, multiple sets of reporter reagents can be generated and/or used, each set containing the same elemental tag (e.g., non-isotopically different elemental tags), but different reporter antibodies coupled to the elemental tags. Thus, while multiple reporter antibodies of a set of reporter reagents can target multiple different target analytes, they will all use the same elemental tag associated with all reporter reagents to tag those multiple target analytes. Alternatively or additionally, the reporter reagent may comprise high-sensitivity and low-sensitivity elemental tags, as further described herein.

In one example, a kit can be provided having a set of 10-12 different barcoding reagents, the barcoding reagents can be provided each with a unique assay barcode, a set of 6 different elemental tags can be provided separately for subsequent combination with the barcoding reagents and/or sample, a barcoding reagent reporter can be provided that is included with the barcoding reagents or separate from the barcoding reagents and has its own unique elemental tag (e.g., a reporter that targets a barcoding reagent) and a set of 30-40 cellular reporters can be provided with each cellular reporter having a unique elemental tag (e.g., a reporter that targets a specific cell type, respectively). The number of each reagent, barcode and/or reporter molecule can be adjusted as desired.

In some cases, the barcode reagent and/or barcode may be freeze-dried and included in a freeze-dried group, such as the freeze-dried groups described herein.

In some cases, the barcoded reagents may be provided in a preconfigured form by preparing the barcoded reagents with some unique combination of the assay barcode and the sample barcode. In this case, each unique barcoded reagent may be stored in a different container, such as a different well of a well plate. In one example, a well plate can be set up such that all wells along a particular column (or row) share the same assay barcode, whereas all wells along a particular row (or column) share the same sample barcode. In another example, a well plate can be set up such that each filled well contains barcoded reagents with a particular unique sample barcode and a different combination of multiple assay barcodes. Thus, the first well may contain barcoding reagents each having a first sample barcode but a different assay barcode, and the second well may contain barcoding reagents each having a second barcode but a different assay barcode. In some cases, pre-configured barcoded reagents may require the production of thousands of unique sets of beads.

In some cases, barcoding reagents (e.g., beads) can be provided in a semi-configured form by preparing a barcoding reagent having a unique assay barcode and a surface functionalized to bind the sample barcode. In these cases, each set of barcoding reagents may be coupled to a moiety (e.g., an antibody) having a targeting function associated with the assay, while the assay is associated with the assay barcode of that set of barcoding reagents.

When a semi-configured (semi-configured) barcoded reagent is provided, the sample barcode may be bound to the barcoded reagent prior to mixing the barcoded reagent with the sample. In one example, different barcoding reagents may be mixed together and then placed in a set of receptacles (e.g., wells in a well plate). A unique sample barcode may then be added to each container, and the results may be mixed with the unique sample to perform an assay-barcode-identifiable assay on the sample and simultaneously label the sample with the sample barcode.

When a semi-configured barcoded reagent is provided, the sample barcode may be bound to the barcoded reagent after the barcoded reagent is mixed with the sample. In one example, the semi-configured barcoding reagents may be provided together or otherwise mixed together. A barcoding reagent may then be added to each of the set of samples. Separately, a unique sample barcode may be mixed with each of a set of samples before or after addition of the barcoding reagent. The sample barcode may label the barcoded reagents and/or cells or particles of the sample.

In an example case, the barcoding reagent may comprise an assay barcoded bead functionalized with polydopamine for attachment of a capture antibody. Another molecule (e.g., avidin) can be added as well as the capture antibody. After addition of the capture antibody to the assay barcoded beads, the beads may be mixed and aliquoted for each sample. For sample barcodes, a unique combination of elemental tags functionalized (e.g., with biotin) to bind the molecules may be added.

In some cases, the sample barcode may be coupled to a reporter molecule such that the reporter molecule is capable of functioning as both a reporter molecule and a sample recognition molecule. For example, an antibody used to report a particular cell type may also act to identify the sample to which the cell type is associated. In this case, multiple copies of the antibody and its unique element tag may be generated, with each copy receiving a unique sample barcode. Each copy may then be provided to each of a plurality of samples. After washing and investigation, detection of the unique sample barcode may indicate the presence of a particular sample, and for that antibody, detection of the unique element tag may indicate the presence of its target in that sample. In some cases, multiple antibody sets (e.g., freeze-dried antibody sets) may be generated, where each antibody set is associated with a particular sample barcode by coupling each antibody of the set to a sample barcode that shares the same unique isotope or combination of the same unique isotopes. In use, each sample-barcoded antibody set can be combined separately with a different sample, and then the samples can be washed and mixed together before being studied using elemental analysis.

In some cases, the sample-specific antibody set may include antibodies with elemental tags such that, after staining with the sample-specific antibody set, the combination of isotopes present in the sample is unique to that sample. In other words, the sample barcode may be introduced into the antibody panel by partitioning unique isotopic combinations among the plurality of antibodies of the antibody panel.

In certain aspects, a live cell barcode (e.g., a thiol-reactive tellurium-based barcode or an element-tagged antibody that broadly expresses a surface marker) can be used, which can increase the benefit of also barcoding live cells in a sample (e.g., fresh blood). For example, a live cell barcode may include an element-tagged antibody that specifically binds CD 45. This method can be performed in conjunction with stimulation of living cells (e.g., PBMCs) or another treatment. In some cases, the sample barcode may be capable of barcoding live cells. In some cases, the sample barcode may be non-destructive to living cells, e.g., non-toxic to living cells.

Certain aspects of the present disclosure may be useful for applications other than cell counting. For example, both the assay and the sample barcode may be encoded in oligonucleotides attached to antibody-bound beads, and a reporter antibody with oligonucleotides may be used to detect analytes bound to the beads. The beads can be separated in the droplets and from the reaction product formed from the assay/sample barcode oligonucleotide on the bead and the oligonucleotide on the reporter antibody. Such reaction products can be detected by sequencing, allowing for the detection of higher numbers of samples and the determination of barcodes.

Certain aspects of the present disclosure allow for benefits not available with existing assays, such as deep immunotyping; t cell profiling; extensive coverage of other immune lineages due to the ability to capture cell frequency for other leukocytes (e.g., B cells, NK cells, dendritic cells, and monocytes); maximization of the amount of information at the single cell level, in particular using all markers combined and thus without the need to make inferences between multiple tubes; ability to control the degree of technical differentiation by using sample barcodes; the ability to delve into the function of interest by adding any group or subset of groups required for the function of interest; the ability to provide a proven mixture of antibodies that covers the needs of most consumers, thus reducing or eliminating the time and effort wasted in validating a slightly different mixture; and the ability to adjust data analysis for specific purposes by enabling rapid understanding without operator bias.

In the case of example use, multiple enterprises compete first to market next generation products or marker amplifications. Their strategy is to conduct early clinical trials in immuno-oncology (I/O) as "basket trials" (basketrials) in which single or combination therapies are applied to multiple indications (e.g., cancer types) and patients are scrutinized for weak signs of clinical benefit. In these accelerated assay settings, each sample is particularly valuable and it is crucial to maximize the amount of information for each analytical method, including flow cytometry. In addition, adverse events of the immunotherapy category are immune-mediated, with potential outcome information present in comparison of baseline to treated immunophenotypes. When used in combination, these adverse events are compounded, as in this class is almost universally accepted as a future standard, and a large number of clinical trials have been initiated to test those combinations. Accordingly, aspects of the present disclosure are particularly useful for these enterprises.

In the case of example use, the contract research organization has served the clinical trials of multiple enterprises, including the selected execution channel of at least most, if not all, flow cytometry tests. Aspects of the present disclosure may be particularly useful for these research organizations, particularly due to the cost and time efficiencies achieved, as well as the long-term stability of the freeze-dried groups as described herein.

In another example use case, a large cancer research center may be used as a phase I/II clinical trial site, but patients are usually treated with approved drugs. These centers can gather additional information to support research in developing new biomarkers and diagnostic tests. Aspects of the present disclosure may be particularly useful for these research centers, particularly due to the cost and time efficiencies achieved, as well as the long-term stability of the freeze-dried groups as described herein. In some cases, certain aspects of the present disclosure may be used as part of laboratory-developed tests that may be validated and used in their institutions.

These illustrative examples are provided to introduce the reader to the general subject matter discussed herein and are not intended to limit the scope of the disclosed concepts. The following section describes various other features and examples with reference to the drawings in which like numerals represent like elements, and directional descriptions are used to describe the illustrative embodiments, but similar to the illustrative embodiments, should not be used to limit the present disclosure. In this document, elements included in the description may not be drawn to scale.

FIG. 1 is a schematic illustration of an element tagged portion 102 in accordance with certain aspects of the present disclosure. The element-tagged portion 102 shown in fig. 1 is an antibody 104 that is tagged with an element tag 106, although any suitable portion may be used when tagged with an element tag 106. The element tag 106 may be an isotope or combination of isotopes. In some cases, the element tag 106 can be a polymer chain having a variety of metal-containing pendant groups, such as some chelating groups. In some cases, multiple copies of a unique isotope may be contained within a single element tag 106. In some cases, multiple copies of a unique isotope combination may be contained within a single element tag 106, although this need not be the case.

The element-tagged portion 102 can be part of a freeze-dried antibody panel as disclosed herein. The antibody 104 may be any suitable antibody having a targeting function for a target of interest. By appropriately mixing the element-tagged portion 102 with the sample, the antibody 104 can bind to any target in the sample, thus labeling or tagging the sample with the element tag 106. This stained sample can then be studied using elemental analysis after washing to remove any unbound elemental tags. Detection of the unique isotope or combination of unique isotopes by the element analyzer can indicate the presence of the element tag 106, and thus the element tagged portion 102, and thus the presence of any target to which the element tagged portion 102 binds.

Fig. 2A is a schematic diagram of a barcoding reagent 208 and a separate sample barcode 212 in accordance with certain aspects of the present disclosure. The barcoded reagents 208 may be beads 214, although other barcoded reagents may be used. The beads 214 may contain an assay barcode 210 located within the beads 214, such as incorporated into the core of the beads 214. The assay barcode 210 may be a type of elemental tag that includes a unique isotope or unique combination of isotopes that are distinguishable by elemental analysis. In some cases, the bead 214 may include a surface 216 that contains, binds, or is functionalized to bind a plurality of moieties, such as a sample barcode 212, a capture antibody 218, and/or an assay-specific biomolecule 250.

The surface 216 of the beads 214 may be bound or functionalized to bind to an affinity reagent of a particular assay, such as a particular capture antibody 218 or a particular assay-specific biomolecule 250, which may be a non-antibody biomolecule. The affinity reagent may bind to a particular target (e.g., a protein or other structure), thus tagging the target with the assay barcode 210. Thus, after washing to remove any unbound barcoded reagents, detection of the assay barcode 210 indicates the presence of the target to which the affinity reagent (e.g., the capture antibody 218 or the assay-specific biomolecule 250) is bound.

In some cases, the surface 216 of the bead 214 may be functionalized to bind the sample barcode 212. The sample barcode 212 may be an elemental tag containing a unique isotope or unique combination of isotopes that is distinguishable using elemental analysis. There may be multiple sets of sample barcodes, where all sample barcodes are from one set having the same isotope or combination of isotopes, and all sample barcodes are from different sets that are unique and distinguishable from each other using elemental analysis. Thus, a set of any number of different barcoded reagents may be tagged with sample barcodes from the same set, thus associating each of those barcoded reagents with the sample associated with the set of sample barcodes. Separately, any number of different sets of barcoded reagents may be tagged with a sample barcode from another set, thus associating the different sets of barcoded reagents with another set of sample barcodes.

The sample barcode 212 may be provided separately from the barcoding reagent 208, as in a kit containing a plurality of different sample barcodes. Thus, when an assay using the barcoded reagent 208 is prepared or performed, the sample barcode 212 may be mixed with the barcoded reagent 208 either prior to the assay (e.g., by mixing the sample barcode 212 and the beads 214 before mixing both with the sample) or during the assay (e.g., by mixing the sample barcode 212 with the sample and then mixing the beads 214 therein).

Fig. 2B is a schematic illustration of the barcoding reagent 208 shown in fig. 2A after attachment of the sample barcode 212, in accordance with certain aspects of the present disclosure. In some cases, the attachment of the sample barcode 212 can occur during production. In these cases, a barcoded reagent 208 may be provided, such as part of a kit, in which the sample barcode 212 is bound to or otherwise contained within the surface 216 of the bead 214. In some cases, the sample barcode 212 may be coupled to the barcoding reagent 208 prior to attachment of the capture antibody 218 and/or the assay-specific biomolecule 250.

In some cases, attachment of the sample barcode 212 can be performed as part of preparing to perform the assay (e.g., by mixing the sample barcode 212 and the beads 214 before mixing both with the sample) or as part of performing the assay (e.g., by mixing the sample barcode 212 with the sample and then mixing the beads 214 therein).

Fig. 3 is a schematic diagram of a barcoding reagent 308 and an integrated sample barcode 312 in accordance with certain aspects of the present disclosure. The barcoded reagents 308 may be beads 314, although other barcoded reagents may be used. The beads 314 can contain both the assay barcode 310 and the sample barcode 312 within the beads 314, such as incorporated into the core of the beads 314. The assay barcode 310 and the sample barcode 312 can each be a type of elemental tag that includes its own unique isotope or unique combination of isotopes that is discernible by elemental analysis.

In some cases, the bead 314 may include a surface 316 that contains, binds, or is functionalized to bind various attachments, such as capture antibodies 318 and/or assay-specific biomolecules 350. The surface 316 of the beads 314 may be bound or functionalized to bind affinity reagents of a particular assay, such as a particular capture antibody 318 or a particular assay-specific biomolecule 350, which may be a non-antibody biomolecule. The affinity reagent can bind to a particular target (e.g., a protein or other structure), thus tagging the target with the assay barcode 310. Thus, after washing to remove any unbound barcoded reagent, detection of the assay barcode 310 indicates the presence of the target to which the affinity reagent (e.g., the capture antibody 318 or the assay-specific biomolecule 350) is bound.

Fig. 4 is a schematic diagram of a system for assaying one or more samples 420 using an elemental analyzer 424 and a freeze-dried antibody panel 422, according to certain aspects of the present disclosure. The sample 420 may be any suitable sample, including whole blood or human peripheral blood, although any suitable sample may be used. The sample 420 may be a biological sample. The freeze-dried antibody panel 422 can be a freeze-dried antibody panel or subset of panels as disclosed herein. The freeze-dried antibody panel 422 can contain a variety of element-tagged antibodies, such as the element-tagged antibody 104 shown in fig. 1.

The freeze-dried antibody panel 422 may be mixed with the sample 420 according to an appropriate protocol, thereby resuspending the freeze-dried antibody panel 422. In some cases, the barcoding reagent 408 may optionally be mixed with the freeze-dried antibody set 422 and the sample 420. Examples of suitable barcoding reagents 408 include sample barcoding reagents and assay barcoding reagents. In some cases, each of the plurality of samples 420 can be mixed with a respective set of barcoding reagents 408, wherein each barcoding reagent in a set contains the same sample barcode, thus applying a unique sample barcode to each of the plurality of samples 420.

After mixing with the lyophilized antibody set 422, the sample 420 can be studied using an elemental analyzer 424, such as a mass spectrometer. In some cases, as shown in fig. 4, the elemental analyzer 424 may comprise an ICP-MS including an inductively coupled plasma torch 426 and a mass detector 428, although other elemental analyzers may be used. The induction coupled plasma torch 426 can receive cells or particles of the stained and/or element tagged sample 420 and ionize the sample. The generated ion stream may optionally be processed (e.g., filtered) and conveyed to a mass detector 428. The mass detector 428 may be a time-of-flight detector or other suitable mass detector. The mass detector 428 may determine the presence and amount of multiple isotopes in the ion stream based on the ion mass. The mass analyzer 428 may output data indicative of the presence and amount of the plurality of isotopes over time. The elemental analyzer 424 may thus output elemental data, which may include data indicative of the presence and amount of the various isotopes over time. In some cases, the element data may be stored as element data 482 in a data store (datastore), which may then be accessed by the element data processor 430 for further analysis. In some cases, the element data may be directed to the element data processor 430 for direct analysis. In some cases, the element data processor 430 may be incorporated into an element analyzer, such as a software component of an element analyzer, although in some cases the element data processor 430 may be incorporated into a separate computing device.

Elemental analyzer 424 can obtain elemental data relating to the presence and amounts of various isotopes over time. In some cases, such elemental data may be processed and segmented into time periods such that all of the elemental data within a particular time period (e.g., a time window) may be correlated to a single cell, bead, or particle detected by the elemental analyzer 424. For example, an elemental analyzer 424 analyzing a sample 420 containing one hundred cells may generate elemental data covering at least one hundred different time periods. Thus, the set of all isotopes identified within a particular window can be used to resolve which targets or barcodes are detected that bind to a particular cell, bead or particle associated with the particular window.

In some cases, the positioning (mapping) data 384 may be stored in a data store, which may be the same or different data store as the data store in which the element data 482 is stored. The positioning data 484 may include information relating isotopes or combinations of isotopes to a particular target or object. The localization data 484 may include information identifying the expression of targets that differentiate cell types, thus facilitating automated cell type identification. For example, for element-tagged antibodies targeting CD20, CD45, CD14, CD16, CD8, CD3, and CD4, localization data 484 may identify the respective isotope of each of these element-tagged antibodies as used to tag ¹⁴⁷Sm、¹⁵⁴Sm、¹⁶⁰Gd、¹⁶⁵Ho、¹⁶⁸Er、¹⁷⁰Er and¹⁷⁴yb, thus localizing each isotope to its respective target. The positioning data 484 may be accessed by the element data processor 430.

In some cases, the localization data 484 can include localization of any number of sets of lyophilized antibodies. The set of freeze-dried antibodies (e.g., freeze-dried antibody set 422) being used can be detected manually (e.g., by user selection) or automatically (e.g., by direct adoption of the set itself or by detection of a unique barcode within the stained sample), thus informing the elemental data processor 430 of which location to use. In some cases, the elemental data 482 may include other metadata, such as identification of which freeze-dried antibody panel to use. In addition to localizing the elemental tag of the lyophilized antibody panel to the target, localization data 484 can include localization of other elemental tags to the target, such as localization of the sample barcode to the sample, localization of the assay barcode to the assay, and localization of the reporter barcode to the reporter target. The use of these other locations can be determined in the same manner as how the location of the freeze-dried antibody panel is determined.

The element data processor 430 may accept element data 482 and positioning data 484. The elemental data processor 430 may analyze the elemental data 482 using the positioning data 484 to obtain information about the sample, such as cell count information about cells or other particles within the sample. In some cases, the elemental data processor 430 may separate the results by sample using different sample barcodes. In some cases, the elemental data processor 430 may identify and quantify cell types based on the targets of the freeze-dried antibody panel 422. In some cases, identifying and quantifying cell types may include applying a gating scheme to the elemental data, as disclosed in further detail herein.

The element data processor 430 may output the results in any suitable manner, such as those described herein. Example output may include storage to a data store, presentation on a display; or input for further processing. The element data processor 430 may output any suitable type of information, such as those identified herein. For example, the element data processor 430 may output cell type results (e.g., relative quantification); marker intensity (e.g., visual output showing an intensity "color" for each cell type in a phenotypic tree); marker frequency, marker intensity and cell count; dot plots of any two markers and/or histograms of any marker; or other suitable information.

Fig. 5 is a schematic diagram illustrating analysis and processing of unknown particles 520, 521, according to certain aspects of the present disclosure. Elemental analysis 524 is performed on the first unknown particle 520 and the second unknown particle 521. The time arrow is shown to show the analysis of the first unknown particle 520 before the second unknown particle 521. The first and second unknown particles 520, 521 may be sample particles, such as the sample 420 shown in fig. 4, after mixing with a suitable set of antibodies and/or reagents.

Element analysis 524 may result in element data 582 being separable into a first time window 586 and a second time window 587. Elemental data 582 may contain information about isotopes detected by the elemental analyzer during elemental analysis 524. For example, the elemental data 582 may contain isotopes in the first time window 586¹⁹⁷Au、¹³⁹La、¹⁹⁷Au、¹⁰⁶Pd and¹⁶⁵ho and isotopes in a second time window 587¹⁷⁰Er、¹⁴⁵Nd、¹⁰⁸Pd、¹⁶⁵Ho and¹⁵⁴sm. The isotopes identified in the first time window 586 may be attributable to ionization and detection of the first unknown particle 520. The isotopes identified in the second time window 587 may be attributable to ionization and detection of the second unknown particle 521.

The element data 582 may be processed 530 to produce output results (e.g., a first result 588 and a second result 589). The positioning data 584 may be utilized by a process 530 that may occur using an element data processor, such as the element data processor 430 shown in FIG. 4.

The positioning data 584 may contain information that identifies the target or barcode based on the detected element tag. As shown in FIG. 5, in a given element tag, for illustrative purposesCertain example isotopes are used, although other elemental tags and other isotopes may be used. In one example, the cell type localization data can include a number of targets (e.g., CD3, CD4, CD61, CD45) and their respective associated element tags (e.g., ¹⁷⁰Er、¹⁴⁵Nd、¹⁶⁵Ho、¹⁵⁴Sm); the sample barcode data can include a number of samples (e.g., first, second, third, fourth) and their respective associated elemental tags (e.g.,¹⁰⁶Pd、¹⁰⁸Pd、¹³⁰Te、¹²⁶te); assay barcode data may include a number of assays (e.g., A, B, C, D) and their respective associated element labels (e.g.,¹³⁹la and¹⁴⁰Ce、¹³⁹la and¹⁵³Eu、¹³⁹la and¹⁶⁵Ho、¹³⁹la and¹⁷⁵lu); and the reporter antibody data may include a number of targets (e.g., analyte 1, analyte 2, analyte 3, and analyte 4) and their common related element tags (e.g.,¹⁹⁷au). In certain aspects, the reporter antibody can include a high sensitivity element tag (e.g., a gold nanoparticle) and a low sensitivity element tag (e.g., a polymer chelated to a lanthanide atom).

Isotopes from the first time window 586 include detected isotopes¹⁹⁷Au, and thus it can be concluded that the first unknown particle 520 is the target analyte (e.g., one of analyte 1, analyte 2, analyte 3, analyte 4). Such reporter reagents may be used to target only the target analytes that the assay beads will target. Thus, it can be concluded that the first unknown particle 520 is not a cell, but an assay bead (e.g., an assay bead coupled to a target analyte). The other detected isotope in the first time window 586 comprises ¹³⁹La and¹⁶⁵ho, which indicates that the first unknown particle 520 may be an assay bead C. In addition, the isotopes detected in the first time window 586 comprise¹⁰⁶Pd, which indicates that the first unknown particle 520 is part of the first sample. This information may be provided as part of the first result 588.

Isotopes from the second time window 587 are not included with a reporter antibody (the reporter)The antibody is associated with the target of the assay bead). However, isotopes from the second time window 587 include¹⁷⁰Er、¹⁴⁵Nd、¹⁶⁵Ho and¹⁵⁴sm, indicating that the second unknown particle 521 contains CD3, CD4, CD61, and CD45, respectively. Furthermore, it can be concluded that the second unknown particle 521 is a cell. Inferences can be made regarding cell types with sufficient target information for other cell types, as disclosed in further detail herein. Although both the first time window 586 and the second time window 587 include¹⁶⁵Ho, but the absence of the reporter antibody and optionally the presence of other cell type targets (e.g., CD3, CD4, CD45) indicates that detection is indicated¹⁶⁵The Ho isotope comes from the cell type antibody, not from the assay barcode. Finally, in a second time window 587¹⁰⁸The presence of the Pd isotope indicates that the second unknown particle 521 is part of the second sample. This information may be provided as part of the second result 589.

Thus, although the first and second unknown particles 520, 521 are sequentially analyzed by the elemental analyzer, the processing of the elemental data can still distinguish the particles 520, 521 into their respective particle types and their respective samples.

FIG. 6 is a schematic diagram showing 3 example element tags 632, 634, 636 and diagrams 638, 640, 642 showing their respective element data, according to certain aspects of the present disclosure. Each element tag 632, 634, 636 can be an element tag as used in an element-tagged portion (e.g., the element-tagged portion 102 shown in fig. 1), a sample barcode (e.g., the sample barcode 212 shown in fig. 2), or an assay barcode (e.g., the assay barcode 210 shown in fig. 2). The plots 638, 640, 642 associated with the respective element tags 632, 634, 636 show the true or relative intensity of expression of a particular element as detected by an element analyzer (e.g., the element analyzer 424 shown in fig. 4) studying a sample containing the respective element tags 632, 634, 636. As used herein, the term "element a", "element B", or "element C" refers to a general element, such as a metal, that can be used to tag an element tag.

Element tag 632 is an example of a single isotope element tag. The element tag 632 includes element a 644 and does not include other elements for distinguishing the element tag from other element tags (e.g., the element tag 632 may include other elements, such as carbon and hydrogen, but these other elements are structural, rather than for distinguishing purposes). The element tag 632 may include one or more instances of element a 644, such as n different copies of the isotope. Through investigation by the element analyzer, the element data of the element tag 632 may show the relative expression of element a at a level corresponding to the n copies of element a 644 in the element tag 632.

The element tag 634 is an example of a single isotope element tag that is distinguishable from the element tag 632. Element tag 634 includes element B646. The element tag 634 may include one or more instances of element B646, such as y different copies of a bitmap. Through investigation by the element analyzer, the element data of element tag 634 may show the relative expression of element B at a level corresponding to y copies of element B646 in element tag 634. Thus, the element tag 634 is isotopically distinguishable from the element tag 634 because a different isotope is detected and optionally because a different isotope is detected in a different relative expression.

The element tag 637 is an example of a plurality of isotope element tags distinguishable from the element tags 632, 634. The element tag 637 includes element D645, element E647, and element F649. The element tag 637 may include one or more instances of each of the element D645, the element E647, and the element F649. As shown in fig. 6, the element tag 637 contains y different copies of element D645, element E647, and element F649. Through element analyzer studies, the element data of the element tag 637 may show the presence (e.g., optionally relative expressions) of the elements D, E and F in the element tag 637. Thus, the element tag 637 is isotopically distinguishable from the element tags 634, 636 due to the detection of unique isotopic combinations. Unique combinations may be identified based on a series of detected isotopes (e.g., elements D, E and F), optionally based on relative expression between detected isotopes in an element tag and/or optionally based on the true expression levels of detected isotopes when compared to other element tags.

The element tag 636 is an example of a plurality of isotope element tags that are distinguishable from the element tags 632, 634, 637. The element tag 636 includes element a 644, element B646, and element C648. The element tag 636 can include one or more instances of element a 644, element B646, and element C648, such as n different copies of element a 644, y different copies of element B646, and z different copies of element C648. Through elemental analyzer studies, the element data of the element tag 636 can show the relative expression of the elements A, B and C at levels corresponding to the n, y, and z copies of the respective elements in the element tag 636. Thus, the element tag 636 is isotopically distinguishable from the element tags 634, 636, 637 due to the detection of unique isotopic combinations. Unique combinations may be identified based on a series of detected isotopes (e.g., elements A, B and C), optionally based on relative expression between detected isotopes in an element tag (e.g., element C expression is less than element a expression, element a expression is less than element B expression), and/or optionally based on the true expression levels of detected isotopes when compared to other element tags (e.g., a: n, B: y, C: n may be associated with element tag 636, while another element tag may be associated with a: z, B: n, C: y).

Thus, multiple unique elemental tags can be generated by unique individual isotopes or unique combinations of isotopes. In some cases, a combination of isotopes may represent a combination comprising a single isotope at a particular expression level that may be distinguishable from a different combination of isotopes comprising the same single isotope at a different and distinguishable expression level.

Each unique isotope or combination of isotopes can be considered an elemental barcode that can be used to identify any moiety to which it is coupled. Thus, when coupled to moieties having targeting functions for a particular target, the elemental barcode can be used to identify the target after the element-tagged moiety has contacted a sample containing the target or the unbound element-tagged moiety has been washed away.

Based on the type of elemental analysis, a particular number of isotopes may be reliably distinguishable. For example, an example ICP-MS system may be able to distinguish up to x different mass channels, and thus be able to reliably distinguish x different isotopes (e.g., isotopes 1, 2, 3, 4, 5, … …, x). Thus, the pair of at least 2 may be based solely on determining the presence of a detectable isotope in the elemental tag ^xDifferent combinations of seed isotopes produce unique barcodes. In these 2^xIn various combinations, it may be desirable to remove certain isotopic combinations that may be detected in the sample. For example, if a set of isotopes or isotope combinations is used as an elemental tag in a freeze-dried antibody set or in a set of possible freeze-dried antibody sets, it may be desirable to use entirely different isotopes for sample barcoding and/or assay barcoding to avoid the possibility of misinterpreting simultaneous or near-simultaneous detection of antibody combinations as different isotope combinations. For example, the element tag 636 may include element C648, so that even if the element tags 632, 634 are detected at or near the same time, the analyzer or processor will not erroneously interpret the detection of elements a and B as element tags 636 because element C was not detected. In some cases, the element tag 636 may be further protected from the possibility of false identifications by not using any isotopes from the element tags 632, 634.

In some cases, the assay barcode and/or the sample barcode are configured to have non-overlapping isotopes, although this need not always be the case. In some cases, the assay barcode and/or the sample barcode may include a unique combination of some isotopes from a set of possible isotopes. For example, if a total of 6 different isotopes are used for sample barcoding, each sample barcode may include a unique combination of 3 of the 6 total isotopes, thus resulting in the generation of 20 different unique sample barcodes. For example, when the total number of possible unique isotopes used for a barcode is n and the number of unique isotopes selected for each barcode is k, then the total number of possible unique barcodes is n

In some cases, the elemental tag used for cell type identification contains one or more single isotopes, such as elemental tags 632, 634, since typically an assay will involve detection of multiple tags all coupled to a single cell. In some cases, the element tags used for assay bead barcoding contain unique isotope combinations because the probability of detecting multiple assay beads within a single time window, and thus the probability of detecting overlapping element tags, is minimal. The use of unique isotope combinations for the assay beads allows for more unique elemental tags with fewer unique isotopes.

Fig. 7 is a schematic illustration of a sample cell 720 tagged with an element-tagged portion 702, a sample barcode 712, and a barcoding reagent 708, according to certain aspects of the present disclosure. Sample cells 720 may be any cells of the sample, such as blood cells. In some cases, instead of sample cells 720, another particle or target may be used, such as a protein present in blood plasma (e.g., albumin). The sample cell 720 shown in fig. 7 is depicted as being labeled by the element-labeled portion 702, the sample barcode 712, and the barcoding reagent 708, although in various uses described herein, such a sample may be labeled with any combination of one, two, all three, or more of these objects.

The element-tagged portion 702 can be any suitable portion (e.g., the element-tagged portion 102 shown in fig. 1), such as an element-tagged antibody of a freeze-dried antibody panel. The antibody 704 can be bound to an element tag 706. The antibody 704 may bind to the sample cell 720 by any suitable means, such as to an antigen on the surface of the sample cell 720. In some cases, the element-tagged portions can infiltrate the sample cells 720 (e.g., permeabilized sample cells 720) and bind to intracellular targets.

The element tag 706 associated with the element-tagged portion 702 can be the same for all element-tagged portions 702 having the same antibody 704, but the element-tagged portions having different antibodies (e.g., targeting different targets) can have unique element tags.

The sample barcode 712 can be an elemental tag that binds to the sample cell 720 directly or through an intermediate moiety (e.g., an antibody). The sample barcode 712 can be designed to bind to all, most, or most of the cells or particles of the sample. Each sample barcode 712 may have the same isotope or combination of isotopes in all sample barcodes 712.

Fig. 8 is a schematic diagram showing a sample cell 820 tagged with a multi-isotope sample barcode 812, in accordance with certain aspects of the present disclosure. The sample cell 820 may be the sample cell 720 shown in fig. 7. The sample barcode 812 can be coupled to a sample cell 820. The sample barcode 812 can include an antibody 852 with a targeting function for the sample cell 820 (e.g., a targeting function for a cell surface target of an intracellular target). The antibody 852 of the sample barcode 812 can be coupled to an element tag 832 that comprises a unique isotopic combination associated with the sample barcode 812. For example, the sample barcode 812 can have a hypothetical barcode of "A, B, C", where A, B and C represent unique isotopes (e.g.,¹⁰²Pd、¹⁰⁴Pd、¹⁰⁵Pd)。

fig. 9 is a schematic diagram showing a sample cell 920 tagged with an assigned sample barcode portion 911, 912, 913, in accordance with certain aspects of the present disclosure. The sample cell 920 may be the sample cell 720 shown in fig. 7. The assigned sample barcode portions 911, 912, 913 of the sample cell 920 can result in the same overall barcode applied to the sample cell 920 as the sample barcode 812 applied to the sample cell 820 shown in fig. 8.

The sample cell 920 can include a plurality of assigned sample barcode portions 911, 912, 913 coupled thereto. Each sample barcode moiety 911, 912, 913 can include a respective antibody 952, 954, 956 coupled to a respective element tag 932, 934, 936, respectively. Each antibody 952, 954, 956 in the assigned sample barcode system may be the same or may be different, as long as the antibodies all have targeting functions associated with the same sample cell 920. For example, antibody 952 may target CD45, antibody 954 may target CD298, and antibody 956 may target b2 m. Isotopes of the entire sample barcode (e.g., the hypothetical isotopes "A, B and C") can be distributed among multiple barcode portions 911, 912, 913. Thus, element tag 932 may contain isotope a, element tag 934 may contain isotope B and element tag 936 may contain isotope C. Thus, when the sample cell 920 is studied using an elemental analyzer, the same "A, B, C" entire sample barcode will be detected, although the entire sample barcode is distributed among multiple barcode portions 911, 912, 913.

Fig. 10 is a schematic illustration of a barcoding reagent 1008 and a reporter antibody 1019 according to certain aspects of the present disclosure. The barcoding reagent 1008 may be beads 1014, although other barcoding reagents may be used. Bead 1014 may contain an assay barcode 1010 located within bead 1014, such as incorporated into the core of bead 1014. The assay barcode 1010 may be a type of elemental tag that includes a unique isotope or unique combination of isotopes that is distinguishable by elemental analysis. In some cases, beads 1014 can include a surface 1016 containing, bound to, or functionalized to bind various attachments, such as a sample barcode 1012, a capture antibody 1018, and/or an assay specific biomolecule.

The surfaces 1016 of the beads 1014 may be bound or functionalized to bind affinity reagents of a particular assay, such as a particular capture antibody 1018 or a particular assay-specific biomolecule, which may be a non-antibody biomolecule. The affinity reagent can bind to a particular target analyte 1020 (e.g., a protein or other structure), thus tagging the target analyte 1020 with the assay barcode 1010. Thus, after washing to remove any unbound barcoded reagent, detection of the assay barcode 1010 indicates the presence of the target analyte 1020 to which the affinity reagent (e.g., the capture antibody 1018 or the assay-specific biomolecule 1050) is bound.

In some cases, the surface 1016 of the bead 1014 can be bound, contained, or functionalized to bind to the sample barcode 1012. The sample barcode 1012 may be an elemental tag containing a unique isotope or unique combination of isotopes that is distinguishable using elemental analysis. The particular sample barcode 1012 bound to the bead 1014 may be specific to the sample from which the target analyte 1020 originates. By using multiple sets of sample barcodes having unique isotopes or isotope combinations, each original sample can be assayed using its own set of sample barcodes (e.g., mixed with the sample or coupled to an assay reagent 1008, which is then mixed with the sample), and thus detection of a particular sample barcode 1012 can be used to identify from which sample the target analyte 1020 originates.

In some cases, a reporting agent 1090 may be used. The reporter reagent 1090 can include a reporter antibody 1019 having a targeting function for the target analyte 1020. Reporter antibody 1019 can be conjugated to an element tag 1032 containing an isotope or unique combination of isotopes. Detection of the isotope or unique combination of isotopes associated with the elemental tag 1032 can be indicative of a particle detected by the elemental analyzer, and thus another isotope detected within a particular time window, being associated with the target analyte 1020, and thus the assay bead 1014.

A plurality of different reporter agents 1090 can be used, each agent having a different type of reporter antibody with targeting functions for a plurality of different target analytes, although all reporter agents 1090 can include the same type of elemental tag (e.g., the same isotope or combination of isotopes). Thus, each reporting agent 1090 will have the same elemental signature 1032. Since the purpose of the reporting agent 1090 is only to determine whether a particular object (e.g., assay bead 1014) is being studied by the elemental analyzer, there is no need to distinguish between different reporting agents 1090.

In some cases, reporter antibody 1019 can be the same type of antibody (e.g., monoclonal antibody) as capture antibody 1018 of assay bead 1014. In some cases, the reporter antibody 1019 is a different type of antibody (e.g., a polyclonal antibody) than the capture antibody 1018 of the assay bead 1014, although both the reporter antibody 1019 and the capture antibody 1018 have targeting functions for the target analyte 1020.

Fig. 11 is a schematic diagram showing the preparation of a freeze-dried antibody panel 1166, according to certain aspects of the present disclosure. The freeze-dried antibody set 1168 may first begin as a set 1100 of element-tagged antibody sets 1152, 1154, 1156, 1158. Panel 1100 may include any number of antibody panels. Each antibody group can include one or more element-tagged antibodies that share the same antibody and the same element tag. For groups targeting 6 different targets, group 1100 may comprise 6 different antibody groups, where each group contains a plurality of single element tagged antibodies.

The element-tagged antibody sets 1152, 1154, 1156, 1158 of set 1100 can be prepared as desired, as described herein. For example, each of the elemental-tagged antibody sets 1152, 1154, 1156, 1158 can be individually titrated and diluted in a stabilizing agent to achieve the desired concentration. The resulting solutions can be separately mixed with excipients and mixed together to form a mixture 1164, which can be placed in a container 1162, such as a tube. In some cases, supplemental reagent 1160 may be added to mixture 1164. These supplemental reagents 1160 may include any reagents that are capable of being lyophilized. For example, a lysis reagent (e.g., red blood cell lysis reagent), a wash buffer, a fixation reagent, and/or a permeabilization reagent may be added as the supplemental reagent 1160. In some cases, a barcoding reagent, such as an assay barcoding reagent or a sample barcoding reagent (e.g., a sample barcode), can be added to the mixture 1164 as a supplemental reagent 1160. In some cases, one or more calibration materials (e.g., calibration beads) may be added as a supplemental reagent 1160 or as a supplemental material.

As disclosed herein, the mixture 1164 may be subjected to a freeze-drying process. For example, the mixture 1164 may be subjected to a heating phase, a vacuuming phase, a drying phase, and a holding phase. During the heating phase, the temperature may be maintained at a temperature between-60 and 0 ℃ for a duration of time while the vacuum is maintained in a range between 100 and 500 torr. During the evacuation phase, the vacuum may be reduced to a range between 100 and 500 mTorr. During the drying stage, the temperature can be manipulated in the range between-50 to 30 ℃ while maintaining the vacuum in the range of 10-150 mtorr. During the hold phase, the temperature may be maintained at a temperature between 10 and 30 ℃ while the vacuum is raised to a range between 100 and 500 mTorr. After the hold stage, the container 1162 may be returned, which may include increasing the pressure in the container, such as in a range of up to 200 torr to 760 torr, although in some cases, the return occurs at a pressure below ambient pressure. In some cases, the temperature of the mixture 1162 does not rise above the glass transition temperature of the mixture 1162 during all stages of freeze-drying. During or after refilling, the container 1162 may be filled with an internal atmosphere 1170 of inert gas or dry air and then sealed.

After freeze-drying, the container 1162 may contain the freeze-dried mixture 1168 that makes up the freeze-dried group 1166. The freeze-dried group 1166 can be stored and then resuspended for staining samples.

Fig. 12 is a schematic diagram of a method 1200 for preparing barcode reagents 1276, 1278 according to certain aspects of the present disclosure. The method 1200 may begin with an element tagged core 1272. The element tagged core 1272 may include any suitable element tag 1210, such as an element tag 1210 containing a combination of isotopes selected from La, Ce, Eu, Ho, and Lu. The element-tagged core 1272 may comprise an isotope as part of a solid metal core or a polymer core that chelates or otherwise embeds a metal isotope.

The polymer precursors 1273 (e.g., subunits) can react with the element-tagged core 1272 to produce element-tagged beads 1274 comprising the element-tagged core 1272 surrounded by a polymer shell 1275. Such element-tagged beads 1274 may react with the capture antibody 1218 or the assay-specific biomolecule 1250.

In some cases, the element-tagged beads 1274 with the polymeric shell 1275 may react with the capture antibody 1218 with exposed amine groups (e.g., on the Fc region), allowing the capture antibody 1218 to form a covalent bond with the polymeric shell 1275, thus resulting in the production of an antibody-based element-tagged barcode reagent 1276.

In some cases, the element-tagged beads 1274 with the polymeric shell 1275 may react with the assay-specific biomolecules 1220 with exposed amine groups, allowing the assay-specific biomolecules 1220 to form covalent bonds with the polymeric shell 1275, thus resulting in the production of biomolecule-based element-tagged barcoded reagents 1278.

In some cases, the antibody-based elemental tagged barcoding reagent 1276 and/or the biomolecule-based elemental tagged barcoding reagent 1278 may be further tagged with a sample barcode, such as with a sample barcode containing a highly reactive functional group capable of binding to the polymeric housing 1275, displacing the antibody 1218 or biomolecule 1250 from the polymeric housing 1275, or binding to other functional groups present on the barcoding reagents 1276, 1278.

Fig. 13 is a flow chart illustrating a method 1300 for staining and analyzing a blood sample, according to certain aspects of the present disclosure. Although the method 1300 is disclosed for analysis of a whole blood sample, aspects of the method 1300 may be used to analyze other samples, as appropriate.

At block 1302, a whole blood sample is provided. The whole blood may be human peripheral blood, whether fresh or frozen. In some cases, at optional block 1304, whole blood may be labeled with a sample barcode 1304.

At block 1306, PBMCs from the whole blood sample may be isolated. Any suitable technique may be used, such as separation of PBMCs by centrifugation. In some cases, the PBMCs may optionally be tagged with a sample barcode at block 1308.

At block 1310, PBMCs may be stained using a freeze-dried panel, such as a freeze-dried antibody panel or subset of panels disclosed herein. In some cases, staining PBMCs with freeze-dried groups may include recording type information of the freeze-dried groups used, which may be retrieved to determine the location of the groups between the elemental tags and the targets. Staining PBMCs may comprise mixing the freeze-dried population with PBMCs for a period of time at a temperature and then washing unbound antibody from the PBMCs. In some cases, at block 1312, the stained sample may optionally be tagged with a sample barcode.

At optional block 1313, further cell processing may occur. Further cell processing may include other processing and/or staining steps, such as cell fixation, cell permeabilization, and/or intracellular staining. Further cell processing at block 1313 may occur after the barcoding of the sample at block 1312, although this need not always be the case.

At block 1314, plasma may be separated from the whole blood sample. Any suitable technique may be used, such as separating plasma by centrifugation. In some cases, the plasma may optionally be tagged with a sample barcode at block 1316.

At block 1318, free analyte bead assay with reporter antibody is added to the plasma. The free analyte bead assay may include one or more barcoded reagents with an assay barcode and a capture antibody for targeting free analyte of plasma or assay specific biomolecules. Thus, capture antibodies or assay-specific biomolecules that bind to free analytes can tag those free analytes with their respective assay barcodes. The use of a reporter antibody may be optional. The reporter antibody can be used to identify the presence of the bead or analyte. The reporter antibody can be selected to bind to all, most, or most of the beads or analytes. All reporter antibodies of a particular subject may share the same elemental tag, such that detection of the elemental tag indicates the presence of that subject type (e.g., bead or analyte). Adding the free analyte bead assay at block 1318 may include mixing the plasma with the free analyte bead assay for a period of time at a temperature, and then washing unbound beads from the plasma. In some cases, the stained plasma may optionally be tagged with a sample barcode at block 1320.

At optional block 1322, the PBMC sample and the plasma sample may be mixed together. In some cases, if sample barcoding is applied at blocks 1304, 1308, 1312, 1316, and/or 1320, the stained sample (e.g., PBMC and/or plasma) from the whole blood of block 1302 may be mixed with stained samples from other whole blood sources (e.g., whole blood from a different patient or whole blood from the same patient, but collected at a different time than the whole blood from block 1302). Because of sample barcoding, the study of mixed samples can result in data containing a unique sample barcode that can be used to separate data by original sample source.

At block 1324, the stained sample (e.g., PBMCs and/or plasma), optionally mixed or not mixed at block 1322, can be studied by an elemental analyzer, such as a mass spectrometer (e.g., ICP-MS). The study of block 1324 may result in the generation of elemental data for the sample.

At block 1326, the elemental data of the sample may be automatically analyzed. The automatic analysis of block 1326 can include accessing a mapping of elemental tags to targets. The automated analysis of block 1326 may include identifying cells or particles of the sample. The automated analysis of block 1326 may include correlating the identified cells or particles with a particular sample based on the detected elemental signature associated with the sample barcode. The automated analysis of block 1326 may include correlating the identified cells or particles with a particular assay based on the detected assay barcode. The automated analysis of block 1326 may include correlating the identified cells or particles with one or more specific markers based on detecting an element tag associated with the one or more specific markers. In some cases, the automated analysis of block 1326 may include identifying a class of cells or particles based on the relevant markers. In some cases, the automated analysis of block 1326 may include generating an output containing information related to the quantification or identification of cells or cell types in the sample. The automatic analysis of block 1326 may include generating any suitable output from the elemental data as disclosed herein.

Certain methods and kits may include only a subset of the boxes described in fig. 13. For example, sample barcoding may not be performed, although cells and assay beads from the same sample may still be mixed prior to the study. Alternatively, sample barcoding of cells and/or assay beads (e.g., individually or mixed) can be performed at any step prior to the study, and mixing of cells and/or assay beads from different samples can be performed at any point after sample barcoding and prior to the study. For example, the sample barcode can be introduced at any step and the mixing of the sample can be performed at any subsequent step. PBMCs in serum and plasma may not be separated and may instead be applied to a lyophilized mixture of element-tagged antibodies and assay barcoded beads, allowing for a more streamlined workflow (although the quality of cell staining may be reduced due to the abundance of free analyte). The inclusion of the sample barcode may be mixed with the freeze-dried mixture or the sample barcode may be provided separately (for addition to the sample before or after staining with the freeze-dried set). In certain aspects, a tissue sample or cell culture may be provided in step 1302 in place of whole blood, and cell separation in step 1306 is optional. In certain aspects, step 1318 is the addition of a reporter reagent comprising a biomolecule other than an antibody. In examples where cells are studied instead of assay beads, the method may (sequentially) comprise steps 1302, 1308, 1310, optionally 1313, 1324 and 1326. In an example where assay beads are studied rather than cells, the method may (in turn) comprise steps 1302, 1314, 1316, 1322 and 1324. In some examples, the reporter antibody can be added separately (before or after) the addition of the bead assay, such as with a washing step in between after the addition of the bead assay. In some examples, staining of the permeabilized and freeze-dried set of cells prior to block 1310 includes antibodies against both extracellular and intracellular targets. In certain examples, cells (e.g., PBMCs) and free analyte (e.g., plasma) are separated prior to adding the freeze-dried sets and bead assays, respectively, and combined prior to sample barcoding and mixing between samples. In some cases, the cell samples were barcoded and mixed prior to addition to the freeze-dried group.

FIG. 14 is a schematic diagram illustrating an example gating strategy 1400 for automatically analyzing element data, in accordance with certain aspects of the present disclosure. Example gating strategy 1400 includes 3-level gating, although any number of levels may be used. Various 2-dimensional spaces are used to display cells or particles detected in a sample studied using elemental analysis. The higher the presence of isotopes or markers with a cell or particle, the more right (on the x-axis) or above (on the y-axis) in space the point of the cell or particle is shown to occur. The spaces shown in fig. 14 are labeled with isotopes on the x and y axes, although it is understood that each isotope may represent a particular marker, such as an antibody or target of an antibody. In addition, isotopic compositions may be used along the axis, rather than a single isotope, such as when isotopic compositions are used to label a particular marker. Although these 2-dimensional spaces are depicted as point diagrams for illustrative purposes, the information may be stored and manipulated in any suitable manner to achieve a desired gating strategy.

In the first layer, elemental data from the sample is shown in 2-dimensional space 1402, with isotope a on the x-axis and isotope B on the y-axis. The space 1402 may include two doors 1414, 1416. Objects (e.g., cells or particles) associated with high values of isotope a and isotope B may fall within gate 1414, while objects associated with low values of isotope a and isotope B may fall within gate 1416. In some cases, objects within gate 1414 and/or objects within gate 1416 may be labeled as cells or objects of a particular type.

In the second level, objects within the portal 1414 may be plotted in space 1404, while objects from portal 1416 are plotted in space 1410. The space 1404 may map the isotope C on the x-axis and the isotope D on the y-axis. Space 1404 may be gated to separate into gate 1418 objects having a high value for isotope D and a low value for isotope C, and to separate into gate 1420 objects having a low value for isotope D and a high value for isotope C. In space 1410, objects from space 1416 may be mapped, with isotope E on the x-axis and isotope F on the y-axis. Space 1410 may contain a single gate 1440 that is associated with objects having high values for both isotopes E and F. In some cases, objects within doors 1418, 1420, and/or 1440 may be labeled as cells or particles of a particular type.

Objects within doors 1418, 1420, and/or 1440 may pass to the third tier. In the third level, objects within gate 1418 are plotted on space 1406, objects within gate 1420 are plotted on space 1408, and objects within gate 1440 are plotted on space 1412. In space 1406, the object may be mapped with isotope G on the x-axis and isotope H on the y-axis. These objects may be gated, with gate 1422 being related to those objects having high values for both isotopes G and H, and gate 1424 being related to those objects having high values for isotope H and low values for isotope H. Objects in gates 1422 and/or 1424 may be marked as objects of a particular type. In space 1408, the object from space 1420 is plotted with isotope C on the x-axis and isotope B on the y-axis. These objects may be gated, with gate 1426 being related to those objects having a high value for isotope B and a median value for isotope C, gate 1428 being related to those objects having a medium-high value for isotope B and a low value for isotope C, and gate 1430 being related to those objects having a median value for isotope B and a low value for isotope C. Objects in doors 1426, 1428, and/or 1430 may be marked as objects of a particular type. In space 1412, the object from space 1440 is plotted, with isotope F on the x-axis and isotope D on the y-axis. These objects may be gated, with gate 1442 being related to those objects having high values for isotopes F and D, gate 1444 being related to those objects having medium-high values for isotope D and high values for isotope F, gate 1446 being related to those objects having medium-low values for isotope D and high values for isotope F, and gate 1448 being related to those objects having low values for isotopes D and F. Objects in gates 1442, 1444, 1446, and/or 1448 may be labeled as cells or objects of a particular type.

Accordingly, a gating scheme may include determining a defined region in a multidimensional space (e.g., a 2-dimensional space) where data may be attributable to a particular outcome. The gating scheme may include subsequently applying successive gates to the data within the defined area based on a new multidimensional space (e.g., a different 2-dimensional space). This process can be repeated as necessary to achieve the desired level of discrimination. Thus, successive gating levels can help narrow down a particular cell type or object type based on the presence or absence of multiple markers. Since unique cell types or object types will have different surface markers and/or intracellular marker combinations, gating strategies can be defined to identify these multiple cell types or particle types based on the presence or absence of multiple markers.

In some cases, instead of defining a region, cells or particles may be gated on the basis of marker expression with a number greater or less than a threshold number. For example, an indication of high expression for a given target (e.g., CD45) may be represented as any expression at or above a particular threshold. In some cases, different thresholds may be used for the same target at different gating scheme levels, or at the same gating scheme level when originating from different previous gates.

In one example, the element data can be converted to a target expression using localization data. For cell samples, data can be analyzed in a 2-dimensional space where the target of CD45 is expressed on the y-axis and the target of CD66b is expressed on the x-axis. The region in the upper left corner of the space indicates high expression of CD45 and low or no expression of CD66 b. Cells falling in the upper left corner can be further analyzed in a new space based on target expression of CD56 and CD 14. Cells falling in the lower left corner of the space may indicate little to no expression of either CD56 or CD 14. Cells falling within this region can be further analyzed in a new space based on target expression of CD19 on the y-axis and CD3 on the x-axis. Cells that fall in the upper left axis can indicate high CD19 expression and low or no CD3 expression. Cells that fall in this region can be identified as B cells. This example gating strategy may be denoted as CD45+ CD66 b-; CD56-CD 14-; CD19+ CD 3-. In some cases, the cells in the region may be further analyzed along other dimensions to determine other characteristics about those cells.

Using the gating strategy representations from the above examples, other gating strategies suitable for use with the lyophilized antibody panel include the following strategies for identifying the following cell types. For CD 8T cells (total; CD161 lo/-): CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ TCRgd-; CD4-CD8 +; CD8+ CD161 lo/-. For CD 4T cells (total): CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ TCRgd-; CD4+ CD 8-. For tregs: CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ TCRgd-; CD4+ CD 8-; CD4+ CCR4 +; CD45RA-CD45RO +; CD25hicD127 lo/-. For γ - δ T cells, CD4-CD 8-: CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD4-CD 8-; CD3+ TCRgd +. For total B cells: CD45+ CD66 b-; CD56-CD 14-; CD19+ CD 3-. For total NK cells: CD45+ CD66 b-; CD19-CD 20-; CD3-CD 14-; CD45RA + CD 123-; CD45+ CD56 +. For neutrophils: CD45loCD66b +; CD294-CD16 +. For total monocytes: CD45+ CD66 b-; CD19-CD 20-; CD3-CD 56-; CD11c + HLA-DR +; CD14+/-CD11c +. For plasmacytoid dendritic cells: CD45+ CD66 b-; CD19-CD 20-; CD3-CD 14-; HLA-DR + CD56 +/-; CD123+ CD11 c-. For naive CD 8T cells: CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ TCRgd-; CD4-CD8 +; CD8+ CD161 lo/-; CD8+ CCR7 hi; CD45RA + CD45 RO-. For naive CD 4T cells: CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ TCRgd-; CD4+ CD 8-; CD4+ CCR7 hi; CD45RA + CD45 RO-. For Th 1-like: CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ TCRgd-; CD4+ CD 8-; CD4+ CXCR 5-; CD4+ CCR 4-; CD45RA-CD45RO +; CXCR3+ CCR 6-. For MAIT/NKT CD 4-cells: CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ CD 4-; CD28+ CD161 hi. For naive B cells: CD45+ CD66 b-; CD56-CD 14-; CD19+ CD 3-; CD19+ CD27 +. For early NK cells: CD45+ CD66 b-; CD19-CD 20-; CD3-CD 14-; CD45RA + CD 123-; CD45+ CD56 +; CD56+ CD 57-. For eosinophils: CD45loCD66b +; CD294+ CD 16-. For classical monocytes: CD45+ CD66 b-; CD19-CD 20-; CD3-CD 56-; CD11c + HLA-DR +; CD14+/-CD11c +; CD38+ CD14 hi. For spinal cord dendritic cells: CD45+ CD66 b-; CD19-CD 20-; CD3-CD 14-; HLA-DR + CD56 +/-; CD123-CD11c +; CD11c + CD38 +. For central memory CD 8T cells: CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ TCRgd-; CD4-CD8 +; CD8+ CD161 lo/-; CD8+ CCR7 hi; CD45RA-CD45RO +. For central memory CD 4T cells: CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ TCRgd-; CD4+ CD 8-; CD4+ CCR7 hi; CD45RA-CD45RO +. For Th 2-like: CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ TCRgd-; CD4+ CD 8-; CD4+ CXCR 5-; CD45RA-CCR4 +; CXCR3-CCR 6-. For memory B cells: CD45+ CD66 b-; CD56-CD 14-; CD19+ CD 3-; CD19+ CD27 +. For late NK cells: CD45+ CD66 b-; CD19-CD 20-; CD3-CD 14-; CD45RA + CD 123-; CD45+ CD56 +; CD56+ CD57 +. For basophils: CD45+ CD66 b-; CD19-CD 20-; CD3-CD 56-; HLA-DR-CD11 c-; CD123+ CD294 +. For intermediate monocytes: CD45+ CD66 b-; CD19-CD 20-; CD3-CD 56-; CD11c + HLA-DR +; CD14+/-CD11c +; CD38lo/-CD14 int. For effector memory CD 8T cells: CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ TCRgd-; CD4-CD8 +; CD8+ CD161 lo/-; CD8+ CCR7 lo/-; CD8+ CD27 +. For effector memory CD 4T cells: CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ TCRgd-; CD4+ CD 8-; CD4+ CCR7 lo/-; CD45RA-CD45RO +; CD45RO + CD27 +. For Th 17-like: CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ TCRgd-; CD4+ CD 8-; CD4+ CXCR 5-; CD45RA-CCR4 +; CXCR3-CCR6 +. For plasmablasts: CD45+ CD66 b-; CD56-CD 14-; CD19+ CD 3-; CD19+ CD27 +; CD38+ CD 20-. For non-classical monocytes: CD45+ CD66 b-; CD19-CD 20-; CD3-CD 56-; CD11c + HLA-DR +; CD14+/-CD11c +; CD38-CD 14-. For terminal effect CD 8T cells: CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ TCRgd-; CD4-CD8 +; CD8+ CD161 lo/-; CD8+ CCR7 lo/-; CD8+ CD 27-. For terminal effect CD 4T cells: CD45+ CD66 b-; CD19-CD 20-; CD14-CD11 c-; CD45+ CD3 +; CD3+ TCRgd-; CD4+ CD 8-; CD4+ CCR7 lo/-; CD45RA-CD45RO +; CD45RO + CD 27-.

Fig. 15 is a schematic diagram illustrating a technique 1500 for sample labeling of barcoded reagents and analysis of a set of samples, according to certain aspects of the present disclosure. A set of barcoding reagents 1592 is provided. A set of barcoding reagents 1592 may include any number of barcoding reagents (e.g., as barcoding reagents 208 in fig. 2) optionally tagged with assay barcode elements for any number of assays. A set of barcoding reagents 1592 may include barcoding reagents with a variety of different capture antibodies and/or assay specific biomolecules. 3 sets of sample barcodes 1586, 1588, 1590 may be provided. Although fig. 15 depicts the use of 3 sets of sample barcodes 1586, 1588, 1590, any number of sample barcode sets may be used, which may be determined in advance by the number of sample barcode sets available and/or the number of different samples being assayed. Each of the sets of sample barcodes 1586, 1588, 1590 can contain some of the same sample barcodes, such that the sample barcodes of the first set of sample barcodes 1586 are all identical and all unique from the second set of sample barcodes 1588, which are themselves all identical and all unique from the third set of sample barcodes 1590, which are themselves all identical. The 3 sets of sample barcodes 1586, 1588, 1590 and/or 1 set of barcoding reagents 1592 may be provided as a kit or as part of a kit containing a freeze-dried set.

The sets of sample barcodes 1586, 1588, 1590 can be combined separately with aliquots of the set of barcoding reagents 1592. Each aliquot of the set of barcoded reagents 1592 may contain a mixture of all of the different types of barcoded reagents from the set of barcoded reagents 1592. Due to the combination of the sets of sample barcodes 1586, 1588, 1590 and the sets of barcoding reagents 1592 in the respective aliquots, 3 sets of sample-encoded barcoding reagents 1593, 1594, 1595 can be generated. The sample barcodes 1586, 1588, 1590 may be coupled to the barcoding reagent 1592 or may be mixed with only the barcoding reagent 1592. Thus, the first set of sample encoded barcoding reagents 1593 may contain barcoding reagents that are all tagged or mixed with the sample barcodes of the first set of sample barcodes 1586; the second set of sample encoded barcoding reagents 1594 may contain barcoding reagents that are all tagged or mixed with the sample barcodes of the second set of sample barcodes 1588; and the third set of sample encoded barcoding reagents 1595 may contain barcoding reagents that are all tagged with or mixed with the sample barcodes of the third set of sample barcodes 1590. When the sample barcodes 1586, 1588, 1590 are used to label the barcoding reagent 1592, excess sample barcodes may be washed from each mixture.

Each of the sets of sample-encoded barcoding reagents 1593, 1594, 1595 may be mixed with a respective sample 1596, 1597, 1598 to label the sample 1596, 1597, 1598. After washing the unbound barcoded reagent (and optionally, unbound sample barcode), samples 1596, 1597, 1598 may be combined into a mixed sample 1599.

The mixed sample 1599 can be studied using elemental analysis, such as using an elemental analyzer (e.g., elemental analyzer 424 shown in fig. 4). The generated elemental data 1582 can be analyzed to detect the presence of elemental tags associated with sample barcodes from the 3 sets of sample barcodes 1586, 1588, 1590, and thus identify the individual sample (e.g., sample 1596, 1597, 1598, respectively) to which the sample barcode is associated. An automated analyzer or processor (e.g., the element data processor 430 of fig. 4) can automatically separate the element data 1582 collected by the element analyzer into sample a data 1585 (e.g., the element data related to sample a 1596, which is tagged with a sample barcode from the set of sample barcodes 1586), sample B data 1587 (e.g., the element data related to sample B1597, which is tagged with a sample barcode from the set of sample barcodes 1588), and sample C data 1589 (e.g., the element data related to sample C1598, which is tagged with a sample barcode from the set of sample barcodes 1590).

Thus, multiple samples can be combined and analyzed simultaneously, which can improve overall study efficiency and can help improve the reliability of the data between samples, as they are all studied during the same run.

Fig. 16 is a schematic diagram illustrating a technique 1600 for sample labeling and analysis of a set of samples, in accordance with certain aspects of the present disclosure. A set of barcoded reagents 1692 is provided. A set of barcoding reagents 1692 may include any number of barcoding reagents (e.g., as barcoding reagents 208 in fig. 2) optionally tagged with assay barcode elements for any number of assays. The set of barcoding reagents 1692 may include barcoding reagents with a plurality of different capture antibodies and/or assay-specific biomolecules. 3 sets of sample barcodes 1686, 1688, 1690 can be provided. Although fig. 16 depicts the use of 3 sets of sample barcodes 1686, 1688, 1690, any number of sample barcode sets may be used, which may be determined in advance by the number of sample barcode sets available and/or the number of different samples being assayed. Each of the sets of sample barcodes 1686, 1688, 1690 can contain some of the same sample barcodes, such that the sample barcodes of the first set of sample barcodes 1686 are all identical and all unique to the second set of sample barcodes 1688, the second set of sample barcodes are themselves all identical and all unique to the third set of sample barcodes 1690, and the third set of sample barcodes are themselves all identical. The 3 sets of sample barcodes 1686, 1688, 1690, and/or 1 set of barcoding reagents 1692 may be provided as a kit or as part of a kit comprising a freeze-dried set.

The sets of sample barcodes 1686, 1688, 1690 can be individually combined with the respective samples 1696, 1697, 1698. In some cases, such as if the sample barcode is designed to bind to cells or particles of the sample itself, the sample 1696, 1697, 1698 may be washed to remove any unbound sample barcode. The samples 1696, 1697, 1698 may then be mixed together into a mixed sample 1699, which may be combined with a barcoding reagent 1692. The unbound barcoded reagent may then be washed and the mixed sample 169 may be studied using elemental analysis, such as using an elemental analyzer (e.g., elemental analyzer 424 shown in fig. 4). The generated elemental data 1682 can be analyzed to detect the presence of elemental tags associated with sample barcodes from the 3 sets of sample barcodes 1686, 1688, 1690, and thus identify the individual sample (e.g., samples 1696, 1697, 1698, respectively) to which the sample barcode is associated. An automated analyzer or processor (e.g., the element data processor 430 of fig. 4) can automatically separate the element data 1682 collected by the element analyzer into sample a data 1685 (e.g., the element data associated with sample a 1696, which is labeled with a sample barcode from the set of sample barcodes 1686), sample B data 1687 (e.g., the element data associated with sample B1697, which is labeled with a sample barcode from the set of sample barcodes 1688), and sample C data 1689 (e.g., the element data associated with sample C1698, which is labeled with a sample barcode from the set of sample barcodes 1690).

Thus, multiple samples can be pooled together for assay (e.g., assayed using a barcoded reagent from set of barcoded reagents 1692) and analyzed simultaneously, which can improve assay efficiency, overall study efficiency, and can help improve the reliability of the data between samples, as they are all assayed and studied during the same run.

In some cases, similar to the technique 1500 described with reference to fig. 15, each combination of sample barcodes from 3 sets of sample barcodes 1686, 1688, 1690 and their respective samples 1696, 1697, 1698 can be combined separately with an aliquot of a set of barcoding reagents 1692 before being washed and then mixed into a mixed sample 1699.

Fig. 17 is a schematic diagram showing a technique 1700 of preparing a set of preconfigured sample barcode labeled barcoding reagents, in accordance with certain aspects of the present disclosure. A set of barcoding reagents 1792 is provided. A set of barcoding reagents 1792 can include any number of barcoding reagents (e.g., as barcoding reagents 208 in fig. 2) optionally tagged with assay barcode elements for any number of assays. A set of barcoding reagents 1792 may include barcoding reagents with a plurality of different capture antibodies and/or assay-specific biomolecules. 3 sets of sample barcodes 1786, 1788, 1790 may be provided. Although fig. 17 depicts the use of 3 sets of sample barcodes 1786, 1788, 1790, any number of sample barcode sets may be used, which may be determined in advance by the number of sample barcode sets available and/or the number of different samples being assayed. Each of the sets of sample barcodes 1786, 1788, 1790 can contain some of the same sample barcodes, such that the sample barcodes of the first set of sample barcodes 1786 are all identical and all unique to the second set of sample barcodes 1788, which are themselves all identical and all unique to the third set of sample barcodes 1790, which are themselves all identical. The 3 sets of sample barcodes 1786, 1788, 1790 and/or 1 set of barcoding reagents 1792 may be provided as a kit or as part of a kit comprising a freeze-dried set.

As shown in fig. 17, the set of barcode reagents 1792 contains 7 different types of barcode reagents (e.g., reagents t-z). Each different type of barcoding reagent represents a barcoding reagent with a unique assay barcode and a unique capture antibody or assay specific biomolecule. Each different type of barcoded reagent of the set of barcoded reagents 1792 may be distributed in some wells along a respective column of the well plate 1785. Thus, each well along the column shares the same type of barcoding reagent, whereas each well between rows contains a different barcoding reagent.

Each set of sample barcodes 1786, 1788, 1790 can be distributed along a respective row of the well plate 1785. Thus, each well shares the same sample barcode along the rows, whereas each well contains a different sample barcode along the columns.

Due to the combination of the sample barcode and barcoded reagent in the wells of the well plate 1785, each well will contain a unique combination of sample barcode and barcoded reagent (e.g., t: A; u: B to z: C). The sample barcode may optionally be bound to a barcoded reagent within the same well, or may simply be maintained in a mixture for future binding to the sample cells or particles.

Thus, a unique set of combinations of sample barcodes and barcoding reagents may be generated. Based on the user's needs, some or all of the set of unique combinations of sample barcodes and barcoding reagents may be provided to one or more samples to label the samples and perform the assay as desired.

The foregoing description of embodiments, including illustrated embodiments, has been presented for the purposes of illustration and description only and is not intended to be exhaustive or limited to the precise forms disclosed. Many modifications, improvements and uses thereof will be apparent to those skilled in the art.

As used below, any reference to a series of embodiments will be understood as a reference to each of those embodiments individually (e.g., "embodiments 1-4" will be understood as "embodiments 1, 2, 3, or 4").

Example 1 is a panel for elemental analysis comprising: a plurality of conjugated antibodies, wherein each of the plurality of conjugated antibodies is tagged with a different elemental tag, wherein each different elemental tag is distinguishable based on its isotopic composition, and wherein the plurality of conjugated antibodies are in a lyophilized mixture.

Embodiment 2 is the set of embodiment 1, wherein the plurality of conjugated antibodies comprises two or more antibodies from the list comprising: cd45, Cd45RA, Cd45RO, Cd123, Cd4, Cd8a, Cd11C, Cd57, CXCR3, Cd185, Cd38, Cd56, Cd3, Cd20, Cd66b, HLA-DR, IgD, Cd27, Cd28, Cd127, Cd19, Cd16, Cd161, Cd194, Cd25, Cd294, Cd197, Cd14, CCR6, and TCR δ γ.

Example 3 is the set of examples 1 or 2, wherein a majority of the conjugated antibodies are specific for cell types in human peripheral blood.

Example 4 is the set of examples 1 to 3, wherein a majority of the conjugated antibodies are specific for cell surface markers.

Embodiment 5 is the set of embodiments 1-4, wherein the plurality of conjugated antibodies comprises ten or more conjugated antibodies in the lyophilized mixture.

Embodiment 6 is the set of embodiments 1-5, wherein each different element tag comprises a plurality of element atoms of an isotope.

Example 7 is the set of examples 1 to 6, wherein at least two conjugated antibodies of the plurality of conjugated antibodies are tagged with different element tags having different isotopes of a single element.

Embodiment 8 is the set of embodiments 1-7, further comprising a biomolecule coupled to other element tags, wherein the biomolecule is not an antibody, and wherein the other element tags are distinguishable from each different element tag based on their isotopic composition.

Example 9 is the set of examples 1-8, further comprising a non-antibody metal-containing moiety comprising a metal isotope distinguishable from each different elemental tag based on its isotopic composition.

Embodiment 10 is the set of embodiments 1-9, wherein each different elemental tag comprises a metallic element having an atomic weight greater than 80 amu.

Embodiment 11 is the set of embodiments 1-10, wherein each different element tag comprises a chelated metal.

Embodiment 12 is the set of embodiments 1-11, wherein each different element tag comprises an element that is not endogenous to human peripheral blood.

Embodiment 13 is the set of embodiments 1-12, wherein the set has a moisture content at or less than 5% by weight.

Embodiment 14 is the set of embodiments 1-12, wherein the set has a moisture content at or less than 3% by weight.

Embodiment 15 is the set of embodiments 1-12, wherein the set has a moisture content at or less than 1% by weight.

Embodiment 16 is the set of embodiments 1-12, wherein the set has a moisture content at or between 0.05 to 1% by weight.

Embodiment 17 is the set of embodiments 1-16, further comprising a freeze-dried intercalating agent, wherein the freeze-dried intercalating agent is included in the freeze-dried mixture.

Embodiment 18 is the set of embodiments 1-17, further comprising a freeze-dried calibration material, wherein the freeze-dried calibration material comprises a known amount of one or more known isotopes, and wherein the freeze-dried calibration material is contained in the freeze-dried mixture.

Embodiment 19 is the set of embodiments 1-18, further comprising a supplemental reagent, wherein the supplemental reagent is useful for performing an assay using a plurality of conjugated antibodies, wherein the supplemental reagent is lyophilized, and wherein the supplemental reagent is contained in the lyophilized mixture.

Example 20 is an assay kit for use with elemental analysis comprising: a closed container; and the group of embodiments 1 to 19, wherein the freeze-dried mixture of the group is stored within the closed container.

Embodiment 21 is the assay kit of embodiment 20, wherein the closed container comprises an internal atmosphere of inert gas or dry air.

Embodiment 22 is the assay kit of embodiment 20 or 21, wherein the plurality of conjugated antibodies comprises a first antibody specific for a cell surface marker and a second antibody specific for an intracellular target.

Embodiment 23 is the assay kit of embodiments 20-22, further comprising: other closed containers; and a set of other antibodies comprising at least one other conjugated antibody tagged with a different element tag distinguishable from each different element tag based on its isotopic composition, wherein the other conjugated antibody is freeze-dried and stored within a further closed container.

Embodiment 24 is the assay kit of embodiment 23, wherein the plurality of conjugated antibodies comprises antibodies specific for one or more cell surface markers, and wherein the other conjugated antibodies are specific for an intracellular target.

Example 25 is the assay kit of examples 20-24, further comprising an intercalating agent comprising additional different elemental tags distinguishable from each different elemental tag based on their isotopic composition.

Embodiment 26 is the assay kit of embodiments 20-25, further comprising a plurality of sample barcoding reagents for labeling a plurality of samples, wherein each of the plurality of sample barcoding reagents comprises a different combination of isotopes.

Embodiment 27 is the assay kit of embodiment 26, further comprising a plurality of containers, wherein each of the plurality of sample barcoding reagents is contained within a different container of the plurality of containers.

Embodiment 28 is the assay kit of embodiment 26 or 27, wherein each of the plurality of sample barcoding reagents binds to a majority of cells in the sample.

Embodiment 29 is the assay kit of embodiments 26-28, wherein each of the plurality of sample barcoding reagents comprises an elemental tag functionalized to covalently bind on or within a cell of the sample.

Embodiment 30 is the assay kit of embodiments 26-29, wherein each of the plurality of sample barcoding reagents comprises a sample barcoded antibody that specifically binds a target present on a majority of cells in the sample or multiple targets that together bind a majority of cells in the sample.

Embodiment 31 is the assay kit of embodiment 30, wherein each of the sample barcoded antibodies specifically binds to one or more of CD45, CD298, and b2 m.

Embodiment 32 is the assay kit of embodiment 30 or 31, wherein the element tag of the sample barcoded antibody provides a weaker signal than the element tag of a majority of the other antibodies in the set when analyzed using an element analyzer.

Embodiment 33 is the assay kit of embodiments 26-32, wherein the different combinations of isotopes comprise cadmium.

Embodiment 34 is the assay kit of embodiments 26-33, wherein the different combinations of isotopes comprise platinum in cisplatin.

Embodiment 35 is the assay kit of embodiments 26-34, wherein each of the plurality of sample barcoding reagents comprises a set of sample barcoded antibodies, wherein each sample barcoded antibody comprises all isotopes of a different combination of the isotopes.

Embodiment 36 is the assay kit of embodiments 26-35, wherein each of the plurality of sample barcoding reagents is capable of barcoding a living cell, and wherein each of the plurality of sample barcoding reagents is non-toxic to the living cell.

Embodiment 37 is the assay kit of embodiments 20-36, further comprising assay barcoding reagents comprising additional antibodies for detecting different analytes, wherein each assay barcoding reagent comprises a different combination of isotopes.

Embodiment 38 is the assay kit of embodiment 37, wherein each assay barcoded reagent is an assay barcoded bead comprising a different combination of the isotopes.

Embodiment 39 is the assay kit of embodiment 38, wherein the assay barcoding reagents are contained in a lyophilized mixture of the set.

Embodiment 40 is the assay kit of embodiment 38 or 39, wherein each assay barcoded bead comprises a unique combination of isotopes present within the assay barcoded bead.

Embodiment 41 is the assay kit of embodiments 37-40, wherein the assay barcoding reagents comprise at least ten assay barcoding reagents for barcoding at least ten different analytes, and wherein the assay barcoding reagents are provided in a mixture.

Embodiment 42 is the assay kit of embodiments 37-41, wherein the different analyte is free analyte in human peripheral blood.

Embodiment 43 is the assay kit of embodiments 37-42, further comprising a combination of reporter antibodies that specifically bind the different analytes, wherein each reporter antibody comprises an element tag detectable by elemental analysis.

Embodiment 44 is the assay kit of embodiment 43, wherein each of the elemental tags of the combination of reporter antibodies comprises an isotopically identical element detectable by elemental analysis.

Embodiment 45 is the assay kit of embodiments 37-44, wherein each assay barcode reagent is functionalized to attach to a sample barcode of a sample barcoding composition comprising an isotope.

Embodiment 46 is the assay kit of embodiment 45, further comprising a sample barcode, the sample barcode comprising the isotopic sample barcoded composition.

Embodiment 47 is the assay kit of embodiment 45 or 46, wherein the sample barcode can bind to cells of the sample stained by the freeze-dried group.

Embodiment 48 is the assay kit of embodiments 20-47 further comprising barcoding reagents, wherein each barcoding reagent comprises an isotopic assay barcoding composition and an isotopic sample barcoding composition, wherein each different isotopic assay barcoding composition is associated with a different analyte, and wherein each different isotopic sample barcoding composition is associated with a different sample.

Embodiment 49 is the assay kit of embodiment 48, wherein each barcoded reagent is a bead, and wherein the sample barcoded composition of isotopes is located inside the bead.

Embodiment 50 is the assay kit of embodiment 48, wherein each barcoded reagent is a bead, and wherein the sample barcoded composition of isotopes is located on the surface of the bead.

Embodiment 51 is the assay kit of embodiments 20-50, further comprising an anticoagulant.

Embodiment 52 is the assay kit of embodiments 20-51, further comprising a calibration material, wherein the calibration material comprises a known amount of a known isotope.

Example 53 is an assay kit for use with elemental analysis comprising: a plurality of closed containers; and the group according to embodiments 1 to 19, wherein the freeze-dried mixture of the group is distributed in a plurality of closed containers.

Embodiment 54 is the assay kit of embodiment 53, further comprising a plurality of sample barcoding reagents for labeling a plurality of samples, wherein each of the plurality of sample barcoding reagents comprises a different combination of isotopes, and wherein each of the plurality of sample barcoding reagents is contained within a different container of the plurality of closed containers.

Embodiment 55 is a barcoded system, comprising: a barcode reagent comprising an assay barcode, wherein the assay barcode comprises an isotopic composition associated with a target analyte, wherein the isotopic composition are distinguishable by elemental analysis, wherein the barcode reagent comprises one of a plurality of sample barcodes or at least one functionalized to bind to a plurality of sample barcodes, wherein each sample barcode of the plurality of sample barcodes comprises a unique other composition of isotopes distinguishable from the assay barcode composition of isotopes by elemental analysis, wherein each sample barcode of the plurality of sample barcodes can be associated with a different sample. The barcoding system may optionally further comprise a group from example 1 or a related example.

Embodiment 56 is the system of embodiment 55, wherein the barcoded reagent is a bead, and optionally wherein the sample barcode is present inside the bead.

Embodiment 57 is the system of embodiment 55, wherein the barcoding reagent is a bead, and wherein the surface of the bead is functionalized to bind to a variety of sample barcodes.

Embodiment 58 is the system of embodiments 55-57, wherein the barcoded reagent is a bead, and wherein a surface of the bead comprises one of a plurality of sample barcodes.

Embodiment 59 is the system of embodiments 55-58, wherein the barcoding reagent is functionalized to bind to a plurality of sample barcodes, and optionally wherein the system further comprises each sample barcode of the plurality of sample barcodes in a separate container.

Embodiment 60 is the system of embodiments 55-59, further comprising a sample barcoding reagent, the sample barcoding reagent comprising at least one of a plurality of sample barcodes, and optionally wherein the sample barcoding reagent can bind both the barcoding reagent and the cells of the sample.

Embodiment 61 is the system of embodiments 55-60, wherein the barcoding reagent is a bead, and wherein the assay barcode is present inside the bead, and optionally wherein the inside of the bead comprises a solid metal core, inside a metal chelating polymer, inside a nanocomposite, or inside a hybrid material (hybrid).

Embodiment 62 is the system of embodiment 56-58 or 61, wherein the bead has a solid metallic core and a polymeric surface.

Embodiment 63 is the system of embodiment 62, wherein the polymer surface is bound to an antibody, and the antibody is bound to the target analyte.

Embodiment 64 is the system of embodiment 63, wherein the target analyte is a free analyte present in blood.

Embodiment 65 is the system of embodiments 55-64, further comprising a reporter antibody that specifically binds to a target analyte and comprises an element tag or a combination of a high intensity element tag and a low intensity element tag.

Embodiment 66 is the system of embodiment 65, wherein the assay barcoding reagents comprise at least ten assay barcoding reagents for barcoding at least ten different analytes, and optionally wherein separate mixtures of the plurality of assay barcoding reagents each comprise an isotopically distinguishable sample barcode.

Embodiment 67 is the system of embodiment 65 or 66, further comprising reporter biomolecules that specifically bind to a target analyte of the assay barcoding reagent, wherein the reporter biomolecules comprise affinity reagents or oligonucleotides, respectively, and wherein each reporter biomolecule comprises an element tag or a combination of a high signal element tag and a low signal element tag.

Embodiment 68 is the system of embodiments 65-67, wherein at least some of the reporter biomolecules that specifically bind different target analytes comprise the same elemental signature.

Embodiment 69 is a method comprising: providing a plurality of antibodies; conjugating each of the plurality of antibodies with a different element tag, wherein each different element tag is distinguishable based on its isotopic composition, and wherein each of the plurality of antibodies is distinguishable by its different element tag; mixing a plurality of conjugated antibodies together into a mixture; and freeze-drying the mixture.

Embodiment 70 is the method of embodiment 69, further comprising spin filtering the plurality of conjugated antibodies.

Embodiment 71 is the method of embodiment 69 or 70, further comprising: selecting a study protocol for studying the sample; and selecting a plurality of antibodies based on the selected study protocol.

Embodiment 72 is the method of embodiments 69-71, wherein providing a plurality of antibodies comprises providing two or more antibodies from the list comprising Cd45, Cd45RA, Cd45RO, Cd123, Cd4, Cd8a, Cd11C, Cd57, CXCR3, Cd185, Cd38, Cd56, Cd3, Cd20, Cd66b, HLA-DR, IgD, Cd27, Cd28, Cd127, Cd19, Cd16, Cd161, Cd194, Cd25, Cd294, Cd197, Cd14, CCR6, and TCR δ γ.

Embodiment 73 is the method of embodiments 69-72, wherein each of the plurality of antibodies is specific for a cell type in human peripheral blood.

Embodiment 74 is the method of embodiments 69-73, wherein each of the plurality of antibodies is specific for a cell surface marker.

Embodiment 75 is the method of embodiments 69-74, wherein mixing a plurality of conjugated antibodies together comprises mixing ten or more antibodies together.

Embodiment 76 is the method of embodiments 69-75, wherein each different element tag comprises a plurality of element atoms of an isotope.

Embodiment 77 is the method of embodiments 69-76, wherein at least two of the different element tags have different isotopes of a single element.

Embodiment 78 is the method of embodiments 69-77, further comprising providing a biomolecule comprising other element tags, wherein the other element tags are distinguishable from each different element tag based on its isotopic composition, wherein the biomolecule is not an antibody, and wherein mixing the plurality of conjugated antibodies together further comprises mixing the biomolecule with the plurality of conjugated antibodies.

Embodiment 79 is the method of embodiments 69-78, wherein each different element tag comprises a metallic element having an atomic weight greater than 80 amu.

Embodiment 80 is the method of embodiments 69-79, wherein each different element tag comprises a chelating metal.

Embodiment 81 is the method of embodiments 69-80, wherein each different element tag comprises elements that are not endogenous to human peripheral blood.

Embodiment 82 is the method of embodiments 69-81, wherein freeze drying the mixture comprises reducing the water content to 5% by weight or less.

Embodiment 83 is the method of embodiments 69-82, further comprising mixing an intercalator into the mixture prior to lyophilizing the mixture, wherein the intercalator comprises other elemental tags distinguishable from each different elemental tag based on their isotopic composition.

Embodiment 84 is the method of embodiments 69-83, further comprising mixing a calibration material into the mixture prior to freezing the dried mixture, wherein the calibration material comprises a known amount of a known isotope.

Embodiment 85 is the method of embodiments 69-84, further comprising mixing a supplemental reagent into the mixture prior to lyophilizing the mixture, wherein the supplemental reagent is useful to facilitate performing an assay using the plurality of conjugated antibodies.

Embodiment 86 is the method of embodiments 69-85, wherein freeze-drying the mixture further comprises storing the freeze-dried mixture in a closed container having an internal atmosphere of inert gas or dry air.

Embodiment 87 is the method of embodiments 69-86, wherein the plurality of antibodies comprises a first antibody specific for a cell surface marker and a second antibody specific for an intracellular target.

Embodiment 88 is the method of embodiments 69-87, further comprising: providing at least one additional antibody; conjugating the at least one other antibody with at least one other distinct elemental tag distinguishable from each distinct elemental tag based on its isotopic composition; freeze-drying the at least one additional antibody; and storing the freeze-dried at least one other antibody separately from the freeze-dried mixture.

Embodiment 89 is the method of embodiment 88, wherein the plurality of antibodies comprises antibodies specific for one or more cell surface markers, and wherein the other antibodies are specific for an intracellular target.

Embodiment 90 is the method of embodiments 69-89, further comprising providing a plurality of sample barcoding reagents for labeling a plurality of samples, wherein each of the plurality of sample barcoding reagents comprises a different combination of isotopes.

Embodiment 91 is the method of embodiment 90, further comprising: providing a plurality of containers; and storing each of the plurality of sample barcoded reagents in a different container of the plurality of containers.

Embodiment 92 is the method of embodiment 90 or 91, wherein each of the plurality of sample barcoding reagents binds to a majority of cells in the sample.

Embodiment 93 is the method of embodiments 90-92, wherein each of the plurality of sample barcoding reagents comprises an elemental tag functionalized to covalently or otherwise permanently bind on or within cells of the sample.

Embodiment 94 is the method of embodiments 90-93, wherein each of the plurality of sample barcoding reagents comprises a sample barcoded antibody that specifically binds a target present in a majority of cells in the sample.

Embodiment 95 is the method of embodiment 94, wherein each of the sample barcoded antibodies specifically binds one or more of CD45, CD298, and b2 m.

Embodiment 96 is the method of embodiment 94 or 95, wherein each of the sample barcoded antibodies specifically binds to a target present in the sample selected to result in a mass signal of a single sample barcoded antibody that is weaker than the mass signal of a majority of the plurality of conjugated antibodies.

Embodiment 97 is the method of embodiments 90-96, wherein the different combinations of isotopes comprise cadmium.

Embodiment 98 is the method of embodiments 90-97, wherein the different combinations of isotopes comprise platinum in cisplatin.

Embodiment 99 is the method of embodiments 90-98, wherein each of the plurality of sample barcoding reagents comprises a set of sample barcoded antibodies, wherein each sample barcoded antibody comprises all isotopes of a different combination of the isotopes.

Embodiment 100 is the method of embodiments 90-99, wherein each of the plurality of sample barcoding reagents is capable of barcoding a living cell, and wherein each of the plurality of sample barcoding reagents is non-toxic to the living cell.

Embodiment 101 is the method of embodiments 69-100, further comprising: assay barcoded reagents are provided, the assay barcoded reagents comprising additional antibodies for detecting different analytes, wherein each assay barcoded reagent comprises a different combination of isotopes.

Embodiment 102 is the method of embodiment 101, wherein each assay barcoded reagent is an assay barcoded bead containing a different combination of isotopes.

Embodiment 103 is the method of embodiment 102, wherein each assay barcoded bead comprises a unique combination of isotopes present within the interior of the assay barcoded bead.

Embodiment 104 is the method of embodiments 101-103, wherein the assay barcoding reagents comprise at least ten assay barcoding reagents for barcoding at least ten different analytes, and wherein the assay barcoding reagents are provided in a mixture.

Embodiment 105 is the method of embodiments 101-104, wherein the different analyte is a free analyte in human peripheral blood.

Embodiment 106 is the method of embodiments 101-105, further comprising providing a combination of reporter antibodies that specifically bind the different analytes, wherein each reporter antibody comprises an element tag detectable by elemental analysis.

Embodiment 107 is the method of embodiment 106, wherein each of the elemental tags of the combination of reporter antibodies comprises an isotopically identical element detectable by elemental analysis.

Embodiment 108 is the method of embodiments 101-107, further comprising functionalizing each assay barcode reagent to attach to a sample barcode of the sample barcode composition comprising an isotope.

Embodiment 109 is the method of embodiment 108, wherein the sample barcode binds cells of the sample determined by freeze-drying the mixture.

Embodiment 110 is the method of embodiment 108 or 109, further comprising providing a sample barcode, the sample barcode comprising a sample barcoded composition of isotopes.

Embodiment 111 is the method of embodiments 69-110, further comprising providing barcoding reagents, wherein each barcoding reagent comprises an isotopic assay barcoding composition and an isotopic sample barcoding composition, wherein each different isotopic assay barcoding composition is associated with a different analyte, and wherein each different isotopic sample barcoding composition is associated with a different sample.

Embodiment 112 is the method of embodiment 111, wherein each barcoded reagent is a bead, and wherein the sample barcoded composition of isotopes is located inside the bead.

Embodiment 113 is the method of embodiment 111 or 112, wherein each barcoded reagent is a bead, and wherein the sample barcoded composition of isotopes is located on a surface of the bead.

Embodiment 114 is the method of embodiments 69-113, further comprising providing an anticoagulant.

Embodiment 115 is the method of embodiments 69-114, further comprising titrating and diluting each of the plurality of conjugated antibodies to a predetermined concentration prior to mixing the plurality of conjugated antibodies.

Embodiment 116 is the method of embodiments 69-115, further comprising mixing the plurality of conjugated antibodies with an excipient prior to lyophilizing the mixture.

Embodiment 117 is the method of embodiment 116, wherein the excipient comprises a sugar and bovine serum albumin.

Embodiment 118 is the method of embodiment 116 or 117, further comprising mixing the plurality of conjugated antibodies with a viability stain prior to lyophilizing the mixture.

Embodiment 119 is the method of embodiment 118, wherein the viability stain is a rhodium intercalator.

Embodiment 120 is a method, comprising: preparing a sample; providing a set of lyophilized antibodies comprising a plurality of conjugated antibodies, wherein each of the plurality of conjugated antibodies is labeled with a different elemental label, and wherein each different elemental label is distinguishable based on its isotopic composition; performing surface staining of cells of the sample using a freeze-dried antibody panel; and studying the sample using elemental analysis to detect the presence of the different elemental tags.

Embodiment 121 is the method of embodiment 120, wherein studying the sample using elemental analysis comprises processing the sample on an inductively coupled plasma mass spectrometer to detect the presence of different elemental tags of a freeze-dried set of antibodies.

Embodiment 122 is the method of embodiment 120 or 121, further comprising staining the sample of cells with a viability stain.

Embodiment 123 is the method of embodiments 120-122, wherein the viability stain is provided as part of a freeze-dried antibody panel.

Embodiment 124 is the method of embodiment 123, wherein the viability stain is rhodium.

Embodiment 125 is the method of embodiments 120-124, further comprising performing FcR blocking of the sample of cells.

Embodiment 126 is the method of embodiments 120-125, further comprising fixing the sample after performing the surface staining.

Embodiment 127 is the method of embodiments 120-126, further comprising staining the sample with an intercalating agent, wherein the intercalating agent comprises other distinct elemental tags distinguishable from each distinct elemental tag based on their isotopic composition.

Embodiment 128 is the method of embodiment 127, wherein staining of the sample occurs by an intercalating agent after permeabilizing the sample.

Embodiment 129 is the method of embodiments 120-128, further comprising permeabilizing the sample and performing intracellular staining of the sample of cells with at least one other antibody, wherein the at least one other antibody is labeled with a further different elemental label that is distinguishable from each different elemental label based on its isotopic composition.

Embodiment 130 is the method of embodiments 120-129, wherein preparing the sample comprises collecting whole blood.

Embodiment 131 is the method of embodiment 130, wherein preparing the sample comprises isolating peripheral blood mononuclear cells from whole blood.

Embodiment 132 is the method of embodiments 120-131, further comprising labeling the sample with a sample barcoding reagent, wherein the sample barcoding reagent comprises different combinations of isotopes available for distinguishing the sample barcoding reagent from other sample barcoding reagents.

Embodiment 133 is the method of embodiment 132, wherein investigating the sample comprises acquiring data by elemental analysis, identifying a sample barcoding reagent by different combinations of the isotopes in the acquired data, and correlating the acquired data with the sample.

Embodiment 134 is the method of embodiment 133, wherein studying the sample further comprises mixing the sample with other samples prior to acquiring data by elemental analysis.

Embodiment 135 is the method of embodiments 120-134, wherein performing surface staining comprises: adding a suspension of cells of the sample to the freeze-dried antibody panel or a re-suspension of the freeze-dried antibody panel; and removing unbound antibody.

Embodiment 136 is the method of embodiments 120-135, further comprising: providing assay barcoding reagents comprising other antibodies for detecting different analytes, wherein each assay barcoding reagent comprises a different combination of isotopes; and mixing an assay barcoding reagent with the sample and removing unbound antibody prior to investigating the sample.

Embodiment 137 is the method of embodiment 136, wherein the sample comprises plasma, and wherein the different analyte is a free analyte within the plasma.

Embodiment 138 is the method of embodiment 136 or 137, wherein providing the assay barcoded reagent and providing the freeze-dried antibody panel occur by providing a mixture of a freeze-dried antibody panel and an assay barcoded reagent.

Embodiment 139 is the method of embodiments 136-138, wherein preparing the sample comprises collecting whole blood.

Embodiment 140 is the method of embodiment 139, wherein preparing the sample further comprises separating peripheral blood mononuclear cells and plasma, wherein performing surface staining comprises mixing a freeze-dried antibody panel with the peripheral blood mononuclear cells and removing unbound antibodies; and wherein determining mixing of the barcoded reagent with the sample comprises determining mixing of the barcoded reagent with the plasma and removing unbound antibody.

Embodiment 141 is the method of embodiments 120-140, wherein studying the sample further comprises automatically identifying cell viability.

Embodiment 142 is the method of embodiments 120-141, wherein studying the sample further comprises automatically identifying a population of cells.

Embodiment 143 is the method of embodiment 142, wherein studying the sample further comprises identifying characteristics of the automatically identified cell population.

Embodiment 144 is the method of embodiment 143, wherein studying the sample further comprises comparing the identified features for the entire identified cell populations or comparing the identified features associated with one of the identified cell populations to other identified characteristics associated with the same one of the identified cell populations from other samples.

Embodiment 145 is the method of embodiment 143 or 144, wherein identifying the characteristic of the automatically identified cell population comprises determining an abundance of one or more targets on or in a cell of the identified cell population.

Embodiment 146 is the method of embodiments 143-145, wherein the identified characteristic comprises a percentage of cells in the population of cells.

Embodiment 147 is the method of embodiments 120-146, wherein studying the sample further comprises generating at least one of a histogram, a 2D dot plot, and a tSNE plot based on known targets of the freeze-dried panel of antibodies.

Embodiment 148 is the method of embodiment 147, further comprising automatically accessing a stored localization map of a known target of the freeze-dried set of antibodies, wherein the stored localization map is to be moved to the target in association with the associated mass channel.

Embodiment 149 is the method of embodiments 136-148, further comprising: labeling the sample with a sample barcoding reagent, wherein the sample barcoding reagent comprises different combinations of isotopes that distinguish the sample barcoding reagent from other sample barcoding reagents; providing a further sample; labeling the other sample with an other sample barcoding reagent; performing additional surface staining of additional cells of the additional sample using the freeze-dried antibody panel; mixing an assay barcoding reagent with the further sample and removing unbound antibody; and mixing the sample with the other sample prior to studying the sample, wherein studying the sample comprises studying a mixture of the sample and the other sample.

Embodiment 150 is the method of embodiment 149, wherein mixing the sample with the other sample occurs before performing the surface staining.

Embodiment 151 is the method of embodiment 149 or 150, wherein functionalizing the assay barcode reagent to bind to the sample barcode reagent, wherein labeling the sample with the sample barcode reagent comprises binding the sample barcode reagent to a first portion of the assay barcode reagent such that a second portion of the assay barcode reagent is free of the sample barcode reagent, wherein mixing the assay barcode reagent with the other sample comprises mixing the second portion of the assay barcode reagent with the other sample, and wherein mixing the sample with the other sample occurs after mixing the assay barcode reagent with the other sample.

Embodiment 152 is the method of embodiments 120-151, wherein investigating the sample comprises obtaining data relating to the sample using an elemental analysis device, wherein the method further comprises automatically analyzing the data.

Embodiment 153 is the method of embodiment 152, wherein automatically analyzing the data comprises applying a cleanup model (cleanup model) to the data, wherein applying the cleanup model comprises accessing gaussian-type measurements produced by an elemental analysis device related to ionization of the sample.

Embodiment 154 is the method of embodiment 152 or 153, wherein automatically analyzing the data comprises: accessing an element tag specification model, wherein the element tag specification model comprises information associating each of the different element tags of the freeze-dried antibody panel with a cell type; identifying presence information of different elemental tags of the freeze-dried antibody panel; and for each cell of the sample, determining the cell type using the identified presence information for the different element tag and the element tag designation model.

Embodiment 155 is a barcoded kit for elemental analysis, comprising: a plurality of sample barcodes for labeling a plurality of samples, wherein each of the sample barcodes comprises a different combination of isotopes distinguishable by elemental analysis, and wherein each of the sample barcodes is stored in a different container; and a set of biomolecules that can be bound to the plurality of samples, wherein the biomolecules of the set comprise or are functionalized to bind to the plurality of sample barcodes.

Embodiment 156 is a barcoding kit of embodiment 155, wherein each of the biomolecules of the set comprises a unique sample barcode of the plurality of sample barcodes.

Embodiment 157 is the barcode kit of embodiment 155 or 156, wherein each of the set of biomolecules is functionalized to bind to a plurality of sample barcodes, and wherein the set of biomolecules is stored independently from the plurality of sample barcodes.

Embodiment 158 is a barcode kit according to embodiment 155-157, wherein the biomolecules of the set comprise a plurality of beads.

Embodiment 159 is a barcoding kit according to embodiment 158, wherein each bead comprises an outer surface functionalized to bind to a plurality of sample barcodes.

Embodiment 160 is a barcoding kit of embodiment 159, wherein each bead comprises an assay barcode located on an interior of the bead, wherein each assay barcode comprises a combination of other isotopes distinguishable from a different combination of isotopes of the sample barcode by elemental analysis.

Embodiment 161 is a method, comprising: providing a plurality of samples comprising a first sample and a second sample; providing a plurality of sample barcodes comprising a first sample barcode and a second sample barcode, wherein each of the sample barcodes comprises a different combination of isotopes distinguishable by elemental analysis; providing a plurality of biomolecules that can be bound to a plurality of samples, wherein each biomolecule comprises one of the plurality of sample barcodes or is functionalized to bind to the plurality of sample barcodes, and wherein the plurality of biomolecules comprises a first biomolecule and a second biomolecule; mixing the first biomolecule with the first sample; mixing the second biomolecule with the second sample; removing any unbound biomolecules; studying a plurality of samples using elemental analysis to obtain elemental data; detecting the presence of each different combination of isotopes in the elemental data; and correlating the elemental data with one of the plurality of samples using the presence of each different combination of isotopes detected.

Embodiment 162 is the method of embodiment 161, further comprising: mixing the first sample barcode with a mixture comprising the first biomolecule and the first sample; and mixing the second sample barcode with a mixture comprising the second biomolecule and the second sample.

Embodiment 163 is the method of embodiment 161, further comprising: mixing the first sample barcode with the first biomolecule prior to mixing the first biomolecule with the first sample; and mixing the second sample barcode with the second biomolecule prior to mixing the second biomolecule with the second sample.

Embodiment 164 is the method of embodiments 161-163, further comprising mixing the first sample and the second sample prior to studying the plurality of samples.

Embodiment 165 is the method of embodiments 161-164, wherein the plurality of biomolecules comprises a plurality of beads.

Embodiment 166 is the method of embodiment 165, wherein each bead comprises an outer surface functionalized to bind to a plurality of sample barcodes.

Embodiment 167 is the method of embodiment 166, wherein each bead comprises an assay barcode located inside the bead, wherein each assay barcode comprises a combination of other isotopes distinguishable from a different combination of isotopes of the sample barcode by elemental analysis.