WO2019194994A1 - Détection d'analyte - Google Patents

Détection d'analyte Download PDF

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Publication number
WO2019194994A1
WO2019194994A1 PCT/US2019/023647 US2019023647W WO2019194994A1 WO 2019194994 A1 WO2019194994 A1 WO 2019194994A1 US 2019023647 W US2019023647 W US 2019023647W WO 2019194994 A1 WO2019194994 A1 WO 2019194994A1
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WO
WIPO (PCT)
Prior art keywords
antigen retrieval
sample
hapten
biomarker
fraction
Prior art date
Application number
PCT/US2019/023647
Other languages
English (en)
Inventor
Daniel E. CAMPTON
Arturo B. RAMIREZ
Melinda R. DUPLESSIS
Original Assignee
Rarecyte, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US15/946,434 external-priority patent/US10274498B1/en
Priority claimed from US15/946,536 external-priority patent/US10267805B1/en
Priority claimed from US15/946,498 external-priority patent/US10330684B1/en
Application filed by Rarecyte, Inc. filed Critical Rarecyte, Inc.
Publication of WO2019194994A1 publication Critical patent/WO2019194994A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells

Definitions

  • This disclosure relates generally to detection and, in particular, to detecting objects within a sample or fraction thereof.
  • Samples often include objects that are to be imaged for analysis. These objects may include a plurality of biomarkers and/or components for which it is desirous to detect and image. As a result, practitioners, researchers, and those working with samples continue to seek systems and methods to more efficiently and accurately detect and image objects of a sample.
  • Figure 1 shows an example method including two antigen retrieval steps.
  • Figure 2 shows example images for example objects.
  • the term“light” is used to describe various uses and aspects of multiplexing and imaging.
  • the term light is not intended to be limited to describing electromagnetic radiation in the visible portion of the electromagnetic spectrum, but is also intended to describe radiation in the ultraviolet and infrared portions of the electromagnetic spectrum.
  • sample is used to describe a biological fluid, a biological semi- solid, a biological solid (which may remain solid, such as tissue, or may be liquefied in any appropriate manner), a suspension, a portion of the suspension, a component of the suspension, or the like.
  • the sample is the anticoagulated whole blood (i.e. a suspension), the buffy coat (i.e. a portion of the suspension), or a circulating tumor cell (i.e. a component of the suspension).
  • sample referenced is whole blood, though it should be understood that the method and system described and discussed herein is used with any appropriate sample, such as urine, blood, bone marrow, buffy coat, cystic fluid, ascites fluid, stool, semen, cerebrospinal fluid, nipple aspirate fluid, saliva, amniotic fluid, mucus membrane secretions, aqueous humor, vitreous humor, vomit, vaginal fluid, and any other physiological fluid or semi-solid.
  • sample such as urine, blood, bone marrow, buffy coat, cystic fluid, ascites fluid, stool, semen, cerebrospinal fluid, nipple aspirate fluid, saliva, amniotic fluid, mucus membrane secretions, aqueous humor, vitreous humor, vomit, vaginal fluid, and any other physiological fluid or semi-solid.
  • the sample is a tissue sample or a material from adipose tissue, an adrenal gland, bone marrow, a breast, a caudate, a cerebellum, a cerebral cortex, a cervix, a uterus, a colon, an endometrium, an esophagus, a fallopian tube, a heart muscle, a hippocampus, a hypothalamus, a kidney, a liver, a lung, a lymph node, an ovary, a pancreas, a pituitary gland, a prostate, a salivary gland, a skeletal muscle, skin, a small intestine, a large intestine, a spleen, a stomach, a testicle, a thyroid gland, or a bladder.
  • target analyte or“target material” are used to describe a biological material of interest.
  • the target analyte can be a fraction of a sample, such as buffy coat, a cell, such as ova, fetal material (such as trophoblasts, nucleated red blood cells, fetal red blood cells, fetal white blood cells, fetal DNA, fetal RNA, or the like), a circulating tumor cell (“CTC”), a circulating endothelial cell, an immune cell (i.e.
  • naive or memory B cells or naive or memory T cells a mesenchymal cell
  • stem cell a vesicle, such as an exosome, a liposome, a protein, a nucleic acid, a biological molecule, a naturally occurring or artificially prepared microscopic unit having an enclosed membrane, parasites (e.g. spirochetes, such as Borrelia burgdorferi which cause Lyme disease; malaria-inducing agents), microorganisms, viruses, or inflammatory cells.
  • parasites e.g. spirochetes, such as Borrelia burgdorferi which cause Lyme disease; malaria-inducing agents
  • microorganisms e.g. spirochetes, such as Borrelia burgdorferi which cause Lyme disease; malaria-inducing agents
  • viruses e.g. spirochetes, such as Borrelia burgdorferi which cause Lyme disease; malaria-inducing agents
  • the target analyte is a tumor cell from adipose tissue, an adrenal gland, bone marrow, a breast, a caudate, a cerebellum, a cerebral cortex, a cervix, a uterus, a colon, an endometrium, an esophagus, a fallopian tube, a heart muscle, a hippocampus, a hypothalamus, a kidney, a liver, a lung, a lymph node, an ovary, a pancreas, a pituitary gland, a prostate, a salivary gland, a skeletal muscle, skin, a small intestine, a large intestine, a spleen, a stomach, a testicle, a thyroid gland, or a bladder.
  • non-target analyte is used to describe a biological material which is not a target analyte.
  • biomarker is used to describe a substance that is present on or within the target analyte or target material (i.e. intracellular or extracellular the target analyte; internalized, such as through phagocytosis, within the target analyte; or the like).
  • Biomarkers include, but are not limited to, peptides, proteins, subunits, domains, motifs, epitopes, isoforms, DNA, RNA, or the like.
  • the biomarker may be a target molecule for drug delivery.
  • affinity molecule is used to describe any molecule that is capable of binding or interacting with a biomarker.
  • the interaction or binding can be covalent or non-covalent.
  • the affinity molecule includes, but is not limited to, an antibody, a hapten, a protein, an aptamer, an oligonucleotide, a polynucleotide, or any appropriate molecule for interacting with or binding to the biomarker.
  • the term“detection moiety” is used to describe a compound or substance which provides a signal for detection, thereby indicating the presence of another compound or substance, an analyte, or the like within a sample or specimen.
  • the detection moiety can be fluorescent, such as a fluorescent probe, or chromogenic, such as a chromogenic dye.
  • the term“channel” is used to describe a color or color range based on the signal provided by one or more detection moieties. The color or color range is obtained based on the filters chosen and/or the wavelength of the signal(s). For example, a channel may be violet, blue, green, yellow, orange, red, dark red, or the like.
  • each channel has a specific color or color range.
  • a first channel may be green and a second channel may be orange.
  • two or more detection moieties may provide signals having different wavelengths, the signals can be in the same channel based on the filter set used.
  • a first detection moiety provides signal having a wavelength of 488 and a second detection moiety provides a signal having a wavelength of 500.
  • the filter set in one of the channels passes wavelengths of both 488 nm and 500 nm, which permits both to be imaged at the same time, thereby producing a single image including the 488 and 500 emissions.
  • the terms “stain” or “label,” which are used interchangeably, are used to describe an affinity molecule bound to or interacted with a detection moiety.
  • the binding or interaction can be direct or indirect.
  • Direct binding or interaction includes covalent or non-covalent interactions between the biomarker and the detection moiety.
  • Indirect binding or interaction includes the use of at least first and second complementary molecules which form binding pairs.
  • the first and second complementary molecules are, in combination, binding pairs which binds or interacts in at least one of the following manners: hydrophobic interactions, ionic interactions, hydrogen bonding interactions, non-covalent interactions, covalent interactions, affinity interactions, or the like.
  • the binding pairs include, but are not limited to, immune-type binding-pairs, such as, antigen-antibody, antigen-antibody fragment, hapten-anti-hapten, or primary antibody- secondary antibody; nonimmune-type binding-pairs, such as biotin-avidin, biotin- streptavidin, folic acid-folate binding protein, hormone-hormone receptor, lectin- specific carbohydrate, enzyme-enzyme, enzyme-substrate, enzyme-substrate analog, enzyme-pseudo- substrate (substrate analogs that cannot be catalyzed by the enzymatic activity), enzyme-cofactor, enzyme-modulator, enzyme-inhibitor, or vitamin B l2-intrinsic factor.
  • immune-type binding-pairs such as, antigen-antibody, antigen-antibody fragment, hapten-anti-hapten, or primary antibody- secondary antibody
  • nonimmune-type binding-pairs such as biotin-avidin,
  • binding pairs include complementary nucleic acid fragments (including complementary nucleotides, oligonucleotides, or polynucleotides); Protein A-antibody; Protein G-antibody; nucleic acid-nucleic acid binding protein; polymeric linkers (e.g., polyethylene glycol); or polynucleotide-polynucleotide binding protein.
  • the binding pairs can be included within or used as amplification techniques. Amplification techniques are also implemented to increase the number of detection moieties bound to or interacted with the biomarker to increase a signal.
  • the stain when binding pairs are used, can be pre-conjugated, such that, during a labeling, staining, or adding step, the affinity molecule is already bound to or interacted with a detection moiety when added to the sample.
  • the stain when binding pairs are used, can be conjugated in the sample, such that the labeling, staining, or adding step includes at least two sub-steps including introducing (in any desired or appropriate order) an affinity molecule-first binding molecule conjugate and a second binding pair molecule-detection moiety conjugate, wherein the first and second binding pair molecules are complementary and bind to or interact with each other.
  • a plurality of stains can be used to describe two or more stains in which the affinity molecules and/or the detection moieties are different.
  • anti-CK- Alexa 647 is different than anti-EpCAM-Alexa 647.
  • anti-CK-Alexa 647 is different than anti-CK-Alexa 488.
  • the terms“permeabilize” or“permeabilization” are used to describe the dissolution or removal of a portion of a plasma membrane of a target or non target analyte by chemical or other means, such that at least an IgG antibody is capable of crossing the plasma membrane.
  • the term“conjugate” is used to describe a first chemical, molecule, moiety, or the like bound to or interacted with a second chemical, molecule, moiety, or the like.
  • the binding or interaction is direct or indirect.
  • Direct binding or interaction includes covalent or non-covalent interactions between the biomarker and the detection moiety.
  • Indirect binding or interaction includes the use of at least first and second complementary molecules which form binding pairs.
  • the first and second complementary molecules are, in combination, binding pairs which binds or interacts in at least one of the following manners: hydrophobic interactions, ionic interactions, hydrogen bonding interactions, non-covalent interactions, covalent interactions, affinity interactions, or the like.
  • the binding pairs include, but are not limited to, immune-type binding -pairs, such as, antigen-antibody, antigen-antibody fragment, hapten-anti-hapten, or primary antibody- secondary antibody; nonimmune-type binding- pairs, such as biotin-avidin, biotin-streptavidin, folic acid-folate binding protein, hormone- hormone receptor, lectin- specific carbohydrate, enzyme-enzyme, enzyme-substrate, enzyme- substrate analog, enzyme-pseudo-substrate (substrate analogs that cannot be catalyzed by the enzymatic activity), enzyme-cofactor, enzyme-modulator, enzyme-inhibitor, or vitamin B 12- intrinsic factor.
  • immune-type binding -pairs such as, antigen-antibody, antigen-antibody fragment, hapten-anti-hapten, or primary antibody- secondary antibody
  • nonimmune-type binding- pairs such as biotin-avidin, biotin-stre
  • binding pairs include complementary nucleic acid fragments (including complementary nucleotides, oligonucleotides, or polynucleotides); Protein A- antibody; Protein G-antibody; nucleic acid-nucleic acid binding protein; polymeric linkers (e.g., polyethylene glycol); or polynucleotide-polynucleotide binding protein.
  • a sample such as blood
  • the sample is suspected of including at least one target analyte.
  • Suitable devices, systems, and/or methods of sample collection and/or processing may include those described in one or more of the following U.S.
  • Suitable devices, systems, and/or methods for target analyte retrieval, isolation, or picking may include those described in one or more of the following U.S. patents and published applications, each of which is hereby incorporated by reference in its entirety: 9,222,953; 9,440,234; 9,519,002; 9,810,605; 2017/0219463; 2017/0276575.
  • the sample is then dispensed onto or into at least one analysis platform.
  • the analysis platform is a microscope slide, a positively charged microscope slide, a negatively charged microscope slide, a coated microscope slide, a porous slide, a micro-well slide, a well plate, a coverslip, a cell microarray, or the like.
  • the analysis platform can be any appropriate material, including, but not limited to, glass, plastic, ceramic, metal, or the like.
  • the sample is re-suspended in an attachment solution in a vessel prior to being dispensed onto the analysis platform.
  • the attachment solution is added to or mixed with the sample.
  • the re-suspended sample which includes at least a portion of the attachment solution is dispensed onto or into the analysis platform by a dispenser, such as a pipet or repeating pipet.
  • the attachment solution includes an alcohol (such as ethanol or methanol) and a non-steroidal anti-inflammatory drug (such as aspirin).
  • the attachment solution includes an alcohol (such as ethanol or methanol) and an anti-coagulant (such as heparin).
  • the attachment solution includes an alcohol (such as ethanol or methanol), an anti-coagulant (such as heparin), and a non-steroidal anti inflammatory drug (such as aspirin).
  • the sample is spread across the analysis platform.
  • the sample is spread across the analysis platform by a spreader, such as a squeegee, a pipet tip, a blade, a two-piece spreader including a blade and a base.
  • the sample is spread across the analysis platform by centrifuging, wetting, or nutating the analysis platform.
  • the re-suspended sample is cured to adhere the re-suspended sample to the analysis platform.
  • the re-suspended sample is dispensed onto the analysis platform and cured without being spread across the analysis platform. Curing occurs in air, such as at room temperature; in an environmentally-controlled chamber, such as at 37°C; or the like.
  • the sample may undergo an additional fixation step, such as in treatment with formalin or any appropriate fixative, after the curing step has been completed.
  • autofluorescence is reduced or eliminated.
  • the autofluorescence is reduced or eliminated by bleaching the sample with a chemical (such as hydrogen peroxide), an enzyme, light, heat, or the like.
  • the sample then undergoes staining.
  • At least one stain is added to the sample for labeling, such as by an autostainer or manually by an operator.
  • the at least one target analyte is stained.
  • at least one non-target analyte or non-target material is stained.
  • the at least one target analyte and the at least one non target analyte or materials are stained.
  • the biomarker or biomarkers include, but are not limited to: 17-IA, 4-1BB, 4Dc, 6-keto-PGFla, 8-iso-PGF2a, 8-oxo-dG, Al Adenosine Receptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RUB, ADAM, ADAM 10, ADAM 12, ADAM 15, ADAM 17/T ACE, ADAM 8, ADAM9, ADAMTS, AD AMTS 4, AD AMTS 5, Addressins, aFGF, ALCAM, ALK, ALK-l, ALK-7, alpha- 1 -antitrypsin, alpha- V/beta-l antagonist, ANG, Ang, APAF-l, APE, APJ, APP, APRIL, AR
  • the detection moiety is a compound or substance which provides a signal for detection, thereby indicating the presence of another compound or substance, an analyte, or the like within a sample or specimen.
  • the detection moiety can be used as a tracer, as a label for certain structures, as a label for biomarkers, or the like.
  • the detection moiety can be distributed or can label the appropriate structure or biomarkers in manners including, but not limited to, uptake, selective uptake, diffusion, and attachment to a linking molecule.
  • the detection moiety is bound to the biomarker via an affinity molecule (i.e. an antibody capable of binding or interacting with the biomarker; or,“direct labeling”) or to the biomarker via an affinity molecule and at least one linking molecules extending from the affinity molecule to the detection moiety (i.e.“indirect labeling”).
  • the detection moiety can be fluorescent, such as a fluorescent probe, or chromogenic, such as a chromogenic dye.
  • the chromogenic dye which can be used with various enzyme labels (e.g. horseradish peroxidase and alkaline phosphate), includes, but is not limited to, 3,3'-Diaminobenzidine (DAB), 3-Amino-9-Ethylcarbazole (AEC), 4-Chloro-l-Naphtol (CN), P- Phenylenediamine Dihydrochloride/pyrocatechol (Hanker- Yates reagent), Fast Red TR, New Fuchsin, Fast Blue BB, or the like.
  • DAB 3,3'-Diaminobenzidine
  • AEC 3-Amino-9-Ethylcarbazole
  • CN 4-Chloro-l-Naphtol
  • P- Phenylenediamine Dihydrochloride/pyrocatechol Hanker- Yates reagent
  • the fluorescent probe can be a reactive dye, an organic dye, a fluorescent protein, a quantum dot, non-protein organic molecules, a nanoparticle (e.g., nanodiamond), or the like.
  • Fluorescent probes include, but are not limited to 1,5 IAEDANS; 1,8- ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5- FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5- Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-
  • Fluorosensor (FiCRhR); CyQuant Cell Proliferation Assay; Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS; DiA (4-DM6-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Fipophilic Tracer; DiD (DiICl8(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DiICl8(3)); Dinitrophenol; DiO (DiOCl8(3)); DiR; DiR (DiICl8(7)); DM-NERF (high pH); DNP; Dopamine;
  • the detection moiety such as organic fluorophore
  • the detection moiety can have a molecule weight of at least 1 kD, including, without limitation, at least 10 kD, at least 25 kD, at least 50 kD, at least 75 kD, at least 100 kD, at least 150 kD, at least 200 kD, at least 250 kD, at least 300 kD, at least 340 kD, at least 350 kD, at least 500 kD, and at least 750 kD.
  • a nuclear stain such as Sytox
  • the quantum dot has an emission peak less than or equal to 520 nm. In one embodiment, the quantum dot has an emission peak greater than or equal to 520 nm.
  • the sample is imaged, whereby the sample is illuminated with one or more wavelengths of excitation light from a light source, such as infrared, red, blue, green, and/or ultraviolet.
  • a light source such as infrared, red, blue, green, and/or ultraviolet.
  • the imaging can be done with a flow cytometer or a microscope, such as a fluorescent microscope, a scanner, or the like. Imaging can be done in brightfield and/or darkfield illumination, phase contrast, differential interference contrast, fluorescence, light sheet microscopy, super resolution microscopy, confocal microscopy, and Hoffman modulation contrast.
  • the images formed can be overlaid when a plurality of detection moieties are used. Emission, reflection, diffraction, scatter, and combinations thereof are used in for detection/imaging.
  • the images are analyzed to detect, enumerate, and locate the target analyte. Imaging is performed in a tube, on a microscope slide, or in any appropriate vessel or substrate for imaging.
  • the target analyte can be retrieved from the rest of the sample.
  • the target analyte undergoes enrichment and/or isolation.
  • the target analyte is isolated from rest of the sample, whether with or without prior enrichment, by selecting the target analyte at a time with any appropriate device or system.
  • Imaging the analysis platform as discussed above, is performed to aid in isolation by providing location and characterization information for isolation purposes.
  • the target analyte can undergo post-processing analysis, such as sequencing, by using any appropriate method or technique, though more specifically extracellular and intracellular analysis including intracellular protein labeling; nucleic acid analysis, including, but not limited to, DNA arrays, expression arrays, protein arrays, and DNA hybridization arrays; or in situ hybridization (“ISH”— a tool for analyzing DNA and/or RNA, such as gene copy number changes); polymerase chain reaction (“PCR”); reverse transcription PCR. Sequencing is done on the entire genome, the transcriptome, or cDNA.
  • post-processing analysis such as sequencing, by using any appropriate method or technique, though more specifically extracellular and intracellular analysis including intracellular protein labeling
  • nucleic acid analysis including, but not limited to, DNA arrays, expression arrays, protein arrays, and DNA hybridization arrays
  • ISH in situ hybridization
  • PCR polymerase chain reaction
  • Sequencing is done on the entire genome, the transcriptome, or cDNA.
  • the steps of the method described above can be performed by at least one of an imaging microscope, a scanner, a flow cytometer, or a microfluidic device, such as a chip or a microchannel, or the method can be performed by any combination of the above.
  • the flow cytometer is used for the collecting step, where enrichment via fluorescent-activated cell sorting is appropriate
  • the microfluidic device is used for the retrieving step.
  • the methods, though described to include one device per method may be performed such that a combination of devices are used.
  • the devices are capable of being used in any appropriate transmitted light modality.
  • the devices are capable of being used in at least one of brightfield and/or darkfield illumination, phase contrast, differential interference contrast, fluorescence, and Hoffman modulation contrast imaging or detection.
  • the patient sample is a fraction of a suspension, such that the sample is obtained through enrichment, including positive and/or negative enrichment and/or density-based enrichment.
  • the enriched fraction is suspected of including at least one target analyte.
  • the sample is enriched by any appropriate enrichment process including, but not limited to, sequential density fractionation, magnetic-activated cell sorting, fluorescence-activated cell sorting, differential lysis, depletion filters, microfluidic device separation, or the like.
  • Sequential density fractionation is a process by which a patient sample is divided into fractions or a fraction of a sample is divided into sub-fractions by a step-wise or sequential process, such that each step or sequence results in the collection or separation of a different fraction or sub-fraction from the preceding and successive steps or sequences.
  • sequential density fractionation provides individual sub-populations of a population or individual sub-sub-populations of a sub population of a population through a series of steps.
  • separation fluids are used whereby each separation fluid has a different density, thereby separating a fraction of a sample into sub-fractions based on the densities of the respective sub-fractions via the different density separating fluids.
  • any number of biomarkers can be labeled for multiplexing purposes, which allows, for example, multiple biomarkers to be identified using a single image. For example, it can be desirous to detect more than one biomarker of a given target analyte.
  • a set of stains which includes at least two stains and can include up to 4, 6, 8, 10, or more, is used to multiplex the target analyte, such that each stain labels a different biomarker of the target analyte. This allows for detection of a plurality of biomarkers on the target material at any given time.
  • a plurality of detection moieties is used. For example, Alexa 488 and Alex 647 are used.
  • a plurality of types of detection moieties are used.
  • Alexa 488 and QD800 are used.
  • a plurality of detection moieties and a plurality of types of detection moieties are used.
  • at least two stains are in the same channel and at least one stain is in at least one other channel.
  • affinity molecules used are selected based on the desired characteristics of the target analyte or in trying to determine a particular characteristic of the target analyte. For example, mesenchymal, epithelial, and confirmatory/characterization (i.e. cancer cell type (e.g., prostate, breast, colon, lung, etc.)) affinity molecules can be selected to characterize a given target analyte into various cell types.
  • a plurality of affinity molecules i.e.
  • first and second affinity molecules is selected to bind to or interact with a plurality of biomarkers of the target analyte, where each of the plurality of affinity molecules is: (1) directed to a different biomarker of the target analyte; and (2) bound to detection moieties which provide signals in different channels.
  • the detection moieties may be the same or different types. Accordingly, any number of affinity molecules and corresponding detection moieties are implemented whereby the complementary molecules or complexes are specific to each other.
  • an EpCAM antibody is selected as the first affinity molecule to bind or interact with an EpCAM biomarker of the target analyte
  • a HER2 antibody is selected as the second affinity molecule to bind or interact with a HER2 biomarker of the target analyte.
  • the EpCAM antibody is bound to or interacted with a first detection moiety and the HER2 antibody is bound to or interacted with a second detection moiety, such that the first and second detection moieties emit in a different channel.
  • a plurality of affinity molecules is selected to bind to or interact with a plurality of biomarkers on the target analyte, where each of the plurality of affinity molecules is: (1) directed to a different biomarker of the target analyte; and (2) bound to detection moieties which provide a signal in the same channel.
  • the detection moieties of the first and second affinity molecules may be the same or may be different but provide signals within the same channel.
  • an EpCAM antibody is selected as the first affinity molecule to bind or interact with an EpCAM biomarker of the target analyte
  • a cytokeratin antibody is selected as the second affinity molecule to bind or interact with a cytokeratin biomarker of the target analyte.
  • the EpCAM antibody and the cytokeratin antibody are bound to detection moieties which provide a signal in, for example, the green channel.
  • a plurality of affinity molecules i.e. first and second affinity molecules
  • each of the plurality of affinity molecules is: (a) directed to a different biomarker of the target analyte, and (b) bound to the same type of detection moiety
  • at least one affinity molecule i.e. a third affinity molecule
  • the detection moieties bound to or interacted with the first and second affinity molecules provide signals within the same channel
  • the detection moiety bound to or interacted with the third affinity molecules provides a signal in a different channel.
  • a plurality of stains can be used such that each stain includes a different binding pair. In one embodiment, a plurality of stains can be used such that at least stains include the same binding pair.
  • multiple rounds of labeling is performed for cyclic labeling purposes.
  • a target analyte is labeled with a first set of labels, the first set of labels are imaged, the signal provided by the stains are reduced or eliminated (such as with heat (between 50-110 °C, including 70 °C, 80 °C, 90 °C, 95 °C, l00°C, or l05°C), light, chemicals, enzymes, higher affinity molecules to remove the detection moiety, or any appropriate manner in which to reduce or eliminate the signal, such as by degrading, cleaving, or modifying), the target analyte is labeled with a second set of labels, and so on until a desired number of biomarkers have been labeled and/or a desired number of rounds have been performed.
  • the heating step is performed in any device or system appropriate for heating the sample, including, but not limited to, an autostainer, a steamer (such as a vegetable steamer or a bottle sanitizer), a pressure cooker, an autoclave, a water bath, a hot plate, a crockpot, a hair dryer, an incubator, a microwave, or a combination thereof.
  • a steamer such as a vegetable steamer or a bottle sanitizer
  • a pressure cooker such as a vegetable steamer or a bottle sanitizer
  • an autoclave such as a vegetable steamer or a bottle sanitizer
  • a pressure cooker such as a vegetable steamer or a bottle sanitizer
  • an autoclave such as a vegetable steamer or a bottle sanitizer
  • a pressure cooker such as a vegetable steamer or a bottle sanitizer
  • an autoclave such as a vegetable steamer or a bottle sanitizer
  • a single stain is used for each round of labeling, such that five stains are used individually across five rounds of labeling.
  • a plurality of stains is used for each round of labeling, such that each round of labeling uses a plurality of stains.
  • at least one round of labeling includes a plurality of stains and at least one round of labeling includes a single stain.
  • amplification techniques are used to increase the signal-to- noise ratio within at least one channel or for at least one stain.
  • the amplification techniques include, but are not limited to, addition of amplification moieties such as haptens and anti-haptens (such as DNP and anti-DNP; DIG and anti-DIG; FITC and anti-FITC; HQ and anti-HQ; and biotin), primary and secondary antibodies, horseradish peroxidase (HRP) and tyramide, HRP or alkaline phosphatase and 3,3' diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC), and complementary molecules (such as biotin and an avidin).
  • HRP horseradish peroxidase
  • DAB 3,3' diaminobenzidine
  • AEC 3-amino-9-ethylcarbazole
  • complementary molecules such as biotin and an avidin.
  • amplification is performed on a single biomarker. In one embodiment, amplification is performed on two or more biomarkers. In one embodiment, amplification performed on two or more biomarkers uses different types of amplification techniques. In one embodiment, amplification performed on two or more biomarkers uses the same type of amplification technique. For example, tyramide signal amplification, which includes at least one horseradish peroxidase molecule (which can be directly or indirectly bound to or interacted with the affinity molecule bound to or interacted with the biomarker) and at least one tyramide-detection moiety conjugate, can be used to amplify multiple biomarkers, whether they are in the same channel or a different channel.
  • multiple haptens can be used to amplify multiple biomarkers, whether they are in the same channel or a different channel.
  • the same type of haptens can be used to amplify different biomarkers; different types of haptens can be used to amplify different biomarkers; or two or more biomarkers may be amplified by a first type of hapten and one or more biomarkers may be amplified by a second type of hapten.
  • an amplification moiety (e.g., HRP) denaturing step can be implemented, such as by adding hydrogen peroxide.
  • the amplification moiety denaturing step eliminates potential cross -reactivity of affinity molecules while allowing for the amplification of a species-independent channel. In other words, the amplification moiety denaturing step opens up an imaging channel for detection of an additional biomarker.
  • the amplification moiety denaturing step is performed before an antigen retrieval step, such as the second antigen retrieval step when two antigen retrievals are performed.
  • the amplification moiety denaturing step is performed after an antigen retrieval step, such as the second antigen retrieval step when two antigen retrievals are performed.
  • the species of that affinity molecule can be independent of any other species implemented in another channel.
  • Species include, but are not limited to a cat, chicken, cow, dog, donkey, goat, guinea pig, hamster, horse, human, llama, monkey, mouse, pig, rabbit, rat, or sheep.
  • Antigen retrieval is a process by which epitope detectability on a sample is increased.
  • the sample is incubated with a solution, such as a buffer (e.g., tris-based buffer, Cell Conditioning 1 from Ventana, or the like), for a given amount of time (e.g., 1 min to 24 hours, including, without limitation, 2 min, 3 min, 4 min, 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, and 18 hours) at a given temperature (50-110 °C, including at 50-90 °C, 50-85 °C, 60-80 °C, 65-85 °C, 70 °C, 80 °C, 85 °C, or at 90 °C).
  • a buffer e.g., tris-based buffer, Cell Conditioning 1 from Ventana, or the like
  • the time and temperature are selected based on the desired epitope or biomarker to be retrieved and appropriate conditions for renaturing the desired epitope or biomarker.
  • Antigen retrieval unmasks epitopes that otherwise would not be detectable to a specific antibody due to sample processing, such as fixation.
  • antigen retrieval is implemented a single time.
  • multiple antigen retrievals e.g., 2, 3, 4, 5, up to 10, up to 25, up to 50, up to 100, or any desirable or appropriate amount
  • each successive antigen retrievals can use a higher temperature than the previous antigen retrievals.
  • each successive antigen retrievals can use a lower temperature than the previous antigen retrievals.
  • successive antigen retrievals can use a higher or lower temperature than the previous antigen retrieval based on the appropriate conditions for antigen retrieval of the desired or targeted epitope/biomarker.
  • At least two separate antigen retrievals can be implemented to prevent a reduction or elimination of efficacy of an antibody, detection moiety, reagent, or the like (such as by denaturing), where a particular condition (such as temperature) affects the efficacy of the antibody, detection moiety, reagent, or the like.
  • a first antigen retrieval is performed at approximately 50-110 °C, including at 50-90 °C, 50-85 °C, 60- 80 °C, 65-85 °C, 70 °C, 80 °C, 85 °C, or at 90 °C.
  • the first antigen retrieval step is performed on or for an epitope or biomarker which can be denatured or have its detectability reduced when exposed to an antigen retrieval step above a given temperature. Labeling of at least one biomarker is then performed.
  • the labeling can include at least one amplification technique discussed above.
  • the labeling step such as with the amplification step, is performed to ensure detectability of the epitope or biomarker when exposed to a second antigen retrieval at or above a given temperature. For example, tyramide signal amplification can be used to detect a first biomarker.
  • an activated tyramide-detection moiety conjugate (by way of an enzymatic reaction with horseradish peroxidase) forms a covalent bond with a tyrosine located at or near the first biomarker.
  • the covalent bond is not broken during the second antigen retrieval step, thereby allowing for detection of the first biomarker which could typically be denatured during the second antigen retrieval at or above the given temperature.
  • a second antigen retrieval is then performed at approximately 50-110 °C, including at 80-110 °C or, more specifically, at 100 °C.
  • the second antigen retrieval is generally performed at a higher temperature than the first antigen retrieval.
  • the second antigen retrieval step is performed on or for an epitope or biomarker which is renatured (or more optimally renatured) at a temperature which can denature at least the first biomarker, as noted above. Labeling of at least one biomarker is then performed.
  • the labeling can include at least one amplification technique discussed above. It should be noted, however, that when antigen retrieval is performed above 100 °C, a reagent can be added to increase the boiling point of the sample, thereby reducing or eliminating any damage to the sample.
  • the reagent can include, but is not limited to, a glycol-based solution (e.g., propylene glycol, ethylene glycol, polyethylene glycol), glycerol or a glycerol-based solution, a solution including at least one ionic compound (e.g., a salt solution), or the like.
  • a glycol-based solution e.g., propylene glycol, ethylene glycol, polyethylene glycol
  • glycerol or a glycerol-based solution e.g., a ionic compound
  • a salt solution e.g., a salt solution
  • a sample undergoes a first antigen retrieval process at 50-90 °C, 50- 85 °C, or 70 °C.
  • CD45, a granulocyte marker (CD1 lb, CD66b, or CD15), and an endothelial cell marker (CD 105, CD31, or CD 144) then undergo labeling.
  • the labeling incorporates an amplification technique (e.g. horseradish peroxidase and tyramide).
  • the sample then undergoes a second antigen retrieval process at 50-110 °C, 80-100 °C, or 100 °C.
  • the granulocyte marker can be denatured above 90 °C.
  • the first antigen retrieval step is performed at 50-90 °C.
  • the granulocyte marker is labeled.
  • the CD45 and the endothelial cell marker can also be labeled with by tyramide signal amplification.
  • the tyramide signal amplification allows for subsequent detection because the second biomarker, such as vimentin, requires antigen retrieval performed at a temperature above 90 °C.
  • each biomarker is to be retrieved or unmasked at different temperatures. Starting with the lowest temperature-dependent antigen retrieval step and progressing, ultimately, to the highest temperature-dependent antigen retrieval step. This allows for antigen retrieval of different biomarkers with at least one different antigen retrieval condition.
  • Some biomarkers or epitopes can be denatured by antigen retrieval processes where the temperature is greater than or equal to about 80 °C.
  • the HRP-tyramide amplification results in activated tyramide, via an enzymatic reaction.
  • the activated tyramide (which is bound to a detection moiety) is deposited in the vicinity of the HRP-biomarker site and forms covalent bonds with particular proteins in the area of deposition.
  • the covalent bonds between the activated tyramides and the proteins are not affected by increased temperatures which may occur in or are required by subsequent antigen retrieval processes, thereby allowing for detection even with subsequently elevated temperatures.
  • a target or non-target analyte can have a biomarker which is both intracellular and extracellular.
  • the ratio of one biomarker to another biomarker can be of clinical utility.
  • the target analyte and/or non-target analyte, where appropriate or desirous
  • it can be desirous to only label the extracellular epitope of the biomarker.
  • An antigen retrieval step is performed at 70-110 °C, and typically at 70-90 °C.
  • Labeling is then performed using at least one stain including an affinity molecule directed to the biomarker linked via a linker to a detection moiety.
  • the detection can be a quantum dot.
  • the quantum dot has a diameter that is greater than or equal to 2 nanometers.
  • the quantum dot has a shell that is hydrophilic (e.g., polyethylene glycol (PEG)) or hydrophobic (e.g., a linker containing an alkane chain of at least four carbon atoms). In one embodiment, the quantum dot does not have a shell.
  • the detection moiety can be an organic fluorophore.
  • the organic fluorophore can have a molecule weight of at least at least 1 kD, including, without limitation, at least 10 kD, at least 25 kD, at least 50 kD, at least 75 kD, at least 100 kD, at least 150 kD, at least 200 kD, at least 250 kD, at least 300 kD, at least 340 kD, at least 350 kD, at least 500 kD, and at least 750 kD.
  • the linker can be hydrophilic (e.g., polyethylene glycol (PEG)) or hydrophobic (e.g., a linker containing an alkane chain of at least four carbon atoms).
  • the affinity molecule, the hydrophilicity of the linker, and the size of the quantum dot directs the stain to the extracellular epitope of the biomarker and inhibits permeation of the stain across the plasma membrane. Therefore, despite permeabilization of the target analyte, the stain preferentially labels the extracellular epitope of the biomarker, rather than the intracellular epitope.
  • a stain including a vimentin antibody is linked to QD800 with a PEG linker is added to a sample.
  • a cell, having been permeabilized can include an intracellular epitope of vimentin and an extracellular epitope of vimentin.
  • the characteristics of the stain, as noted above i.e., hydrophilicity of the linker and size of the quantum dot
  • the detector transmits the data back to the computer or is retained within the scanner.
  • the image data may then be compiled into images, processed and analyzed by a computer or associated software or programs, whereby the target analyte may be characterized, such as by biomarkers present, morphology, clustering, and/or the like.
  • the images formed due to the emission lights may be overlaid when a plurality of fluorescent probes, having bound themselves to the target analyte, are excited and emit light.
  • the computer or scanner may then implement a non-transitory computer-readable medium process to analyze and process the data.
  • the non-transitory computer-readable medium process may detect a candidate object.
  • target analytes are found using at least one image processing technique, including, but not limited to, thresholding (manual or automatic), template matching, watershedding, cross-correlation, convolutional neural networks, region growing, and edge detection.
  • non-target analytes within the sample can be labeled and detected for removal (i.e. negative marker, dumping, or dump channel) from analysis of the remainder of the sample, such as by image processing and/or algorithm-based methods (i.e. a non- transitory computer-readable medium process).
  • removal i.e. negative marker, dumping, or dump channel
  • algorithm-based methods i.e. a non- transitory computer-readable medium process
  • stains including affinity molecules for white blood cell biomarkers can be used to label and detect the white blood cells so as to be removed from subsequent analysis.
  • white blood cell biomarkers such as CD45, a granulocyte marker (CDl lb, CD66b, or CD15), and an endothelial cell marker (CD105, CD31, or CD144)
  • CD45 a granulocyte marker
  • CD66b a granulocyte marker
  • CD105 CD31, or CD144
  • a single affinity molecule e.g. antibody directed to a single biomarker of a non-target analyte
  • the single affinity molecule can be directly or indirectly labeled with a detection moiety.
  • An amplification technique can also be implemented.
  • a plurality of affinity molecules directed to a plurality of biomarkers of a non-target analyte can be added to the sample.
  • the plurality of affinity molecules can be directly or indirectly labeled with a detection moiety.
  • each affinity molecule of the plurality of affinity molecules is labeled with a different detection moiety.
  • at least two affinity molecules of the plurality of affinity molecules are labeled with either: (1) detection moieties providing a signal in the same channel (e.g., a first affinity molecule is bound to or interacted with Alexa 488 and a second affinity molecule is bound to or interacted with QD500), or (2) the same detection moiety (e.g., the affinity molecules are bound to or interacted with Alexa 488).
  • At least one amplification technique can also be implemented for one or more of the biomarkers.
  • the non-target analyte can express the mesenchymal cell marker.
  • Figure 2 shows example images for objects, such that the term“object” or “candidate object” are terms (which are interchangeable) used to describe a target analyte or non target analyte before performing analysis and/or scoring used to determine whether the object is a target analyte or non-target analyte.
  • Figure 2 shows labeled biomarkers (columns) of the various objects (rows).
  • a process incorporates a plurality of sub steps (i.e. multiplexing, amplification, cyclic labeling, antigen retrieval, non-target analyte dumping, etc.) into the general method.
  • the general method can incorporate any number of sub steps described herein.
  • the incorporation of these sub-steps allows for the detection of a target analyte having a given definition (for example, a circulating tumor cell that is (EPCAM+ or CK+)/CD45-/nuclear+) and/or a target analyte which does not fall within the given definition (for example, (EPCAM or CK)+/mesenchymal+ or (EPCAM or CK)-/mesenchymal+).
  • confirmation of these target analytes is done by sequencing.
  • CD 105 CD31, or CD 144
  • references to a structure or feature that is disposed“adjacent” another feature may have portions that overlap or underlie the adjacent feature.
  • spatially relative terms such as“under”,“below”,“lower”,“over”,“upper” and the like, may be used herein for ease of description to describe one element or feature’s relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as“under” or“beneath” other elements or features would then be oriented“over” the other elements or features. Thus, the exemplary term“under” can encompass both an orientation of over and under.
  • the device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
  • the terms“upwardly”,“downwardly”,“vertical”,“horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.
  • first and second may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.
  • the word“comprise”, and variations such as“comprises” and“comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods).
  • the term“comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.
  • a numeric value may have a value that is +/- 0.1% of the stated value (or range of values), +/- 1% of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), +/- 10% of the stated value (or range of values), etc.
  • Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value“10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub ranges subsumed therein.

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Abstract

La présente invention se rapporte globalement à une détection et, en particulier, à une détection d'objets dans un échantillon ou dans une fraction de ce dernier.
PCT/US2019/023647 2018-04-05 2019-03-22 Détection d'analyte WO2019194994A1 (fr)

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WO2008063378A2 (fr) * 2006-11-01 2008-05-29 Ventana Medical Systems, Inc. Haptènes, conjugués de haptène, compositions de haptène, procédé de fabrication et utilisation
WO2009110936A2 (fr) * 2007-12-28 2009-09-11 Spring Bioscience Corporation Procédés d’extraction d’antigène pour l’immunohistochimie
WO2010028277A1 (fr) * 2008-09-04 2010-03-11 Beckman Coulter, Inc. Activation de la pan-kinase et évaluation de voies de signalisation
WO2014138183A1 (fr) * 2013-03-05 2014-09-12 Board Of Regents, The University Of Texas System Outil de détection spécifique pour cellules tumorales circulantes transformées mésenchymateuses et épithéliales-mésenchymateuses

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WO2008063378A2 (fr) * 2006-11-01 2008-05-29 Ventana Medical Systems, Inc. Haptènes, conjugués de haptène, compositions de haptène, procédé de fabrication et utilisation
WO2009110936A2 (fr) * 2007-12-28 2009-09-11 Spring Bioscience Corporation Procédés d’extraction d’antigène pour l’immunohistochimie
WO2010028277A1 (fr) * 2008-09-04 2010-03-11 Beckman Coulter, Inc. Activation de la pan-kinase et évaluation de voies de signalisation
WO2014138183A1 (fr) * 2013-03-05 2014-09-12 Board Of Regents, The University Of Texas System Outil de détection spécifique pour cellules tumorales circulantes transformées mésenchymateuses et épithéliales-mésenchymateuses

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