WO2016089966A1 - Solution et procédé pour faire adhérer des constituants de suspension à un substrat - Google Patents

Solution et procédé pour faire adhérer des constituants de suspension à un substrat Download PDF

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Publication number
WO2016089966A1
WO2016089966A1 PCT/US2015/063379 US2015063379W WO2016089966A1 WO 2016089966 A1 WO2016089966 A1 WO 2016089966A1 US 2015063379 W US2015063379 W US 2015063379W WO 2016089966 A1 WO2016089966 A1 WO 2016089966A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample
solution
substrate
attachment
heparin
Prior art date
Application number
PCT/US2015/063379
Other languages
English (en)
Inventor
Daniel CAMPTON
Joshua Nordberg
Steve QUARRE
Ronald Seubert
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
Application filed by Rarecyte, Inc. filed Critical Rarecyte, Inc.
Publication of WO2016089966A1 publication Critical patent/WO2016089966A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G01N1/312—Apparatus therefor for samples mounted on planar substrates

Definitions

  • This disclosure relates generally to analyzing a suspension and, in particular, to a solution and method for adhering a component of a suspension to a substrate.
  • Figure 1 shows a flowchart of an example method for making an example attachment solution.
  • Figure 2 shows a flowchart of an example method for adhering a sample to a substrate.
  • Figure 3A shows an example sample having been re-suspended in an example attachment solution.
  • Figure 3B shows the example sample mixing with the attach solution to form a re-suspended cytological sample.
  • Figure 3C shows the re-suspended sample being deposited on an example analysis platform.
  • Figure 3D shows the re-suspended sample having been spread on and adhered to the analysis platform.
  • the attachment solution includes an attachment base, an anti-coagulant, and a nonsteroidal anti-inflammatory drug.
  • the attachment base may be an alcohol, an acid, an oxidizer, an organohalogen, a ketone, such as acetone, or any combination thereof, such as Carnoy's solution.
  • sample is used to describe a specimen to be analyzed.
  • the specimen may .be a suspension, a portion of the suspension, or a component of the suspension.
  • the sample when the suspension is anticoagulated whole blood, the sample may be 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).
  • the attachment solution adheres a sample to a substrate.
  • the sample can be a bufry coat of a blood sample and the substrate can be the surface of a microscope slide.
  • the attachment solution includes an attachment base, an anti-coagulant, and a nonsteroidal anti-inflammatory drug.
  • the attachment solution may also include water or a buffered solution, such as phosphate buffered saline.
  • the attachment base may include an alcohol, such as ethanol, methanol, propanol, isopropanol, butanol, and an acid, an oxidizer, an organohalogen, a ketone, such as acetone, or any combination thereof, such as Carnoy's solution.
  • the attachment base may be capable of fixing sample components, such as cells, without cross-linking other sample components, such as proteins. Preventing cross-linking of the other sample components, such as proteins, may avoid an additional processing step, such as antigen retrieval. In other words, fixing sample components to a substrate with the attachment base eliminates the process of antigen retrieval.
  • the attachment base may also have a fast (i.e. less than one hour) cure time.
  • the attachment base may have a final concentration of approximately 40-90% by volume of the attachment solution.
  • the final concentration of the attachment base may be determined by a measured property of the sample to be adhered including, but not limited to, sample volume, packed cell volume, hematocrit level, blood type, sample type (i.e. blood, urine, buffy coat, single cell, etc.), or the like.
  • the final concentration of the attachment base in the solution-sample mixture may be approximately 55-70% by volume.
  • the anti-coagulant portion of the attachment solution may be, but is not limited to, heparin, heparin sodium, heparin/dextrose, ethylene-diamine-tetra-acetic acid, dalteparin sodium, argatroban, bivalirudin, lepirudin, or title like.
  • the anti-coagulant prevents coagulation (i.e. clumping, clotting or solidification) of the sample.
  • the anticoagulant may have a final concentration of approximately 10-1000 ug/mL.
  • the nonsteroidal anti-inflammatory drug may be, but is not limited to, acetylsalicylic acid, celecoxib (Celebrex), diclofenac (Voltaren), diflunisal (Dolobid), etodolac (Lodine), ibuprofen (Motrin), indomethacin (Indocin), ketoprofen (Orudis), ketorolac (Toradol), nabumetone (Relafen), naproxen (Aleve, Naprosyn), oxaprozin (Daypro), piroxicam (Feldene), salsalate (Amigesic), sulindac (Clinoril), tolmetin (Tolectin), or the like.
  • the nonsteroidal anti-inflammatory drug may act as an antithrombotic (i.e. reducing blood clotting, such as by reducing the blood clotting function of various blood components).
  • the nonsteroidal anti-inflammatory drug may have a final concentration of approximately 100-1000 ug/mL.
  • the attachment solution may also include a biological polymer to increase the adhesion between the sample and the substrate.
  • the biological polymer may include, but is not limited to, biomimetic adhesive polymers (such as polyphenolic protein from marine life; 3,4-dihydroxyphenyialanine), fibrin glue, gelatin-resorcinol-formaldehyde, poly-L-lysine, poly-D-lysine, or the like.
  • Heparin 2000 ⁇ g/mL Prepared by dissolving 20mg of heparin sodium salt (Sigma H3393) in lOmL of water
  • BD Cell-Tak BD Cell-Tak® Cell and Tissue Adhesive (BD 354241) - approximately 1500 ⁇ g/mL, stock concentration
  • Cell-Tak Buffer 5X Prepared by dissolving 21g sodium bicarbonate (NaHCO 3 ) into 500mL of water
  • Cell-Tak Buffer 1X Prepared by dissolving 4,2g sodium bicarbonate (NaHCO 3 ) into 500mL of water and adding 2.5 mL 1M hydrochloric acid (HC1)
  • Figure 2 shows an example method for adhering a sample to a substrate.
  • a sample is obtained.
  • the sample may be withdrawn directly from a subject or the sample may undergo enrichment and/or isolation from the suspension.
  • the sample may be 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, or the like.
  • Sequential density fractionation is a process by which a suspension is divided into fractions or a fraction of a suspension 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.
  • the sample may be obtained from other suspension components by selecting the sample with a device for picking, such as a cell picker, a pipet, a syringe, or the like.
  • the sample 304 is re-suspended in an attachment solution 306 in a vessel 302, as shown in Figure 3A.
  • the attachment solution may be added to or mixed with the sample.
  • a dispenser such as a pipet or repeating pipet
  • the sample 308 is spread across the analysis platform 310 by a spreader 312, such as a squeegee, a pipet tip, a blade, a two-piece spreader including a blade and a base.
  • the sample 308 may be spread across the analysis platform 310 by centrifuging, wetting, or nutating the analysis platform 310.
  • the re-suspended sample 308 is cured, as shown in Figure 3D, to adhere the re-suspended sample 308 to the analysis platform 310.
  • the re-suspended sample 308 may be dispensed onto the analysis platform 310 and cured without being spread across the analysis platform 310. Curing may occur 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 formalin or any appropriate fixative, after the curing step has been completed.
  • the attachment solution may be compatible with any appropriate analysis method or technique, though more specifically extracellular and intracellular analysis including immunofluorescent labeling and imaging; intracellular protein labeling; chromogenic staining; molecular analysis; genomic analysis or nucleic acid analysis, including, but not limited to, genomic sequencing, DNA arrays, expression arrays, protein arrays, and DNA hybridization arrays; 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; or branched DNA (“bDNA”— a tool for analyzing DNA and/or RNA, such as mRNA expression levels) analysis.
  • ISH in situ hybridization
  • PCR polymerase chain reaction
  • bDNA branched DNA
  • intracellular proteins which may be labeled include, but are not limited to, cytokeratin ("CK"), actin, Arp2/3, coronin, dystrophin, FtsZ, myosin, spectrin, tubulin, collagen, cathepsin D, ALDH, PBGD, Aktl, Akt2, c-myc, caspases, survivin, p27 ldp , FOXC2, BRAF, Phospho-Aktl and 2, Phospho-Erkl/2, Erkl/2, P38 MAPK, Vimentin, ER, PgR, PI3K, pFAK, KRAS, ALKH1, Twistl, Snaill, ZEB1, Fibronectin, Slug, Ki-67, M30, MAGEA3, phosphorylated receptor kinases, modified histones, chromatin-associated proteins, and MAGE.
  • CK cytokeratin
  • actin actin
  • Arp2/3, coronin coron
  • the analysis platform 310 may be a microscope slide, a positively 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 310 may be any appropriate material, including, but not limited to, glass, plastic, ceramic, metal, or the like.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé et une solution de fixation pour faire adhérer un échantillon cytologique ou histologique, tel qu'une couche leuco-plaquettaire, à un substrat, tel qu'une lame de microscope. La solution de fixation comprend une base de fixation, un anticoagulant, et un médicament anti-inflammatoire non stéroïdien. La base de fixation peut être un alcool, un acide, un oxydant, un organo-halogène, une cétone, ou n'importe quelle combinaison de ceux-ci. Une fois qu'un échantillon est obtenu, l'échantillon peut être remis en suspension dans la solution de fixation ou la solution de fixation peut être ajoutée à l'échantillon. L'échantillon peut ensuite être distribué sur une plate-forme d'analyse sous la forme d'une ou de plusieurs gouttelettes et durci.
PCT/US2015/063379 2014-12-02 2015-12-02 Solution et procédé pour faire adhérer des constituants de suspension à un substrat WO2016089966A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462086309P 2014-12-02 2014-12-02
US62/086,309 2014-12-02

Publications (1)

Publication Number Publication Date
WO2016089966A1 true WO2016089966A1 (fr) 2016-06-09

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019079363A1 (fr) * 2017-10-18 2019-04-25 Rarecyte, Inc. Solution et procédé pour faire adhérer des constituants de suspension à un substrat

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5532311A (en) * 1995-02-01 1996-07-02 Minnesota Mining And Manufacturing Company Process for modifying surfaces
US5811151A (en) * 1996-05-31 1998-09-22 Medtronic, Inc. Method of modifying the surface of a medical device
US6146771A (en) * 1997-07-01 2000-11-14 Terumo Cardiovascular Systems Corporation Process for modifying surfaces using the reaction product of a water-insoluble polymer and a polyalkylene imine
US20020115836A1 (en) * 1998-09-22 2002-08-22 Ray Tsang Non-thrombogenic coating composition and methods for using same
WO2010120253A1 (fr) * 2009-04-17 2010-10-21 Mustafa Nevzat Ilac Sanayii A.S. Combinaisons de thiocolchicoside et de médicament anti-inflammatoire non stéroïdien

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5532311A (en) * 1995-02-01 1996-07-02 Minnesota Mining And Manufacturing Company Process for modifying surfaces
US5811151A (en) * 1996-05-31 1998-09-22 Medtronic, Inc. Method of modifying the surface of a medical device
US6146771A (en) * 1997-07-01 2000-11-14 Terumo Cardiovascular Systems Corporation Process for modifying surfaces using the reaction product of a water-insoluble polymer and a polyalkylene imine
US20020115836A1 (en) * 1998-09-22 2002-08-22 Ray Tsang Non-thrombogenic coating composition and methods for using same
WO2010120253A1 (fr) * 2009-04-17 2010-10-21 Mustafa Nevzat Ilac Sanayii A.S. Combinaisons de thiocolchicoside et de médicament anti-inflammatoire non stéroïdien

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019079363A1 (fr) * 2017-10-18 2019-04-25 Rarecyte, Inc. Solution et procédé pour faire adhérer des constituants de suspension à un substrat
CN111226105A (zh) * 2017-10-18 2020-06-02 瑞尔赛特股份有限公司 将悬浮液组分粘附到基材的溶液和方法
EP3698120A4 (fr) * 2017-10-18 2021-07-21 Rarecyte, Inc. Solution et procédé pour faire adhérer des constituants de suspension à un substrat

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