CN111226105A

CN111226105A - Solution and method for adhering suspension components to a substrate

Info

Publication number: CN111226105A
Application number: CN201880067717.4A
Authority: CN
Inventors: D·E·坎普顿; J·诺德伯格; R·C·苏伯特; S·卡雷
Original assignee: Rarecyte Inc
Current assignee: Rarecyte Inc
Priority date: 2017-10-18
Filing date: 2018-10-17
Publication date: 2020-06-02
Also published as: JP2021500553A; WO2019079363A1; EP3698120A1; EP3698120A4

Abstract

The present disclosure relates to a method and an attachment solution for adhering a cytological or histological sample (such as a buffy coat) to a substrate (such as a microscope slide). The attachment solution includes an attachment matrix, an anticoagulant, and a non-steroidal anti-inflammatory drug. The attachment substrate may be an alcohol, an acid, an oxidizing agent, an organic halide, a ketone, or any combination thereof. Once the sample is obtained, the sample can be resuspended in the attachment solution, or the attachment solution can be added to the sample. The sample may then be dispensed as one or more droplets onto the analytical platform and allowed to solidify.

Description

Solution and method for adhering suspension components to a substrate

Cross Reference to Related Applications

The present application is a partial continuation application with application number 14/956,622 filed on day 12, month 2 in 2015, which claims the benefit of provisional application number 62/086,309 filed on day 12, month 2 in 2014, is a partial continuation application with application number 14/317,683 filed on day 27 in 2014, which claims the benefit of provisional application number 61/856,498 filed on day 7, month 19 in 2013 and the benefit of provisional application number 61/856,506 filed on day 7, month 19 in 2013.

Technical Field

The present disclosure relates generally to analyzing suspensions, and in particular to solutions and methods for adhering suspension components to a substrate.

Background

Current diagnostic methods and techniques rely on the interpretation of histological and cytological samples. An important aspect in these explanations is: the sample remains adhered to the substrate (e.g., microscope slide) on which it is to be analyzed and processed. Valuable diagnostic information can then be obtained from the sample in subsequent processing due to the adherence of the sample.

Drawings

FIG. 1 shows a flow diagram of an example method for making an example attachment solution.

Fig. 2 shows a flow diagram of an example method for adhering a sample to a substrate.

Fig. 3A shows an example sample that has been resuspended in an example attachment solution.

Figure 3B shows an exemplary sample mixed with an attachment solution to form a resuspended cytological sample.

Figure 3C shows a resuspended sample deposited on an example analytical platform.

Figure 3D shows the resuspended sample that has been spread and adhered to the analysis platform.

Detailed Description

The present disclosure relates to methods and attachment solutions for adhering a cytological or histological sample to a substrate. The attachment solution includes an attachment matrix and a non-steroidal anti-inflammatory drug. The attachment matrix may be an alcohol, an acid, an oxidizing agent, an organic halogen, a ketone (e.g., acetone), or any combination thereof, such as Carnoy's solution. The attachment solution may also include an anticoagulant. Once the sample is obtained, the sample can be resuspended in the attachment solution, or the attachment solution can be added to the sample. The sample is dispensed in the form of one or more droplets onto the analytical platform and solidified. The sample may then be fixed, permeabilized, labeled, blocked, and washed. The sample may then be imaged and analyzed.

In the following description, the term "sample" is used to describe the sample to be analyzed. The sample may be a suspension, a portion of a suspension, or a component of a suspension. For example, when the suspension is anticoagulated whole blood, the sample can be anticoagulated whole blood (i.e., the suspension), buffy coat (i.e., a portion of the suspension), or circulating tumor cells (i.e., a component of the suspension).

Adhesion solution

The attachment solution can adhere the sample to the substrate. The sample may be a buffy coat of a blood sample, and the substrate may be a surface of a microscope slide. The attachment solution includes an attachment matrix and a non-steroidal anti-inflammatory drug. The adhesion solution may also comprise water. Instead of or in addition to water, buffers may also be included or buffer solutions may be used. The buffer of the buffer or buffer solution may include, but is not limited to, ACES, ADA, AMP, AMPD, AMPSO, BES, Bicine, BIS-TRIS, BIS-TRIS propane, CABS, CAPS, CAPSO, CHES, DIPSO, EDTA, HEPPS (EPPS), Gly-Gly, HEPBS, HEPES, MES, MOPS, MOPSO, PIPES, POPSO, potassium phosphate, sodium phosphate (mono-and/or di-basic), TAPS, TAPSO, TES, Tricine, TRIS,

and combinations thereof. The pH of the buffer or buffer solution may range from 5 to 12.

It should be noted that in the first embodiment, the adherent solution comprises a salt, such as a salt in a physiological saline solution, wherein the adherent solution remains hypotonic relative to the sample, as isotonic or hypertonic adherent solutions may cause the sample to rupture when dried or cured. Alternatively, the adhering solution does not contain salts, such as those contained in physiological saline solution, as salts may cause the sample to break when dried or cured.

The attachment matrix may include an alcohol, such as ethanol, methanol, propanol, isopropanol, butanol, and an acid, an oxidizing agent, an organic halide, a ketone, such as acetone, or any combination thereof, such as a carnot solution. The attachment matrix body may be capable of immobilizing sample components, such as cells, without cross-linking other sample components, such as proteins. Preventing cross-linking of other sample components (e.g., proteins) may avoid other processing steps, such as antigen retrieval. In other words, immobilization of the sample components on the substrate via the attachment matrix precludes the process of antigen retrieval. The attachment matrix may also have a fast (i.e., less than one hour) cure time. For example, when the attachment solution and sample have been mixed, the final concentration of the attachment matrix can be 60-70 vol%, more specifically 63-68 vol%. Concentrations with attached matrix outside the desired range may affect the sample or a part thereof, fail to adhere to the substrate, etc., due to e.g. lysis, clumping. The final concentration of the attachment matrix can be determined from the measured characteristics of the sample to be adhered, including but not limited to sample volume, hematocrit level, blood type, sample type (i.e., blood, urine, buffy coat, individual cells, etc.).

The non-steroidal 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 (oridis), ketorolac (Toradol), nabumetone (Relafen), naproxen (Aleve, Naprosyn), oxaprozin (Daypro), piroxicam (Feldene), salsalate (amigesic), sulindac (Clinoril), tolmetin (Tolectin), and the like. Non-steroidal anti-inflammatory drugs can act as anti-thrombotic agents (i.e., reduce blood clotting, for example, by reducing the blood clotting function of various blood components). The final concentration of the NSAID may be 100 and 1000 ug/mL.

The attachment solution may also comprise an anticoagulant, which may be, but is not limited to, heparin sodium, heparin/dextrose, ethylenediaminetetraacetic acid, dalteparin sodium, argatroban, bivalirudin, lepirudin (lepirudin), and the like. Anticoagulants prevent coagulation (i.e., caking, coagulation, or coagulation) of the sample. The final concentration of anticoagulant may be 10-1000 ug/mL.

The attachment solution may also include a biopolymer to increase adhesion between the sample and the substrate. Biopolymers may include, but are not limited to, biomimetic adhesive polymers (e.g., polyphenoproteins from marine organisms; 3, 4-dihydroxyphenylalanine), fibrin glue, gelatin-resorcinol-formaldehyde, poly-L-lysine, poly-D-lysine, and the like.

Method for preparing adhesion solution

The following are exemplary reagents for making the attachment solution:

1. heparin 2000. mu.g/mL: prepared by dissolving 20mg of heparin sodium salt (Sigma H3393) in 10mL of water

2.BD Cell-Tak:BD

Cell and Tissue Adhesive (Tissue Adhesive) (BD354241) -about 1500 μ g/mL stock solution concentration

Cell-Tak buffer 5 Xby dissolving 21g sodium bicarbonate (NaHCO)₃) Preparation into 500mL of water

Cell-Tak buffer 1 Xby dissolving 4.2g sodium bicarbonate (NaHCO)₃) Prepared by adding to 500mL of water and adding 2.5mL of 1M hydrochloric acid (HCl)

Sigma Aldrich 494437 methanol (about 99.93 vol%)

The following are exemplary formulations for the attachment solution, as shown in FIG. 1:

1. methanol 70 vol%, 100ug/mL heparin, 300ug/mL acetylsalicylic acid, 50ug/mL

A. Mix 350uL

And 650uL Cell-Tak buffer 1X

B. Mixing thoroughly

C. 1.5mL of water was added

D. 500uL heparin 2000ug/mL was added

E. 7.0mL of methanol was added

F. Adding 3000ug of acetylsalicylic acid

G. Mixing thoroughly

2. Methanol 60 vol%, 300ug/mL acetylsalicylic acid:

A. 6000uL of methanol was added to 4000uL of water

B. Adding 3000ug of acetylsalicylic acid

C. Mixing thoroughly

Method of using adhesion solution

Fig. 2 shows an exemplary method for adhering a sample to a substrate. In block 202, a sample is obtained. To obtain a sample, the sample may be removed directly from the subject, or the sample may be enriched and/or isolated from the suspension. The sample may be enriched by any suitable enrichment method including, but not limited to, sequential density fractionation, magnetic activated cell sorting, fluorescence activated cell sorting, differential lysis, depletion filters (depletion filters), and the like. Sequential density fractionation is a process of dividing a suspension into several fractions or dividing a fraction of a suspension into multiple sub-fractions by a stepwise or sequential process, such that each step or sequence results in the collection or separation of different fractions or sub-fractions from previous and subsequent steps or sequences. Samples can be obtained from other suspension components by selecting the sample using a device for picking up (e.g., a cell harvester, pipette, syringe, etc.).

Referring back to fig. 2, in block 204, the sample 304 is resuspended in the attachment solution 306 in the container 302, as shown in fig. 3A. Alternatively, the attachment solution may be added to or mixed with the sample. In block 206, a resuspended sample 308 (as shown in fig. 3B) comprising at least a portion of the attachment solution is dispensed by a dispenser (e.g., a pipette or repeat pipette) onto or into the analysis platform 310 and spread throughout the analysis platform. In fig. 3C, the sample 308 is spread on the analysis platform 310 by a spreader 312 such as a scraper, pipette tip, blade, two-piece spreader comprising a blade and a base. Alternatively, the sample 308 may be spread on the analysis platform 310 by centrifuging, wetting or nutating the analysis platform 310. As shown in fig. 3D, the resuspended sample 308 is cured to adhere the resuspended sample 308 to the analytical platform 310. Alternatively, the resuspended sample 308 can be dispensed onto the analysis platform 310 and cured without spreading over the analysis platform 310. Curing may take place in air, e.g., at room temperature, in an environmental control room, e.g., at 37 ℃; or similar environment. Furthermore, after the curing step is complete, the sample may be subjected to an additional fixation step, for example in formalin or any suitable fixative.

The attachment solution may be compatible with any suitable analytical method or technique, although more specifically, extracellular and intracellular assays include immunofluorescent labeling and imaging; intracellular protein labeling; carrying out color development and dyeing; performing molecular analysis; genomic 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 means of analyzing DNA and/or RNA, e.g., gene copy number variation); polymerase chain reaction ("PCR"); performing reverse transcription PCR; or branched DNA ("bDNA" -a means for analyzing the expression level of DNA and/or RNA, e.g., mRNA) analysis. Some intracellular proteins that 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, Akt1, Akt2, c-myc, caspase, survivin (survivin), p27^kipFOXC2, BRAF, Phospho-Aktl and 2, Phospho-Erk1/2, Erk1/2, P38 MAPK, vimentin, ER, PgR, PI3K, pFAK, KRAS, ALKH1, Twist1, Snail1, ZEB1, fibronectin, Slug, Ki-67, M30, MAGEA3, phosphorylated receptor kinases, modified histones, chromatin-related proteins and MAGE.

The analysis platform 310 can be a microscope slide, a positively charged microscope slide, a coated microscope slide, a multi-well slide, a micro-well slide, a well plate, a coverslip, a cellular microarray, or the like. The analysis platform 310 may be any suitable material including, but not limited to, glass, plastic, ceramic, metal, and the like.

The foregoing description, for purposes of explanation, used specific nomenclature to provide a thorough understanding of the disclosure. It will be apparent, however, to one skilled in the art that the systems and methods described herein may be practiced without specific details. The foregoing descriptions of specific embodiments have been presented by way of illustration and description. They are not intended to be exhaustive or to limit the disclosure to the precise forms described. Many modifications and variations are possible in light of the above teaching. The embodiments are shown and described in order to best explain the principles of the disclosure and the practical application, to thereby enable others skilled in the art to best utilize the disclosure and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of the disclosure be defined by the following claims and their equivalents.

Claims

1. A solution, comprising:

an alcohol; and

the final concentration is 100-1000ug/mL of the NSAID,

wherein the final concentration is the concentration of the NSAID when mixed with the sample.

2. The solution of claim 1, wherein the alcohol is methanol.

3. The solution of claim 2, wherein the non-steroidal anti-inflammatory drug is acetylsalicylic acid.

4. The solution of claim 3, further comprising a buffer.

5. The solution of claim 3, further comprising a salt, wherein the solution is hypotonic relative to the sample.

6. The solution of claim 1, wherein the final concentration of the alcohol is 60-70% by volume when mixed with the sample.

7. The solution of claim 1, further comprising water.

8. The solution of claim 1, further comprising a buffer solution.

9. The solution of claim 1, further comprising an anticoagulant.

10. The solution of claim 9, wherein the anticoagulant is heparin.