Solution and method for histoprocessing of biological samples

[Cross-reference regarding related applications]

This application is filed on June 17, 2011, US Provisional Patent Application No. 61 / 498,159, US Provisional Patent Application No. 61 / 505,011 filed on July 6, 2011, and July 21, 2011. Claims priority under US Provisional Patent Application No. 61 / 510,180 filed on the same day. Each previously filed application is incorporated herein by reference in its entirety.

The present disclosure relates to formulations for the preparation of biological specimens, more specifically to fixation, staining and rinsing formulations.

Over the years, to improve the contrast of specimens under examination, laboratory techniques have used dyes and dyes such as those used for Romanosufky staining to prepare biological specimens. Such examinations typically utilize a microscope, an automated device that captures an image of the specimen, or, in other cases, a macroscopic visual examination. Several different systems and methods are known for preparing test specimens. For example, Patent Documents 1 to 6 relate to a mechanism and a method for staining a substrate during sample processing. These publications provide various details regarding staining and preparation of laboratory specimens.

<p><patcit num="1"><text>U.S. Pat. No. 6,096,271</text></patcit><patcit num="2"><text>U.S. Pat. No. 7,318,913</text></patcit><patcit num="3"><text>U.S. Pat. No. 5,419,279</text></patcit><patcit num="4"><text>U.S. Pat. No. 5,948,360</text></patcit><patcit num="5"><text>U.S. Patent Publication No. 2008/010 2006</text></patcit><patcit num="6"><text>U.S. Patent Publication No. 2006/0073074</text></patcit></p>

The present disclosure relates to formulations, systems and methods for preparing test specimens. Examples of the sample include erythrocyte cells derived from a biological sample such as a blood sample, a blood sample containing white blood cells and platelets, which is applied to a substrate such as a microscope slide or coverslip. Various embodiments can be used to prepare specimens from biological samples such as bone marrow, urine, vaginal tissue, epithelial tissue, tumors, semen, saliva and other body fluids. Further aspects of the present disclosure include systems and methods for immobilizing, staining, rinsing and stirring specimens using the formulations of the present disclosure. Generally, the formulations, systems and methods disclosed herein use as little liquid volume as possible to provide rapid, efficient and highly uniform specimen preparation and processing. The method comprises one or more fixation, staining and rinsing steps, eg, one or more stirring steps after each of one or more fixation, staining and rinsing steps. The system can be run as a stand-alone device or as a component in a larger system for preparing and inspecting specimens.

In a first aspect, the disclosure is characterized by a cell fixative containing Azure B; surfactant; methanol; and ethylene glycol.

For example, the cell fixation solution is about 0.5 g / L to about 5.0 g / L of Azure B; about 0.5 mL / L to about 2.0 mL / L of polysolvate 20; about 5 mL / L to about 50 mL / L of ethylene glycol, It may contain propylene glycol or polypropylene glycol; about 0.1 g / L to about 10 g / L of HEPES sodium salt; and methanol.

As another example, the cell fixation solution is about 0.8 g / L to about 1.2 g / L of Azure B; about 0.8 mL / L to about 1.2 mL / L of polysorbate 20; about 9 mL / L to about 11 mL / L. Ethylene glycol; about 0.25 g / L to about 0.38 g / L of HEPES sodium salt; and methanol may be included.

For example, the fixative may contain about 1 g / L of Azure B; about 1 mL / L of polysorbate 20; about 10 mL / L of ethylene glycol; about 0.32 g / L of HEPES sodium salt; and methanol.

As another example, the cell fixation solution contains about 1 g / L of Azure B; about 0.5 mL / L of polysorbate 20; about 10 mL / L of propylene glycol; about 0.32 g / L of HEPES sodium salt; and methanol. obtain.

As a further example, the fixative may contain about 1 g / L of Azure B; about 1.5 mL / L of polysorbate 20; about 10 mL / L of polypropylene glycol; about 0.32 g / L of HEPES sodium salt; and methanol. ..

In a second aspect, the disclosure features a first cell stain comprising eosin Y; buffer; surfactant; sodium chloride; ethylene glycol; and water.

For example, the first cell stain is about 0.5 g / L to about 5.0 g / L of eosin Y; about 5 mM to about 250 mM Bis-Tris buffer or phosphate buffer; about 0.5 mL / L to about 2.0. mL / L polysolvate 20; about 1 g / L to about 20 g / L sodium chloride; about 5 mL / L to about 50 mL / L ethylene glycol; about 0.2 ppm to about 50 ppm ProClin 300®; acetic acid; and water The pH of the solution is about 5.8 to about 6.2.

As another example, the first cell stain is about 0.6 g / L to about 0.9 g / L of eosin Y; about 45 mM to about 55 mM Bis-Tris buffer; about 0.8 mL / L to about 1.2 mL. / L Polysorbate 20; Approximately 3 g / L to Approximately 5 g / L Sodium Chloride; Approximately 9 mL / L to Approximately 11 mL / L of Ethylene Glycol; Approximately 10 ppm to Approximately 20 ppm of ProClin 300®; Acetic Acid; The pH of the solution is about 5.8 to about 6.2.

For example, the first cell stain is about 0.75 g / L of eosin Y; about 50 mM bis-tris buffer; about 1 mL / L of polysorbate 20; about 4 g / L of sodium chloride; about 10 mL / L of ethylene. Glycol; about 15 ppm ProClin 300®; acetic acid; and water can be included, the pH of the solution being about 5.8 to about 6.2.

As another example, the first cell stain is about 0.75 g / L of eosin Y; about 50 mM bis-tris buffer; about 0.5 mL / L of polysorbate 20; about 6 g / L of sodium chloride; about It can contain 20 mL / L ethylene glycol; about 15 ppm ProClin 300®; acetic acid; and water, the pH of the solution being about 5.8 to about 6.2.

As a further example, the first cell stain is about 0.75 g / L of eosin Y; about 50 mM phosphate buffer; about 2.0 mL / L of polysorbate 20; about 4 g / L of sodium chloride; about 50 mL / It can contain L ethylene glycol; about 15 ppm ProClin 300®; acetic acid; and water, the pH of the solution being about 5.8 to about 6.2.

In a third aspect, the disclosure features a second cell stain containing Azure B; methylene blue; buffer; surfactant; sodium chloride; and water.

For example, the second cell stain is about 0.25 to about 2.5 g / L of Azure B; about 0.25 g / L to about 2.5 g / L of methylene blue; about 5 mM to about 250 mM Bis-Tris buffer or HEPES buffer. Liquid; containing about 0.5 mL / L to about 2.0 mL / L of polybuffer 20, about 1 g / L to about 20 g / L of sodium chloride, and about 0.2 ppm to about 50 ppm of ProClin 300®; acetic acid; and water. The pH of the solution can be from about 6.8 to about 7.2.

As another example, the second cell stain is about 0.4-about 0.6 g / L Azure B; about 0.4 g / L-about 0.5 g / L methylene blue; about 45 mM-about 55 mM Bis-Tris buffer. Liquid; about 0.8 mL / L to about 1.2 mL / L of polysorbate 20; about 1.8 g / L to about 2.2 g / L of sodium chloride, and about 10 ppm to about 20 ppm of ProClin 300®; acetic acid; and water. It can be included and the pH of the solution is from about 6.8 to about 7.2.

For example, the second cell stain is about 0.5 g / L Azure B; about 0.45 g / L methylene blue; about 50 mM Bis-Tris buffer; about 1 mL / L polysorbate 20; about 2 g / L chloride. It can contain sodium, and about 15 ppm ProClin 300®; acetic acid; and water, the pH of the solution being about 6.8 to about 7.2.

As another example, the second cell stain is about 0.5 g / L of Azure B; about 0.45 g / L of methylene blue; about 50 mM HEPES buffer; about 0.5 mL / L of polysorbate 20, about 2 g / It can contain L sodium chloride, and about 15 ppm ProClin 300®; acetic acid; and water, the pH of the solution being about 6.8 to about 7.2.

As a further example, the second cell stain is about 0.5 g / L of Azure B; about 0.45 g / L of methylene blue; about 50 mM HEPES buffer; about 1 mL / L of polysolvate 20, about 1 g / L. It can contain sodium chloride, and about 15 ppm ProClin 300®; acetic acid; and water, the pH of the solution being about 6.8 to about 7.2.

In a fourth aspect, the disclosure features a rinse solution for an automated sample preparation device containing polyethylene glycol; buffer; surfactant; methanol; and water.

For example, the rinse solution is about 0.2 g / L to about 10 g / L of polyethylene glycol; about 1 mM to about 250 mM HEPES, MES or Bis-Tris buffer; about 0.1 mL / L to about 2.40 mL / L of polysorbate 20. It can contain from about 0.04 ppm to about 50 ppm ProClin 300®; about 9 mL / L to about 200 mL / L of methanol; and water, the pH of the rinse solution being about 6.6 to about 7.0.

For example, the rinse solution is about 1 g / L to about 10 g / L of polyethylene glycol; about 5 mM to about 250 mM HEPES, MES or Bis-Tris buffer; about 0.5 mL / L to about 2.0 mL / L of polysorbate 20; It can contain from about 0.2 ppm to about 50 ppm ProClin 300®; about 10 mL / L to about 200 mL / L of methanol; and water, the pH of the rinse solution being about 6.6 to about 7.0.

As another example, the rinse solution is about 4.5 g / L to about 5.5 g / L polyethylene glycol; about 45 mM to about 55 mM HEPES buffer; about 0.8 mL / L to about 1.2 mL / L polysorbate 20; It can contain from about 10 ppm to about 20 ppm ProClin 300®; about 45 mL / L to about 55 mL / L of methanol; and water, the pH of the rinsing solution being about 6.6 to about 7.0.

For example, the rinse solution should contain about 5 g / L polyethylene glycol; about 50 mM HEPES buffer; about 1 mL / L polysorbate 20; about 15 ppm ProClin 300®; about 50 mL / L methanol; and water. The pH of the rinse solution is about 6.6 to about 7.0.

As another example, the rinse solution is about 10 g / L polyethylene glycol; about 50 mM MES buffer; about 1 mL / L polysorbate 20; about 15 ppm ProClin 300®; about 50 mL / L methanol; and It can contain water and the pH of the rinse solution is about 6.6 to about 7.0.

As a further example, the rinse solution is about 10 g / L polyethylene glycol; about 50 mM bis-tris buffer; about 0.5 mL / L polysorbate 20; about 15 ppm ProClin 300®; about 50 mL / L methanol. And can contain water, the pH of the rinse solution is about 6.6 to about 7.0.

For example, the rinse solution is about 0.2 g / L to about 2 g / L of polyethylene glycol; about 1 mM to about 50 mM HEPES buffer; about 0.16 mL / L to about 0.24 mL / L of polysorbate 20; about 0.04 ppm to about. It can contain 10 ppm ProClin 300®; about 9 mL / L to about 11 mL / L of methanol; and water, the pH of the rinse solution being about 6.6 to about 7.0.

As another example, the rinse solution is about 0.9 g / L to about 1.1 g / L of polyethylene glycol; about 9 mM to about 11 mM HEPES buffer; about 0.16 mL / L to about 0.24 mL / L of polysorbate 20; It can contain from about 2 ppm to about 10 ppm ProClin 300®; about 9 mL / L to about 11 mL / L of methanol; and water, the pH of the rinse solution being about 6.6 to about 7.0.

As another example, the rinse solution is about 1 g / L polyethylene glycol; about 10 mM HEPES buffer; about 0.2 mL / L polysorbate 20; about 3 ppm ProClin 300®; about 10 mL / L methanol. And can contain water, the pH of the rinse solution is about 6.6 to about 7.0.

In a fifth aspect, the disclosure features a specimen preparation kit comprising a fixative; a first cell stain; a second cell stain; and a rinse solution. In some embodiments, the specimen preparation kit is a separately packaged cell fixative; a separately packaged first cell stain; a separately packaged second cell stain; and individually packaged. Contains the rinse solution.

In a sixth aspect, the present disclosure is a method of preparing a specimen on an inspection substrate, wherein (a) the specimen faces a surface and the substrate is at least between its surface and at least one portion of the substrate. Position the substrate with respect to its surface so that it forms a separation of approximately 100 microns; (b) (i) Specimen and surface contact during separation between substrate and surface. Distribute a sufficient amount to (ii) at least in the first stirring step, the distance between the substrate and the surface while the fixation liquid is in contact with the specimen during the first stirring step. Perform a first stirring step to change; and (iii) perform a fixation step that involves separating and removing the fixation from the specimen; (c) perform a first staining step; (d) perform a second It features a method comprising performing a staining step of; and (e) performing a first rinsing step.

In a seventh aspect, the present disclosure provides an automated sample analyzer and a system for imaging specimens containing cell fixatives; first cell stains; second cell stains; and / or rinses. It is a feature. In some embodiments, the system for imaging the specimen is prepared with an automated sample analyzer and a cell fixative; a first cell stain; a second cell stain; and / or a rinse solution. Includes specimens.

The cell fixatives, first cell stains, second cell stains, rinses, methods, kits and systems described herein may include any one or more of the following features:

In some embodiments, the fixative may contain from about 0.5 to about 5 g / L of Azure B (eg, about 1 g / L of Azure B). For example, a fixative diluted 1: 1000 with water has a UV absorbance of about 0.1 to about 1 at a peak wavelength of about 640 to about 650 nm.

In some embodiments, the fixative contains one (or more) red dye (eg, one or more conventional red dyes known in the art, such as eosin Y, and fluorescein derivatives). Not included or substantially not included.

The fixative should be about 0.05 to about 0.5 volume or weight% of surfactant (eg, about 0.05 to about 0.3 volume or weight% of surfactant, about 0.05 to about 0.1 volume or weight% of surfactant). Can include. The surfactant can be selected from the group consisting of nonionic, cationic, anionic and zwitterionic surfactants. In some embodiments, the surfactant is nonionic, such as polysorbate 20. For example, the fixative may contain from about 0.5 mL / L to about 2 mL / L (eg, about 0.5 mL / L to about 1.5 mL / L, about 1 mL / L) of polysorbate 20.

In some embodiments, the cell fixation solution may further comprise a buffer such as Bis-Tris buffer, Phosphate buffer, HEPES buffer, MES buffer and / or Tris buffer. For example, a cell fixative diluted 1:10 with water may have a pH of about 6 to about 8 (eg, about 6.7 to about 7.3) and may contain about 0.5 mM to about 10 mM HEPES.

The fixative may contain from about 0.5 to about 5% by volume (eg, about 1% by volume) of ethylene glycol.

In some embodiments, the fixative comprises about 1 g / L of Azure B; about 1 mL / L of polysorbate 20; about 10 mL / L of ethylene glycol; about 0.32 g / L of HEPES sodium salt; and methanol. obtain.

In some embodiments, the first cell stain may contain an antibacterial agent (eg, about 0.2 to about 50 ppm of antibacterial agent), such as benzalkonium chloride, 5-chloro-2. -Methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, ProClin® (eg, ProClin300®), azide, merthiolate, antibiotics, and any of them. Combinations can be mentioned. For example, antibacterial agents include 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one. In some embodiments, the antibacterial agent is ProClin 300®. For example, the first cell stain may contain approximately 15 ppm ProClin 300®.

The first cell stain may contain from about 0.5 to about 1 g / L (eg, about 0.75 g / L) of eosin Y. For example, a solution diluted 1: 500 with water has a UV absorbance of about 0.1 to about 1 at a peak wavelength of about 510 to about 530 nm.

In some embodiments, the first cell stain is one (or more) other red dye (eg, one or more conventional red dyes known in the art, eg, other). Fluorescein Derivatives) Free or Substantially Free.

In some embodiments, the first cell stain is one (or more) blue dye (eg, one or more conventional blue dyes known in the art, such as Azure B, methylene blue and / Or other thiazine dyes) free or substantially free.

In some embodiments, the first cell stain is one (or more) other red dye (eg, one or more conventional red dyes known in the art, eg, other). The first cell stain, which is free or substantially free of (fluorescein derivatives), is one (or more) blue dye (eg, one or more conventional known in the art). Free or substantially free of blue dyes such as Azure B, methylene blue and / or other thiazine dyes.

The first cell stain may contain from about 0.5 to about 5% by volume (eg, about 1% by volume) of ethylene glycol.

In some embodiments, the first cell stain may have a pH of about 5 to about 8 and a buffer concentration of about 5 to about 250 mM.

Buffering agents include bis-tris, phosphoric acid, HEPES, MES, tris and any mixture thereof. For example, the first cell stain can have a pH of about 5.8 to about 6.2 and a Bis-Tris buffer concentration of about 5 mM to about 250 mM. In some embodiments, the first cell stain may further contain acetic acid (eg, about 2-3 mL / L of acetic acid).

In some embodiments, the first cell stain has a surface activity of about 0.05 to about 0.5 volume or weight% (eg, about 0.05 to about 0.3 volume or weight%, about 0.05 to about 0.1 volume or weight%). May include agents. The surfactant can be selected from the group consisting of nonionic, cationic, anionic and zwitterionic surfactants. For example, the surfactant can be nonionic, such as polysorbate 20. In some embodiments, the first cell stain is about 0.5 mL / L to about 2 mL / L (eg, about 0.5 mL / L to about 1.5 mL / L, about 1 mL / L) of polysorbate 20. Can include.

In some embodiments, the first cell stain may contain from about 1 to about 20 g / L of sodium chloride.

In some embodiments, the first cell stain is about 0.75 g / L eosin Y; about 50 mM bis-tris buffer; about 1 mL / L polysorbate 20; about 4 g / L sodium chloride; about 4 g / L sodium chloride. It can contain 10 mL / L ethylene glycol; about 15 ppm ProClin 300®; acetic acid; and water, the pH of the solution being about 5.8 to about 6.2.

In some embodiments, the second cell stain can contain an antibacterial agent (eg, about 0.2 to about 50 ppm of antibacterial agent), such as benzalkonium chloride, 5-chloro. -2-Methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, ProClin® (eg, ProClin300®), azide, merthiolate, antibiotics, and theirs. Any combination can be mentioned. For example, antibacterial agents include 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one. In some embodiments, the second cell stain may include ProClin 300® (eg, about 15 ppm ProClin 300®).

In some embodiments, the second cell stain may contain from about 0.25 to about 1 g / L (eg, about 0.5 g / L) of Azure B. The second cell stain may contain from about 0.25 to about 1 g / L (eg, about 0.45 g / L) methylene blue. For example, a second cell stain diluted 1: 1000 with water can have a UV absorbance of about 0.1 to about 1 at a peak wavelength of about 640 to about 660 nm.

In some embodiments, the second cell stain is one (or more) other blue dye (eg, one or more conventional other blue dyes known in the art, eg Azure B. , Methylene blue and / or other thiazine dyes) free or substantially free.

In some embodiments, the second cell stain is free of one (or more) red dye (eg, one or more conventional red dyes known in the art, eg, fluorescein derivatives). Or substantially not included.

In some embodiments, the second cell stain is one (or more) other blue dye (eg, one or more conventional other blue dyes known in the art, eg, other). The second cell stain, which is free or substantially free of thiazine dyes), is one (or more) red dye (eg, one or more conventional red dyes known in the art). , For example fluorescein derivatives) are free or substantially free.

In some embodiments, the second cell stain has a pH of about 5 to about 8 and a buffer concentration of about 5 mM to about 250 mM (eg, a pH of about 6.8 to about 7.2 and a bis of about 25 mM to about 100 mM). -Tris buffer concentration). Buffers include Bis-Tris buffer, phosphate buffer, HEPES buffer, MES buffer, Tris buffer and any mixture thereof. In some embodiments, the second cell stain may further contain acetic acid.

In some embodiments, the second cell stain has a surface activity of about 0.05 to about 0.5 volume or weight% (eg, about 0.05 to about 0.3 volume or weight%, about 0.05 to about 0.1 volume or weight%). Agents may be included, and surfactants include nonionic, cationic, anionic and zwitterionic surfactants. In some embodiments, the detergent is nonionic and can contain polysorbate 20, and the second cell stain is from about 0.5 mL / L to about 2 mL / L (eg, about 0.5 mL). / L ~ about 1.5 mL / L, about 1 mL / L) may contain polysorbate 20.

In some embodiments, the second cell stain may contain from about 1 to about 20 g / L of sodium chloride.

In some embodiments, the second cell stain is about 0.5 g / L of Azure B; about 0.45 g / L of methylene blue; about 50 mM Bis-Tris buffer; about 1 mL / L of polysorbate 20; about It may contain 2 g / L sodium chloride; and about 15 ppm ProClin 300®; acetic acid; and water. This solution can have a pH of about 6.8 to about 7.2.

In some embodiments, the rinse solution can contain an antibacterial agent (eg, about 0.2 to about 50 ppm of antibacterial agent, or about 0.04 to about 10 ppm of antibacterial agent), such as benzalkonium chloride. Luconium, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, ProClin® (eg, ProClin300®), azide, merthiolate, Antibiotics, and any combination thereof, may be mentioned. In some embodiments, antibacterial agents include 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one. For example, the rinse solution may contain ProClin 300® (eg, about 15 ppm ProClin 300® to about 3 ppm ProClin 300®).

The rinse solution may contain from about 1 to about 10 g / L (eg, about 5 g / L) of polyethylene glycol.

The rinse solution may contain from about 0.2 to about 2 g / L (eg, about 1 g / L) of polyethylene glycol.

The rinse solution can have a pH of 5-8 and a buffer concentration of about 5 mM to about 250 mM (eg, a pH of about 5 to about 8 and a HEPES buffer concentration of about 50 mM). Buffers include Bis-Tris buffer, phosphate buffer, HEPES buffer, MES buffer, Tris buffer and any mixture thereof.

The rinse solution can have a pH of 5-8 and a buffer concentration of about 1 mM to about 50 mM (eg, a pH of about 5-8 and a HEPES buffer concentration of about 10 mM). Buffers include Bis-Tris buffer, phosphate buffer, HEPES buffer, MES buffer, Tris buffer and any mixture thereof.

In some embodiments, the rinse solution comprises from about 0.05 to about 0.5 volume or weight% (eg, about 0.05 to about 0.3 volume or weight%, about 0.05 to about 0.1 volume or weight%) of surfactant. The surfactant can be selected from the group consisting of nonionic, cationic, anionic and zwitterionic surfactants. For example, the surfactant is nonionic and may contain polysorbate 20 (eg, about 0.5 mL / L to about 2 mL / L, about 0.5 mL / L to about 1.5 mL / L of polysorbate 20).

In some embodiments, the rinse solution comprises from about 0.01 to about 0.1 volume or weight% (eg, about 0.01 to about 0.06 volume or weight%, about 0.01 to about 0.02 volume or weight%) of surfactant. The surfactant can be selected from the group consisting of nonionic, cationic, anionic and zwitterionic surfactants. For example, the surfactant can be nonionic and can contain polysorbate 20 (eg, about 0.1 mL / L to about 0.4 mL / L, about 0.1 mL / L to about 0.3 mL / L of polysorbate 20).

In some embodiments, the rinse solution comprises from about 45 to about 55 mL / L (eg, about 50 mL / L) of methanol.

In some embodiments, the rinse solution comprises from about 9 to about 11 mL / L (eg, about 10 mL / L) of methanol.

In some embodiments, the rinse solution is about 5 g / L polyethylene glycol; about 50 mM HEPES buffer; about 1 mL / L polysorbate 20; about 15 ppm ProClin 300®; about 50 mL / L methanol; and water. including. The pH of the rinse solution can be from about 6.6 to about 7.0.

In some embodiments, the rinse solution is about 1 g / L polyethylene glycol; about 10 mM HEPES buffer; about 0.2 mL / L polysorbate 20; about 3 ppm ProClin 300®; about 10 mL / L methanol. ; And contains water. The pH of the rinse solution can be from about 6.6 to about 7.0.

In some embodiments, the fixative does not contain the Dye Patent Blue VF (eg, at all or more than the amount of Patent Blue VF present in the formulation disclosed in WO 2007/117798. Including small amounts).

In some embodiments, the first cell stain does not contain Dye Patent Blue VF (eg, the amount of Patent Blue that is completely free or is present in the formulation disclosed in WO 2007/117798). Includes less than VF).

In some embodiments, the second cell stain does not contain Dye Patent Blue VF (eg, the amount of Patent Blue that is completely free or is present in the formulation disclosed in WO 2007/117798). Includes less than VF).

In some embodiments, the rinse solution is free of Dye Patent Blue VF (eg, no or less than the amount of Patent Blue VF present in the formulation disclosed in WO 2007/117798. Including quantity).

In some embodiments, the fixative is free of proteolytic enzymes (eg, no proteolytic enzyme or less than the amount of proteolytic enzyme present in the formulation disclosed in WO 2007/117798). Including quantity).

In some embodiments, the first cell stain is free of proteolytic enzymes (eg, no proteolytic enzymes, or the amount of proteolytic enzymes present in the formulation disclosed in WO 2007/117798). Including less than the amount).

In some embodiments, the second cell stain is free of proteolytic enzymes (eg, no proteolytic enzymes or the amount of proteolytic enzymes present in the formulation disclosed in WO 2007/117798). Including less than the amount).

In some embodiments, the rinse solution is free of proteolytic enzymes (eg, no amount or less than the amount of proteolytic enzyme present in the formulation disclosed in WO 2007/117798). including).

In some embodiments, performing the first staining step is (i) in an amount sufficient to allow the first cell stain to be in contact with the specimen and surface for separation between the substrate and the surface. Distributing; (ii) Perform at least a second stirring step (the second stirring step is the distance between the substrate and the surface, the first stain is with the specimen during the second stirring step. Includes contacting changes); and (iii) involves separating and removing the first stain from the specimen.

In some embodiments, performing the second staining step is (i) in an amount sufficient to allow the second cell stain solution to come into contact with the specimen and surface for separation between the substrate and the surface. Distributing; (ii) Perform at least a third stirring step (the third stirring step is the distance between the substrate and the surface, and the second stain is with the specimen during the third stirring step. Includes contacting changes); and (iii) involves separating and removing the second stain from the specimen.

In some embodiments, performing the first rinsing step is: (i) Distributing the rinse solution in an amount sufficient to contact the specimen and the surface for separation between the substrate and the surface; (ii) Perform at least a fourth stirring step (the fourth stirring step changes the distance between the substrate and the surface while the rinse solution is in contact with the specimen during the fourth stirring step. Includes); and (iii) Includes separation and removal of the rinse solution from the specimen.

In some embodiments, the automated sample analyzer is a substrate arm that includes a substrate grip; a first configuration that is connected to a substrate arm and is configured to move the substrate arm between an open position and a sample processing position. Actuator; A second actuator arranged and set to agitate the substrate gripped on the substrate arm by the substrate grip; a platform with an upper surface that faces the substrate when the substrate arm is in the sample processing position; And two or more offsets placed on the top surface of the platform, where the substrate is in contact with all offsets at the substrate processing position, the top surface of the substrate and platform is substantially parallel, at least about 50 microns. Includes two or more offsets arranged to form a separation.

Embodiments of an automated sample inspection system include any one or more features disclosed herein as appropriate, such as any one or more features of the sample preparation device disclosed herein. May include features.

In embodiments of the methods, kits and systems disclosed herein, one, two, three or four (eg, 4) of (A), (B), (C) and (D). One) can be applied.

(A) The fixative contains Azure B; surfactant; methanol and ethylene glycol.

For example, the cell fixation solution is about 0.5 g / L to about 5.0 g / L of Azure B; about 0.5 mL / L to about 2.0 mL / L of polysolvate 20; about 5 mL / L to about 50 mL / L of ethylene glycol, It may contain propylene glycol, or polypropylene glycol; about 0.1 g / L to about 10 g / L of HEPES sodium salt; and methanol.

As another example, the cell fixation solution is about 0.8 g / L to about 1.2 g / L of Azure B; about 0.8 mL / L to about 1.2 mL / L of polysorbate 20; about 9 mL / L to about 11 mL / L. It may contain ethylene glycol; about 0.25 g / L to about 0.38 g / L of HEPES sodium salt; and methanol.

For example, the fixative may contain about 1 g / L of Azure B; about 1 mL / L of polysorbate 20; about 10 mL / L of ethylene glycol; about 0.32 g / L of HEPES sodium salt; and methanol.

As another example, the cell fixation solution contains about 1 g / L of Azure B; about 0.5 mL / L of polysorbate 20; about 10 mL / L of propylene glycol; about 0.32 g / L of HEPES sodium salt; and methanol. obtain.

As a further example, the fixative may contain about 1 g / L of Azure B; about 1.5 mL / L of polysorbate 20; about 10 mL / L of polypropylene glycol; about 0.32 g / L of HEPES sodium salt; and methanol. ..

Embodiments may include any one or more of the features described above and / or in the detailed description of the invention and / or in the claims.

(B) The first cell stain contains eosin Y; buffer; surfactant; sodium chloride; ethylene glycol; and water.

For example, the first cell stain is about 0.5 g / L to about 5.0 g / L of eosin Y; about 5 mM to about 250 mM Bis-Tris buffer or phosphate buffer; about 0.5 mL / L to about 2.0. mL / L polysolvate 20; about 1 g / L to about 20 g / L sodium chloride; about 5 mL / L to about 50 mL / L ethylene glycol; about 0.2 ppm to about 50 ppm ProClin 300®; acetic acid; and water The pH of the solution is about 5.8 to about 6.2.

As another example, the first cell stain is about 0.6 g / L to about 0.9 g / L of eosin Y; about 45 mM to about 55 mM Bis-Tris buffer; about 0.8 mL / L to about 1.2 mL. / L Polysorbate 20; Approximately 3 g / L to Approximately 5 g / L Sodium Chloride; Approximately 9 mL / L to Approximately 11 mL / L Ethylene Glycol; Approximately 10 ppm to Approximately 20 ppm ProClin 300®; Acetic Acid; The pH of the solution can be from about 5.8 to about 6.2.

For example, the first cell stain is about 0.75 g / L of eosin Y; about 50 mM bis-tris buffer; about 1 mL / L of polysorbate 20; about 4 g / L of sodium chloride; about 10 mL / L of ethylene. Glycol; about 15 ppm ProClin 300®; acetic acid; and water can be included, the pH of the solution being about 5.8 to about 6.2.

As another example, the first cell stain is about 0.75 g / L of eosin Y; about 50 mM bis-tris buffer; about 0.5 mL / L of polysorbate 20; about 6 g / L of sodium chloride; about It can contain 20 mL / L ethylene glycol; about 15 ppm ProClin 300®; acetic acid; and water, the pH of the solution being about 5.8 to about 6.2.

As a further example, the first cell stain is about 0.75 g / L eosin Y; about 50 mM phosphate buffer, about 2.0 mL / L polysorbate 20; about 4 g / L sodium chloride; about 50 mL / It can contain L ethylene glycol; about 15 ppm ProClin 300®; acetic acid; and water, the pH of the solution being about 5.8 to about 6.2.

Embodiments may include any one or more of the features described above and / or in the detailed description of the invention and / or in the claims.

(C) The second cell stain contains Azure B; methylene blue; buffer; surfactant; sodium chloride; and water.

For example, the second cell stain is about 0.25 to about 2.5 g / L of Azure B; about 0.25 g / L to about 2.5 g / L of methylene blue; about 5 mM to about 250 mM Bis-Tris or HEPES buffer; May contain about 0.5 mL / L to about 2.0 mL / L of polysolvate 20; about 1 g / L to about 20 g / L of sodium chloride; and about 0.2 ppm to about 50 ppm of ProClin 300®; acetic acid; and water. The pH of the solution is about 6.8 to about 7.2.

As another example, the second cell stain is about 0.4-about 0.6 g / L Azure B; about 0.4 g / L-about 0.5 g / L methylene blue; about 45 mM-about 55 mM Bis-Tris buffer. Liquid; about 0.8 mL / L to about 1.2 mL / L of polysorbate 20; about 1.8 g / L to about 2.2 g / L of sodium chloride; and about 10 ppm to about 20 ppm of ProClin 300®; acetic acid; and water. It can be included and the pH of the solution is from about 6.8 to about 7.2.

For example, the second cell stain is about 0.5 g / L Azure B; about 0.45 g / L methylene blue; about 50 mM Bis-Tris buffer; about 1 mL / L polysorbate 20; about 2 g / L chloride. It can contain sodium, and about 15 ppm ProClin 300®; acetic acid; and water, the pH of the solution being about 6.8 to about 7.2.

As another example, the second cell stain is about 0.5 g / L of Azure B; about 0.45 g / L of methylene blue; about 50 mM HEPES buffer; about 0.5 mL / L of polysorbate 20; about 2 g / It can contain L sodium chloride, and about 15 ppm ProClin 300®; acetic acid; and water, the pH of the solution being about 6.8 to about 7.2.

As a further example, the second cell stain is about 0.5 g / L of Azure B; about 0.45 g / L of methylene blue; about 50 mM HEPES buffer; about 1 mL / L of polysolvate 20, about 1 g / L. It can contain sodium chloride, and about 15 ppm ProClin 300®; acetic acid; and water, the pH of the solution being about 6.8 to about 7.2.

The solution can have a pH of about 6.8 to about 7.2.

Embodiments may include any one or more of the features described above and / or in the detailed description of the invention and / or in the claims.

(D) The rinse solution for the automatic sample preparation device contains polyethylene glycol; buffer; surfactant; methanol; and water.

For example, the rinse solution is about 0.2 g / L to about 10 g / L of polyethylene glycol; about 1 mM to about 250 mM HEPES, MES or Bis-Tris buffer; about 0.1 mL / L to about 2.40 mL / L of polysorbate 20. It can contain from about 0.04 ppm to about 50 ppm ProClin 300®; about 9 mL / L to about 200 mL / L of methanol; and water, the pH of the rinse solution being about 6.6 to about 7.0.

As another example, the rinse solution is about 1 g / L to about 10 g / L polyethylene glycol; about 5 mM to about 250 mM HEPES, MES or Bis-Tris buffer; about 0.5 mL / L to about 2.0 mL / L. Polysolvate 20; about 0.2 ppm to about 50 ppm of ProClin 300®; about 10 mL / L to about 200 mL / L of methanol; and water, the pH of the rinse solution is about 6.6 to about 7.0. is there.

As a further example, the rinse solution is about 4.5 g / L to about 5.5 g / L polyethylene glycol; about 45 mM to about 55 mM HEPES buffer; about 0.8 mL / L to about 1.2 mL / L polysorbate 20; about. It can contain from 10 ppm to about 20 ppm ProClin 300®; about 45 mL / L to about 55 mL / L of methanol; and water, the pH of the rinsing solution being about 6.6 to about 7.0.

For example, the rinse solution should contain about 5 g / L polyethylene glycol; about 50 mM HEPES buffer; about 1 mL / L polysorbate 20; about 15 ppm ProClin 300®; about 50 mL / L methanol; and water. The pH of the rinse solution is about 6.6 to about 7.0.

As another example, the rinse solution is about 10 g / L polyethylene glycol; about 50 mM MES buffer; about 1 mL / L polysorbate 20; about 15 ppm ProClin 300®; about 50 mL / L methanol; It can be included and the pH of the rinse solution is from about 6.6 to about 7.0.

As a further example, the rinse solution is about 10 g / L polyethylene glycol; about 50 mM bis-tris buffer; about 0.5 mL / L polysorbate 20; about 15 ppm ProClin 300®; about 50 mL / L methanol. And can contain water, the pH of the rinse solution is about 6.6 to about 7.0.

For example, the rinse solution is about 0.2 g / L to about 2 g / L of polyethylene glycol; about 1 mM to about 50 mM HEPES buffer; about 0.16 mL / L to about 0.24 mL / L of polysorbate 20; about 0.04 ppm to about. It can contain 10 ppm ProClin 300®; about 9 mL / L to about 11 mL / L of methanol; and water, the pH of the rinse solution being about 6.6 to about 7.0.

As another example, the rinse solution is about 0.9 g / L to about 1.1 g / L of polyethylene glycol; about 9 mM to about 11 mM HEPES buffer; about 0.16 mL / L to about 0.24 mL / L of polysorbate 20; It can contain from about 2 ppm to about 10 ppm ProClin 300®; about 9 mL / L to about 11 mL / L of methanol; and water, the pH of the rinsing solution being about 6.6 to about 7.0.

As another example, the rinse solution is about 1 g / L polyethylene glycol; about 10 mM HEPES buffer; about 0.2 mL / L polysorbate 20; about 3 ppm ProClin 300®; about 10 mL / L methanol. And can contain water, the pH of the rinse solution is about 6.6 to about 7.0.

As used herein, when a solution is said to be "free" of one or more substances, this is when the solution is measured by HPLC, UV spectroscopy, electrophoresis, and / or enzyme assay detection. Means containing less than 5% of that one or more substances (eg, less than 4%, less than 3%, less than 2%, less than 1%, 0% (w / w or w / v or v / v)) ..

Unless defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Methods and materials similar to or equivalent to those described herein can be used in the practice or inspection of the present invention, but suitable methods and materials are described below. All publications, patent applications, patents, and other references referred to herein are incorporated by reference in their entirety. In case of conflict, this specification, including definitions, will be considered. Also, the materials, methods and examples are merely exemplary and are not intended to be limiting.

Other features and advantages of the present invention will become apparent from the following detailed description and claims.

<figref num="1">FIG. 5 is an HPLC chromatograph of an embodiment of a fixative.</figref><figref num="2">It is an ultraviolet-visible absorption spectrum of one embodiment of a fixative.</figref><figref num="3">It is an HPLC chromatograph of one Embodiment of the 1st staining liquid.</figref><figref num="4">It is an ultraviolet-visible absorption spectrum of one Embodiment of the 1st staining liquid.</figref><figref num="5">It is an HPLC chromatograph of one Embodiment of the 2nd staining liquid.</figref><figref num="6">It is an ultraviolet-visible absorption spectrum of one embodiment of the second staining liquid.</figref><figref num="7">FIG. 5 is a perspective view of an embodiment of an apparatus for preparing a test specimen provided with both sample grips 20A and 20B in the open position.</figref><figref num="8">Another perspective view of a portion of the device described in FIG. 7 (the substrate arm and sample grip are not shown).</figref><figref num="9A">Another perspective view of the apparatus of FIG. 7 with a sample grip 20A in the open position and a sample grip 20B in the closed (sample processing) position.</figref><figref num="9B">It is a perspective view of the indexing mechanism of the apparatus shown in FIG.</figref><figref num="10">It is a perspective view of the device of FIG. 7 which shows the connection between a device and a liquid container by a plurality of liquid routes.</figref><figref num="11">FIG. 5 is a perspective view of a sample inspection system including an automated substrate moving means and an embodiment of a sample preparation device described herein.</figref><figref num="12A">It is an enlarged perspective view of a part of the apparatus of FIG. 7 which shows the specimen gripping part 20B, the platform 60B and the block 80B in detail.</figref><figref num="12B">It is a perspective view of the ball joint mechanism of the apparatus of FIG.</figref><figref num="12C">It is sectional drawing of the ball joint mechanism of FIG. 12B.</figref><figref num="13A">It is a flowchart which shows a series of steps for moving a substrate arm from an open position to a closed (sample processing) position.</figref><figref num="13B">It is the schematic of one Embodiment of the sample preparation apparatus described in this specification.</figref><figref num="14A">It is a flowchart which shows another series of steps for moving a substrate arm from an open position to a sample processing position.</figref><figref num="14B">It is the schematic of the apparatus for preparing the inspection specimen containing two actuators.</figref><figref num="15">It is a flowchart which shows a series of steps for applying a fixative to a specimen.</figref><figref num="16">It is a flowchart which shows a series of steps for applying a dyeing agent to a specimen.</figref><figref num="17A">It is a flowchart which shows a series of steps for removing excess liquid from a substrate.</figref><figref num="17B">It is a flowchart which shows another series of steps for removing excess liquid from a substrate.</figref><figref num="18">It is a flowchart which shows a series of steps for rinsing a sample.</figref><figref num="19">It is a flowchart which shows a series of steps for stirring a sample.</figref><figref num="20">It is a flowchart which shows a series of steps for drying a sample.</figref><figref num="21">FIG. 3 is a perspective view of a sample preparation device when used in a larger sample inspection system.</figref><figref num="22">It is a flowchart which shows a series of steps for processing a sample placed on a substrate.</figref><figref num="23">In the flowchart of FIG. 22, it is a graph showing the volume of liquid consumed as a function of time.</figref><figref num="24A">It is a perspective view of the apparatus of FIG. 7 which shows the installation of a substrate on a substrate arm by an automatic substrate moving means.</figref><figref num="24B">It is a perspective view of the apparatus of FIG. 7 which shows the installation of a substrate on a substrate arm by an automatic substrate moving means.</figref><figref num="25">It is an embodiment of a kit containing a fixative, a first stain, a second stain and a rinse.</figref>

Similar reference numerals in the various figures indicate similar elements.

Disclosed herein are formulations, methods and systems for automated sampling. The automated sampling methods and systems described herein are the application of stains, immobilizers and other reagents when processed manually or by other systems, as opposed to manual and other automated methods. It offers the advantage of increased processing speed with the least amount of reagent, with highly uniform staining while significantly reducing variability associated with. Fixatives, stains and rinses enhance sample contrast and can be easily dispensed from automated sample processing systems. In addition, fixatives, stains and rinses can provide improved cell identification and cell classification compared to conventional fixatives, stains and rinses.

Conventional fixation, stain and rinse solutions usually provide unfavorable staining results, such as light-colored staining or non-specific staining of biological samples (eg, non-specific staining of cytoplasm), or large amounts of treatment. Need liquid. The formulations disclosed herein allow for high contrast and selective staining of biological samples with low doses of treatment, for example when used in automated specimen processing systems.

Further, the conventional automatic processing method usually has a relatively high throughput and consumes a large amount of processing liquid at the same time, or has a relatively low throughput but consumes a small amount of processing liquid. However, for many applications, both high-throughput operation and low liquid consumption are desirable. By maintaining high throughput, samples can be processed and inspected efficiently. By keeping the liquid consumption low, the amount of processing waste is reduced along with the required amount of processing reagents, and the operating cost is kept low. The formulations, systems and methods disclosed herein are rapid with low doses of treatment (eg, less than about 1 mL of liquid per specimen, less than about 1.5 mL of liquid per specimen, less than about 2 mL of liquid per specimen). It allows automatic processing of specimens (for example, more than 100 specimens per hour on a single machine), while producing highly uniform and reproducible results.

Formulations for use in sample preparation systems Fixatives, one or more stains and rinses are provided herein and they can be used in automated sample preparation systems. Each solution may have a particular formulation. For example, fixatives can include cytological dyes, surfactants, organic solvents, agents to minimize non-specific binding, and buffers. The first aqueous staining solution may contain cytological dyes, buffers, surfactants, salts, agents for minimizing non-specific binding, and antibacterial agents. The second aqueous staining solution may contain one or more cytological dyes, buffers, surfactants, salts and antibacterial agents. The rinse solution may contain buffers, surfactants, antibacterial agents, organic solvents and water soluble polymers. The formulation composition can be analyzed for purity and / or purified by HPLC. The formulation may also include an acid for adjusting the pH. Each formulation is described in more detail below.

Fixative Fixatives that can be included in the fixative include chemicals used to protect biological samples from spoilage. Such immobilizers can prevent the biological reaction that occurs in the specimen and increase the mechanical strength and stability of the specimen. Various fixatives can be included in the fixative, including, for example, methanol, ethanol, isopropanol, acetone, formaldehyde, glutaraldehyde, EDTA, surfactants, metal salts, metal ions, urea and amino compounds. In some embodiments, the fixative comprises a fixative capable of precipitating, cross-linking or preserving an organic solvent or intracellular component (eg, a protein) for imaging purposes. For example, immobilizers include organic solvents such as one or more alcohols such as methanol, ethanol and isopropanol, or other organic solvents such as acetone and ether. In some embodiments, the fixative comprises an organic solvent such as methanol. The organic solvent can have an ACS grade of less than about 0.1% water (eg, less than about 0.05% water, less than about 0.01% water). The organic solvent may be present in the solution in the remaining amount after the other components have been added to the fixative. The organic solvent can also act as a desiccant for the specimen and can remove water from the specimen.

In some embodiments, the fixative comprises a cytological dye. Dyes can increase staining of specimens, including, for example, Azure B, methylene blue, eosin, thiazine stains, and fluorescein derivatives. The dye can have a purity of about 80% or higher (eg, a purity of about 85% or higher, a purity of about 90% or higher, a purity of about 95% or higher, or a purity of about 99% or higher). A typical HPLC chromatogram of Azure B is shown, for example, in FIG. Dyes range from approximately 0.5 g / L (eg, approximately 0.75 g / L, approximately 1 g / L, approximately 2 g / L, approximately 3 g / L, approximately 4 g / L) to approximately 5 g / L (eg, approximately 4 g / L) in the fixative. It can have a concentration of about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, or about 0.75 g / L). For example, fixative may contain approximately 1 g / L of Azure B. In some embodiments, the fixative may contain approximately 0.8 g / L of Azure B.

The fixative may contain a surfactant. Although not bound by any theory, surfactants reduce the surface tension of the solvent and, as shown in FIG. 13B, good coatability of the solution onto the sample substrate at separation 92. Is considered to provide. In some embodiments, the surfactant is nonionic and can minimize the potential for precipitation from solution that can result from ionic interactions with components within the formulation, such as ionically charged dyes. .. A solution containing little or no precipitate can be more easily expelled from the distribution nozzle, reducing the possibility of clogging of the distribution nozzle and the gradual diminishing of liquid flow. In some embodiments, the surfactant can reduce the likelihood of non-selective binding of the components in the fixation solution to the specimen, thereby minimizing image artifacts.

Fixatives are by volume% for liquid surfactants or by weight% for solid surfactants, from approximately 0.05% (eg, from approximately 0.075%, from approximately 0.1%, from approximately 0.2%, to approximately 0.2%). It may contain from 0.3% or from about 0.4%, about 0.5% (eg, about 0.4%, about 0.3%, about 0.2%, about 0.1%, or about 0.075%) of surfactant. In some embodiments, the fixative may contain from approximately 0.5 to approximately 2 mL / L of surfactant (eg, nonionic surfactant). For example, fixative may contain approximately 1 mL / L of polysorbate 20 (eg, Tween 20).

The surfactant may be nonionic, cationic, anionic or zwitterionic. A mixture of surfactants may be used. Specific classifications of surfactants include alcohol ether sulfates, alcohol sulfates, alkanolamides, alkylsulfonates, amine oxides, amphoteric surfactants, anionic surfactants, betaine derivatives, cationic surfactants, and di. Sulfonate, dodecylbenzene, sulfonic acid, ethoxylated alcohol, alkylphenol ethoxylated, ethoxylated fatty acid, glycerol ester hydrotropes, lauryl sulphate, mono and diglycerides, nonionic surfactants, phosphates, 4th Examples include class surfactants and sorbitan derivatives.

Specific nonionic surfactants include, for example, BigCHAP (N, N-bis [3- (D-gluconamide) propyl] colamide), bis (polyethylene glycol bis [imidazolylcarbonyl]), Brij (registered). Trademarks) 30 (Polyoxyethylene 4 Lauryl Ether), Brij® 35 (Polyoxyethylene 23 Lauryl Ether), Brij® 52 (Polyoxyethylene 2 Cetyl Ether), Brij® 56 (Poly) Oxyethylene 10 Cetyl Ether), Brij® 58 (Polyoxyethylene 20 Cetyl Ether), Brij® 72 (Polyoxyethylene 2 Stearyl Ether), Brij® 76 (Polyoxyethylene 10 Stearyl Ether) ), Brij® 78 (Polyoxyethylene 20 Stearyl Ether), Brij® 92 (Polyoxyethylene 2 Oleyl Ether), Brij® 97 (Polyoxyethylene 10 Oleyl Ether), Brij (Registered) Trademarks) 98 (Polyoxyethylene 20 Oleyl Ether), Brij® 700 (Polyoxyethylene 100 Stearyl Ether), Cremophor® EL (Himasi Oil / Polyethylene Oxide Polyether), Decaethylene Glycol Monododecyl Ether, Octanoyl -N-Methylglucamide (MECA-8), Decanoyl-N-Methylglucamide (MECA-10), n-octylglucoside, n-dodecylglucoside, isotridecylpoly (ethylene glycol ether)_n, N-decanoyl-N-methylglucamine, n-decyl α-D glucopyranoside, decyl β-D-maltopyranoside, n-dodecanoyl-N-methylglucamide, n-dodecyl α-D-maltoside, n-dodecyl β- D-maltoside, heptaethylene glycol monodecyl ether, heptaethylene glycol monotetradecyl ether, n-hexadecyl β-D-maltoside, hexaethylene glycol monododecyl ether, hexaethylene glycol monohexadecyl ether, hexaethylene glycol monooctadecyl ether, Hexaethylene glycol monotetradecyl ether, Igepal® CA-630 (octylphenyl-polyethylene glycol), Igepal® CA-210 (polyoxyethylene (2) isooctylphenyl ether), Igepal® CA-520 (polyoxyethylene (5) isooctylphenyl ether), Igepal® CO-630 (polyoxyethylene (9) nonylphenyl ether), Igepal® CO-720 (polyoxyethylene (12) ) Nonylphenyl ether), Igepal® CO-890 (polyoxyethylene (40) nonylphenyl ether), Igepal® CO-990 (polyoxyethylene (100) nonylphenyl ether), Igepal® ) DM-970 (polyoxyethylene (150) dinonylphenyl ether), methyl-6-O- (N-heptylcarbamoyl) -α-D-glucopyranoside, nonaethylene glycol monododecyl ether, N-nonanoyl-N-methyl Glucamine, Octaethylene Glycol Monodecyl Ether, Octaethylene Glycol Monododecyl Ether, Octaethylene Glycol Monohexadecyl Ether, Octaethylene Glycol Monooctadecyl Ether, Octaethylene Glycol Monotetradecyl Ether, Octyl-β-D-Glucopyranoside, Pentaethylene Glycol monoDecyl ether, pentaethylene glycol monododecyl ether, pentaethylene glycol monohexadecyl ether, pentaethylene glycol monohexyl ether, pentaethylene glycol monooctadecyl ether, pentaethylene glycol monooctyl ether, polyethylene glycol diglycidyl ether, polyethylene glycol ether W- 1, Polyoxyethylene 10 tridecyl ether, polyoxyethylene 100 stearate, polyoxyethylene 20 isohexadecyl ether, polyoxyethylene 20 oleyl ether, polyoxyethylene 40 stearate, polyoxyethylene 50 stearate, polyoxyethylene 8 stearate, polyoxyethylene bis (imidazolylcarbonyl), polyoxyethylene 25 propylene glycol stearate, saponin, Span® 20 (sorbitan lauric acid monoester), Span® 40 (sorbitan palmitate monoester) ), Span® 60 (sorbitan stearate monoester), Span® 65 (sorbitan stearate triester), Span® 80 (sorbitan oleic acid monoester), Span® 85 (Sorbitan oleic acid triester), Tergitol in any form (15-S-5, 15-S-7, 15-S-9, 15-S-12, 15-S-30, NP-4, NP -7, NP-9, NP-10, NP-40, NPX (Imbentin-N / 63), TMN-3 (polyethylene glycol trimethylnonyl ether), TMN-6 (polyethylene glycol trimethylnonyl ether), TMN-10 ( Polyethylene Glycol Trimethyl Nonyl Ether), MIN FOAM lx, and MIN FOAM2x etc.), Tetradecyl-β-D-maltoside, Tetraethylene glycol monodecyl ether, Tetraethylene glycol monododecyl ether, Tetraethylene glycol monotetradecyl ether, Triethylene glycol monodecyl ether, Triethylene glycol monododecyl ether, Tri Polyethylene Glycol Monohexadecyl Ether, Triethylene Glycol Monooctyl Ether, Triethylene Glycol Monotetradecyl Ether, Triton® CF-21, Triton® CF-32, Triton® DF-12, Triton (Registered Trademarks) DF-16, Triton (Registered Trademarks) GR-5M, Triton (Registered Trademarks) N-101 (Polyoxyethylene Branched Nonylphenyl Ether), Triton (Registered Trademarks) QS-15, Triton (Registered Trademarks) QS -44, Triton® RW-75 (polyethylene glycol 260 mono (hexadecyl / octadecyl) ether and 1-octadecanol), Triton® X-100 (polyethylene glycol tert-octylphenyl ether), Triton ( Registered Trademarks) X-102, Triton® X-15, Triton® X-151, Triton® X-200, Triton® X-207, Triton® X- 114, Triton® X-165, Triton® X-305, Triton® X-405 (polyoxyethylene (40) isooctylphenyl ether), Triton® X-405 reduction (Polyoxyethylene (40) isooctylcyclohexyl ether), Triton® X-45 (polyethylene glycol 4-tert-octylphenyl ether), Triton® X-705-70, any form of TWEEN ( Registered Trademark) (TWEEN® 20 (Polyoxyethylene sorbitan lauric acid monoester,That is, polysorbate 20), TWEEN (registered trademark) 21 (polyoxyethylene sorbitan lauric acid monoester), TWEEN (registered trademark) 40 (polyoxyethylene (20) sorbitan palmitate monoester) TWEEN (registered trademark) 60 (polyethylene glycol). Sorbitane stearate monoester), TWEEN® 61 (polyethylene glycol sorbitan stearate monoester), TWEEN® 65 (polyoxyethylene sorbitan stearate triester), TWEEN® 80 (polyoxyethylene) Sorbitane oleic acid monoester), TWEEN® 81 (polyoxyethylene sorbitan oleic acid monoester) and TWEEN® 85 (polyoxyethylene (20) sorbitan oleic acid triester), etc.), Tyloxapol (with formaldehyde) 4- (1,1,3,3-tetramethylbutyl) phenol polymer with oxylan) and n-undecyl β-D-glucopyranoside.3-Tetramethylbutyl) phenol polymer), and n-undecylic β-D-glucopyranoside.3-Tetramethylbutyl) phenol polymer), and n-undecylic β-D-glucopyranoside.

Specific anionic surfactants include kenodeoxycholic acid, sulfonic acid, dehydrocholic acid, deoxycholic acid, digitonine, digitoxygenin, N, N-dimethyldodecylamine N-oxide, docusate sodium, sodium glycokenodeoxycholate, glyco. Sulfonic Acid, Glycodeoxysulfonic Acid, Glycolithocolic Acid 3-Sodium Sulfonic Acid, Ethyl Glycolitholic Sulfonic Acid, N-Lauroyl Sulfonic Acid, Lithium Dodecyl Sulfonate, Lugol (Potato Iodoiodate), Niaproof (Sodium 2-Ethylhexyl Sulfonate) , Niaproof4 (7-ethyl-2-methyl-4-undecylsulfonic acid sodium salt), optionally substituted alkyl sulfonate (1-butane sulfonic acid salt, pentan sulfonic acid salt, hexane sulfonic acid salt, 1 -Octane sulfonic acid salt, 1-decane sulfonic acid salt, 1-dodecane sulfonic acid salt, 1-heptane sulfonic acid salt, 1-heptane sulfonic acid salt, 1-nonane sulfonic acid salt, 1-propane Sulfonic acid salts, and 2-bromoethanesulfonic acid salts, especially sodium salts), sodium cholate, sodium deoxycholate, optionally substituted sodium dodecyl sulphate, sodium octyl sulphate, sodium taurocholate, taurokenodeoxycholic Examples thereof include sodium acid, sodium taurohiodeoxycholic acid, disodium 3-sulfate taurolithocorate, sodium tauroursodeoxycholic acid, Trizma® dodecylsulfate, and ursodeoxycholic acid. Anionic surfactants may be provided in the form of acids and / or salts.

Specific cationic surfactants include alkyltrimethylammonium bromide, benzalkonium chloride, benzyldimethylhexadecylammonium chloride, benzyldimethyltetradecylammonium chloride, benzyldodecyldimethylammonium bromide, and benzyltrimethylammonium tetrachloroiodate. , Dimethyldioctadecylammonium bromide, dodecylethyldimethylammonium bromide, dodecyltrimethylammonium bromide, ethylhexadecyldimethylammonium bromide, girard reagent T, hexadecyltrimethylammonium bromide, N, N', N'-polyoxy Examples include ethyleneon (10) -N-taro-1,3-diaminopropane, tonzonium bromide, and trimethyl (tetradecyl) ammonium bromide.

Specific amphoteric surfactants include CHAPS (3-{(3-cholamidepropyl) dimethylammonio} -I-propane) and CHAPSO (3-{(3-cholamidepropyl) sulfonic acid). Dimethylammonio} -2-Hydroxy-1-propane), sulfonic acid 3- (decyldimethylammonio) propane, sulfonic acid 3- (dodecyldimethylammonio) propane, sulfonic acid 3- (N, N-dimethylmyristyl ammoni) E) Propyl, 3- (N, N-dimethyloctadecyl ammonio) sulfonic acid, 3- (N, N-dimethyloctyl ammonio) sulfonic acid, and 3- (N, N-dimethylpalmityl ammoni) sulfonic acid E) Propyl can be mentioned.

Surfactants include those known or those found in the art. Surfactants can be synthesized or purchased from any variety of sellers (eg, Sigma-Aldrich, St. Louis, MO, USA, www.sigmaaldrich.com). Certain surfactants are known to reduce contaminating artifacts from samples treated with test surfactants to samples treated without surfactant treatment for their ability to reduce artifacts. It may be investigated by evaluating against surfactants or other positive controls. A given surfactant is visually inspected for its ability to reduce the precipitation of components in the formulation after allowing the solution containing the surfactant to stand against the detergent-free solution for at least 2 weeks. You may check by doing.

The fixative may contain agents for minimizing the non-specific binding of the dye to the cellular components, such as ethylene glycol, propylene glycol and / or polyethylene glycol. In some embodiments, the fixative is approximately 0.5% by volume in the case of a liquid non-specific binding minimizing agent or by weight% in the case of a solid non-specific binding minimizing agent (eg,). From about 0.75%, about 1%, about 2%, about 3%, or about 4% (for example, about 4%, about 3%, about 2%, about 1%, or about 0.75%) May include non-specific binding minimizing agents. For example, fixatives are approximately 5 mL / L (eg, approximately 10 mL / L, approximately 15 mL / L, approximately 20 mL / L, approximately 25 mL / L, approximately 30 mL / L, approximately 35 mL / L, approximately 40 mL / L, or approximately. From 45 mL / L) to about 50 mL / L (eg, about 45 mL / L, about 40 mL / L, about 35 mL / L, about 30 mL / L, about 25 mL / L, about 20 mL / L, about 15 mL / L, or about It may contain 10 mL / L) of ethylene glycol. In some embodiments, the fixative comprises about 10 mL / L (eg, about 15 mL / L, about 20 mL / L, or about 25 mL / L) of ethylene glycol.

The fixative may contain a buffer. Examples of buffers include HEPES buffers (eg, HEPES sodium salts and / or HEPES free acids), MES, bis-tris, and other organic buffers. Fixatives range from approximately 0.5 mM (eg, approximately 1 mM, approximately 3 mM, approximately 5 mM, approximately 7 mM, or approximately 9 mM) to approximately 10 mM (eg, approximately 9 mM, approximately 7 mM, approximately 5 mM, approximately 3 mM, or approximately 1 mM). May include buffer. For example, the fixative may contain approximately 1.5 mM (eg, approximately 2 mM or approximately 1 mM) HEPES sodium salt. The fixative contains approximately 0.1 g / L to approximately 10 g / L of HEPES (eg, approximately 0.5 g / L to approximately 10 g / L, approximately 0.25 g / L to approximately 0.38 g / L, approximately 0.32 g / L). obtain.

The fixed solution has a pH of about 6 to about 8 (eg, about 6.3 to about 7.7, about 6.5 to about 7.5, about 6.7 to) when diluted in water at a ratio of about 1:10 to the fixed solution to water. It can have a pH of about 7.3, a pH of about 6.8 to about 7.2, a pH of about 6.9 to about 7.1, or a pH of about 7.0). In some embodiments, the fixative is about 0.1 to 1 (eg, about 0.1) at a peak wavelength of about 640 to about 650 nm (eg, about 646 to about 648 nm), with a dilution of fixative to water 1: 1000. Has an absorbance of ~ 0.8, about 0.1 ~ 0.7, about 0.1 ~ 0.5, about 0.1 ~ 0.4, about 0.1 ~ 0.3, about 0.15 ~ 0.3, about 0.15 ~ 0.2, about 0.185 ~ 0.205, about 0.19, or about 0.2) obtain.

As an example, to make a fixative, an organic solvent such as methanol can first be added to the mixing vessel to less than 100% (eg, approximately 90%) of the final desired volume. Calculated amounts of non-specific binding minimizers (eg ethylene glycol), surfactants (eg polysorbate 20), cytological dyes (eg Azure B) and buffers (eg HEPES sodium salt) Can be added to methanol. In addition, methanol can be added to bring the solution to its final desired volume. The mixture can be mixed on a magnetic stir plate / stir bar and / or impeller for a minimum of about 30 minutes. After mixing, a fixative diluted 1:10 with distilled water is prepared and pH measurements can be made using a pH meter (eg, a METTLER pH meter). In some embodiments, if the pH is not within the desired range, then a further buffer is added to the undiluted fixed solution until a 1:10 dilution of the fixed solution into distilled water reaches the desired pH. Can be added.

The absorbance of the fixation solution can be measured using a UV-spectrophotometer (for example, Hitachi UV-spectrophotometer). First the baseline can be run to the spectrophotometer. For example, a 10 mL fixed solution sample can be filtered through a 0.45 μm syringe filter, the solution can be diluted 1: 1000 with distilled water and spectrophotometered between approximately 500 and approximately 700 nm. Absorbance at about 640 to about 650 nm can be recorded. A typical UV-visible absorption spectrum of a fixative containing Azure B is shown, for example, in FIG. If desired, additional cytological dyes can be added to the fixation solution to bring the absorbance to the desired range. When additional cytological dyes are added to the fixation solution, the solution is mixed for a minimum of about 30 minutes and the measurement process (eg, filter 10 mL aliquots and dilute the aliquots with distilled water at a ratio of 1: 1000). , And the absorbance is measured) is repeated. The pH of the solution can also be measured again and adjusted (if necessary) by the method described above.

Finally, the fixative may be filtered through a 0.45 μm filter to remove any particulate matter prior to bottling. In some embodiments, a fine filter or a coarse filter may be used. For example, a 0.1-1 μm filter (eg, a 0.2 μm filter, a 0.4 μm filter, or a 0.8 μm filter) can be used to remove any microorganisms and / or particulate matter in the fixation solution. In some embodiments, the immobilizing agent can be stored in a 500 mL bottle and, as measured by a scale, can be filled up to 396 g ± 1 g. The pH of the final product can be measured as needed. In some embodiments, an HPLC chromatogram of fixative can be obtained, for example, to assess solution purity.

In some embodiments, the fixative is soluble in methanol, compatible with Azure B, and / or the pH can be adjusted to about 7.8, so HEPES buffer (eg, HEPES sodium salt and / or HEPES free acid). )including. Azure B can preferentially stain specific cellular components (eg, cell nuclei, basophils, cytoplasm, granulocytes, etc.) to improve contrast. The polysorbate 20 can improve the applicability of the fixate onto a substrate (eg, a microscope slide) and / or can prepare the nozzle for subsequent delivery of the aqueous solution. Ethylene glycol is non-specific of dye to cellular components (eg, chromatin to provide nucleoli stained to a lighter color compared to the nucleus) to provide good contrast in stained specimens. Bonding can be reduced.

For example, the cell fixation solution is about 0.5 g / L to about 5.0 g / L of Azure B; about 0.5 mL / L to about 2.0 mL / L of polysolvate 20; about 5 mL / L to about 50 mL / L of ethylene glycol, It may contain propylene glycol or polypropylene glycol; about 0.1 g / L to about 10 g / L of HEPES sodium salt; and methanol.

As another example, the cell fixation solution is about 0.8 g / L to about 1.2 g / L of Azure B; about 0.8 mL / L to about 1.2 mL / L of polysorbate 20; about 9 mL / L to about 11 mL / L. It may contain ethylene glycol; about 0.25 g / L to about 0.38 g / L of HEPES sodium salt; and methanol.

For example, the fixative may contain about 1 g / L of Azure B; about 1 mL / L of polysorbate 20; about 10 mL / L of ethylene glycol; about 0.32 g / L of HEPES sodium salt; and methanol.

As another example, the cell fixation solution may contain about 1 g / L of Azure B; about 0.5 mL / L of polysorbate 20; about 10 mL / L of propylene glycol; about 0.32 g / L of HEPES sodium salt; and methanol. ..

As another example, the fixative may contain about 1 g / L of Azure B; about 1.5 mL / L of polysorbate 20; about 10 mL / L of polypropylene glycol; about 0.32 g / L of HEPES sodium salt; and methanol. ..

1st and 2nd stains Usually, the first stain can be an aqueous solution. Examples of the solvent include distilled water and deionized water. The first stain contains a cytological dye. The first stain may provide a red stain. Dyes can improve the staining of specimens, such as eosin Y and fluorescein derivatives. In some embodiments, eosin Y can stain the cytoplasm and nucleus of cells within a specimen prepared, for example, from a blood sample. In some embodiments, eosinophils and neutrophils are preferentially stained with eosin Y. The dye can have a purity of about 80% or higher (eg, a purity of about 85% or higher, a purity of about 90% or higher, a purity of about 95% or higher, or a purity of about 100%). A typical HPLC chromatogram of eosin Y is shown, for example, in FIG. Dyes range from about 0.5 g / L (eg, about 0.75 g / L, about 1 g / L, about 2 g / L, about 3 g / L, or about 4 g / L) to about 5 g / L in the first dye solution. It can have a concentration of L (eg, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, or about 0.75 g / L). For example, the first stain may contain approximately 0.75 g / L (eg, approximately 1 g / L, approximately 2 g / L, approximately 3 g / L, approximately 4 g / L, or 5 g / L) of eosin Y.

Usually, the second stain can be an aqueous solution. Examples of the solvent include distilled water and deionized water. The second stain can contain more than one cytological dye. The second stain may provide a blue dye. The dye can improve the staining of the specimen and may include, for example, at least two of Azure B, methylene blue, and / or other thiazine dyes. In some embodiments, Azure B can preferentially stain cell nuclei, while methylene blue can preferentially stain cytoplasm in specimens prepared from, for example, blood samples, nuclei. Can be dyed slightly. Each dye can independently have a purity of about 80% or higher (eg, a purity of about 85% or higher, a purity of about 90% or higher, a purity of about 95% or higher, or a purity of about 100%). Each dye independently in the second stain is approximately 0.25 g / L (eg, approximately 0.3 g / L, approximately 0.4 g / L, approximately 0.45 g / L, approximately 0.5 g / L, approximately 0.75 g). From / L, about 1g / L, about 1.5g / L, or about 2g / L) to about 2.5g / L (for example, about 2g / L, about 1.5g / L, about 1g / L, about 0.75g / L) It can have concentrations of L, 0.5 g / L, about 0.45 g / L, about 0.4 g / L, or about 0.3 g / L). For example, the second stain may contain approximately 0.5 g / L of Azure B and approximately 0.45 g / L of methylene blue hydrate. In some embodiments, the second stain is assessed by the maximum HPLC peak area of each dye if the absorbance is monitored at wavelengths corresponding to the maximum absorbance of each dye (eg, about 630 nm and about 660 nm). If so, it may contain two dyes in a ratio of about 1: 4 to about 4: 1 (eg, about 3: 1, about 2: 1, about 1: 1, about 1: 2, or about 1: 3). .. A typical HPLC chromatograph of the second stain containing Azure B to methylene blue at a ratio of approximately 1: 1 is shown, for example, in FIG.

The first and second stains may each independently contain a surfactant. Although not bound by any particular theory, it is believed that surfactants can reduce the surface tension of the solvent and provide good coatability of the solution onto the sample substrate. In some embodiments, the surfactant is nonionic, minimizing the possibility of precipitation from solution that can result from ionic interactions with components in the formulation, such as ionically charged dyes. be able to. A solution containing little or no sediment can more easily be expelled from the distribution nozzle, reducing the possibility of clogging the distribution nozzle and diminishing the flow of liquid. In some embodiments, the surfactant can reduce the likelihood of non-specific binding or minimize the artifacts that can result from non-specific binding. A solution with little or no precipitation can also minimize the artifacts that may be present in the sample.

The first and second stains are independently about 0.05% by volume in the case of liquid surfactants or by weight% in the case of solid surfactants (eg, about 0.075%, about 0.1). Contains about 0.5% (eg, about 0.4%, about 0.3%, about 0.2%, about 0.1%, or about 0.075%) surfactant from%, about 0.2%, about 0.3%, or about 0.4%) obtain. In some embodiments, the first and second stains may each independently contain from about 0.5 to about 2 mL / L of surfactant (eg, nonionic surfactant). For example, the first and second stains may each independently contain approximately 1 mL / L of polysorbate 20 (eg, Tween 20).

The surfactant may be nonionic, cationic, anionic or zwitterionic. A mixture of surfactants can also be used. Specific classifications of surfactants include alcohol ether sulfates, alcohol sulfates, alkanolamides, alkylsulfonates, amine oxides, amphoteric surfactants, anionic surfactants, betaine derivatives, cationic surfactants, and di. Sulfonate, dodecylbenzene, sulfonic acid, ethoxylated alcohol, alkylphenol ethoxylated, ethoxylated fatty acid, glycerol ester hydrotropy, lauryl sulfate, mono and diglyceride, nonionic surfactant, phosphate ester, quaternary surfactant Agents and sorbitan derivatives include. Specific surfactants have already been described in the "Fixing Solution" section.

The first and second stains may each independently contain agents for minimizing non-specific binding of the dye to cellular components, such as ethylene glycol, propylene glycol, polyethylene glycol. In some embodiments, the first and second stains are independent, by volume% in the case of a liquid non-specific binding minimizing agent, or in the case of a solid non-specific binding minimizing agent. Is about 0.5% by weight (for example, about 0.75%, about 1%, about 2%, about 3%, or about 4%) to about 5% (for example, about 4%, about 3%, about 2). %, Approximately 1%, or approximately 0.75%) of non-specific binding minimizing agents may be included. For example, the first and second stains are independently about 5 mL / L (eg, about 10 mL / L, about 15 mL / L, about 20 mL / L, about 25 mL / L, about 30 mL / L, about 30 mL / L, respectively. From 35 mL / L, approximately 40 mL / L, or approximately 45 mL / L, to approximately 50 mL / L (eg, approximately 45 mL / L, approximately 40 mL / L, approximately 35 mL / L, approximately 30 mL / L, approximately 25 mL / L, approximately 25 mL / L) It may contain 20 mL / L, approximately 15 mL / L, or approximately 10 mL / L) of ethylene glycol. In some embodiments, the first and second stains each independently contain about 10 mL / L (eg, about 15 mL / L, about 20 mL / L, or about 25 mL / L) of ethylene glycol. ..

The first and second stains may each independently contain a buffer. Examples of buffers include Bis-Tris buffer, Phosphate buffer, HEPES, MES, Tris, and other organic buffers having a pH between about 5 and about 8. The first and second stains are independently from about 5 mM (eg, about 25 mM, about 50 mM, about 100 mM, about 150 mM, or about 200 mM) to about 250 mM (eg, about 200 mM, about 150 mM, about 150 mM). It may contain a buffer of 100 mM, about 50 mM, or about 25 mM). For example, the first and second stains can each independently contain approximately 50 mM bis-tris. In some embodiments, the buffer in the second stain is the same as the buffer in the first stain to increase the compatibility between the stains.

The first and second stains may independently contain salts such as sodium chloride, potassium chloride, sodium acetate, calcium chloride, magnesium chloride, sodium sulfate, magnesium sulfate and ammonium sulfate. The first and second stains, respectively, range from approximately 1 g / L (eg, approximately 2 g / L, approximately 5 g / L, approximately 10 g / L, or approximately 15 g / L) to approximately 20 g / L (eg, approximately 2 g / L, approximately 5 g / L, or approximately 15 g / L). For example, it can contain salt concentrations of about 15 g / L, about 10 g / L, about 5 g / L, or about 2 g / L). For example, the first and second stains may each independently contain a salt such as about 4 g / L (about 3 g / L or about 2 g / L) of sodium chloride. For example, the second stain may contain approximately 2 g / L (eg, approximately 3 g / L, or approximately 4 g / L) salts such as sodium chloride. The salt can eliminate the non-specific binding of the cytological dye to the specimen. Although not desired to be constrained by any theory, non-specific charges between the dye and the specimen, such as salt anions and cations binding to charged species in the specimen- Charge interaction can be shielded.

The first and second stains may each independently contain an antibacterial agent. Antibacterial agents can inhibit the growth of microorganisms and extend the shelf life of the stain. Antibacterial agents include benzalkonium chloride, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, ProClin® (for example, ProClin300® )), Azido, merthiolate and / or antibiotics. In some embodiments, the antibacterial agent comprises 5-chloro 2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one. For example, the antibacterial agent is about 2.3% 5-chloro-2-methyl-4-isothiazolin-3-one and about 0.7% 2-methyl-4-isothiazolin-3-one, available from Sigma-Aldrich. It can be ProClin® 300, which can be contained in an inert solvent (eg, modified glycol and alkylcarboxylate). The first and second stains, respectively, independently range from approximately 0.2 ppm (eg, approximately 1 ppm, approximately 5 ppm, approximately 10 ppm, approximately 20 ppm, approximately 30 ppm, or approximately 40 ppm) to approximately 50 ppm (eg, approximately 40 ppm). Antibacterial agents may be included at concentrations of about 30 ppm, about 20 ppm, about 10 ppm, about 5 ppm, or about 1 ppm). In some embodiments, the first and second stains may each independently contain approximately 15 ppm (approximately 10 ppm, approximately 5 ppm, or approximately 2 ppm) of ProClin 300®.

In some embodiments, the first and second stains may each independently contain an acid to further adjust the pH. The acid can be any acid traditionally used to regulate the pH of a solution. For example, acetic acid, nitric acid, hydrochloric acid, phosphoric acid, formic acid, sulfonic acid, or citric acid can be used.

The first and second stains independently have a pH of approximately 5 to approximately 8 (eg, approximately 5.5 to approximately 8, approximately 5.5 to approximately 7.5, approximately 5.5 to approximately 7, approximately 5.5 to approximately 6, approximately 5.5 to approximately 6). It can have a pH of 5.8 to about 6.2, about 5.9 to about 6.1, a pH of about 6, or a pH of about 7). In some embodiments, the first stain has a maximum of about 510 to about 530 nm (eg, about 510 to about 520 nm, or about 515 to about 517 nm) at a dilution of 1: 500 of the first stain versus water. At peak wavelength, about 0.1 to about 1 (for example, about 0.1 to about 0.8, about 0.1 to about 0.7, about 0.1 to about 0.5, about 0.1 to about 0.4, about 0.1 to about 0.3, about 0.15 to about 0.3, about 0.15 It can have an absorbance of ~ about 0.2, about 0.185 ~ about 0.205, about 0.19, or about 0.2). In some embodiments, the second stain is about 630 to about 660 nm (eg, about 640 to about 660 nm, about 640 to about 655 nm, about 645 to) at a dilution of 1: 1000 of the second stain to water. At peak wavelengths of about 655 nm, about 650 to about 655 nm, or about 650.5 to about 652.5 nm, about 0.1 to about 1 (eg, about 0.1 to about 0.8, about 0.1 to about 0.7, about 0.1 to about 0.5, about 0.1). It can have an absorbance of ~ about 0.4, about 0.1 ~ about 0.3, about 0.15 ~ about 0.3, about 0.15 ~ about 0.2, about 0.185 ~ 0.205, about 0.19, or about 0.2).

In some embodiments, distilled or deionized water is first placed in a mixing vessel to less than 100% (eg, approximately 90%) of the final desired volume to make a first or second stain. Add. Non-specific binding minimizers (eg ethylene glycol), surfactants (eg polysorbate 20), one or more cytological dyes (eg eosin Y, or Azure B and methylene blue), salts (eg NaCl) ), Antibacterial agents (eg, ProClin300®), acids (eg, acetic acid), and / or buffers (eg, bistris) can be added to the water in calculated amounts. In addition, water can be added to bring the solution to its final desired volume. The mixture can be mixed on a magnetic stir plate / stir bar and / or wing for a minimum of about 30 minutes. After mixing, pH measurements can be made on an aliquot of the first or second stain using a pH meter (eg, a METTLER pH meter). In some embodiments, if the pH is not within the desired range (eg, 5-8), then additional acid may be added to the first or second stain until the desired pH is obtained. it can.

The absorbance of the first staining solution can be measured using a UV-spectrophotometer (for example, Hitachi UV-spectrophotometer). First the baseline can be run to the spectrophotometer. For example, a sample of 10 mL of the first stain can be filtered through a 0.45 μm syringe filter, the solution can be diluted 1: 500 with distilled water and then spectrophotometered between 500 and 700 nm. it can. Absorbance at 510-530 nm can be recorded. A typical ultraviolet-visible absorption spectrum of the first stain containing eosin Y is shown, for example, in FIG. If necessary, additional cytological dyes can be added to the first stain to bring the absorbance to the desired range. If an additional cytological dye (eg, eosin) was added to the first stain, the solution was mixed for a minimum of about 30 minutes and the measurement process (eg, 10 mL aliquots were filtered and the aliquots were diluted with distilled water). Dilute at a ratio of 1: 500 and measure the absorbance) is repeated. The pH of the solution can also be measured again and adjusted to pH 5-8 by the method described above (if necessary).

The absorbance of the second staining solution can be measured using a UV-spectrophotometer (for example, Hitachi UV-spectrophotometer). First the baseline can be run to the spectrophotometer. For example, a 10 mL second stain sample can be filtered through a 0.45 μm syringe filter, the solution can be diluted 1: 1000 with distilled water and then spectrophotometered between 500 and 700 nm. it can. Absorbance at 630-660 nm can be recorded. A typical UV-visible absorption spectrum of the second stain containing 1: 1 methylene blue and Azure B is shown, for example, in FIG. If necessary, additional cytological dyes can be added to the second stain to bring the absorbance to the desired range. A portion of the filtered sample can be chromatographed using high performance liquid chromatography (HPLC) to record the area of the highest absorbance peak corresponding to each dye in the sample. The peak area can be approximately even for different dye components.

If the HPLC peak areas for the dyes in the second stain are not uniform, then additional cytological dyes (eg, Azure B and / or methylene blue) were used, and the solution evaluated each dye in approximately equal amounts (by peak area). As such) it can be added to the second stain until it can be included. After adding one or more dyes, the second stain is mixed for a minimum of about 30 minutes, the measurement process (eg, filter 10 mL aliquots, dilute the aliquots with distilled water at a ratio of 1: 500, and Absorbance measurement and HPLC measurement) can be repeated. The pH of the solution can also be measured again and adjusted to the desired range by the method described above (if necessary).

Finally, the first and second stains can be independently filtered through a 0.45 μm filter to remove any particulate matter prior to bottling. In some embodiments, a fine filter or a coarse filter may be used. For example, a 0.1-1 μm filter (eg, a 0.2 μm filter, a 0.4 μm filter, or a 0.8 μm filter) can be used to remove any microorganisms and / or particulate matter in the first or second stain. In some embodiments, the first or second stain can be stored independently in 250 mL bottles and can be filled up to 250 g ± 1 g as measured by a scale. The pH of the final product can be measured as needed. In some embodiments, an HPLC chromatogram of the first stain can be obtained, for example, to assess solution purity. In some embodiments, an HPLC chromatogram of the second stain can be obtained, for example, to assess solution purity and / or to obtain the proportion of dye in the solution.

In some embodiments, the first stain can, for example, stain specific cells of a specimen prepared from a blood sample (eg, basophils, neutrophils). The first stain can include a Bis-Tris buffer, the resulting stain can provide a reddish tint and a more visually pleasing stained specimen. Bis-Tris buffer can be compatible with eosin Y. In some embodiments, Azure B is included in more than one formulation, such as in a fixation solution and a second stain. It is believed that if a specimen is exposed to Azure B more than once, better staining of the specimen may occur compared to a single exposure to Azure B.

The first and second stains can independently contain NaCl and ethylene glycol, which together can provide a synergistic shielding effect on non-specific binding. That is, formulations containing both NaCl and ethylene glycol may have fewer non-specific bonds than formulations containing the corresponding concentrations of either NaCl or ethylene glycol. For example, formulations containing both NaCl and ethylene glycol can color the nucleoli lighter than the remaining nuclei, while formulations without NaCl and ethylene glycol can give uniformly dark nuclei. In some embodiments, the NaCl-containing stain can reduce background staining as compared to the NaCl-free stain.

For example, the first cell stain is about 0.5 g / L to about 5.0 g / L of eosin Y; about 5 mM to about 250 mM bis-tris or phosphate buffer; about 0.5 mL / L to about 2.0 mL / L Polysorbate 20; Approximately 1 g / L to Approximately 20 g / L Sodium Chloride; Approximately 5 mL / L to Approximately 50 mL / L of Ethylene Glycol; Approximately 0.2 ppm to Approximately 50 ppm of ProClin 300®; Acetic Acid; The pH of this solution can be from about 5.8 to about 6.2.

As another example, the first cell stain is about 0.6 g / L to about 0.9 g / L of eosin Y; about 45 mM to about 55 mM Bis-Tris buffer; about 0.8 mL / L to about 1.2 mL / Polysorbate 20 of L; about 3 g / L to about 5 g / L of sodium chloride; about 9 mL / L to about 11 mL / L of ethylene glycol; about 10 ppm to about 20 ppm of ProClin 300®; acetic acid; and water. The pH of this solution is about 5.8 to about 6.2.

For example, the first cell stain is about 0.75 g / L of eosin Y; about 50 mM bis-tris buffer; about 1 mL / L of polysorbate 20; about 4 g / L of sodium chloride; about 10 mL / L of ethylene. Glycol; about 15 ppm ProClin 300®; acetic acid; and water can be included and the pH of this solution is about 5.8 to about 6.2.

As another example, the first cell stain is about 0.75 g / L eosin Y; about 50 mM bis-tris buffer; about 0.5 mL / L polysorbate 20; about 6 g / L sodium chloride; about 20 mL. It can contain / L ethylene glycol; about 15 ppm ProClin 300®; acetic acid; and water, and the pH of this solution is about 5.8 to about 6.2.

As another example, the first cell stain is about 0.75 g / L of eosin Y; about 50 mM phosphate buffer; about 2.0 mL / L of polysorbate 20; about 4 g / L of sodium chloride; about 50 mL / It can contain L ethylene glycol; about 15 ppm ProClin 300®; acetic acid; and water, and the pH of this solution is about 5.8 to about 6.2.

For example, the second cell stain is about 0.25 g / L to about 2.5 g / L of Azure B; about 0.25 g / L to about 0.5 g / L of methylene blue; about 5 mM to about 250 mM Bis-Tris or HEPES. Buffer solution; about 0.5 mL / L to about 2.0 mL / L of polysolvate 20; about 1 g / L to about 20 g / L of sodium chloride; about 0.2 ppm to about 50 ppm of ProClin 300®; acetic acid; and water The pH of this solution can be from about 6.8 to about 7.2.

As another example, the second cell stain is about 0.4 g / L to about 0.6 g / L of Azure B; about 0.4 g / L to about 0.5 g / L of methylene blue; about 45 mM to about 55 mM bis-. Tris buffer; about 0.8 mL / L to about 1.2 mL / L of polysorbate 20; about 1.8 g / L to about 2.2 g / L of sodium chloride; and about 10 ppm to about 20 ppm of ProClin 300®; acetic acid; and It can contain water and the pH of this solution is from about 6.8 to about 7.2.

For example, the second cell stain is about 0.5 g / L Azure B; about 0.45 g / L methylene blue; about 50 mM Bis-Tris buffer; about 1 mL / L polysorbate 20; about 2 g / L chloride. It can contain sodium; and about 15 ppm ProClin 300®; acetic acid; and water, and the pH of this solution is about 6.8 to about 7.2.

As another example, the second cell stain is about 0.5 g / L Azure B; about 0.45 g / L methylene blue; about 50 mM HEPES buffer; about 0.5 mL / L polysolvate 20; about 2 g / L. Sodium chloride; and about 15 ppm ProClin 300®; acetic acid; and water can be included, and the pH of this solution is about 6.8 to about 7.2.

As another example, the second cell stain is about 0.5 g / L of Azure B; about 0.45 g / L of methylene blue; about 50 mM HEPES buffer; about 1 mL / L of polysorbate 20; about 1 g / L. Sodium chloride; and about 15 ppm ProClin 300®; acetic acid; and water can be included, and the pH of this solution is about 6.8 to about 7.2.

Rinse liquid Usually, the rinse solution can be an aqueous solution. Examples of the solvent include distilled water and deionized water. The rinse solution may contain non-specific binding minimizing agents such as polyethylene glycol, polyvinylpyrrolidone, polyacrylic acid, polyvinyl alcohol, polysaccharides and / or other hydrophilic water-soluble polymers. In some embodiments, the non-specific bond minimizing agent may include ethylene glycol or propylene glycol. Although not bound by any theory, it is believed that non-specific binding minimizers can prepare the specimen for the drying process during specimen preparation. Non-specific binding minimizing agents can provide a lacquer on the surface of the specimen and improve the visual appearance of the specimen. Non-specific binding minimizers in the rinse solution are approximately 1 g / L (eg, approximately 2 g / L, approximately 3 g / L, approximately 4 g / L, approximately 5 g / L, approximately 6 g / L, approximately 7 g / L, From about 8g / L, or about 9g / L) to about 10g / L (eg, about 9g / L, about 8g / L, about 7g / L, about 6g / L, about 5g / L, about 4g / L, It can have a concentration of about 3 g / L, or about 2 g / L). For example, the rinse solution may contain approximately 5 g / L of polyethylene glycol (eg, polyethylene glycol 1450).

The rinse solution may contain a surfactant. Although not bound by any particular theory, it is believed that surfactants can reduce the surface tension of the solvent and provide good coatability of the solution onto the sample substrate. In some embodiments, the surfactant is nonionic, minimizing the possibility of precipitation of solution components that may result from ionic interactions with components in the formulation, such as ionically charged dyes. Can be done. A solution containing little or no precipitate can be more easily expelled from the distribution nozzle, reducing the possibility of clogging the distribution nozzle and diminishing the flow of liquid. In some embodiments, the surfactant can reduce the likelihood of non-specific binding or minimize the artifacts that can result from non-specific binding. A solution with little or no precipitation can minimize the artifacts that can be present in the specimen.

The rinse solution is approximately 0.05% by volume for liquid surfactants or% by weight for solid surfactants (eg, approximately 0.075%, approximately 0.1%, approximately 0.2%, approximately 0.3%, or It may contain from about 0.4%) to about 0.5% (eg, about 0.4%, about 0.3%, about 0.2%, about 0.1%, or about 0.075%) of surfactant. In some embodiments, the rinse solution may contain from about 0.5 to about 2 mL / L of surfactant (eg, nonionic surfactant). For example, the rinse solution may contain approximately 1 mL / L of polysorbate 20 (eg, Tween 20).

The surfactant may be nonionic, cationic, anionic or zwitterionic. A mixture of surfactants can also be used. Specific classifications of surfactants include alcohol ether sulfates, alcohol sulfates, alkanolamides, alkylsulfonates, amine oxides, amphoteric surfactants, anionic surfactants, betaine derivatives, cationic surfactants, and di. Sulfonate, dodecylbenzene, sulfonic acid, ethoxylated alcohol, alkylphenol ethoxylated, ethoxylated fatty acid, glycerol ester hydrotropy, lauryl sulfate, mono and diglyceride, nonionic surfactant, phosphate ester, quaternary surfactant Agents and sorbitan derivatives include. Specific surfactants have already been described in the "Fixing Solution" section.

The rinse solution may contain a buffer. Examples of buffers include HEPES buffer (eg, HEPES sodium salt and / or HEPES free acid), Bis-Tris buffer, phosphate buffer, MES, Tris and pH between about 5 and about 8. Other organic buffers that have. Rinse fluid buffers from approximately 5 mM (eg, approximately 25 mM, approximately 50 mM, approximately 100 mM, approximately 150 mM, or approximately 200 mM) to approximately 250 mM (eg, approximately 200 mM, approximately 150 mM, approximately 100 mM, approximately 50 mM, or approximately 25 mM). May include agents. For example, the rinse solution may contain approximately 50 mM HEPES.

The rinse solution may contain an antibacterial agent. Antibacterial agents can inhibit the growth of microorganisms and extend the shelf life of the rinse solution. Antibacterial agents include benzalkonium chloride, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, ProClin® (ProClin 300®). , Azido, merthiolate and / or antibiotics. In some embodiments, the antibacterial agent comprises 5-chloro 2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one. For example, the antibacterial agent is about 2.3% 5-chloro-2-methyl-4-isothiazolin-3-one and about 0.7% 2-methyl-4-isothiazolin-3-one, available from Sigma-Aldrich. It can be ProClin® 300 contained in an inert solvent (eg, modified glycol and alkylcarboxylate). Antibacterial agents range from approximately 0.2 ppm (eg, approximately 1 ppm, approximately 5 ppm, approximately 10 ppm, approximately 20 ppm, approximately 30 ppm, or approximately 40 ppm) to approximately 50 ppm (eg, approximately 40 ppm, approximately 30 ppm, approximately 20 ppm, approximately 10 ppm, approximately 5 ppm). , Or at a concentration of approximately 1 ppm). In some embodiments, the rinse solution may contain approximately 15 ppm (approximately 10 ppm, approximately 5 ppm, or approximately 2 ppm) of ProClin 300®.

In some embodiments, the rinse solution may further contain an acid to adjust the pH. The acid can be any acid traditionally used to regulate the pH of a solution. For example, acetic acid, nitric acid, hydrochloric acid, phosphoric acid, formic acid, sulfonic acid, or citric acid can be used.

The rinse solution may contain alcohols such as methanol, ethanol or propanol. Alcohol can remove excess dye and promote fast drying of the rinse solution on the specimen and / or substrate. In some embodiments, DMSO or other organic solvent in which the dye dissolves can be used in the rinsing solution, resulting in a complete vacuum evacuation of the liquid from the distribution nozzle. For example, rinse fluids can start from approximately 10 mL / L (eg, approximately 25 mL / L, approximately 50 mL / L, approximately 75 mL / L, approximately 100 mL / L, approximately 125 mL / L, approximately 150 mL / L, or approximately 175 mL / L). , About 200 mL / L (eg, about 175 mL / L, about 150 mL / L, about 125 mL / L, about 100 mL / L, about 75 mL / L, about 50 mL / L, or about 25 mL / L). In some embodiments, the rinse solution comprises approximately 50 mL / L of methanol. In some embodiments, the rinse solution can be up to approximately 200 mL / L of organic solvent so that the rinse solution cannot be washed from a biological sample stained with an excess amount of the desired dye.

The pH of the rinse solution is about 5 to about 8 (eg, about 5.5 to about 8, about 5.5 to about 7.5, about 5.5 to about 7, about 5.5 to about 6, about 5.8 to about 6.2, about 5.9 to about 6.1, It can be about 7 or about 6). For example, the pH of the rinse solution can be approximately 7.0.

To make the rinse solution, distilled or deionized water can be added to the mixing vessel to less than 100% (eg, approximately 90%) of the final desired volume. Alcohols (eg methanol), non-specific binding minimizers (eg PEG), buffers (eg HEPES sodium salts and HEPES free acids), surfactants (eg polysorbate 20), acids (if used) And a calculated amount of antibacterial agent (eg, ProClin 300®) can be added to the water. In addition, water can be added to bring the solution to its final desired volume. The mixture can be mixed on a magnetic stir plate / stir bar and / or wing for a minimum of about 30 minutes. After mixing, pH measurements can be made to aliquot the rinse solution using a pH meter (eg, Mettler pH meter). In some embodiments, if the pH is not within the desired range (eg, about 5 to about 8), then additional acid can be added to the rinse solution until the desired pH is obtained.

Finally, the rinse solution can be filtered through a 0.45 μm filter to remove any particulate matter prior to bottling. In some embodiments, a fine filter or a coarse filter may be used. For example, a 0.1-1 μm filter (eg, a 0.2 μm filter, a 0.4 μm filter, or a 0.8 μm filter) can be used to remove any microorganisms and / or particulate matter in the rinse solution. In some embodiments, the rinse solution can be stored in a 500 mL bottle and, as measured by a scale, can be filled up to 500 g ± 1 g. The pH of the final product can be measured as needed.

In some embodiments, the polyethylene glycol-containing rinse solution applied at the end of the stain application can improve the visual appearance of the sample as compared to a sample treated with a polyethylene glycol-free rinse solution. .. In some embodiments, a rinse solution containing HEPES buffer and approximately pH 6.8 improves the appearance of cells, such as red blood cells, by providing a good color balance between red and blue. Can be done.

In some embodiments, the components of the rinse solution may be present at lower (eg, one-fifth) concentrations than those described above.

The polyethylene glycol component of the rinse solution is approximately 0.2 g / L (eg, approximately 0.4 g / L, approximately 0.6 g / L, approximately 0.8 g / L, approximately 1 g / L, approximately 1.2 g / L, approximately 1.4 g / L). , Approximately 1.6 g / L, or approximately 1.8 g / L), and can have concentrations of approximately 2 g / L. For example, the rinse solution may contain approximately 1 g / L of polyethylene glycol (eg, polyethylene glycol 1450).

The rinse solution is approximately 0.01% by volume for liquid surfactants or% by weight for solid surfactants (eg, approximately 0.015%, approximately 0.02%, approximately 0.04%, approximately 0.06%, or From about 0.8%), it may contain about 0.1% surfactant. In some embodiments, the rinse solution may contain approximately 0.0501 to 0.306% of surfactant. In some embodiments, the rinse solution may contain from approximately 0.1 to approximately 0.4 mL / L of surfactant (eg, nonionic surfactant). For example, the rinse solution may contain approximately 0.2 mL / L of polysorbate 20 (eg, Tween 20).

The rinse solution may contain from approximately 1 mM (eg, approximately 5 mM, approximately 10 mM, approximately 20 mM, approximately 30 mM, or approximately 40 mM) to approximately 50 mM buffer. For example, the rinse solution may contain approximately 10 mM HEPES.

Antibacterial agents can be present in concentrations from approximately 0.04 ppm (eg, approximately 0.2 ppm, approximately 1 ppm, approximately 2 ppm, approximately 5 ppm, approximately 6 ppm, or approximately 8 ppm) to approximately 10 ppm. In some embodiments, the rinse solution comprises approximately 3 ppm (approximately 2 ppm, approximately 1 ppm, or approximately 0.4 ppm) of ProClin 300®.

Rinse fluids range from approximately 2 mL / L (eg, approximately 5 mL / L, approximately 10 mL / L, approximately 15 mL / L, approximately 20 mL / L, approximately 25 mL / L, approximately 30 mL / L, or approximately 35 mL / L) to approximately. May contain 40 mL / L alcohol. In some embodiments, the rinse solution contains approximately 10 mL / L of alcohol. In some embodiments, the rinse solution can be up to approximately 40 mL / L of organic solvent so that the rinse solution cannot be washed from a biological sample stained with an excess amount of the desired dye.

For example, the rinse solution is about 0.2 g / L to about 10 g / L of polyethylene glycol; about 1 mM to about 250 mM HEPES, MES or Bis-Tris buffer; about 0.1 mL / L to about 2.40 mL / L of polysorbate 20. It can contain from about 0.04 ppm to about 50 ppm ProClin 300®; about 9 mL / L to about 200 mL / L of methanol; and water, the pH of the rinse solution being about 6.6 to about 7.0.

For example, the rinse solution is about 1 g / L to about 10 g / L of polyethylene glycol; about 5 mM to about 250 mM HEPES, MES or Bis-Tris buffer; about 0.5 mL / L to about 2.0 mL / L of polysorbate 20; It can contain from about 0.2 ppm to about 50 ppm ProClin 300®; about 10 mL / L to about 200 mL / L of methanol; and water, the pH of the rinse solution being about 6.6 to about 7.0.

As another example, the rinse solution is about 4.5 g / L to about 5.5 g / L polyethylene glycol; about 45 mM to about 55 mM HEPES buffer; about 0.8 mL / L to about 1.2 mL / L polysorbate 20; about It can contain from 10 ppm to about 20 ppm ProClin 300®; about 45 mL / L to about 55 mL / L of methanol; and water, the pH of the rinsing solution being about 6.6 to about 7.0.

For example, the rinse solution should contain about 5 g / L polyethylene glycol; about 50 mM HEPES buffer; about 1 mL / L polysorbate 20; about 15 ppm ProClin 300®; about 50 mL / L methanol; and water. The pH of the rinse solution is about 6.6 to about 7.0.

As another example, the rinse solution is about 10 g / L polyethylene glycol; about 50 mM HEPES buffer; about 1 mL / L polysorbate 20; about 15 ppm ProClin 300®; about 50 mL / L methanol; and water. The pH of the rinse solution is about 6.6 to about 7.0.

As a further example, the rinse solution is about 10 g / L polyethylene glycol; about 50 mM bis-tris buffer; about 0.5 mL / L polysorbate 20; about 15 ppm ProClin 300®; about 50 mL / L methanol; And water can be included, and the pH of the rinse solution is about 6.6 to about 7.0.

For example, the rinse solution is about 0.2 g / L to about 2 g / L of polyethylene glycol; about 1 mM to about 50 mM HEPES buffer; about 0.16 mL / L to about 0.24 mL / L of polysorbate 20; about 0.04 ppm to about. It can contain 10 ppm ProClin 300®; about 9 mL / L to about 11 mL / L of methanol; and water, the pH of the rinse solution being about 6.6 to about 7.0.

As another example, the rinse solution is about 0.9 g / L to about 1.1 g / L polyethylene glycol; about 9 mM to about 11 mM HEPES buffer; about 0.16 mL / L to about 0.24 mL / L polysorbate 20; about It can contain 2 ppm to about 10 ppm of ProClin 300®; about 9 mL / L to about 11 mL / L of methanol; and water, the pH of the rinsing solution being about 6.6 to about 7.0.

As yet another example, the rinse solution is about 1 g / L polyethylene glycol; about 10 mM HEPES buffer; about 0.2 mL / L polysorbate 20; about 3 ppm ProClin 300®; about 10 mL / L methanol; And water can be included, and the pH of the rinse solution is about 6.6 to about 7.0.

Kit In some embodiments, the kit may include one or more bottles for each of the fixation solution, the first stain solution, the second stain solution and / or the rinse solution. The kit may include an instruction manual and a label. The label may include, for example, lot information and expiration date. FIG. 25 shows an example of a kit containing a fixation solution, first and second stains and a rinse solution.

In some embodiments, the treatment step is to apply a fixative, a fixative, a first stain, a second stain, a rinse. This includes applying a rinse solution.

Specimen preparation system and method The fixation, staining, and rinsing formulations described above are incorporated by device or machine for specimen preparation (eg, US Patent Application No. 13/293050, filed November 9, 2011, incorporated herein by reference in its entirety. Can be used to improve the visual appearance of certain features in the specimen). FIG. 7 illustrates an embodiment of an apparatus or mechanism 1 for preparing a specimen on a substrate 2 such as a microscope slide, coverslip or other transparent surface for inspection or imaging. Mechanism 1 can be incorporated into a comprehensive system for preparing and analyzing specimens containing cell-containing bodily fluids or other biological samples, such as System 2000, shown in FIG. 21 and described below. Mechanism 1 is usually the first step of obtaining the sample, the second step of applying the sample to the substrate, the third and fourth steps of fixing and staining the sample, respectively, and the fifth step of drying the sample. , A system comprising, a sixth step of taking a specimen, and a seventh step of analyzing images and data obtained from the specimen can be included or part of it. Some embodiments of Mechanism 1 are compatible with System 2000; some embodiments of Mechanism 1 may be used in other sample preparation systems and / or as stand-alone devices.

Generally, mechanism 1 may provide one or more (eg, 2, 3, 4, 5, or more than 5) platforms 60A and 60B for sampling as shown in FIGS. 7-9. As shown in FIG. 8, platform 60A may include sides to support the top surface of the platform. The shield 100 shown in FIGS. 7 and 9 may be placed between platforms 60A and 60B to prevent liquid from splashing between platforms 60. In some embodiments, the shield 100 can be formed from a transparent material that prevents liquids from one of the platforms 60A and 60B from entering the other platform. In certain embodiments, the shield 100 can be formed from a translucent or opaque material. In FIGS. 7 and 9, the shield 100 is depicted as being made of a transparent material so that other components located behind the shield 100 can be shown in the same figure. The shield 100 can also be shown to be made of opaque material, in which case some of the components such as platform 60A and block 80A will not be well visible.

FIG. 9B shows an indexing mechanism 50A that can be used to move mechanism 1 to provide substrate 2 to a position for sample processing from each of the substrate grips 20A, 20B. The indexing mechanism 50A can be of many types, such as electromechanical devices (eg, rack and pinion gears with electric motors), linear actuators (eg, pneumatic actuators, hydraulic actuators, or electromagnetic actuators). In the embodiments described, the indexing mechanism 50A linearly moves mechanism 1 between two positions, but other movement paths are also circular or semi-circular, depending on the number of platforms included in mechanism 1. This is possible due to their configuration and layout, such as (for example, an indexing table that can move in a bow-shaped path). As shown, the indexing mechanism 50A may include a gear rack 50B attached to the base 50C of mechanism 1 and a pinion gear 50D attached to an electric motor 50E fixed to the base 50C. The mechanism 1 can be attached to the base 50C using one or more slide devices 50F so that the mechanism 1 can move smoothly as it is moved by the indexing mechanism 50A. During use, the indexing mechanism 50A deposits on the substrate so that the plurality of substrate grips 20A and / or 20B of mechanism 1 can receive the substrate 2 from the substrate moving means 120 (shown in FIG. 11). A mechanism so that the sample can be prepared by the mechanism 1, and once prepared, the substrate gripper 20A and / or 20B can provide the substrate 2 with the prepared sample to the substrate moving means 120 for sample processing. 1 can be moved.

Due to the mechanism having the two platforms 60A and 60B, the substrates 2 are typically provided alternately to and from the substrate moving means 120, as in the embodiments described. In some embodiments, the first substrate 2 is provided from the substrate moving means 120 to the first substrate grip 20A for processing on the first platform 60A, while the mechanism 1 is in the initial position. is there. While the first substrate 2 is being processed by the first platform 60A, the indexing mechanism 50A is provided with the substrate moving means 120 to the second so that the second substrate gripping portion 20B is processed by the second platform 60B. Mechanism 1 can be moved to a second position so that it can receive the substrate of. While the second substrate is being processed on the second platform 60B, the indexing mechanism 50A allows the substrate moving means 120 to remove the first substrate 2 from the first substrate grip 20A. Can be moved back to its initial position. Once the substrate 2 has been removed from the first gripping platform 20A, the next substrate may be provided to the first gripping platform 20A. This method for alternately providing substrates to the gripping platform can be performed on more than two (eg, more than 3, 4, 5 or 5) platforms, thereby preparing specimens for further evaluation. The processing capacity can be increased.

Platforms 60A and 60B are typically formed from one or more materials that are relatively chemically inert to the liquid used during sampling and provide adequate surface tension. .. As specific materials that can be used to form the platforms 60A and 60B, they uniformly distribute, confine, and top the treatment solution in the space between substrate 2 and the platform. Polyoxymethylene (eg, DuPont Delrin), provided, manufactured and / or processed to provide the appropriate surface tension that acts to help ensure proper vacuum exhaust. (Registered Trademarks)), high molecular weight fluorocarbons such as polytetrafluoroethylene (PTFE) (eg, DuPont Teflon®), and metals such as aluminum, steel and titanium. .. With proper material selection, the platform can also advantageously reduce or eliminate the formation of bubbles or spaces in the liquid when distributed, while at the same time the liquid out of the separation between the platform and substrate 2. Sufficient surface tension can also be maintained to reduce or reduce leakage.

Generally, the surface areas of platforms 60A and 60B can be selected as desired for substrate handling and liquid delivery. Factors such as the surface area of platforms 60A and 60B can also affect the selected surface area of substrate 2. For example, in some embodiments, the surface area of platform 60A (eg, the area of the surface of platform 60A in contact with substrate 2) is slightly smaller than the area of the surface of substrate 2 in contact with platform 60A. By maintaining such a relationship between the contact area of platform 60A and substrate 2, liquid leakage from the area between the surfaces can be reduced or eliminated. Typically, for example, the surface area of platform 60A in contact with substrate 2 is 2% or more (eg, 3% or more, 5% or more, 7% or more, 10% or more, 15) or more than the surface area of substrate 2. % Or more, 20% or more, 25% or more, 30% or more) smaller.

Platforms 60A and 60B can be attached to blocks 80A and 80B, respectively. Block 80A includes sides 81A-84A that support the top surface 85A, as shown in FIG. Blocks 80A and 80B can be made of the same or similar materials used for platforms, such as metals, ceramics, and / or plastics. Therefore, materials such as Delrin® can be used to form blocks 80A and 80B, especially in embodiments where Romanosufky staining of specimens is performed. Other materials that can be used in embodiments include metals and Teflon® brand polytetrafluoroethylene coated aluminum, steel or titanium.

In some embodiments, platforms 60A and / or 60B can be lifted as shown in Figures 7-9. Alternatively, in certain embodiments, platforms 60A and / or 60B can be integrated with the top surfaces of blocks 80A and 80B, respectively. In each case, the particular features of Mechanism 1 as well as the surface tension of the liquid and the surface energy of the platform or block allow excess liquid to flow across the edges of the platform 60A / 60B and / or block 80A / 80B. prevent.

As shown in FIGS. 7 and 8, platform 60A may include offsets 70A-70D to provide separation between platform 60A and substrate 2 and prevent substrate 2 from contacting platform 60A. Platform 60B may include the corresponding offset sets 71A-71D. Offsets include standoffs, pins, nails, rods, beads, walls or other structures that can provide separation between the surfaces of platforms 60A and / or 60B and substrate 2. Offsets 70A-70D and 71A-71D ensure that the surfaces of platforms 60A and 60B and substrate 2 remain substantially parallel when substrate 2 is in contact with the offset. The benefit of keeping these two surfaces parallel is that the volume enclosed between these two surfaces is defined by it and can be precisely controlled. If the two surfaces are not substantially parallel and the angle between them changes, the volume between them also changes, is not fixed and is not precisely controlled. In addition, if such two surfaces are not substantially parallel, the liquid may not be uniformly applicable to the specimen.

As used herein, the phrase "substantially parallel" means that when substrate 2 comes into contact with an offset, the two surfaces can reduce or eliminate imperfections in the surface flatness of substrate 2. It means that they are exactly parallel or almost parallel. For example, even with the utmost care in the manufacture of substrates, some substrates may have defects such as distortion and / or corners not being coplanar. In the systems and methods disclosed herein, the use of offsets improves the surface flatness of substrate 2 if necessary, placing substrate 2 in a substantially parallel relationship to platforms 60A and 60B in the process. Positioning helps correct these shortcomings. The phrase "substantially parallel" is a situation in which the two surfaces are not perfectly flat, but at least the offsets on the substrate surface are coplanar, but the offsets are all the same size or height. Include.

FIG. 12A shows substrate 2, substrate grip 20B, blocks 80A, 80B, platforms 60A, 60B, offsets 70A-70D and 71A-71D, and separation 92 between substrate 2 and platform 60B. Separation 92 moves the liquid between the surface of platform 60B, including ports 40B-45B, and substrate 2. The separation distance required for optimal specimen fixation, staining, and rinsing applies to the flow rate of the liquid distributed from ports 40B-45B (and / or ports 40A-45A), the diameter of the port, and during processing. It depends on the viscosity of the liquid and the amount of suction that can be used to remove the liquid from the substrate, separation and platform.

In some embodiments, for example, an offset that provides a separation 92 of about 100-200 microns between the surface of platform 60B and substrate 2 is from ports 40B-45B to 1 with diameters in the range 500-1500 microns. In embodiments where the liquid can be dispensed at a flow rate in the range of about 50 to about 300 μL per second, it allows for fixation, staining and rinsing of specimens containing red blood cells. Typically, the size or height of the separation 92 is relative to a particular embodiment as long as such embodiments can overcome the surface tension of the liquid in the separation while partitioning and removing the liquid during sample processing. It can vary from about 50 microns to 1000 microns (eg, about 50 to 500 microns, about 75 to 250 microns, about 100 to 200 microns). Moreover, in certain embodiments, the diameter of the port located on platforms 60A and / or 60B can vary from about 125 microns to 5000 microns.

Figures 12B and 6C show a ball joint mechanism 25 that can be used to align the substrate grip 20A parallel to the platform 60A. The ball joint mechanism 25 is fixed to the ball member 25A, the flexible member 25B (for example, a spring), the lower socket 25C, the upper socket 25D, and the lower socket 25C that are firmly connected to the board arm 10A. Can include cap 25E, and set screw 25F, which are (eg, with fasteners). In some embodiments, during the manufacture and / or assembly of mechanism 1 and substrate grip 20A and / or 20B, the ball joint mechanism 25 may be present due to cross-accumulation or molding problems. Can be adjusted to supplement. In some embodiments, the set screw 25F is loosened and the substrate arm 10A is moved to the closed position in order to adjust the ball joint mechanism 25. Since the set screw 25F is loosened, the substrate grip 20A can be placed substantially parallel to the platform 60A while gripping the substrate 2 while the substrate 2 is placed along the contact offset 70. Alternatively, in some embodiments, the number of offsets on platform 60 has been reduced or completely eliminated, and shims with a thickness corresponding to the desired separation distance have separated 92 at the desired distance for sample processing. Can be used temporarily during the setup or calibration of mechanism 1 in conjunction with the ball joint mechanism 25 to configure. The ball joint mechanism 25 is loosened, but the deflection member 25B allows the board grip portion 20A, which is semi-fixed to the board arm 10A, to move independently, but not too loose, and other configurations of mechanism 1. Use force to maintain it so that it does not move freely enough to interfere with or cause damage to the element. When the substrate 2 is pressed and fixed in the closed position, the substrate 2 is substantially parallel to the platform 60A, so that the set screw 25F can be tightened and the ball joint mechanism 25 can be securely fixed. Tighten as shown Then, the set screw 25F applies a downward force on the upper socket 25D, thereby applying a frictional force on the upper part of the ball member 25A via the upper socket 25D. Since the lower socket 25C is fixed to the cap 25E, the force generated by the set screw 25F is applied by the lower socket 25C to the bottom side of the ball member 25A to restrain the ball member 25A in the upper and lower sockets 25C and 25D. Also lift the lower socket 25C to do so. Once restrained by the ball member 25A, the substrate grip portion 20A is fixed to the substrate arm 10A.

Typically, once the board grip 20A is placed and constrained by the set screw 25F, the ball joint mechanism 25 does not need to be readjusted during normal use. However, if the board grip 20A is not aligned correctly and the ball joint mechanism 25 requires adjustment (eg, due to damage, mechanical repair, poor performance, or other reasons), the set screw 25F can be loosened, the substrate grip 20A can be moved to a closed position, the substrate gripped by the substrate grip 20A can be moved to a position substantially parallel to the platform 60A, and then the set screw 25F is tightened. The ball joint mechanism 25 can be securely fixed.

Typically, actuators 30A and / or 30B are configured to adjust the position of board arms 10A and / or 10B to vary the degree of separation between the surface of platforms 60A and / or 60B and substrate 2. Can be done. This change in separation provides great flexibility in embodiments that allow adjustment of the liquid, flow rate, liquid viscosity and exhaust power from platforms 60A and / or 60B assigned to each port. For example, 100 micron separation 92 provides sufficient specimen fixation when the liquid applied from platform 60A is dispensed from ports 40A to 45A with port diameters in the range of 500 to 1500 microns at a flow rate of 115 μL / sec. , Staining and rinsing can be provided. Alternatively, if the separation 92 distance between the surface of platform 60A and substrate 2 is approximately 200 microns, the liquid distributed from ports 40A-45A may use faster flow rates such as 140 μL / sec for sampling. it can.

As mentioned above, mechanism 1 may include a set of ports and tubes for distributing and removing the liquid applied during sampling. The following discussion describes various ports, tubes, and other components in connection with platform 60A, but similar considerations apply to platform 60B and its related components. FIG. 8 shows an enlarged view of the device shown in FIG. 7, detailing ports 40A-45A on platform 60A and tubes 50A-55A connected to block 80A. Distribute certain liquids, such as one or more fixatives, stains and rinses, across the platform, during separation, and on the substrate. As shown in FIG. 8, the top surface of platform 60A includes six ports 40A-45A connected to tubes 50A-55A. The liquid is pumped onto the substrate through tubes and ports by one or more pumps. For example, one or more liquid containers 210A to 213A (first stain container 211A, second stain container 212A, immobilizer container 210A, and rinse liquid container 213A), as shown in FIG. It can be directed onto platform 60A and substrate 2. The diameters of ports 40A-45A shown in FIGS. 7-9 range from approximately 500 microns to 1500 microns, but the diameter can be smaller or larger in certain embodiments. In some embodiments, the diameters of the vacuum ports 40A and 41A are larger than twice the diameter of the liquid ports 42A-45A.

Each port 40A-45A is typically dedicated to a particular liquid or vacuum source. Alternatively, more than one port may be used for each liquid or vacuum source, connecting multiple tubes from various liquids and vacuum sources to a single port located on platform 60A. You may. For example, in some embodiments, only one port on platform 60A may be used for waste removal, but when using a more viscous liquid, a single port may be used. It may not be possible to provide sufficient suction to exhaust the remaining liquid from the platform. Therefore, in one embodiment, two suction ports are provided at different locations on the platform to remove excess stain, fixative and rinse fluids, as shown by ports 40A-41A in FIG. 8 (eg,). , One suction port at each end of the platform) may be desirable. Further emphasizing the variety of liquid-to-port arrangements, in one embodiment a single port on platform 60A may be dedicated to a particular stain, while in other embodiments multiple The port is used to apply the stain during sampling. In fact, the number of ports, port arrangements, and various combinations of liquids assigned to each port and liquid tube can be used in various embodiments of the invention.

Ports 40A-45A can typically be placed on platform 60A as desired to guide the delivery of liquid to substrate 2 and the removal of liquid from substrate 2. Typically, each liquid port is placed on platform 60A so that when the sample is being processed, the port opening is not placed directly adjacent to or directly below sample 3 on substrate 2. For certain combinations of specimens and dyes, for example, if the stain is dispensed from a port located directly adjacent to or beneath a portion of specimen 3, a larger amount of stain will be applied to the other portion of the specimen. It can be applied to cells in that part (the part with the opening of the port) rather than the cell. As a result, cells that receive higher amounts of stain may appear darker in the sample image, and this non-uniform sample cell stain can cause errors in image-based diagnostic measurements and analytical results. .. Therefore, the liquid port that delivers the stain to sample 3 can be placed at a specific distance from the sample-containing area of the substrate (eg, slide) to improve staining results.

In addition, the use of pairs of ports located opposite each other, such as multiple pairs of ports, can also improve dye uniformity. For example, in some embodiments, two ports are used to deliver the stain to Specimen 3. The two ports may be located on platform 60A at a certain distance (eg, offset) from the edge of specimen 3 and face each other in a direction parallel to the short edge of platform 60A. When the stain is dispensed from the two spaced ports, a relatively uniform amount of the stain is deposited on cells in different regions of sample 3, and an improvement in staining uniformity is observed in the sample image. ..

Similarly, ports 40A-45A can typically be arranged as desired to remove excess liquid from the surface of substrate 2 using one or more vacuum sources, but in some embodiments, The port used for liquid removal is placed at a distance from platform 60A, which is directly below the cells in specimen 3 on substrate 2. Placing the waste removal ports in this way reduces the chance of cells from sample 3 being inadvertently drawn into the liquid removal port if such ports are made to act to exhaust liquid from substrate 2. .. In one embodiment, due to the difference in length between the long and short sides of the platform 60A, the waste removal ports are placed away from the edge of the specimen area and along the direction parallel to the long edge of the platform 60A. Placed facing each other.

The mechanism 1 may include or be connected to a control system 5 as shown in FIG. 10, which provides another perspective view of the mechanism 1. The control system 5 may include one or more computers, each including a central processing unit capable of executing software instructions stored on a computer-readable medium such as a hard drive, optical disk or memory. In addition, the control system 5 may include electrical circuits for executing software instructions. The control system 5 may include a user interface for receiving user commands to control the operation of mechanism 1. Software stored in or provided to the computer may include programs that control the operation of components of Mechanism 1 such as liquid pumps and vacuums during sampling. For example, the software may include instructions for the mechanism 1 to apply various immobilizing agents, dyes and rinses to the specimen and to perform several agitation steps during the specimen processing.

In addition, the software may include default settings, and the user interface may include customization features to give the user the ability to change these default settings. For example, the user interface may include customization features to allow the user to customize speed, frequency or immobilization, staining and rinsing step instructions and agitation parameters (further described below). Control system 5 can also communicate over network protocols such as Appletalk®, IPX or TCP / IP. For example, network protocols may use cables (such as twisted pair cables) and / or wireless connections such as WiFi. The control system can be connected to the laboratory information system using a network protocol. The laboratory information system may include a server and / or database for storing information related to the specimen processed by Mechanism 1. For example, a database may contain information about the person or source of a sample (eg, name, date of birth (DOB), address, sampling time, gender, etc.), information about sample processing (processing date ## / ## / ###). #, Specimen number #, etc.), a copy of any pre-existing image of the sample, and a table that provides a copy of any result obtained by analyzing the image may be included.

FIG. 7 also shows that mechanism 1 may include supports 110A and 110B or laboratory workstations for fixing the device in place in the system. Mechanism 1 also includes one or more substrate arms 10A and 10B connected to actuators 30A and 30B at their bases, respectively. The opposite ends of the board arms 10A and 10B include board grips 20A and 20B for receiving and holding the board during sample processing. The substrate grips 20A and 20B receive and hold the substrate 2 while Mechanism 1 completes all sample processing steps (described below). The substrate may be, or include, a microscopic slide, coverslip, or other transparent material suitable for holding the specimen during and post-sampling microscopy. The embodiment of FIG. 7 depicts a substrate 2 containing a glass microscope slide and specimen 3. Using the suction port, the substrate grips 20A and 20B can hold the substrate 2 on the substrate arms 10A and 10B during the sample processing. The suction tube 23 provides suction to the substrate grips 20A and 20B by suction ports 21A and 21B, and 22A and 22B (in FIG. 7, ports 21A and 22A are located behind slide 2 and are shown by dashed lines. Note that).

The embodiment of mechanism 1 shown in FIGS. 7-9 is a dual substrate mechanism capable of holding and processing the substrate on each of the substrate arms 10A and 10B. Another embodiment provides processing of a single substrate or three or more substrates in sequence or simultaneously. Further, while the embodiments depicted in FIGS. 7-12 use suction to attach the substrate 2 to the substrate arms 10A and 10B, an alternative embodiment uses the substrate 2 on the substrate arm 10A during the sampling process. Use various types of clamps, fingers or magnets (if the substrate is magnetized) to attach.

In the embodiments shown in FIGS. 11 and 24A-B, the mechanism 1 receives the substrate 2 carrying the specimen 3 from the automatic substrate moving means 120 or manually from an individual. As an example, the substrate moving means 120 is a device that transfers a substrate between positions (eg, from position 121 to position 122, to position 123, to position 124, and to position 125). FIG. 11 shows a system with a first level leader position 121, an applicator position 122, a staining position 123 including mechanism 1, a camera or imaging position 124, and a second label reader position 125. The first label reader position 121 is set to read information from board 2 such as barcodes and / or "fingerprint" information used to identify a particular board 2 and sample 3 on it. Ru. The second label reader position 125 works in the same way and the information it reads is used to ensure that specimen 3 imaged at position 124 is the same as under the processed substrate. The label.

The board moving means 120 may include a grip portion 127 for holding the board, and a registration circuit or software for allowing the moving means 120 to determine whether the board 2 is mounted on the moving means 120. In one embodiment, the substrate moving means 120 may include a hydraulic cylinder for moving the substrate 2 from the first position 121 to the second position 122. After sample processing, the substrate moving means 120 may remove the treated substrate from the staining position 123 and transfer the substrate 2 to another position for substrate inspection, such as a microscope or position 124. Alternatively, the individual may manually remove the substrate from Mechanism 1 after sampling.

Board arms 10A and 10B rotate around an axis to allow the board to move from the open position to the sample processing position for loading and to return to the open position for unloading after sampling. can do. FIG. 13A shows a flowchart 500 including a series of steps for moving the substrate arm from the open position to the processing position. The flowchart 500 will be further described below with reference to FIG. 13B, which shows a schematic diagram of the mechanism 1.

It should be noted that mechanism 1 in FIG. 7 is configured to accept and inspect two substrates. In the discussions and figures below, references may be made to only one set of components in Mechanism 1 (eg, board grip 20A, actuator 30A, board arm 10A, etc.). However, the same steps, features and properties disclosed in connection with one set of components also apply to other sets of components in Mechanism 1 (eg, board grip 20B, actuator 30B, board arm 10B, etc.). It should be understood that it is applicable. Therefore, while the discussion herein focuses on only one set of components for clarity and simplicity, there are two or even more mechanisms for sampling, such as Mechanism 1. It is understood that the components of a set can be included and that each set has some or all of the features described herein.

Returning to FIGS. 13A and 13B, in the first step 502 of the flowchart 500, the substrate moving means 120 puts the substrate 2 in contact with the substrate gripping portion 20A. In step 504, substrate 2 is placed in the substrate grip in a "specimen up" or "open" position. Next, in step 506, the actuator 30A rotates the substrate arm 10A approximately 180 ° (see FIG. 13B) so that the substrate is in the processing position in step 510 and "specimen down" the substrate 2. Place it directly above platform 60A in the "sample processing" or "closed" position (step 508).

Then, in step 512, mechanism 1 comprises a stain, rinse and immobilizer delivered from containers 210A, 211A, 212A and 213A to contact specimen 3 through ports 42A, 43A, 44A and 45A. Specimen 3 placed on substrate 2 is stained by directing the appropriate liquid. Excess liquid is removed from Specimen 3 by vacuum pump through ports 40A and 41A and collected in waste collectors 230 and 231.

In step 514, following staining of specimen 3, actuator 30A rotates the substrate arm 10 approximately 180 ° (as opposed to rotation in step 506) to return the substrate to the "on specimen" arrangement. Finally, in step 516, the substrate moving means 120 removes the treated substrate from the substrate grip 20A. Other open, or "on-specimen" arrangements can be used as long as the operator or automatic board moving means can be loaded and the board can be unloaded from Mechanism 1. For example, the placement on the sample can be rotated by 100 ° or more (eg, 120 ° or more, 130 ° or more, 140 ° or more) from the sample processing position. In some embodiments, the placement on the sample is less than 100 ° from the sample processing position (eg, less than 90 °, 80 °) if the operator or automatic board moving means can be loaded and the board can be unloaded from Mechanism 1. Less than, less than 70 °) can be rotated.

Actuators 30A and / or 30B may include electric motors, hydraulics, magnetic systems, or other hardware (eg, worm gears) to move the arms 10A and / or 10B. When the board arms 10A and 10B are in the open position as depicted in FIG. 7, the grips 20A and 20B can receive the board 2, respectively. Once loaded onto the substrate grip 20A or 20B, the actuators 30A and / or 30B then immobilize the arms 10A and / or 10B, and thus the substrate 2, from an open ("on specimen") arrangement, including a stirring step. Rotate to the treatment position (as shown in arm 10B in Figure 9 "under specimen") for application of agents, stains and rinses and return to the open position for post-treatment unloading.

With respect to FIG. 9A, the actuator 30B rotates the substrate arm 10B from the open position depicted in FIG. 1 to the "closed" or processed position. FIG. 9A shows a downward orientation in which the substrate 2 on the substrate arm 10B is inverted and approximately 180 ° from the load position shown in FIG. 7 the sample 3 on the substrate 2 is substantially parallel to the surface of the platform 60B. Rotate to the position facing. As discussed in connection with FIG. 13A above, Mechanism 1 applies various immobilizers, stains and rinses to Specimen 3 on Substrate 2 in several processing steps, which steps are more detailed. It will be explained below. To remove substrate 2 from the processing position, the actuator 30B rotates the substrate arm 10B into the open position as shown in FIGS. 7 (both arms) and 9A (only one arm 10A is in the open position). return.

In certain embodiments, control system 5 uses one or more sensors 105A and 105B to detect indicator arms 101A and 101B (as shown in FIGS. 7 and 9) to detect the position of the arm. be able to. The sensors 105A and 105B can be proximity sensors, such as optoelectronic sensors, using, for example, infrared or various other techniques (lasers, motion sensing devices, etc.) to detect the presence or absence of the arm. For example, the proximity sensor 105A or 105B has a detection field, and the sensor has a board arm (eg, arms 10A and / or 10B) or a board grip (eg, grip 20A and / or 20B) in the detection field. Whether or not it can be determined by detecting the indicator arms 101A and / or 101B. The control system 5 can receive information from the sensor to determine the position of the board arm 10. For example, when the substrate arm 10B (not shown in FIG. 9) rotates to the processing position, the proximity sensor 105B on the proximity end of the indicator arm 101B senses the target substrate grip 20B and the substrate arm 10B is in the sample processing position. Notify control system 5 that it will rotate. At this position, the proximity sensor 105B on the distal end of the indicator 101B does not signal the control system 5 because the sensor does not detect any target (eg, the board arm or board grip).

When the board arm 10B is rotated to the open position (as shown in FIG. 7), the proximity sensor 105B on the distal end of the indicator arm 101B senses the target substrate grip 20B and tells control system 5 the board. Notifies that arm 10B has rotated to the open position. In other words, as the board arm 10B rotates away from the sensor 105B, the sensor sends a "non-existent" signal to control system 5. When the arm 10B rotates to the open position, the arm 10B approaches the sensor 105B, which signals the control system 5 to "exist". In an alternative setting, the sensor can detect the presence of the indicator arm 101B with the substrate 10B resting on the open position. In some embodiments, the control system 5 moves the substrate arms 10A and 10B and / or based on control signals and / or feedback received from actuators 30A and 30B to know the open and sample processing positions. It can be used to adjust the position of actuators 30A and 30B to actively monitor the position.

The rotational structure and axes of the substrate arms 10A and 10B in FIG. 7 can be modified in other embodiments of the invention. FIG. 14A shows a flowchart 600 including another sequence of steps for moving the substrate arm from the open position to the processing position. Flowchart 600 is further described below in connection with FIG. 14B. FIG. 14B shows a schematic view of the mechanism 1.

In step 602 of Flowchart 600, the substrate moving means 120 places the substrate 2 on the substrate grip 20A in the "on the specimen" direction. Next, in step 604, the first actuator 30A places the substrate 2 perpendicular to the plane of FIG. 14B so that the substrate 2 remains oriented "on the specimen" above the platform 60A. Rotate board 2 approximately 180 °. In step 606, the second actuator 35A receives the substrate 2 oriented in a "on-specimen" arrangement. Then, in step 608, the second actuator 35A (eg, the position between the substrate arm 10A and the substrate grip 20A) rotates the substrate 2 in the "bottom of specimen" direction. The second actuator 35A can also move the substrate 2 downwards towards the platform 60A so that the substrate 2 contacts the offsets 70A and 70B.

Next, in step 610, the substrate 2 at the processing position is used, and the mechanism 1 applies the staining solution, the fixing solution, and the rinsing solution to the sample 3 on the substrate 2 as described above in relation to step 512 of the flowchart 500. Stain by doing. After the staining is complete, the second actuator 35A rotates substrate 2 from "below the specimen" to "above the specimen" (step 614), and then the first actuator 30A causes the substrate to be "above the specimen". Rotate the substrate 2 approximately 180 ° (eg, in a plane perpendicular to the plane of FIG. 14B, opposite to the rotation applied in step 606) so that it remains oriented in the arrangement. Finally, in step 618, the substrate moving means 120 removes the treated substrate from the substrate grip 20A.

Fixed stage According to FIG. 10, liquid tubes 52A-55A and 52B-55B can be placed to deliver the immobilizing agent to platforms 60A and 60B, Separation 92, Substrate 2 and Specimen 3 during sample processing. One or more liquid tubes 52-55A can be connected to ports within platform 60A and individual immobilizer containers 210A. The liquid tube includes a fitting to a pump 200A and / or a valve that can move the immobilizing agent from the container through ports located on the tube and platform onto the substrate and specimen. As an example, pump 200A delivers the fixation fluid out of block 80A through tube 54A, onto platform 60A through port 44A, and onto substrate 2 containing specimen 3 during separation 92 between platform 60A and substrate 2. Can be exposed. After applying a certain amount of fixation to substrate 2, a vacuum or other suction source 220A and / or 221A removes the remaining fixation from platform 60A, separation 92, and substrate 2 from one or more ports 40A and / or Alternatively, it can be evacuated to waste container 230A and / or 231A via waste tubes 50A and 51A via 41A.

FIG. 15 shows a flowchart 700 including a series of steps for applying a fixation solution to a specimen. In step 702, a pump (eg, pump 200A) directs a fixed solution (eg, methanol) from a storage container (eg, storage container 210A) to a fixed solution tube (eg, tube 54A). In step 704, the fixative is directed into port 44A attached to block 80A. Then, in step 706, the fixative is moved out of port 44A on platform 60A. In step 708, the fixative is transferred out through port 44A and into the separation 92 between substrate 2 and platform 60A. Finally, in step 710, the sample 3 on the substrate 2 is fixed by the fixing liquid.

In some embodiments, the pump 200A delivers the fixation fluid through tube 54A and port 44A onto platform 60A and into separation 92 at least about 50 μL per second (eg, at least 75 μL, at least 100 μL). , 150 μL or more, 200 μL or more, or 250 μL or more) and / or about 300 μL or less (for example, 250 μL or less, 200 μL or less, 150 μL or less, 100 μL or less, or 75 μL or less) at a flow rate of about 2 (for example, 3, 4 or 5). Move for seconds. For example, the flow rate can be about 115 μL / s (about 70 μL / s, about 100 μL / s, about 150 μL / s) for about 2 seconds (eg, 3 or 4 seconds). Vacuum or other suction source 220A and / or 221A then removes the remaining fixation fluid present in separation 92 and / or platform 60A and substrate 2, ports 40A and / or 41A and waste tube 50A and / or. It can be removed using 51A (more on this below). Pump 200A then again pumps fixative through tube 54A and port 44A onto platform 60A over approximately 50 μL or more per second (eg, 75 μL or more, 100 μL or more, 150 μL or more, 200 μL or more, or 250 μL or more) and / Or move at a flow rate of about 300 μL or less (eg, 250 μL or less, 200 μL or less, 150 μL or less, 100 μL or less, or 75 μL or less) for about 2 (eg, 3, 4 or 5) seconds. For example, the flow rate can be about 115 μL / s (about 70 μL / s, about 100 μL / s, about 150 μL / s) for about 2 seconds (eg, 3 or 4 seconds). This immobilization and exhaust process can be repeated once more with the same or different immobilization agents depending on the type of specimen that requires immobilization. In addition, Mechanism 1 can vary the frequency and flow rate for each fixation step. Other flow rates sufficient to overcome the surface tension of the liquid located at Separation 92 can also be used. Fixed phase frequency and / Alternatively, by adjusting the flow rate, Mechanism 1 can achieve optimal immobilization with several different immobilization agents for different specimens. Machine commands for different types of specimens can be integrated or programmed into the hardware in control unit 5 and selected by the system operator as needed.

In general, a wide variety of immobilization agents can be applied to the sample during the immobilization step. For example, 85% methanol can be used as the immobilizing agent. For some dyes, ethyl alcohol or formaldehyde-based fixatives can be used. The fixed solution embodiments disclosed herein can be used for the preparation of test specimens.

Dyeing Stage Mechanism 1 also includes tubes and ports configured to apply one or more dyes or dyes to a sample immobilized on a substrate in one or more dyeing stages. Staining a specimen improves the contrast of the specimen when viewed or photographed under a microscope or other image processing device.

FIG. 16 is a flowchart 800 including a series of steps for applying a stain to a specimen. In step 802, the pump (eg, pump 201A) sends the stain solution from the container (eg, container 211A) to the stain tube (eg, tube 52A). In step 804, the stain is delivered to a port attached to block 80A (eg, port 42A). Next, in step 806, the stain exits port 42A on platform 60A. In step 808, the stain flows into the separation 92 between substrate 2 and platform 60A. Finally, in step 810, the dye is applied to sample 3 on substrate 2.

In some embodiments, multiple tubes and ports can be used to apply the stain to Specimen 3. For example, a second pump (eg, pump 202A) dispenses a stain (eg, the same or different dye as that distributed from container 211A) from container 212A through tube 53A and port 43A onto platform 60A. Can be sent. In certain embodiments, the two or more liquid tubes may be connected to a common stain container or pump and / or valve used to deliver the stain through the port onto the platform. Looking back at FIG. 8, tube 52A can deliver a red dye such as eosin Y to the platform, substrate 3 and specimen 2. Tube 53A may deliver a blue dye such as a thiazine dye (eg, Azure B, methylene blue). In Figures 7-12, the members, arrangement, and size of the ports on the platform 60A are selected to optimize the application of the dye to the specimen anchored to the substrate. If a different dye is selected, the different members, arrangements and sizes of the ports will preferably depend on the viscosity of the dye.

Each port 40A-45A (and 40B-45B) may include both an input channel for receiving the liquid and an output channel for outputting the liquid. In some embodiments, the output channels of the rinse agent 45A, the immobilizing agent 44A and the staining ports 42A-43A are on the top surface of the platform 60A, and the input channels of the vacuum ports 40A and 41A are of the platform 60A. It can be on the opposite edge of the top surface. The input channels of rinsing agent 45A, immobilizing agent 44A and staining ports 42A-43A may be located on the same side of block 80A, and the output channels of vacuum ports 40A and 41A on the opposite side of block 80A. Can be placed on top.

As an example, and with reference to FIGS. 8 and 16, in step 802, control system 5 applies a first stain (eg, a dye containing eosin Y) to a pump (eg, pump 201A) from a dye container to a liquid tube. Send it to 52A. In step 804, the first stain enters port 42 through the liquid tube. Next, in step 806, the first stain exits port 42A, and in step 808, the first stain is about 50 μL per second during separation 92 between platform 60A and substrate 2. Approximately 2 at flow rates greater than or equal to (eg, 75 μL or greater, 100 μL or greater, 150 μL or greater, 200 μL or greater, or 250 μL or greater) and / or about 300 μL or less (eg, 250 μL or less, 200 μL or less, 150 μL or less, 100 μL or less, or 75 μL or less). Allowed for (eg 3, 4 or 5) seconds. For example, the flow velocity can be about 115 μL / s (eg, about 70 μL / s, about 100 μL / s, or 150 μL / s) for about 2 seconds (eg, 3 or 4 seconds). In step 810, the sample 3 on the substrate 2 is stained with the first stain. Following staining, a vacuum or other suction source (eg, pump 220 and / or 221) is then subjected to the first stain remaining on platform 60A and substrate 3 during separation 92, ports 40A-41A and Exhaust can be done using waste tubes 50A-51A.

Mechanism 1 may be programmed to repeat these staining and exhaust steps after a delay (eg, a delay between 3 and 10 seconds, a delay of 5 seconds, etc.) after the first staining step. it can. The second pump 202A, by control system 5, draws the second stain (solution containing thiazine dye) from the stain container through the liquid tube 53A to the outside of the port 43A by about 50 μL or more per second (for example). , 75 μL or more, 100 μL or more, 150 μL or more, 200 μL or more, or 250 μL or more) and / or about 300 μL or less (for example, 250 μL or less, 200 μL or less, 150 μL or less, 100 μL or less, or 75 μL or less) at a flow velocity of about 2 (for example, You will be instructed to send on platform 60A for 3, 4 or 5) seconds. For example, the flow velocity can be about 115 μL / s (eg, about 70 μL / s, about 100 μL / s, or 150 μL / s) for about 2 seconds (eg, 3 or 4 seconds). A vacuum or other suction source (eg, pump 220A and / or 221) will then allow a second stain remaining in separation 92 and / or on platform 60A and / or substrate 2 at ports 40A-41A. And can be exhausted using waste tubes 50A-51A. As with the fixation steps, Mechanism 1 can vary the frequency and flow rate for each staining step. The flow rate can be, for example, in the range of 50-300 μL / sec, and if the flow rate is sufficient to overcome any surface tension of the liquid located in the separation 92, it is better than the outer limit of this range. It may be smaller or larger (eg, 10-500 μL / sec).

Typical stains applicable to specimens include, but are not limited to, Light-Giemsa stain, Giemsa stain, Romanovsky stain, and the first and second stains disclosed herein. Includes embodiments of. Other reagents, such as immunocytochemical reagents, and other markers of specific cellular components can also be applied to the specimen.

Removal of Waste Liquid As mentioned above, the vacuum or other suction source 220 and / or 221 can evacuate residual liquid from substrate 2, separation 92 and platform 60A during or during the fixation and staining steps. According to FIG. 7, one or more waste tubes can be connected to the sides 82A and 84A of the block 80A. Waste or vacuum tubes 50A and 51A are used to collect liquids and small particulate matter away from the waste container or mechanism 1 from platform 60A, separation 92 and substrate 2. With respect to FIG. 8, the waste tubes 51A and 51B may be connected to separate vacuum sources 220 and 221 and waste containers 230 and 231 at the tip of the waste tube. Alternatively, two or more waste tubes can be connected to a single vacuum source and the same waste container as shown in Figure 10. The waste tubes 50A and 50B can be expanded by pinch valves 90A and 90B, respectively.

A vacuum or other suction source for suction (eg, vacuum pump 220 and / or 221) is waste to draw liquid from platforms 60A and / or 60B, separation 92 and substrate 2 into waste containers 230 and 231. Can be connected to one or more of tubes 50A, 50B, 51A and 51B. The vacuum force applied in the waste tube is -1 to 1 to provide sufficient suction to remove the liquid if the separation between substrate 2 and platform is between 100 and 200 microns. It can be equivalent to minus 5 pounds per square inch ("psi"). In general, as used herein, "minus" pressure means pressure below the ambient pressure within Mechanism 1 or the environment surrounding Mechanism 1. For example, in some embodiments, the environment around Mechanism 1 is atmospheric pressure around 1 atmosphere. A "minus" pressure means a pressure below this ambient atmospheric pressure (for example, a pressure of -1 psi applied to a liquid is 1 psi lower than the ambient atmospheric pressure applied to the liquid). Other vacuums in the range of 0.1 psi to 14 psi or higher can be used as long as such vacuum is sufficient to overcome any surface tension of the liquid present during separation. In addition, just before applying a vacuum to evacuate the liquid from the separation, the actuator 30A can lift the adjacent edges of substrate 2 at a distance of 15-35 microns from the sampling position. By increasing the separation between the substrate 2 and the platform 60 in this way, the exhaust of any residual liquid in the separation 92 between the vacuum steps can be improved.

In some embodiments, the control system 5 is configured to vary the frequency and vacuum applied to the removal of the liquid during sampling. FIG. 17A includes a flowchart 900 that characterizes a series of steps for removing excess liquid from the substrate. Following the fixation phase, for example, control system 5 can open the pinch valves 90A and / or 90C in step 902 and evacuate the waste tubes (eg, waste tubes 50A and 51A) for 5 seconds. During this time, fixative is separated, removed from the substrate and platform through ports 40A and 41A (step 904). The liquid proceeds through the waste tube in step 906 and is placed in one or more waste containers (eg, containers 230 and / or 231) in step 908. Once the exhaust period is over, the control system 5 can close the waste tubes 50A and / or 51A to one or more pinch valves 90A, 90C in step 910, thereby further by vacuum 220-221. Prevent exhaust. The control system 5 may allow mechanism 1 to repeat this liquid removal step after each fixation step.

FIG. 17B includes Flowchart 1000 featuring an alternative sequence of steps for removing excess liquid from the substrate. The method of Flowchart 1000 does not use a pinch valve to seal the waste tube. Instead, after the staining step, the suction sources 220 and / or 221 are initialized in step 1002 and enter the active state in step 1004. The suction source applies a pressure of 3 psi to the waste tube 50A and / or 51A for 4 seconds to remove the dye from Separation 92, substrate 2 and platform 60A in step 1006 through ports 40A and 41A. The evacuated liquid travels through the waste tubes 50A and / or 51A in step 1008 and is placed in one or more waste containers 230, 231 in step 1010. Mechanism 1 may repeat this liquid removal step after each staining step. By varying the frequency and pressure applied during the liquid removal step, Mechanism 1 can achieve optimal fixation and staining of the specimen.

Pinch valves 90A, 90B, 90c and 90D close the waste tubes 50A, 50B, 51A and 51B as shown in FIG. Pinch valves 90A-90D can be mechanically, electrically, hydraulically or pneumatically operated by actuators contained within the valve and outside the valve. The pinch valves 90A-90D function to block the flow of liquid through the waste tubes 50A, 50B, 51A and 51B. For example, when replacing or emptying the filled waste container 230 from mechanism 1, the pinch valve (90A-90D) may be closed to prevent leakage of residual liquid present in the waste tube. desirable. Other mechanisms, such as different types of valves or clamps or stoppers, may be used in the embodiment of Mechanism 1 to close the waste tubes 50A, 50B, 51A and 51B.

Rinse Steps Rinse solutions can be applied in one or more rinse steps during sample processing by Mechanism 1. For example, it is desirable to wash substrate 2, separation 92 and platforms 60A and / or 60B between fixation steps, between dyeing steps, and / or between fixing and dyeing steps.

FIG. 18 includes a flowchart 1100 characterizing a series of steps for rinsing a sample. In step 1102, the pump (eg, pump 203A) sends the rinse solution (including, for example, distilled water) from the container (eg, container 213A) to the rinse tube (eg, rinse tube 55A). In step 1104, the rinse solution enters separation 92 between substrate 2 and platform 60A. In step 1110, sample 3 is rinsed. Finally, in step 1112, the vacuum sources 220, 221 aspirate one or more of the waste tubes 50A and 51A to remove the rinse solution from the separation 92 and substrate 2, and the rinse solution is the waste container 230. And / or transferred to 231. In some embodiments, the above steps are repeated in the second rinsing step.

In some embodiments, the control system 5 flushes pump 203A with about 50 μL or more per second (eg, 75 μL or more, 100 μL or more, 150 μL or more, 200 μL or more, or 250 μL or more) and / or about 300 μL. Apply at a flow rate of 250 μL or less, 200 μL or less, 150 μL or less, 100 μL or less, or 75 μL or less for about 2 (eg, 3, 4 or 5) seconds. For example, the flow velocity can be about 115 μL / s (eg, about 70 μL / s, about 100 μL / s, or 150 μL / s) for about 2 seconds (eg, 3 or 4 seconds). As with the fixed stages, the control system 5 can vary the duration and flow rate of each rinse stage and the number of rinse stages. In addition, control system 5 may adjust the placement of one or more rinse steps during sampling. The control system 5 may indicate, for example, that the rinse step occurs once after the completion of all fixation steps and the second rinse step occurs once after the completion of all staining steps. Alternatively, the rinsing step can be incorporated between two or more fixation steps or between two or more staining steps.

In some embodiments, the staining procedure is (1) fixation step, (2) second fixation step, (3) first staining step with first staining solution, (4) second staining. It may include a second staining step with a solution, (5) a rinse step, and (6) a second rinse step. In some embodiments, the various (eg, first and second) staining steps and / or solutions can be used in any order and / or repeated in any order. For example, the first and second staining steps and / or solutions can be replaced. Each step is about 50 μL or more (eg, 75 μL or more, 100 μL or more, 150 μL or more, 200 μL or more, or 250 μL or more) and / or about 300 μL or less (for example, 250 μL or less, 200 μL or less, 150 μL or less, 100 μL or less, per second. Or it may include about 2 (eg, 3, 4 or 5) seconds at a deposition velocity of 75 μL or less. For example, the flow velocity can be about 115 μL / s (eg, about 70 μL / s, about 100 μL / s, or 150 μL / s) for about 2 seconds (eg, 3 or 4 seconds).

Stirring Steps Specimen processing in certain embodiments involves placing a fixative, stain and / or rinse solution anywhere on Separation 92, substrate 2 and platforms 60A and / or 60B during the fixation, staining and / or rinsing steps. Includes one or more stirring steps to disperse. FIG. 19 includes a flowchart 1200 characterizing a series of steps for stirring a sample. Actuators 30A and / or 30B shown in FIG. 9 can provide fine motion adjustment to change the position of substrate 2 with respect to platform 60A and / or 60B.

The control system 5 may include software and / or hardware for instructing actuators 30A and / or 30B to start the stirring step. Actuators 30A and / or 30B can be configured to move the substrate arms 20A and / or 20B up and down in response to a stirring start command from the control system. The stirring step can be repeated for a predetermined number of cycles. As used herein, the term "cycle" means an upward movement from a starting position followed by a downward movement as opposed to an upward movement. In some embodiments, one or more agitation cycles return substrate 2 to the starting position at the end of each cycle, or at least at the end of some cycles. In certain embodiments, substrate 2 does not return to the starting position at the end of some or all agitation cycles, each cycle still comprising an upward movement followed by a downward movement. Actuators 30A and / or 30B typically continue to move substrate 2 in one or more agitation cycles until a stop command is sent from control system 5 to the actuator. The stirring step can temporarily increase the separation size (separation distance) between substrate 2 and the surfaces of platforms 60A and / or 60B, after which the substrate can be returned to the sample processing position. In addition, the agitation step may include a series of movements to move the substrate 2 between the angular position and the sample processing position of platforms 60A and / or 60B with respect to the surface. The surface tension of the liquid distributed during the separation between the platform and substrate 2 causes the redistribution of liquid molecules on the substrate as the substrate moves from the specimen processing position during the agitation step, causing the liquid to spread throughout the specimen. Distribution can be improved advantageously.

Other methods can be used to move substrate 2 against the platform during the stirring phase. For example, in some embodiments, the position of one or more offsets 70A-D and / or 71A-D (eg, the amount by which the offset extends onto the surface of platforms 60A and / or 60B) is in sample 3. Can be adjusted quickly for agitation. In certain embodiments, the positions of platforms 60A and / or 60B can be adjusted to result in agitation of specimen 3. For example, platforms 60A and / or 60B can be moved up and down alternately to result in agitation of sample 3 (eg, corresponding to the direction of movement of substrate 2 described above).

In some embodiments, the agitation of specimen 3 is made of a material that contracts the substrate arm, as described below, with actuators 30A and / or 30B offsetting substrate 2 from 70A to D and / or. It can also be done by changing the degree of movement toward 71A to D. The strain gauge can be used to measure and adjust the frequency of agitation applied to substrate 2 by detecting strain fluctuations in the substrate arm as a function of time.

According to FIG. 19, in the first step 1202, the stirring step is started. In step 1204, control system 5 causes actuator 30A to initiate a stirring cycle. In response to this instruction, the actuator 30A rotates the substrate 2 upward in step 1206 to increase the distance between the substrate 2 and the platform 60A. Next, in step 1208, the actuator 30A rotates the substrate 2 downward toward the platform 60A, reducing the distance between the substrate and the platform 60A. In the determination step 1210, if the stirring step is continued, control returns to step 1204 and the rotation of substrate 2 by the actuator 30A occurs again in another stirring cycle. When the stirring step is completed, control is then passed from step 1210 to step 1212 and the substrate 2 is returned to its initial position upon completion of stirring.

The stirring step may include one or more stirring cycles applied by actuators 30A and / or 30B. In addition, the stirring step can occur once or multiple times between the fixation, staining and / or rinsing steps, and at various frequencies between the fixation, staining and / or rinsing steps, respectively. For example, according to FIG. 9, actuators 30A and / or 30B lift the adjacent edges of substrate 2 vertically at a distance of 35 microns from the sampling position, and then fix substrate 2 to the sampling position three times. It can be returned once after each of the step, staining and rinsing steps. Actuators 30A and / or 30B can complete each agitation cycle in 2 seconds (eg, 1 second to vertically lift the adjacent edges of substrate 2 at a distance of 35 microns from the sampling position, and to the sampling position. 1 second to put the board back). Mechanism 1 can carry out commands to change the agitation frequency and distance for each agitation cycle and / or stage. For example, the agitation step includes actuators 30A and / or 30B that lift the adjacent edges of substrate 2 vertically from the sampling position to a distance of 5 microns 10-20 times per second and then return the substrate to the sampling position. obtain.

Alternative combinations of agitation distance and frequency can also be used. For example, in some embodiments, the stirring distance is 25 microns or more (eg, 50 microns or more, 100 microns or more, 150 microns or more, 200 microns or more, 250 microns or more, 300 microns or more, 500 microns or more, 700 microns or more, 1 mm or more). For example, in some embodiments, the stirring distance is between 35 microns and 350 microns.

In some embodiments, the agitation cycle frequency is greater than or equal to 1 cycle per second (eg, greater than or equal to 2 cycles per second, greater than or equal to 3 cycles per second, greater than or equal to 4 cycles per second, greater than or equal to 5 cycles per second, 7 cycles or more per second, 10 cycles or more per second).

Additional agitation techniques can also be used. For example, in some embodiments, the substrate grip 20A and / or 20B is an actuator that rotates the substrate around an axis perpendicular to the axis of rotation of actuators 30A and / or 30B, as depicted in FIGS. 7 and 9. May include.

Alternatively, platform 60A and / or platform 60B may be equipped with an offset adjuster for raising or lowering one or more offsets 70A-D and / or 71A-D during the fixation, staining and rinsing steps. To incorporate the offset adjuster, platforms 60A and / or 60B may include offsets that are attached to the platform's internal plates. The height of the plate can be varied using an internal actuator, which in turn changes the height of the offset. Alternatively, the positions of offsets 70A-D and 71A-D with respect to substrate 2 can be changed by instructing the actuator to move platforms 60A and / or 60B, or blocks 80A and / or 80B, thereby stirring. The separation distance changes between stages. Control system 5 uses a significantly smaller amount of liquid during the sample preparation process compared to conventional staining and preparation techniques to process the sample more efficiently, with liquid cycle, flow velocity, offset height, separation distance. , And stirring parameters and frequency can be adjusted.

In some embodiments, if the board arm is supported by only two offsets extending from the platform at the sampling position, the platform surface until the actuator or other driving element is supported by all four offsets of the slide. It can be made of a material that shrinks so that the slide can rotate further towards. The change in substrate position between these two positions can achieve sufficient agitation during sample processing. The board arm includes a strain gauge to monitor the strain of the board arm and can be used to inform control system 5 of the position of the board with respect to the platform offset. In addition, the control system may include information corresponding to substrate thickness defects that can be explained when the control system lays the substrate at the sampling position or during the agitation step.

Drying Stage In certain embodiments, the control system 5 can dry the specimen using a dryer 4 attached to mechanism 1. FIG. 20 includes a flowchart 1300 characterizing a series of steps for drying a specimen. Following step 1302, where the completion of staining and other steps (eg, one or more rinsing steps) is confirmed, in step 1304, dryer 4 sends a stream of air throughout the specimen. The drying process continues in step 1306 until a signal is received from the control unit to stop drying. When the signal is received, the dryer stops the flow of air throughout the specimen and the drying step ends at step 1308.

In general, mechanism 1 is controlled to change the temperature of the air, the flow velocity, the duration of the applied air flow, and the steps during sample processing to dry the sample 3. For example, after completing the staining step, dryer 4 can deliver a stream of air over the entire specimen at a flow rate of 10 L / min for 7 seconds at approximately 120 ° F. Also use the temperature of other air (eg, from ambient temperature to 300 ° F), the flow velocity of the air (eg, 1 L / min to 100 L / min), and the duration of the air flow (eg, a few seconds to a few minutes). be able to.

Specimen inspection system The automated sample preparation mechanisms and devices disclosed herein include Mechanism 1, typically US Application Nos. 12/430885 and 13/293050, all of which are incorporated herein by reference. ) Can be used with and / or incorporated into larger specimen inspection systems such as those disclosed herein. For example, FIG. 21 shows a schematic diagram illustrating one possible embodiment of the sample inspection system 2000. System 2000 includes platform 2100, light receiving device 2200, computer 2300, applicator 2400, gas circulation device 2500, light source 2600, dispenser 2800, discharge device 2900, slide labeler 3000, and slide label reader 3100. The Advancer 2110 may be configured to receive one or more slides or other boards 2700. The Advancer 2110 can be mounted on the surface of the platform, the top surface 2101, etc. The Advancer 2110 can be in the shape of a belt and the system can use mechanical arms, gravity, magnetic force, hydraulic pressure, gears, or other movement techniques to move the specimens placed on the substrate along the surface 2101 of the platform. it can.

Platform 2100 may include feeder 2102 and collector 2106, respectively, to feed and retrieve substrates 2700 (eg, slides) from or to a stack or rack. The feeder 2102 may include a feeder propulsion mechanism 2103 (such as a rubber wheel) for pushing the specimen on the advancer 2110. Alternatively, a mechanical arm can be used to grab the board 2700 and place the board directly on the advancer. An alternative mechanism for advancing the substrate out of the feeder 2102 may be magnets, hydraulic pressure, or the like. The feeder may include a sensor for measuring how long the slide is present. The sensor weighs the substrate 2700, for example, to determine how much substrate is present. The collector 2106 can also include a sensor to determine how many substrates are present. The sensor can be configured to notify the computer 2300 as soon as it analyzes the number of samples before setting and / or continuously notify the computer of the receipt of the sample mounted on the board.

The light receiving device 220 can be a microscope (such as a brightfield microscope), a video camera, a still camera, or other optical device that receives light. Embodiments that include a standard brightfield microscope may also include an automatic stage (eg, substrate moving means 2201) and automatic focusing. In some embodiments, the microscope can be fitted with a stage with a motor and a focus motor. The microscope can have an objective lens mount with a motor for selecting lenses of various magnifications under the control of the computer 2300. The filter wheel can be used to allow the computer 2300 to automatically select narrowband color filters in the optical path. The LED certification can be replaced by a filter, and the use of LEDs can reduce the image acquisition time compared to the time required to rotate the filter wheel. For example, a 1600 x 1200 pixel FireWire® (IEEE1394 high speed serial bus) camera can be used to collect narrowband imaging.

In some embodiments, the light receiving device 2200 receives the light reflected from the substrate 2700 and stores one or more images formed by the reflected light. Alternatively, or in addition, in some embodiments, fluorescence emission from the specimen on the substrate can be detected by the light receiving device 2200.

In certain embodiments, the light receiving device 2200 is configured to obtain a transmitted image of the specimen on the substrate. For example, a light source 2600 can be placed beneath the platform to direct light through the platform 2100 and substrate 2700 to the light receiving device 2200.

The light receiving device 2200 and any other component shown in FIG. 21 can be linked to the computer 2300 via a link (2011-2014), thus supplying energy to the computer and directing the component from the computer 2300. It can be given and / or components can be sent information to the computer 2300. Links 2011 to 2014 may be wired links or wireless links.

The light receiving device 2200 is capable of moving in the X, Y and Z axes (in other embodiments, the motorized stage or substrate moving means 2201 is capable of moving in the X, Y and Z axes. obtain). The light receiving device 2200 may include pan, tilt and / or moving actuators so that the computer 2300 can position the light receiving device 2200 in an appropriate position. The light receiving device 2200 may include a lens 2210 that focuses the incident light on the focal point.

The light receiving device 2200 can be selected to capture black and white and / or color images. In some embodiments, two or more light receiving devices can be used to share the processing time associated with image acquisition. For example, a low-magnification contrast step can be followed by a high-magnification contrast step. Similarly, in some embodiments, the system 2000, platform 2100, computer 2300 and / or light receiving device 2200 places the substrate 2700 on the substrate moving means 2201 and all or most of the cells on the substrate or on specific parts of the substrate. Can be moved to ensure the acquisition and storage of one or more images of.

The computer 2300 may be a notebook, server, workstation, or other type of computer device. The computer may include a processor, display 2320, interface 2310, and internal memory and / or disk drive. The computer 2300 may include software stored in memory or on a computer-readable tangible medium such as an optical disc. The software is in the light receiving device 2200, applicator 2400, gas circulation device 2500, platform 2100, advancer 2110, light source 2600, dispenser 2450 and / or 2800, specimen preparation mechanism 1, or one of these components. Alternatively, it may include commands for the computer to operate any connected component. Similarly, the computer is arranged to receive information from any of these components.

For example, software can control the rate of distribution of the boards from the feeder 2102, which can provide information about the number of boards present in the computer. In addition, the computer 2300 can also be responsible for performing the analysis of the images captured by the light receiving device 2200. The analytical process allows the computer to calculate the number of cells of a particular type in a particular blood volume, such as blood, red blood cells, white blood cells and platelets, and complete blood cells such as hemoglobin content, red blood cell morphology or leukocyte percentages that can be calculated. It can be arranged and controlled to calculate other measurements or induced components of the calculation. Image analysis software can analyze each individual region and sum the total red blood cell count and white blood cell count. The number counted on the slide can be multiplied by the dilution ratio and the volume of the subsample to calculate the total count per μL in the patient's blood sample. As a result of counting from the slides, morphological measurements and images of red blood cells and white blood cells can be shown on the display 2320.

In some embodiments, the computer 2300 is configured to display a direct assessment of cell morphology using numerical data, cell population histograms, scatter plots and images of red blood cells displayed on a monitor. The ability to display cell morphology quickly demonstrates the presence or absence of abnormalities in cell morphology that can ensure that additional slides are prepared for manual review by experienced technicians or other professionals. Providing capabilities to users of System 2000. The software can also give the computer instructions to display the image 2331 received from the light receiving device, and can also cause the display 2330 to show the result of the image analysis 2332 (perhaps a chart or graph, for example). Similarly, the computer 2300 is controlled to count the number of cells of a particular type in a particular blood volume, or to count the number of damaged cells, cancer-like cells or lysed cells in a particular blood volume. You can also. The software allows the computer to perform the analytical process. The computer can use one or more magnifications during the analysis.

Although shown as one component, the computer 2300 is a plurality of computers; the first computer can be used to control the components of the system 2000 and the second computer is from the light receiving device 2200. It can be used to process images. Various computers can be linked together to allow the computers to share information. The computer 2300 can also be connected to a network or laboratory information system to allow the computer to send and receive information from other computers.

In certain embodiments, the applicator 2400 may include a syringe, a manual or motor driven pipetter, or a motor controlled pump attached to the pipette tip with a tube. The applicator 2400 applies the sample to substrate 2700 in a controlled manner. Specific features, properties and methods of using the applicator 2400 are disclosed, for example, in US Patent Publication No. 2009/0269799. Specimens include one or more blood components, cells, tissues, or other biological components.

Once the specimen is applied to substrate 2700, the applied specimen is processed by mechanism 1. Mechanism 1 functions as described herein and applies one or more stains, fixatives and / or other solutions to the specimen on the substrate.

In some embodiments, the system 2000 is configured to minimize cell-cell overlap placed on substrate 2799 by placing non-contact rows of cells from the chip of the applicator 2400. Can be done. The increase in viscosity of the diluted liquid, or the type and amount of diluent, can affect the width of the final anchorage position of the sample flow from the applicator. All cells can be counted in all samples by choosing the distance between rows that tolerates typical changes in blood samples.

The gas transfer device 2500 may be a separate device as shown in FIG. 21 or may be incorporated into mechanism 1 as described above, but may include a fan and / or for example. Other gas transfer devices such as compressors and blowers may be included. The gas transfer device 2500 may be connected directly to the computer 2300 or by another component such as the platform 2100 or the applicator 2400. The gas transfer device pushes gas (in some cases, the atmosphere) across the substrate to control the rate at which the specimen on the substrate dries. If there is too much air across the substrate or too fast (ie, the fan speed is too high), rapid drying can cause cells in the specimen to rupture, resulting in too little air or too much substrate. If it crosses too slowly (ie, the fan speed is too slow), the cells may dry too slowly and contract.

The computer 2300 measures the amount of air that traverses the substrate in a given amount of time (eg, cubic feet or centimeters of air), the distance from the substrate of the gas transfer device, the type of liquid analyzed, the width of the flow, and the gas. It can be selected and controlled based on the temperature of the (eg, air) and the average thickness of the flow. The gas transfer device 2500 can be arranged so that the device can deliver the gas so that the gas hits the substrate at an angle of 30 ° to 60 ° (eg, 45 °) for about 15 to 20 seconds. In some embodiments, the computer 2300 can control the humidity and temperature settings near the system to carry out the drying process without the use of the gas transfer device 2500.

The light emitting device 2600 and its various components are described as examples in US Patent Publication No. 2009/0269799. Light of various wavelengths can be generated by the light emitting device 2600 and detected by the light receiving device 2200. Wavelengths, such as 415 nm, are useful for obtaining hemoglobin-only images to assess RBC morphology and hemoglobin content. Light emitted at 600 nm can be useful in providing high contrast images of platelets and nuclei. Other wavelengths may be selected to best distinguish between the colors of basophils, monocytes, lymphocytes (all in blue tones), eosinophils (red) and neutrophils (neutral).

<p> Although the present disclosure will be further described by the following examples, the following examples are not intended to limit the scope of the invention described in the claims.</p><p>Example 1 FIG. 22 is a flowchart 1400 showing a typical sequence of steps for processing a specimen placed on a substrate. The steps in Flowchart 1400 can be used to prepare test specimens. The description of this process sometimes cites a particular process with a particular scope or discloses steps that occur in a particular order, but the description is intended to illustrate only one process example. Is. With reference to FIG. 22, mechanism 1 is connected to control system 5 for commanding the operation of various mechanism components during the processing process. In the sample initiation step, the sample 3 containing red blood cells, white blood cells, and platelets derived from blood aliquots is placed on a substrate 2 consisting of glass microscope slides. This can be done using a variety of steps, such as one or more stations described in Co-pending US Patent Application Publication No. 2008/0102006. In the arrangement step 1402, the substrate 2 including the sample 3 is loaded on the substrate grip portion 20A of the substrate arm 10A as shown in FIG. The control system 5 instructs the suction source 222 to evacuate the air from the substrate grip 20A (step 1404). Suction applied through suction ports 21 and 22 (step 1406) attaches substrate 2 to substrate grip 20A during sample processing. The control system 5 instructs the actuator 30A to rotate the substrate 3 from the open position shown in FIG. 7 to the sample processing position shown in FIG. 9A (step 1408). At the sample processing position, sample 3 is directed to the surface of platform 60A and substrate 2 is supported by offsets 70A-D shown in FIG. This offset prevents substrate 2 from coming into contact with the surface of platform 60A. In this typical process, the separation 92 between the sample contact surface of substrate 2 and the surface of platform 60A is approximately 100 to 200 microns (eg, 200 microns).</p><p> During the first fixation step (step 1412, also see FIG. 15), the pump applies the fixative to sample 3 in step 1414. The pump 200A connected to the liquid tube 54A shown in FIG. 8 allows a fixative containing methanol to pass from the fixative container 210 through the tube 54A, outside port 44A, on platform 60A, on platform 2, and platform 60A and substrate 2. Proceed during separation 92 with. Pump 200A pushes the fixate from port 44A at a flow rate of 115 μL / sec for 2 seconds, thereby moving a total of 230 μL of fixative V1 onto substrate 2.</p><p> Next, in the first agitation step 1416, control system 5 moves actuator 30A so that the adjacent edges of substrate 2 rise a distance of 35 microns vertically from the sample processing position, and the sample is moved to that sample processing position. The substrate is agitated by returning. Mechanism 1 repeats this stirring step four more times. As shown in FIG. 23, the mechanism 1 completes 5 stirring operations in about 10 seconds at T2. After stirring, the control system initiates vacuum step 1420. A vacuum force of -0.10 psi is applied for one and a half seconds, during T3, and the remaining anchorage present on the platform or substrate during separation is passed through ports 40A and 41A, as well as waste tubes 50A and 51A. Evacuate in vacuum (step 1422). The evacuated fixative is collected in waste containers 230 and / or 231.</p><p> Then, in the second fixing step including the second stirring step, the above-mentioned steps of the first fixing step and the first stirring step are repeated.</p><p> After the fixation step, control system 5 initiates the first staining step (step 1424). At that time, the control system 5 causes the mechanism 1 to stain the sample (step 1426). Referring to the flowcharts of FIGS. 8 and 16, the pump 201 connected to the liquid tube 52A stains the first stain containing eosin Y from the stain container 211A outside the port 42A, on the platform 60A, on the substrate 2 and on the platform. Extrude during separation 92 between 60A and substrate 2. Pump 201 injects the first stain into port 42A at a flow rate of 115 μl / sec for 2 seconds, thereby directing 230 μl of the first stain V2 onto the substrate.</p><p> After applying the first stain to Specimen 3, Mechanism 1 raises the second agitation step 1428 to Actuator 30A by raising the adjacent edge of substrate 2 vertically from the specimen processing position to a distance of 35 microns. (Step 1430), followed by returning the sample to its sample processing position.</p><p> The control system 5 causes mechanism 1 to repeat this stirring step two more times, completing three stirrings over a period of approximately 6 seconds T5 shown in FIG.</p><p> Next, a second vacuum step is initiated in step 1432. A 5 psi vacuum was applied in step 1434 for 3 seconds, with the remaining first stain present in separation 92 or on the platform and substrate, ports 40A and / or 41A and waste tubes 50A and 51A. After that, vacuum exhaust. The evacuated first stain is collected in the waste container 230A and / or 231A.</p><p> After staining the specimen with a first stain containing eosin Y, Mechanism 1 initiates a second stain step with a second stain containing Azure B and methylene blue in step 1436. Pump 202 connected to the liquid tube 53A pushes the second stain from the stain container through port 43A onto platform 60A, onto substrate 2 and into the separation 92 between platform 60A and substrate 2 (step). 1438). Mechanism 1 distributes the second stain through port 43A at a rate of 115 μl / sec for 2 seconds, thereby moving the second stain a total of 230 μl onto the V3 substrate.</p><p> After applying the stain to sample 3, mechanism 1 raises the actuator 30A in step 1440 to raise the adjacent edge of substrate 2 to a distance of 35 microns from the sample processing position (step 1442), after which sample 3 is sampled. The third stirring step is started by returning to the processing position. Mechanism 1 repeats this stirring step three more times. The mechanism completes four stirring operations over T8 for about 8 seconds.</p><p> The third vacuum step 1444 is then started. A 5 psi vacuum was applied for 2 seconds at T9, and after stirring, the second stain remaining during separation or on platform 60A and substrate 2 was applied in step 1446 to ports 40A and / or 41A, and waste tube 50A and / Or vacuum exhaust via 51A. The evacuated second stain is collected in the waste container 230A and / or 231A.</p><p> Mechanism 1 then performs two series of rinse-stirring-vacuum steps. The first series of steps is started in step 1448 when the control system 5 instructs mechanism 1 to start the first rinse step. Container 213A containing the rinse solution is connected to pump 203 and liquid tube 55A. Pump 203 moves the rinse solution through the wash tube 55A, which feeds port 45A, into the separation 92, onto platform 60A and substrate 2, and rinses sample 3 in step 1450. Alternatively, in some embodiments, the rinse solution is passed through two or more liquid ports 42A-45A. Pump 203 pumps the rinse fluid out of T10, port 45A for 2 seconds at a flow rate of 115 μl / sec, thereby delivering a total of 230 μl, V4 of water onto the substrate.</p><p> The control system 5 then raises the actuator 30A in step 1452 by raising the adjacent edge of substrate 2 vertically from the sample processing position to a distance of 5 microns (step 1454) and returning the sample to that sample processing position. The fourth stirring step is started. The control system 5 causes mechanism 1 to repeat this stirring step and completes two stirrings at T11 for approximately 4 seconds.</p><p> The vacuum step is then initiated in step 1456. In step 1458, a 5 psi vacuum over T12 for 5 and a half seconds, after agitation, the residual rinse solution present in separation 92, or on platform 60A and substrate 2, ports 40A and / or 41A, and a waste tube. Exhaust after 50A and / or 51A.</p><p> Then, in step 1460, the control system 5 causes mechanism 1 to initiate a second rinse-stirring-vacuum phase by initiating a second rinse step. The second rinse step (steps 1460, 1462), the fifth stirring step (steps 1464, 1466), and the fifth vacuum step (steps 1468, 1470) have described the first rinse-stirring-vacuum step. It is done in the same way as. During the second rinse-stirring-vacuum step, the amount of rinse solution, V5, and treatment time, T13, T14, and T15 are usually the same as in the first rinse-stirring-vacuum step.</p><p> After immobilizing the specimen, staining with a first stain containing eosin Y and a second stain containing Azure B and methylene blue, and rinsing, Mechanism 1 initiates the drying step in step 1472. The dryer 4 sends an air flow of 10 L / min at a flow rate of about 120 ° over the entire sample for 8 seconds in T16 (step 1474).</p><p> After completion of this step, substrate 2 is returned to its original position in step 1476. In this step, the actuator 30A rotates the substrate 2 from the sample processing position to the open position as shown in FIG. Substrate 2 may then be removed by substrate moving means and a new substrate may be loaded to process the new specimen.</p><p> As described above and in the typical sampling process shown in FIG. 23, the systems and methods disclosed herein are more than conventional sampling methods such as automated and manual sampling techniques. It is provided for more efficient sample processing with less reagent consumption. According to FIG. 23, for example, Mechanism 1 consumes less than 1.5 mL of reagents during typical processing steps for specimen fixation, staining and rinsing (eg, fixation solution 460 μL + first stain 230 μL +). 230 μL of second stain + 460 μL of rinse solution = 1380 μL of reagent). In some embodiments, approximately 1380 μL of liquid may be used during sample processing. For example, the amount of liquid used in the processing of the specimen can be approximately 1150 μL (eg, by excluding the rinse step once).</p><p> The volume of the liquid is usually related to the spacing of approximately 100 microns between the substrate and the platform. Larger spacing between the substrate and the platform usually consumes more volume of liquid during sampling. For example, if the spacing is approximately 200 microns, the total volume of liquid consumed would have to be greater than 1380 μL.</p><p> More generally, the total volume of liquid consumed is 500 μL or more (eg, 520 μL or more, 540 μL or more, 560 μL or more, 580 μL or more, 600 μL or more, 650 μL or more, 700 μL or more, 750 μL or more) and / or 2 mL or less (for example). For example, 1.5 mL or less, 1.4 mL or less, 1.3 mL or less, 1.2 mL or less, 1.1 mL or less, 1.0 mL or less, 900 μL or less).</p><p> Again, according to Figure 23, the specimen preparation process took just over a minute (eg, 13.5 seconds between each fixation step, a total of 27 seconds for fixation + 11 seconds between the first staining steps + 12 seconds between the second staining steps + 23 seconds between the rinsing steps + 8 seconds between the drying steps = total elapsed time 81 seconds). In certain embodiments, specimen preparation can be completed in approximately 81 seconds. For example, sampling can be completed in 180 seconds or less (eg, 150 seconds or less, 120 seconds or less, 90 seconds or less, 80 seconds or less, 70 seconds or less, 60 seconds or less, 50 seconds or less, or 40 seconds or less).</p><p> In addition, the typical process described above describes the processing time for a single sample, but is configured to process systems and methods for processing multiple substrates (eg, processing two substrates). Mechanism 1 in Figure 7 and / or a system configured to process three or more substrates is capable of processing more than 100 samples per hour (eg 60 per hour). Between specimens and 120 specimens). Laboratory use of the systems and methods disclosed herein results in faster processing power per specimen, while consuming liquids (eg, fixatives, stains, and rinses). Is reduced compared to traditional automated and manual sampling techniques.</p><p>Example 2 The processing steps described above for Example 1 can be adjusted in other embodiments of the invention as follows. In addition, the fixative, stain and rinse formulations described herein can be used in the treatment steps of the following examples.</p><p> During the first fixation step (step 1412, also see FIG. 22), the pump applies the fixative to sample 3 in step 1414. The pump 200A connected to the liquid tube 54A shown in FIG. 8 transfers a fixative containing methanol from the fixative container 210 through the tube 54A, outside the port 44A, on the platform 60A, on the platform 2, and on the platform. Extrude during separation 92 between 60A and substrate 2. Pump 200A extrudes a fixed solution from port 44A at a flow rate of 115 μL / sec for 2 seconds, thereby delivering a total of 230 μL of fixed solution V1 onto substrate 2.</p><p> Next, in the first agitation step 1416, the control system 5 raises the adjacent edge of the substrate 2 to the actuator 30A (step 1418) by 35 microns perpendicular to the sampling position, and the sample is sampled. The substrate is agitated by returning to the position. Mechanism 1 repeats this stirring step 5 more times. Mechanism 1 completes 6 stirring operations in about 12 seconds. After stirring, the control system initiates vacuum step 1420. A -6 psi vacuum force is applied for one and a half seconds (T3) to evacuate the fixing fluid remaining on the platform or substrate during separation through ports 40A and 41A and exhaust tubes 50A and 51A (step). 1422). The exhausted fixative is collected in waste containers 230 and / or 231.</p><p> Then, in the second fixing step including the second stirring step, the above-mentioned first fixing step and the first stirring step are repeated.</p><p> Following the fixation step, control system 5 initiates a first staining step (step 1424). At that time, the control system 5 causes the mechanism 1 to stain the sample (step 1426). According to the flowcharts of FIGS. 8 and 16, the pump 201 connected to the liquid tube 52A pumps the first stain containing eosin Y from the stain container 211A, outside the port 42A, on the platform 60A, sample 3. Extrude over substrate 2 and during separation 92 between platform 60A and substrate 2. Pump 201 distributes the first stain through port 42A at a flow rate of 115 μL / sec for 2 seconds, T4, thereby delivering 230 μL of the first stain, V2, onto the substrate.</p><p> After applying the first stain to Specimen 3, Mechanism 1 moved the second agitation step 1428 to actuator 30A and in step 1430 the adjacent edges of substrate 2 to a distance of 35 microns perpendicular to the sample processing position. This is done by raising and then returning the sample to its sample processing position. The control system 5 causes the mechanism 1 to repeat this stirring step two more times, and completes three stirrings over T5 for about 6 seconds, as shown in FIG.</p><p> Next, a second vacuum step is initiated in step 1432. A -5 psi vacuum was applied in step 1434 for 3 seconds (T6) and the first stain remaining during separation 92 or on the platform and substrate was passed through ports 40A and / or 41A, and waste tubes 50A and 51A. Vacuum exhaust. The exhausted first stain is collected in the waste container 230A and / or 231A.</p><p> After staining the specimen with a first stain containing eosin Y, Mechanism 1 initiates a second stain step in step 1436 with a second stain containing Azure B and methylene blue. Pump 202, connected to the liquid tube 53A, allows the second stain to be removed from the stain container through port 43A on platform 60A, on substrate 2, and separation between platform 60A and substrate 2 92. Extrude in (step 1438). Mechanism 1 distributes the second stain through port 43A at a flow rate of 115 μL / sec for 2 seconds, T7, thereby delivering 230 μL of the second stain, V3, onto the substrate.</p><p> After applying the stain to specimen 3, mechanism 1 raises the third agitation step to actuator 30A in step 1440 by 35 microns vertically from the specimen processing position on the adjacent edge of substrate 2 (step 1442). , And then start by returning Specimen 3 to its Specimen Processing Position. Mechanism 1 repeats this stirring step two more times. The mechanism completes three stirring operations over T8 for approximately 6 seconds.</p><p> Then the third vacuum step 1444 is started. A -6 psi vacuum was applied for 2 seconds (T9) and in step 1446, the second stain remaining during separation or on platform 60A and substrate 2 was applied to ports 40A and / or 41A, and waste tube 50A and /. Alternatively, after stirring through 51A, evacuate to vacuum. The exhausted second stain is collected in the waste container 230A and / or 231A.</p><p> Mechanism 1 then performs a series of rinse-stirring-vacuum steps twice. The first is started when the control system 5 instructs mechanism 1 to start the first rinse step in step 1448. Container 213A containing the rinse solution is connected to pump 203 and liquid tube 55A. Pump 203 moves the rinse solution through the wash tube 55A, which feeds port 45A, into separation 92 and onto platform 60A and substrate 2 to rinse sample 3 in step 1450. Alternatively, in some embodiments, the rinse solution is fed through two or more liquid ports 42A-45A. Pump 203 moves the rinse fluid out of port 45A at a flow rate of 115 μL / sec for 2 seconds, T10, thereby delivering a total of 230 μL, V4, of water onto the substrate.</p><p> The control system 5 then raises a fourth stirring step to actuator 30A in step 1452 by raising the adjacent edge of substrate 2 by 35 microns vertically from the sampling position (step 1454), and then raising the sample. Start by returning to the sample processing position. The control system 5 then causes mechanism 1 to repeat this stirring step three more times, completing four stirrings at T11 for approximately 8 seconds.</p><p> The vacuum step is then initiated in step 1456. A 5 psi vacuum was applied for 5 and a half seconds (T12), and in step 1458, the rinse solution remaining during separation 92 or on platform 60A and substrate 2 was applied to ports 40A and / or 41A, and waste tube 50A and /. Alternatively, the mixture is stirred through 51A and then evacuated.</p><p> Then, in step 1460, control system 5 causes mechanism 1 to initiate a second rinse-stirring-vacuum step by initiating a second rinse step. The second rinsing step (steps 1460, 1462), the fifth stirring step including 6 agitation completed in approximately 12 seconds, and the fifth vacuum step (steps 1468, 1470) are the first. The rinse-stirring-vacuum step is performed in the same manner as disclosed. During the second rinse-stirring-vacuum step, the amount of rinse solution, V5, and treatment time, T13, T14 and T15 are usually the same as in the first rinse-stirring-vacuum step. In addition, just before the vacuum step, actuator 30A raises the close edge of substrate 2 at a distance of 15-35 microns from the sampling position. This increased separation between substrate 2 and platform 60 improves vacuum evacuation of the liquid remaining in separation 92 during the final vacuum phase.</p><p> After the specimen is fixed, stained and rinsed with a first stain containing eosin Y and a second stain containing Azure B and methylene blue, Mechanism 1 initiates the drying step in step 1472. The dryer 4 flows an air flow of about 120 ° at a flow rate of 10 L / min (step 1474) for 8 seconds at T16 over the entire sample.</p><p> After completion of these steps, substrate 2 is returned to its original position in step 1476. In this step, the actuator 30A rotates the substrate 2 from the sample processing position to the open position shown in FIG. Substrate 2 can then be removed by substrate moving means and a new substrate can be loaded to process the new specimen.</p><p> As described in the exemplary sample processing steps described above, the systems and methods disclosed herein require less reagent consumption compared to conventional sample processing methods such as automated and manual sample preparation techniques. , Provided for more efficient sampling. According to Example 2, Mechanism 1 consumes less than 1.5 mL of reagents during typical treatment steps for specimen fixation, staining and rinsing (eg, fixation solution 460 μL + first stain 230 μL + first stain). 230 μL of stain solution of 2 + 460 μL of rinse solution = 1380 μL of reagent). In some embodiments, approximately 1380 μL of liquid can be used during sample processing. For example, the amount of liquid used in the processing of the specimen can be approximately 1150 μL (eg, by excluding the rinse step once) or less than 1000 μL (eg, by further excluding the fixation step once). ..</p><p> For FIG. 23 with respect to Example 1, Mechanism 1 consumed less than 1 mL of reagent during typical treatment steps for specimen fixation, staining and rinsing (eg, methanol immobilizing agent 140 μL + fluorescein dye 140 μL + thiazine). Dye 140 μL + rinse solution 280 μL = reagent 700 μL). In some embodiments, approximately 700 μL of liquid may be used during sample processing. For example, the amount of liquid used in the processing of the specimen can be approximately 560 μL (eg, by excluding the rinse step once).</p><p> Generally, the total volume of liquid consumed is 500 μL or more (eg, 520 μL or more, 540 μL or more, 560 μL or more, 580 μL or more, 600 μL or more, 650 μL or more, 700 μL or more, 750 μL or more) and / or 2 mL or less (for example, 1.5 mL). Below, 1.4 mL or less, 1.3 mL or less, 1.2 mL or less, 1.1 mL or less, 1.0 mL or less, 900 μL or less).</p><p> According to FIG. 23 and Example 1, the sample preparation process takes just over 1 minute (eg, 13.5 seconds during the fixation step + 11 seconds during the fluorescein staining step + 12 during the thiadin staining step. It is completed in 2 seconds elapsed + 23 seconds elapsed during the rinse stage + 8 seconds elapsed during the drying stage = total elapsed time 67.5 seconds). In certain embodiments, specimen preparation can be completed in approximately 67.5 seconds, as shown in Example 2. For example, sampling can be completed in 180 seconds or less (for example, 150 seconds or less, 120 seconds or less, 90 seconds or less, 80 seconds or less, 70 seconds or less, 60 seconds or less, 50 seconds or less, or 40 seconds or less). it can.</p><p> In addition, the typical process described above describes the processing time for a single sample, but is configured to process systems and methods for processing multiple substrates (eg, processing two substrates). Mechanism 1 in Figure 7 and / or a system configured to process three or more substrates is capable of processing more than 100 samples per hour (eg 60 per hour). Between specimens and 120 specimens). Laboratory use of the systems and methods disclosed herein results in faster processing capacity per specimen, while consuming liquids (eg, immobilization, stain, and rinse). The amount is reduced compared to conventional automated and manual sample preparation techniques.</p><p>Example 3 For each series of experiments in Table 1, blood samples were prepared using, for example, the sample preparation techniques described in US Patent Publication No. 20090269799. The samples were then treated by immobilization, staining, and rinsing, generally according to Example 1. The flow rate was 115 μL / s for 2 seconds for each of the fixation solution, stain solution, and rinse solution. For each set of experiments listed below, substrates (eg, microscope slides) were prepared from at least 5 blood samples. The treated sample is then manually evaluated for sample staining and preparation (eg, overall uniformity of staining, color, differentiation of cellular features, presence or absence of debris in the background, etc.). Quality was evaluated under a microscope at a magnification of at least 10x. Manual evaluation was performed to compare the quality of sample specimen staining and sample preparation to the quality of control specimen staining and sample preparation. Typically, "rolling control" Condition) "was adopted and the previous formulation providing optimal staining and sample preparation was used as a control to compare the new indication with the various formulations. Therefore (unless otherwise specified below), any row in Table 1 typically represents the control conditions for a series of experiments summarized in the next row. How the prescription affects the ability of the imaging device to classify the five types of WBCs present in the specimen, if any, due to the limited number of samples and after manual examination. The samples were processed with an imaging device as described in US Patent Publication No. 20090269799 to determine if. These results are reported in Table 2.</p><p><tables num="1"><img file="JP6495366B2_D0001.tif" /></tables><img file="JP6495366B2_D0002.tif" /><img file="JP6495366B2_D0003.tif" /><img file="JP6495366B2_D0004.tif" /><img file="JP6495366B2_D0005.tif" /></p><p> The WBC classification results listed in Table 2 are the same as the automatic machine WBC classification results as described in US Patent Publication No. 20090269799, which incorporates all of the manual classification of ABCs in the sample herein. Compare. Its percentage reflects the accuracy of automatic machine classification compared to manual inspection and classification of WBCs in the sample. For each of the three experiments (2-1, 2-2 and 2-3), a minimum of 10 samples prepared there using the latest fixative and stain solutions were processed on the system and the WBCs in each sample were processed. Compared with the manual inspection of. Each manual test consisting of WBCs distinction was performed on at least 100 WBCs in the sample.</p><p><tables num="2"><img file="JP6495366B2_D0006.tif" /></tables></p><p>Example 4 Dozens of commercially available blood fixation and staining products were tested in the systems described herein. Such products were generally unsuitable for use in the cell identification, counting, and classification systems described herein. For example, the darkness of staining achieved with these products was compared to that achieved with the formulations described herein. In general, the products examined did not produce optimal sample preparation results as in the formulations described herein (eg, samples were not stained as dark or uniform). Commercially available products produced, for example, lightly stained nuclei and cytoplasm when compared to the formulations described herein.</p><p>Other Embodiments Since the present invention has been described in conjunction with detailed description, the above description is intended as an example and does not limit the scope of disclosure, and the scope of disclosure is the appended claims. It is defined by the range of. Other aspects, advantages, and modifications are within the scope of the following claims.</p><p> All references cited herein, such as patent applications, patent gazettes and patents, are incorporated herein by reference in their entirety.</p>