Staining reagent

Abstract

The invention relates to a staining reagent for carrying out histological and / or hematological stainings, comprising A) at least one solvent in a proportion of 55% by volume to 99.95% by volume, selected from the group consisting of A1) esters of the formula R1-C(=O)-O-R2, A2) ethers of the formula R3-O-R4, A3) sulfoxides of the formula R5-S(=O)-R6, A4) sulfones of the formula R7-S(=O)2-R8, A5) monohydric alcohols in which the alcoholic OH group is bonded to an aliphatic C atom in a hydrocarbon group of 4 to 8 carbon atoms, A6) diols which have 2 OH groups on an aliphatic hydrocarbon radical with 3 to 6 carbon atoms, A7) formals of the formula R9-O-CH2-O-R10, A8) dihydrolevoglucosenone, A9) propylene carbonate, and A10) N-butylpyrrolidone; B) at least one staining pigment in a proportion of 0.05% by volume to 7% by volume; and C) optionally an additional solvent selected from the group consisting of dimethyl sulfoxide, water, monohydric alcohols with 1 to 3 carbon atoms, and sugar alcohols, wherein R1 represents a C4 to C8 alkyl group or a group of the formula R2-O-C(=O)-R-; R2 represents a C1 to C4 alkyl group; R represents a C2 to C6 alkylene group; R3 represents a C2 to C4 alkyl group which can have an OH group; R4 represents a group of the formula -R11-(O-R12-)n-R13; R11 represents a C2 to C4 alkylene group; R12 represents a C1 to C4 alkylene group; R13 represents H, OH or -O-C(=O)-CH3; n represents 0, 1, 2 or 3; and R5 and R6 independently of one another represent a C1 to C4 alkyl group, with the proviso that the sum of the number of carbon atoms in R5 and R6 is 3 or higher, R7 and R8 independently of one another represent a C1 to C4 alkyl group, and R9 and R10 independently of one another represent a C4 to C8 alkyl group.

Description

[0001] Patent application

[0002] Applicant: Bio-Gram Diagnostics GmbH, 67550 Worms

[0003] staining reagent

[0004] The invention relates to a staining reagent for carrying out histological and / or hematological staining.

[0005] In the field of histology, the study of biological tissues, staining methods are used to better distinguish tissue structures during visual examination. Various staining methods are known in this context, such as HE staining (hematoxylin-eosin staining). With this staining, cell nuclei of the tissue under examination appear blue-violet and cytoplasm pink. Intercellular substances also stain, for example, cartilage violet or fibers pink. Staining methods are also used in the field of hematology, the study of blood, bone marrow, and other body fluids and tissues.

[0006] The samples to be examined are applied to a slide and then stained with various staining reagents or staining procedures, depending on the examination method. To achieve optimal staining, it is important that the intended staining procedure, particularly the staining time and the amount of staining reagent applied, are precisely adhered to. Devices are available for this purpose that can automatically stain slides with attached biological samples.

[0007] Such a staining reagent is known from EP 2 457 079 B1. The coloring component of the staining agent is usually a pigment dissolved in a liquid and can thus be conveyed via a peristaltic pump or the like and easily applied to a microscope slide. Depending on the pigment, this pigment attaches to various structures of the object to be stained, so that the structures to be examined stand out more clearly and the objects are easier to examine under the microscope. The staining reagent known from the prior art is an alcoholic solution based on isopropanol, a monohydric alcohol. The use of other monohydric alcohols, for example methanol or ethanol, is also known. A disadvantage of monohydric alcohols is that alcohol vapors are formed during the staining process, which can be harmful to the health of the operating personnel.

[0008] The invention is based on the object of providing a staining reagent for carrying out histological staining which has improved handling.

[0009] The problem is solved by the features of claim 1. The subclaims refer to advantageous embodiments.

[0010] The invention relates to a staining reagent for carrying out histological and / or hematological staining, comprising

[0011] A) at least one solvent in a proportion of 1 vol.% to 99.95 vol.% selected from the group consisting of

[0012] A1 ) Esters of the formula R1-C(=O)-O-R2

[0013] A2) Ethers of the formula R3-O-R4

[0014] A3) Sulfoxides of the formula R5-S(=O)-R6

[0015] A4) Sulfones of the formula R7-S(=O)2-R8

[0016] A5) monohydric alcohols in which the alcoholic OH group is bonded to an aliphatic C atom in a hydrocarbon group of 4 to 8 carbon atoms

[0017] A6) Diols bearing 2 OH groups on an aliphatic hydrocarbon residue with 3 to 6 carbon atoms, A7) Formals of the formula R9-O-CH2-O-R10 A8) Dihydrolevoglucosenone A9) Propylene carbonate,

[0018] A10) N-butylpyrrolidone, and

[0019] B) at least one coloring pigment in a proportion of 0.05 vol.% to 7 vol.%,

[0020] C) optionally a further solvent selected from the group consisting of dimethyl sulfoxide, water, monohydric alcohols having 1 to 3 carbon atoms and sugar alcohols, wherein

[0021] R1 represents a C4 to Cs-alkyl group, or a group of the formula R2-OC(=O)-R-

[0022] R2 represents a Ci to C4 alkyl group, and

[0023] R represents a C2 to Cs-alkylene group

[0024] R3 represents a C2 to C4 alkyl group which may carry an OH group

[0025] R4 represents a group of the formula -R11 -(O-R12-) n -R13

[0026] R11 represents a C2 to C4 alkylene group

[0027] R12 represents a Ci to C4 alkylene group

[0028] R13 is H, OH or -OC(O)-CH3n for 0, 1, 2 or 3

[0029] R5 and R6 independently represent a Ci to C4 alkyl group, with the proviso that the sum of the number of carbon atoms in R5 and R6 is 3 or higher,

[0030] R7 and R8 independently represent a Ci to C4 alkyl group,

[0031] R9 and R10 independently represent a C4 to C8 alkyl group. The staining reagent according to the invention for carrying out histological and / or hematological staining comprises at least one of the components selected from A1 to A10 in a proportion of 55 vol.% to 99.95 vol.% and at least one coloring pigment in a proportion of 0.05 vol.% to 7 vol.%.

[0032] The proportion of the components selected from A1 to A10 is preferably in a range from 70 vol.% to 97 vol.%, more preferably 70 vol.% to 90 vol.%, particularly preferably in a range from 75 vol.% to 85 vol.%, in particular 80 vol.%. The proportion of the coloring pigment is preferably in a range from 1 vol.% to 3 vol.%, in particular 2 vol.%.

[0033] Bulk densities for coloring pigments are generally in the range of 0.7 to 0.8 g / cm 3 , preferably at 0.75 g / cm 3. For the purposes of this invention, a conversion from volume to weight percentage is made with a bulk density of 0.75 g / cm 3 .

[0034] Solvent

[0035] Component A of the coloring reagent according to the invention is a solvent which consists of one or more of the components

[0036] A1 ) Esters of the formula R1-C(=O)-O-R2

[0037] A2) Ethers of the formula R3-O-R4

[0038] A3) Sulfoxides of the formula R5-S(=O)-R6

[0039] A4) Sulfones of the formula R7-S(=O)2-R8

[0040] A5) monohydric alcohols in which the alcoholic OH group is bonded to an aliphatic C atom in a hydrocarbon group of 4 to 8 carbon atoms

[0041] A6) Diols bearing 2 OH groups on an aliphatic hydrocarbon residue with 3 to 6 carbon atoms, A7) Formals of the formula R9-O-CH2-O-R10

[0042] A8) Dihydrolevoglucosenone

[0043] A9) Propylene carbonate, A10) N-butylpyrrolidone. Reference to solvent means one or more of the components A1, A2, A3, A4, A5, A6, A7, A8, A9, and / or A10. In a preferred embodiment, the solvent is a component selected from the group consisting of A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10.

[0044] A1 ) Esters of the formula R1-C(=O)-O-R2

[0045] In one embodiment, the subject matter of the invention is a staining reagent for carrying out histological and / or hematological stainings, comprising at least one ester of the formula R1-C(=O)-O-R2 as component A1 in a proportion of 55 vol.% to 99.95 vol.%, preferably in a range of 70 vol.% to 97 vol.%, more preferably 70 vol.% to 90 vol.%, particularly preferably in a range of 75 vol.% to 85 vol.%, in particular of 80 vol.%, and at least one coloring pigment as component B in a proportion of 0.05 vol.% to 7 vol.%, preferably in a range of 1 vol.% to 3 vol.%, in particular of 2 vol.%, wherein

[0046] R1 represents a C4 to Cs-alkyl group, or a group of the formula R2-OC(=O)-R-,

[0047] R2 represents a Ci to C4 alkyl group, and

[0048] R represents a C2 to Cs alkylene group.

[0049] R1 preferably represents a group of the formula R2-OC(=O)-R-

[0050] R2 preferably represents ethyl or methyl, especially methyl.

[0051] R preferably represents an ethylene, butylene or hexylene group.

[0052] In a preferred embodiment, the ester A1 is a diester. Particularly preferred esters A1 are dimethyl succinate

[0053] Dimethyl glutarate and dimethyl adipate.

[0054] In one embodiment, the subject matter of the invention is a staining reagent for carrying out histological and / or hematological stainings, comprising at least one ether of the formula R3-O-R4 as component A2 in a proportion of 55 vol.% to 99.95 vol.%, preferably in a range of 70 vol.% to 97 vol.%, more preferably 70 vol.% to 90 vol.%, particularly preferably in a range of 75 vol.% to 85 vol.%, in particular of 80 vol.%, and at least one coloring pigment as component B in a proportion of 0.05 vol.% to 7 vol.%, preferably in a range of 1 vol.% to 3 vol.%, in particular of 2 vol.%, wherein

[0055] R3 represents a C2 to C4 alkyl group which may carry an OH group

[0056] R4 represents a group of the formula -R11 -(O-R12-) n -R13

[0057] R11 represents a C2 to C4 alkylene group

[0058] R12 represents a Ci to C4 alkylene group; R13 represents H, OH or -OC(=O)-CH3n represents 0, 1, 2 or 3.

[0059] The ethers A2 of the formula R3-O-R4 are preferably dimers or trimers of ethylene glycol or propylene glycol, in which terminal OH groups may optionally be etherified with a methyl or ethyl group.

[0060] Particularly preferred ethers A2 of the formula R3-O-R4 are diethylene glycol

[0061] Dipropylene glycol monomethyl ether

[0062] Tripropylene glycol

[0063] Diethylene glycol monoethyl ether Diethylene glycol butyl ether acetate

[0064] Tetraoxaundecan

[0065] A3) Sulfoxides of the formula R5-S(=O)-R6

[0066] In one embodiment, the subject matter of the invention is a staining reagent for carrying out histological and / or hematological staining, comprising at least one sulfoxide of the formula R5-S(=O)-R6 as component A3 in a proportion of 55 vol.% to 99.95 vol.%, preferably in a range of 70 vol.% to 97 vol.%, more preferably 70 vol.% to 90 vol.%, particularly preferably in a range of 75 vol.% to 85 vol.%, in particular of 80 vol.%, and at least one coloring pigment as component B in a proportion of 0.05 vol.% to 7 vol.%, preferably in a range of 1 vol.% to 3 vol.%, in particular of 2 vol.%, wherein R5 and R6 independently of one another represent a C1 to C4 alkyl group, with the proviso that the sum of the number of carbon atoms in R5 and R6 is 3 or higher. A4) Sulfones of the formula R7-S 2-R8

[0067] In one embodiment, the subject matter of the invention is a staining reagent for carrying out histological and / or hematological staining, comprising at least one sulfone of the formula R7-S(=O)2-R8 as component A4 in a proportion of 55 vol.% to 99.95 vol.%, preferably in a range of 70 vol.% to 97 vol.%, more preferably 70 vol.% to 90 vol.%, particularly preferably in a range of 75 vol.% to 85 vol.%, in particular of 80 vol.%, and at least one coloring pigment as component B in a proportion of 0.05 vol.% to 7 vol.%, preferably in a range of 1 vol.% to 3 vol.%, in particular of 2 vol.%, wherein R7 and R8 independently of one another represent a Ci to C4 alkyl group.

[0068] It is preferred that R7 and R8 are methyl or ethyl groups, particularly preferably methyl groups. In the particularly preferred embodiment, component A4 is dimethylsulfone

[0069] A5) Monohydric alcohols with 4 to 8 carbon atoms

[0070] In one embodiment, the subject matter of the invention is a staining reagent for carrying out histological and / or hematological stainings, comprising at least one monohydric alcohol as component A5 in a proportion of 55 vol.% to 99.95 vol.%, preferably in a range of 70 vol.% to 97 vol.%, more preferably 70 vol.% to 90 vol.%, particularly preferably in a range of 75 vol.% to 85 vol.%, in particular 80 vol.%, and at least one coloring pigment as component B in a proportion of 0.05 vol.% to 7 vol.%, preferably in a range of 1 vol.% to

[0071] 3 vol.%, in particular 2 vol.%, wherein the alcoholic OH group is bonded to an aliphatic C atom in a hydrocarbon group of 4 to 8 carbon atoms.

[0072] Monohydric alcohols A5 with 4 to 8 carbon atoms are linear, cyclic, or branched alcohols whose hydrocarbon group contains 4 to 8 carbon atoms. The OH group is attached to an aliphatic carbon atom.

[0073] In one embodiment, the monohydric alcohols are alkyl alcohols whose alkyl chain length is 4 to 8 carbon atoms. A chain length of 6 carbon atoms is preferred.

[0074] In a further embodiment, the monohydric alcohol is 2-phenylethanol

[0075] Diols with 3 to 6 carbon atoms

[0076] In one embodiment, the subject matter of the invention is a staining reagent for carrying out histological and / or hematological staining, comprising at least one diol as component A6 in a proportion of 55 vol.% to 99.95 vol.%, preferably in a range of 70 vol.% to 97 vol.%, more preferably 70 vol.% to 90 vol.%, particularly preferably in a range of 75 vol.% to 85 vol.%, in particular of 80 vol.%, and at least one coloring pigment as component B in a proportion of 0.05 vol.% to 7 vol.%, preferably in a range of 1 vol.% to 3 vol.%, in particular of 2 vol.%, wherein the diol carries 2 OH groups on a hydrocarbon radical having 3 to 6 carbon atoms.

[0077] Diols A6 with 3 to 6 carbon atoms are dihydric alcohols that carry two OH groups on a hydrocarbon group with 3 to 6 carbon atoms. The hydrocarbon group can be linear or branched. Diols A6 do not contain ether groups. Examples of diols are 1,6-hexanediol. and 1,2-propanediol

[0078] In one embodiment, the subject matter of the invention is a staining reagent for carrying out histological and / or hematological staining, comprising at least one formula of the formula R9-O-CH2-O-R10 as component A7 in a proportion of 55 vol.% to 99.95 vol.%, preferably in a range of 70 vol.% to 97 vol.%, more preferably 70 vol.% to 90 vol.%, particularly preferably in a range of 75 vol.% to 85 vol.%, in particular of 80 vol.%, and at least one coloring pigment as component B in a proportion of 0.05 vol.% to 7 vol.%, preferably in a range of 1 vol.% to 3 vol.%, in particular of 2 vol.%, wherein R9 and R10 independently of one another represent a C4 to Cs-alkyl group, which may be linear or branched.

[0079] Examples of suitable formals A7 are Butylal and 2-ethylhexylal

[0080] A8) Dihydrolevoglucosenone

[0081] In one embodiment, the subject matter of the invention is a staining reagent for carrying out histological and / or hematological stainings, comprising dihydrolevoglucosenone as component A8 in a proportion of 55 vol.% to 99.95 vol.%, preferably in a range of 70 vol.% to 97 vol.%, more preferably 70 vol.% to 90 vol.%, particularly preferably in a range of 75 vol.% to 85 vol.%, in particular of 80 vol.%, and at least one coloring pigment as component B in a proportion of 0.05 vol.% to 7 vol.%, preferably in a range of 1 vol.% to 3 vol.%, in particular of 2 vol.%.

[0082] Dihydrolevoglucosenone is a compound of the formula A9) Propylene carbonate

[0083] In one embodiment, the subject matter of the invention is a staining reagent for carrying out histological and / or hematological staining, comprising propylene carbonate as component A9 in a proportion of 55 vol.% to 99.95 vol.%, preferably in a range of 70 vol.% to 97 vol.%, more preferably 70 vol.% to 90 vol.%, particularly preferably in a range of 75 vol.% to 85 vol.%, in particular of 80 vol.%, and at least one coloring pigment as component B in a proportion of 0.05 vol.% to 7 vol.%, preferably in a range of 1 vol.% to 3 vol.%, in particular of 2 vol.%.

[0084] Propylene carbonate is a compound of the formula

[0085] A10) N-Butylpyrrolidone

[0086] In one embodiment, the subject matter of the invention is a staining reagent for carrying out histological and / or hematological staining, comprising N-butylpyrrolidone as component A10 in a proportion of 55 vol.% to 99.95 vol.%, preferably in a range of 70 vol.% to 97 vol.%, more preferably 70 vol.% to 90 vol.%, particularly preferably in a range of 75 vol.% to 85 vol.%, in particular of 80 vol.%, and at least one coloring pigment as component B in a proportion of 0.05 vol.% to 7 vol.%, preferably in a range of 1 vol.% to 3 vol.%, in particular of 2 vol.%.

[0087] N-Butylpyrrolidone is a compound of the formula

[0088] An advantageous embodiment of the invention provides that the coloring reagent comprises, in addition to at least one of the components A1, A2, A3, A4, A5, A6, A7, A8, A9, and / or A10 and component B, water. The water is preferably demineralized or deionized water. The addition of water reduces the proportion of solvent, whereby the beneficial effects of the solvent also apply when water is added. At the same time, the manufacturing costs of the coloring reagent can be reduced.

[0089] The proportion of water can be in a range from 0.1 vol.% to 35 vol.%. If water is present, the proportion of solvent is reduced by the proportion of water. The proportion of water is preferably in a range from 10 vol.% to 30 vol.%, particularly preferably in a range from 15 vol.% to 25 vol.%, in particular 20 vol.%. With the aforementioned proportions of water, the proportion of solvent in the coloring reagent is sufficiently large to maintain the positive properties of the solvent with regard to the effectiveness of the coloring pigments in the coloring reagent. If water is present, the proportion of solvent is generally between 55 vol.% and 99.85 vol.%.

[0090] The proportion of lower, monohydric alcohols in the coloring reagent, which comprise 1 to 3 carbon atoms and therefore do not correspond to the solvent A5, is preferably at most 5 vol.%, in particular at most 0.5 vol.%.

[0091] Alcohol is preferably not actively added to the staining reagent during the manufacturing process, but can be included as an accompanying substance in other additives to the staining reagent. Surprisingly, studies have shown that the use of solvents A1 to A10 leads to particularly good staining results, while avoiding the adverse properties of alcohols, particularly with regard to the generation of hazardous alcohol vapors. This improves the handling of the staining reagent, as the proportion of monohydric alcohols commonly used for staining reagents, such as methanol, ethanol, or isopropanol, whose alcohol vapors can be harmful, can be eliminated.

[0092] In this respect, the coloring reagent according to the invention can be free of lower monohydric alcohols which comprise 1 to 3 carbon atoms and do not correspond to the solvent A5.

[0093] The staining reagent may contain alcohols in the form of sugar alcohols. Sugar alcohols are polyhydric alcohols, which are known, for example, as sugar substitutes. The proportion of sugar alcohols in the staining reagent is low, however, and preferably not more than 5 vol.%, in particular between 0.1 and 5 vol.%. Glycerol is particularly suitable as the sugar alcohol. Unlike methanol, ethanol, or isopropanol, glycerol is not a hazardous substance, so the easy handling of the staining reagent according to the invention is not impaired. In particular, staining reagents provided with pigments for Giemsa staining can have a proportion of glycerol added during the manufacturing process. This acts as a stabilizer and improves the Giemsa staining result.

[0094] Apart from polyhydric alcohols, especially sugar alcohols, no other alcohols, especially monohydric alcohols, are preferably added to the coloring reagent. If sugar alcohol is present, the solvent content is between 55% and 99.85% by volume in a preferred embodiment.

[0095] If water and sugar alcohol are present, the solvent content in a preferred embodiment is between 55 vol.% and 99.75 vol.%. Solvents A1 to A10 can have a purity of more than 90%. The staining results of the staining reagent correlate with the purity of the solvent, with the quality of the staining results improving with increasing solvent purity. Good staining results can be achieved at a purity of more than 95%, and such solvents are particularly cost-effective. Better staining results at a reasonable cost can be achieved with a purity of 99%. At a purity of 99.9%, the quality of the staining results improves even further; however, solvents with this level of purity are very expensive. A solvent purity exceeding 99.9% no longer appears economically viable due to the high production costs of such high-purity solvents.

[0096] In one embodiment of the invention, the coloring pigment is dissolved in one or more, preferably one, of the solvents A1 to A10. The coloring reagent is then a solution of the coloring pigment in one or more, preferably one, of the solvents A1 to A10. The coloring reagent according to the invention for carrying out histological and / or hematological staining then comprises at least one of the components selected from A1 to A10 in a proportion of 55 vol.% to 99.95 vol.% and at least one coloring pigment in a proportion of 0.05 vol.% to 7 vol.%. The proportion of the components selected from A1 to A10 in this embodiment is preferably in a range from 70 vol.% to 97 vol.%, more preferably 70 vol.% to 90 vol.%, particularly preferably in a range from 75 vol.% to 85 vol.%, in particular 80 vol.%. The proportion of the coloring pigment is preferably in a range of 1 vol.% to 3 vol.%, in particular 2 vol.%.

[0097] In a further embodiment of the invention, the coloring pigment is dissolved in dimethyl sulfoxide (DMSO), and the resulting solution is then diluted with one or more, preferably one, of the solvents A1 to A10. The coloring reagent is then a solution of the coloring pigment in DMSO and one or more, preferably one, of the solvents A1 to A10. In this embodiment, DMSO replaces part of the solvents A1 to A10. The coloring reagent according to the invention for carrying out histological and / or hematological staining then comprises at least one of the components selected from A1 to A10 in a proportion of 1 vol.% to 89.95 vol.% and at least one coloring pigment in a proportion of 0.05 vol.% to 7 vol.%. The proportion of DMSO is between 10 and 90 vol.%.

[0098] In a further embodiment of the invention, the coloring pigment is dissolved in dimethyl sulfoxide (DMSO), and the resulting solution is then diluted with one or more, preferably one, of the solvents A1 to A10, and with water. The coloring reagent is then a solution of the coloring pigment in DMSO, one or more, preferably one, of the solvents A1 to A10, and water. In this embodiment, water and DMSO replace part of the solvents A1 to A10. The coloring reagent according to the invention for carrying out histological and / or hematological staining then comprises at least one of the components selected from A1 to A10 in a proportion of 1 vol.% to 79.95 vol.% and at least one coloring pigment in a proportion of 0.05 vol.% to 7 vol.%. The proportion of DMSO is between 10 and 80 vol.%. The proportion of water is between 10 and 80 vol.%.

[0099] The preferred mixing ratios of DMSO, preferred solvents A1 to A10, and optionally water are shown in Table 1. The solvents can be used for all coloring pigments.

[0100] The coloring pigments can be selected from eosin and / or erythrosine and / or methylene blue and / or azure I and / or a dye belonging to the thiazine group. The coloring pigments can be selected, in particular, from methylene blue (CAS No. 61-73-4), eosin G (CAS No. 17372-87-1), azure A (CAS No. 531-55-5), and / or 3-methylamino-7-dimethylaminophenothiazin-5-ium chloride. Good coloring results have been achieved with these coloring pigments. The selection of coloring pigments depends on the coloring method. The coloring reagent can contain a stabilizer. The stabilizer serves to extend the shelf life and improve the storage life of the coloring reagent. The stabilizer, in particular, prevents contamination of the coloring reagent, for example, by bacterial and / or fungal attack. This allows good coloring results to be maintained over a long period of time.An antibiotic, such as gentamicin, can be used as a stabilizer. Surfactants are also conceivable as stabilizers. Furthermore, a biocide from the isothiazolinones group can be selected, particularly polyoxyethylene(20) sorbitan monooleate (CAS No. 2682-20-4). The stabilizer content is preferably in the range of 0.05 vol.% to 7 vol.%.

[0101] The coloring reagent may comprise a stabilizing buffer. The stabilizing buffer maintains the pigments dissolved in the coloring reagent within a preselected pH range, preferably within a range of pH 4.5 to pH 6, particularly preferably within a range of pH 6 to pH 6.5. The proportion of the stabilizing buffer is preferably within a range of 0.05 vol.% to 1.5 vol.%. A particularly suitable stabilizing buffer is an amino acid in the form of L-histidine (CAS No. 71-00-1).

[0102] The staining reagent is explained in more detail below. The figures show, schematically:

[0103] Fig. 1 shows a blood smear after Giemsa staining, carried out with a staining reagent according to the invention, the image was taken with an oil immersion objective;

[0104] Fig. 2 shows a blood smear after May-Grünwald staining, carried out with a staining reagent according to the invention, the image was taken with an oil immersion objective;

[0105] Fig. 3 shows a blood smear after Wright staining, performed with a staining reagent according to the invention; the image was taken using an oil immersion objective. The staining reagent contains the components solvent A1, A2, A3, A4, A5, A6, A7, A8, A9 and / or A10 with a purity of more than 90% and a proportion of the staining reagent in a range from 55 vol.% to 99.95 vol.%, as well as color pigments in a range from 0.05 vol.% to 7 vol.%. The proportion of the color pigments in the staining reagent can be between 0.05 vol.% and 0.25 vol.% of the staining reagent. Stirring the components results in a homogeneous mixture without the components reacting chemically with one another. The coloring pigments are initially present as solids and are dissolved in the solvent so that the coloring reagent is present as a solution.

[0106] The coloring pigments can be selected from eosin and / or erythrosine and / or methylene blue and / or azure I and / or a dye belonging to the thiazine group. The coloring pigments can be selected, in particular, from methylene blue (CAS No. 61-73-4), eosin G (CAS No. 17372-87-1), azure A (CAS No. 531-55-5), and / or 3-methylamino-7-dimethylaminophenothiazin-5-ium chloride. The selection of the coloring pigments depends on the dyeing process to be performed.

[0107] The staining reagent may also contain water. The proportion of water in the staining reagent can range from 0.1 vol.% to 35 vol.%.

[0108] The staining reagent may contain polyhydric alcohols in the form of sugar alcohols. The sugar alcohol content is preferably no more than 5% by volume.

[0109] The staining reagent may comprise a stabilizer. The stabilizer content is preferably in a range of 0.05 vol.% to 7 vol.%.

[0110] The staining reagent may comprise a stabilizing buffer. The proportion of the stabilizing buffer is preferably in a range from 0.05 vol.% to 1.5 vol.%. The staining reagent may be used in a staining device. A device for staining biological samples adhering to a slide comprises a staining device for staining the biological samples. The staining device comprises a staining plate or staining chamber into which at least one opening is provided. If the staining reagent is passed through the opening in the direction of a

[0111] As the staining surface is promoted, it is evenly distributed over the surface of the slide and stains the biological structures. The staining procedures are always carried out at room temperature. Specific staining reagent compositions that can achieve good staining results are shown in Table 1 below. The volume fractions of the components refer to a temperature of 21 °C.

[0112] The proportions given in vol.% include the tolerances permissible in the field of staining reagent production.

[0113] Table 1 Very good staining results can be achieved with all of the staining reagents listed in the table, and the staining reagent is equally suitable for all staining methods. Accordingly, the compositions of selected examples are explained in more detail below.

[0114] The coloring pigments are Azure A (CAS No. 531-55-5). Alternatively, the coloring pigments can contain eosin, erythrosine, methylene blue, Azure I, and / or a dye belonging to the thiazine group. The stabilizer is polyoxyethylene(20) sorbitan monooleate (CAS No. 2682-20-4), and the stabilizing buffer is L-histidine. The coloring reagent is free of monohydric alcohols, and in this case, also free of polyhydric alcohols.

[0115] The figures show red and white blood cells 1, 2. The red and white blood cells 1, 2 are disc-shaped. Predominantly red blood cells 1, which lack a nucleus, are present. A few white blood cells 2 are also present. The white blood cells 2 contain a nucleus 3. The white blood cells 2 are approximately twice as large in diameter as the red blood cells 1.

[0116] It is clearly visible that the staining reagent provides particularly strong contours for the edges of the red and white blood cells 1 and 2, allowing for sharp differentiation. Irregularities or fraying in the borders are also clearly visible. Furthermore, the cell nuclei 3 of the white blood cells 2 are sharply demarcated from the white blood cell 2.

Claims

Patent claims 1. Staining reagent for performing histological and / or hematological staining, comprising A) At least one solvent in a proportion of 1 vol.% to 99.95 vol.% selected from the group consisting of A1) Esters of the formula R1 -C(=O)-O-R2 A2) Ethers of the formula R3-O-R4 A3) Sulfoxides of the formula R5-S(=O)-R6 A4) Sulfones of the formula R7-S(=O)2-R8 A5) monohydric alcohols in which the alcoholic OH group is bonded to an aliphatic C atom in a hydrocarbon group of 4 to 8 carbon atoms A6) Diols which carry 2 OH groups on an aliphatic hydrocarbon residue with 3 to 6 carbon atoms, A7) Formal of the formula R9-O-CH2-O-R10 A8) Dihydrolevoglucosenone A9) Propylene carbonate, A10) N-butylpyrrolidone, and B) at least one coloring pigment in a proportion of 0.05 vol.% to 7 vol.%, C) optionally a further solvent selected from the group consisting of dimethyl sulfoxide, water, monohydric alcohols having 1 to 3 carbon atoms and sugar alcohols, wherein R1 represents a C4 to C8 alkyl group, or a group of the formula R2-OC(=O)-R- R2 represents a Ci to C4 alkyl group, and R represents a C2 to C1-alkylene group R3 represents a C2 to C4 alkyl group which may carry an OH group R4 represents a group of the formula -R11 -(O-R12-) n -R13 R11 represents a C2 to C4 alkylene group R12 represents a Ci to C4 alkylene group R13 is H, OH or -OC(=O)-CH3n for 0, 1, 2 or 3 R5 and R6 independently represent a Ci to C4 alkyl group, with the proviso that the sum of the number of carbon atoms in R5 and R6 is 3 or higher, R7 and R8 independently represent a Ci to C4 alkyl group R9 and R10 independently represent a C4 to Cs alkyl group.

2. Staining reagent according to claim 1, characterized in that the staining reagent comprises water.

3. Staining reagent according to claim 2, characterized in that the proportion of water in the staining reagent is in a range from 0.1 vol.% to 35 vol.% and the proportion of the solvent(s) A1 to A10 is between 55 vol.% and 99.85 vol.%.

4. Staining reagent according to one of claims 1 to 3, characterized in that the proportion of alcohols having 1 to 3 carbon atoms in the staining reagent is at most 5 vol.%.

5. Staining reagent according to one of claims 1 to 4, characterized in that the staining reagent contains alcohols in the form of sugar alcohols in an amount of at most 5 vol.%.

6. Staining reagent according to one of claims 1 to 5, characterized in that the solvent has a purity of more than 90%.

7. Coloring reagent according to one of claims 1 to 6, characterized in that the proportion of coloring pigments in the coloring reagent is between 1 vol.% to 3 vol.%.

8. Staining reagent according to one of claims 1 to 7, characterized in that the proportion of solvent in the staining reagent is between 55 vol.% to 97 vol.%.

9. Coloring reagent according to one of claims 1, 2 and 4 to 8, characterized in that it comprises 55 vol.% to 99.5 vol.% of one or more of the solvents A1 to A10, and A) at least one coloring pigment in a proportion of 0.05 vol.% to 7 vol.%.

10. Coloring reagent according to one of claims 1, 2 and 4 to 8, characterized in that it comprises at least one solvent selected from A1 to A10 in a proportion of 1 vol.% to 89.95 vol.%, at least one coloring pigment in a proportion of 0.05 vol.% to 7 vol.% and DMSO in a proportion of between 10 and 90 vol.%.

11. Coloring reagent according to one of claims 1, 2 and 4 to 8, characterized in that it comprises at least one solvent selected from A1 to A10 in a proportion of 1 vol.% to 79.95 vol.%, at least one coloring pigment in a proportion of 0.05 vol.% to 7 vol.%, DMSO in a proportion of between 10 and 80 vol.% and water in a proportion of between 10 and 80 vol.%.

12. Staining reagent according to one of claims 1 to 11, characterized in that the coloring pigments are selected from eosin and / or erythrosin and / or methylene blue and / or azure blue I and / or from a dye belonging to the group of thiazines.

13. Staining reagent according to one of claims 1 to 12, characterized in that the staining reagent comprises a stabilizer.

14. Staining reagent according to one of claims 1 to 13, characterized in that the staining reagent comprises a stabilizing buffer.

15. Staining reagent according to one of claims 1 to 14, characterized in that it contains an ester of the formula R1 -C(=O)-O-R2 as component A1, wherein R1 represents a group of the formula R2-O-C(=O)-R- R2 represents ethyl or methyl, in particular methyl, and R represents an ethylene, butylene or hexylene group.

16. Staining reagent according to claim 15, characterized in that the ester A1 is selected from the group consisting of dimethyl succinate Dimethyl glutarate and dimethyl adipate.

17. Staining reagent according to one of claims 1 to 14, characterized in that it contains an ether of the formula R3-O-R4 as component A2, which is a dimer or trimer of ethylene glycol or propylene glycol, in which terminal OH groups may optionally be etherified with a methyl or ethyl group.

18. Staining reagent according to one of claims 1 to 14, characterized in that the ether A2 is selected from the group consisting of diethylene glycol Dipropylene glycol monomethyl ether Tripropylene glycol Diethylene glycol monoethyl ether Diethylene glycol butyl ether acetate Tetraoxaundecan 19. Staining reagent according to one of claims 1 to 14, characterized in that it contains a sulfoxide of the formula R5-S(=O)-R6 as component A3, wherein R5 and R6 independently of one another represent a C2 to C4 alkyl group.

20. Staining reagent according to one of claims 1 to 14, characterized in that it contains a sulfone of the formula R7-S(=O)2-R8 as component A4, wherein R7 and R8 are methyl or ethyl groups, preferably methyl groups. 21 . Staining reagent according to claim 20, characterized in that component A4 is dimethylsulfone.

22. Staining reagent according to one of claims 1 to 14, characterized in that it contains a monohydric alcohol as component A5, which is a C4 to C5 alkyl alcohol, preferably a Ce alkyl alcohol.

23. Staining reagent according to one of claims 1 to 14, characterized in that it contains a monohydric alcohol as component A5, which is 2-phenylethanol.

24. Staining reagent according to one of claims 1 to 14, characterized in that it contains a diol as component A6, which is selected from 1,6-Hexanediol and 1,2-propanediol 25. Staining reagent according to one of claims 1 to 14, characterized in that it contains a formula of the formula R9-O-CH2-O-R10 as component A7, which is selected from butylal and 2-ethylhexylal is selected.

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