Stabilized Hematoxylin Stabilisiertes Hämatoxylin Hématoxyline Stabilisée

(19) TZZ _¥ZZ ¥_T

(11) EP 2 130 023 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: G01N 1/30 (2006.01) 06.05.2015 Bulletin 2015/19 (86) International application number: (21) Application number: 08743909.7 PCT/US2008/057035

(22) Date of filing: 14.03.2008 (87) International publication number: WO 2008/112993 (18.09.2008 Gazette 2008/38)

(54) STABILIZED HEMATOXYLIN STABILISIERTES HÄMATOXYLIN HÉMATOXYLINE STABILISÉE

(84) Designated Contracting States: •TOWNE,Penny AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Tucson, AZ 85742 (US) HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT • WILLOUGHBY KIVI, Linda RO SE SI SK TR Marana, AZ 85653 (US)

(30) Priority: 15.03.2007 US 895007 P (74) Representative: Müller-Boré & Partner Patentanwälte PartG mbB (43) Date of publication of application: Friedenheimer Brücke 21 09.12.2009 Bulletin 2009/50 80639 München (DE)

(73) Proprietor: Ventana Medical Systems, Inc. (56) References cited: Tucson, Arizona 85755 (US) EP-A- 0 985 929 EP-A- 1 048 289 EP-A- 1 331 252 JP-A- 4 202 119 (72) Inventors: JP-A- 59 051 279 US-A- 4 502 864 • KOSMEDER, Jerome, W. US-A- 4 816 244 US-A- 5 772 699 Tucson, AZ 85750 (US) US-A- 5 788 754 • BIENIARZ, Christopher Tucson, AZ 85718 (US)

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 130 023 B1

Printed by Jouve, 75001 PARIS (FR) 1 EP 2 130 023 B1 2

Description lution wherein the concentration of hematein available for staining is better stabilized over time. [0001] The present invention relates to a composition [0006] In one aspect the present invention provides a and method for histochemical staining of biological sam- stabilized hematoxylin composition including: a solvent; ples. More particularly, the present invention relates to a 5 hematoxylin; an amount of a chemical oxidant sufficient dye formulation that is stabilized against degradation to convert at least a portion of the hematoxylin to he- over time, and use of the formulation to stain biological matein; a mordant; and both of cyclodextrin and hydro- samples. quinone. In a particular embodiment the stabilized he- [0002] Several histochemical staining protocols, in- matoxylin solution includes hematoxylin, water, a polyol, cluding Hematoxylin and Eosin (H&E) staining and Pa- 10 an amount of an oxidant sufficient to convert at least a panicolaou (PAP) staining, rely on the dye hematoxylin portion of the hematoxylin to hematein;a mordant; and to stain cytological and tissue samples. In particular, he- both of cyclodextrin and hydroquinone. matoxylin staining of cell nuclei is used by pathologists [0007] The composition includes: a solvent; hematox- to detect the presence of malignant and/or metastatic ylin; an amount of a chemical and both of cyclodextrin cells in a tumor biopsy sample. 15 and hydroquinone. In a particular embodiment, a and [0003] Hematoxylin is a naturally-occurring compound both of cyclodextrin and hydroquinone. found in the red heartwood of trees of the genus Hema- [0008] In another aspect, the present invention relates toxylin.Hematoxylin itselfis colorlessin aqueous solution to an automated method for histochemical staining of a and is not the active ingredient that stains tissue compo- biological sample. The method includes contacting the nents. Rather, an oxidation product of hematoxylin, he- 20 biological sample with a disclosed hematoxylin compo- matein, becomes the active staining component of a he- sition, and can further include contacting the sample with matoxylin dye solution, particularly upon complexation one or more additional staining compositions, such as with a mordant. Hematein is produced naturally through one or more counter-stains. In a particular embodiment, exposure to air and sunlight. The natural process is the method further includes contacting the sample with termed "ripening," and can take 3 or more months to pro- 25 an eosin composition. In another particular embodiment, vide a solution suitable for staining cells. the method is automated. [0004] In order to accelerate the conversion of hema- [0009] In another aspect, the present invention pro- toxylin to hematein, a chemical oxidant can be utilized. vides a method for making a stabilized hematoxylin com- Unfortunately, the accelerated process often produces position that can be used for histochemical staining. The ineffective reaction products such as oxyhematein and 30 method includes forming a hematein solution, adding a complex polymeric precipitates, and also provides a so- mordant to the hematein solution to form a staining solution that degrades faster than a naturally ripened dye lution, and adding both of cyclodextrin and hydroquinone solution. The exact amount of oxidant needed to quanti- to the staining solution to form a stabilized hematoxylin tatively oxidize hematoxylin to hematein can be used to composition. The hematein solution is formed by dissolv- help avoid over-oxidation to ineffective products, but a 35 ing hematoxylin in a solvent and then adding a chemical partially-oxidized solution is more typically utilized when oxidant to convert at least a portion of the hematoxylin staining is not performed immediately. In a partially-oxi- into hematein. dized solution, natural oxidation of the hematoxylin that is remaining after a chemical oxidation step will continue FIG. 1 is a block diagram outlining an automated to replace any hematein that is either consumed during 40 H&E staining protocol into which the disclosed he- staining or is naturally oxidized further to ineffective prod- matoxylin composition can be incorporated. ucts. Still, the concentration (and amount) of hematein FIG. 2 is diagram showing stability results for several can change over time. embodiments of the disclosed hematoxylin compo- [0005] Since hematein is the active staining composition. nent of a hematoxylin solution, changes in its concentra- 45 FIG. 3 is another diagram showing stability results tion (and/or the concentration of its mordant complexes) for several embodiments of the disclosed hematox- over time leads to staining inconsistencies. In a manual ylin composition. staining procedure, changes in hematein content of a hematoxylin solution can be compensated for by altering [0010] The following description of several embodi- the contact time of a biological sample with the solution 50 ments describes non-limiting examples that further illus- based on visual inspection. For example, an apparently trate the invention. All titles of sections contained herein, under-stained sample can simply be placed back into the including those appearing above, are not to be construed hematoxylin solution for a period of time to increase the as limitations on the invention, but rather are provided to staining intensity. In an automated staining procedure, structure the illustrative description of the invention that however, "visual" inspection and extension of the expo- 55 is provided by the specification. In order to aid the reader sure time in response to under-staining can require costly in understanding the various illustrated embodiments, imaging equipment and can disrupt processing of other explanations of particular terms utilized in the description samples. Therefore, a need exists for a hematoxylin so- are provided, after which an overview of particular em-

2 3 EP 2 130 023 B1 4 bodiments of the invention and specific examples are herein, generically refers both to compositions formed provided. by dissolving hematein (the oxidation product of hematoxylin) directly into a solvent and to compositions formed I. Terms: by dissolving hematoxylin in a solvent and allowing or 5 promoting oxidation of the hematoxylin to hematein. Al- [0011] Unless defined otherwise, all technical and sci- though it is more typical to prepare the disclosed com- entific terms used herein have the same meanings as positions by dissolving hematoxylin in a solvent and con- commonly understood by one skilled in the art to which verting the hematoxylin to hematein (either completely the disclosed invention pertains. or partially) by natural oxidation through contact with air [0012] The singular forms "a," "an," and "the" include 10 or accelerated chemical oxidation, the benefits of the sta- plural referents unless the context clearly indicates oth- bilizing effects of the disclosed composition components erwise. Thus, for example, reference to "a host com- can also be utilized in combination with hematein compound" refers to one or more host compounds, such as positions prepared by directly dissolving hematein in sol- 2 or more host compounds, 3 or more host compounds, vent. Thus, in some embodiments, a "hematoxylin com- or even 4 or more host compounds. 15 position" will include, at least initially, little or no hema- [0013] The term "antioxidant" refers to an atom or mol- toxylin, and consist primarily of hematein. Host com- ecule that has a greater oxidation potential than a second pounds can be included at concentrations ranging from atom or molecule, such that the antioxidant is preferen- about 1 mM to about 1M, for example, from about 5 mM tially oxidized instead of the second atom or molecule. to about 500 mM, such as from about 5 mM to about 25 For example, an antioxidant can have a greater oxidation 20 mM. potential than hematein, and thus help prevent oxidation [0017] The cyclodextrin used in the solution of the of hematein to oxyhematein. Furthermore, an antioxidant present inventioncan include α-cyclodextrin, β-cyclodex- also can function as a reducing agent, for example, a trin, y-cyclodextrin, and δ-cyclodextrin, and derivatives reducing agent that converts oxyhematein back to he- of each of these classes of cyclodextrins. Particular ex- matein. Antioxidants can be present in the disclosed25 amples of cyclodextrin derivatives, include hydroxypro- compositionsat concentrations rangingfrom about about pylated α-cyclodextrin, hydroxypropylated β-cyclodex- 1 mM to about 1M, for example, from about 5 mM to trin, hydroxypropylated γ-cyclodextrin, hydroxyethylated about 500 mM, such as from about 50 mM to about 150 α-cyclodextrin, hydroxyethylated β-cyclodextrin, hydrox- mM. yethylated γ-cyclodextrin, hydroxyisopropylated α-cyclo- [0014] The term "aqueous solvent" refers to a compo- 30 dextrin, hydroxyisopropylated β-cyclodextrin, hydrox- sition having water as the major component and that is yisopropylated γ-cyclodextrin, carboxymethylated α-cy- a liquid at room temperature. Mixtures of water and one clodextrin, carboxymethylated β-cyclodextrin, car- or more lower alkanols or polyols that have 50% or great- boxymethylated γ-cyclodextrin, carboxyethylated α-cy- er water content by volume are examples of aqueous clodextrin, carboxyethylated β-cyclodextrin, carbox- solvents. 35 yethylated γ-cyclodextrin, octyl succinated-α-cyclodex- [0015] The term "biological sample" refers to any sam- trin, octyl succinated-β-cyclodextrin, octyl succinated-γ- ple that is obtained from or otherwise derived from a bi- cyclodextrin, acetylated-α-cyclodextrin, acetylated β -- ological entity such as an animal, for example, a sample cyclodextrin, acetylated γ --cyclodextrin, sulfated-α-cy- obtained from a human or a veterinary animal such as a clodextrin, sulfated-β-cyclodextrin and sulfated-γ-cyclo- dog, cat, horse or cow. Examples of biological samples 40 dextrin. Other particular examples of cyclodextrins deriv- include cytology samples, tissue samples and biological atives include the followingβ -cyclodextrin derivatives: fluids. Non-limiting particular examples of biological sam- 2,3-dimethyl-6-aminomethyl-β-cyclodextrin, 6-Azido-β- ples include blood, urine, pre-ejaculate, nipple aspirates, cyclodextrin, 6-Bromo-β-cyclodextrin, 6A,6B-dibromo-β- semen, milk, sputum, mucus, pleural fluid, pelvic fluid, cyclodextrin, 6A,6B-diiodo-β-cyclodextrin, 6-O-Maltosyl- sinovial fluid, ascites fluid, body cavity washes, eye45 β-cyclodextrin, 6-Iodo-β-cyclodextrin, 6-Tosyl-β-cyclo- brushings, skin scrapings, a buccal swab, a vaginal dextrin, Peracetyl-maltosyl-β-cyclodextrin, 6-t-butyld- swab, a pap smear, a rectal swab, an aspirate, a needle imethylsilyl-β-cyclodextrin, 2,3-diacetyl-6-butyldimethyl- biopsy, a section of tissue obtained for example by sur- silyl-β-cyclodextrin, 2,6-dibutyl-3-acetyl-β-cyclodextrin, gery or autopsy, plasma, serum, spinal fluid, lymph fluid, 2,6-dibutyl-β-cyclodextrin, 2,6-t-butyl-dimethylsilyl-β-cy- sweat, tears, saliva, tumors, organs and samples ob- 50 clodextrin, and 2,6-di-O-methyl-3-allyl-β-cyclodextrin. A tained from in vitro cell or tissue cultures. Typically, the variety of cyclodextrins and cyclodextrin derivatives can sample will be a biopsy sample that has been fixed, proc- be obtained commercially, for example, from CTD, Inc. essed to remove water and embedded in paraffin or an- (High Springs, FL), or they can be synthesized according other suitable waxy substance for cutting into tissue sec- to procedures outlined in the scientific literature, for ex- tions. Biological samples can be mounted on substrates 55 ample, in "Synthesis of Chemically Modified Cyclodex- such as microscope slides for treatment and/or exami- trins," Croft and Bartsch, Tetrahedron, 39: 1417-1474, nation. 1983. [0016] The term "hematoxylin composition," as used [0018] The term "lower alkanol" refers to a compound

3 5 EP 2 130 023 B1 6 having the formula R-OH, where R is an alkyl group hav- hematoxylin, a disclosed hematoxylin composition also ing between 1 and 5 carbon atoms such as a methyl appears to have a higher effective dye concentration that group, an ethyl group, ann -propyl group, an isopropyl permits darker staining of biological samples in a prede- group, an n-butyl group, a sec-butyl group, a t-butyl termined amount of time, which is especially advanta- group, an n-pentyl group, an isopentyl group or a neo- 5 geous in an automated staining method where a biolog- pentyl group. Examples of lower alkanols include meth- ical sample mounted on a microscope slide, and even anol, ethanol and isopropanol. more advantageous if the slide is processed in a hori- [0019] The term"oxidant" refers to an atom or molecule zontal position. All known hematoxylin compositions and having a greater reduction potential than a second mol- all histochemical staining methods utilizing hematoxylin ecule, for example, a greater reduction potential than he- 10 as part of the staining process can benefit from applica- matoxylin such that it will react with and oxidize hema- tion of the teachings of the present disclosure. Further- toxylin to hematein. Oxidants include naturally occurring more the benefits can be extended to "special stains" molecular oxygen in the atmosphere that diffuses to and and the automated application of such special stains to oxidizes hematoxylin and a "chemical oxidant" that is ac- biological samples (such as on the NexES™ Special tively combined with hematoxylin (typically in solution) to 15 Stainer (Ventana Medical Systems, Inc., Tucson, AZ). convert at least a portion of the hematoxylin to hematein. [0024] In one embodiment, the disclosed composition Examples of useful chemical oxidants include one or includes two or more different antioxidants such as two more of an iodate salt (such as sodium iodate and po- or more water-soluble antioxidants. In other even more tassium iodate), mercuric oxide, a permanganate salt particular embodiments, the composition includes one or (such as potassium permanganate), a periodate salt20 more host compounds and one or more antioxidants. (such as sodium periodate and potassium periodate), [0025] In some embodiments, the solvent is an aque- and a peroxide (such as hydrogen peroxide). In particular ous solvent and the antioxidant is a water-soluble anti- embodiments, the chemical oxidant comprises sodium oxidant. Examples of water soluble antioxidants include iodate. hydroquinones; n-alkyl gallates (such as n-propyl, n-oc- [0020] The term "mordant" refers to an ionic metal spe- 25 tyl, and n-dodecyl gallates); reducible sugars such as cies with which a dye (such as hematein) can form a sorbitol and mannitol; benzoates and hydroxybenzoates; complex (such as a cationic complex) that serves to bind sulfites and metabisulfites; certain acids such as citric the dye (such as hematein) to particular cellular compo- acid, tartaric acid, lactic acid, erythorbic acid ascorbic nents such as nuclear DNA, myelin, elastic and collagen acid, uric acid, tannic acid, and salts of such acids (such 30 2+ + + + 2+ fibers, muscle striations and mitochondria. Examples of as Mg , NH4 , Na , K and Ca salts); chelators such mordants include aluminum (for example, in the form of as EDTA that remove metals that function as oxidants; an alum such as aluminum sulphate, aluminum potassi- and choral hydrate. In particular embodiments, the water um sulphate or aluminum ammonium sulphate), iron, soluble antioxidantincludes one or more of hydroquinone tungsten, zirconium, bismuth, molybdenum (phospho- and n-propyl gallate. molybdic acid or molybdic acid), vanadium (vanadate). 35 [0026] Various solvents can be utilized for the compo- [0021] The term "water-soluble antioxidant" refers to sition, but typically the solvent comprises one or more of an antioxidant that has a solubility in water at 25°C that water, a lower alkanol such as ethanol, and a polyol. In is sufficient to provide a concentration of the antioxidant particular embodiments, the solvent comprises an aque- of at least 1mM, such as greater than 5mM, greater than ous solvent wherein the aqueous solvent comprises wa- 10mM, or even greater than 50mM. 40 ter and a polyol. Particular examples of useful polyols include glycerol, ethylene glycol, propylene glycol, poly II. Overview (ethylene glycol), and poly (propylene glycol). Aqueous solvent compositions typically will comprise 5-45% by [0022] A stabilized hematoxylin composition is dis- volume of one or more of ethylene glycol and propylene closed, which composition can be used for staining of a 45 glycol, and more typically 10-30% by volume of one or biological sample, and in particular, for staining the nuclei more of ethylene glycol and propylene glycol. of cells in the biological sample. The composition of the [0027] The amount of chemical oxidant utilized in some invention includes mordant hematein (such as hemalum) embodiments of the composition can be sufficient to com- stabilized by both of cyclodextrin and hydroquinone. The pletely (such as substantially quantitatively) oxidize the hematoxylin compositions of the invention show im-50 hematoxylin to hematein, or sufficient only to partially ox- proved stability over similar hematoxylin hemalum) sta- idize the hematoxylin to hematein. In particular embodi- bilized by both of cyclodextrin and hydroquinone. The ments, more than half of the hematoxylin is oxidized to compositions not including cyclodextrin and hydroqui- hematein by the chemical oxidant, and in others, less none. than half of the hematoxylin is oxidized to hematein by [0023] Likewise, the use of antioxidants and host com- 55 thechemical oxidant. Forexample, between 1%and 50% pounds to increase stability of other histochemical dye of the hematoxylin can be oxidized to hematein by the compositions and their use in histochemical staining chemical oxidant, but more typically, between about 10% methods are contemplated. Furthermore, in the case of and about 30% of the hematoxylin is oxidized to hematein

4 7 EP 2 130 023 B1 8 bythe chemical oxidant.In particularexamples, the molar Oxford: BIOS, ISBN 1859960995, 2002. In other embod- ratio of hematoxylin to oxidant used in the composition iments, contacting the sample with the hematoxylin com- is between 6:1 and 1:1. It should be understood that al- position comprises a progressive hematoxylin staining though the chemical oxidant is considered part of the protocol. In yet others, contacting the sample with the composition, it is converted to its reduction products upon 5 hematoxylin composition comprises a regressive hema- reaction with the hematoxylin, which reduction products toxylin staining protocol. The method can be performed will remain in the composition. on a biological sample that is supported on a substrate [0028] The mordant of the composition can be any such as a microscope slide. In particular embodiments, mordant such as one or more of an aluminum mordant, the method is used to stain a tissue section or a cytology an iron mordant, a bismuth mordant, a copper mordant, 10 sample mounted on a microscope slide. In particular em- a molybdenum mordant, a vanadium mordant, and a zir- bodiments further including a counterstaining step, the conium mordant. In some embodiments, the mordant method can be an H&E staining method or a PAP staining comprises an alum, and in more particular embodiments, method, and more particularly an automated H&E or PAP the mordant comprises aluminum sulphate. The mordant staining method. can be present in the composition at a concentration15 [0031] In a further aspect, a method is disclosed for greater than the concentration of the hematein in the making a stabilized hematoxylin composition for histo- composition (determinable by refractometry, thin-layer chemical staining of a biological sample. In one embod- chromatography or spectroscopy), or it can be present iment, the method for making the stabilized hematoxylin in the composition at a concentration less than the con- solution includes forming a hematein solution, adding a centration of the hematein in the composition. Alterna- 20 mordant to the hematein solution to form a staining so- tively, in some embodiments, the molar ratio of hema- lution, and adding both of cyclodextrin and hydroquinone toxylin to mordant in the composition is between 2:1 and to the staining solution to adding both of cyclodextrin and 1:100, and in particular embodiments, the molar ratio of hydroquinone to the staining solution to form the stabi- hematoxylin to mordant in the composition is between lizedhematoxylin composition. Forming thehematein so- 1:5 and 1:20. 25 lution comprises dissolving hematoxylin in a solvent and [0029] In some embodiments, the composition further adding an amount of a chemical oxidant sufficient to cov- includes an acid such as acetic acid. In other embodi- ert at least a portion of the hematoxylin to hematein. In ments, no acid is added, and the absence of the acid particular embodiments, the solvent used to dissolve the surprisingly still provides a stabilized and effective he- hematoxylin comprises an aqueous composition such as matoxylin composition. In other embodiments, the com- 30 composition including water and a polyol. Useful polyols, position further includes a buffer to control pH, for exam- as indicated before, include glycerol, ethylene glycol and ple, a buffer to control the pH near a pH between 1 and propylene glycol. 4, such as a pH near 2.5. [0030] In some particular embodiments the composi- III. Examples tion comprises a mixture of water and ethylene glycol as 35 the solvent, sodium iodate as the oxidant, aluminium sul- [0032] Although the method and composition of the phate as the mordant cyclodextrin as the host compound disclosure can be applied to any histological staining and hydroquinone as a water soluble antioxidant. In even process (manual or automated) or any slide staining in- more particular embodiments, the mixture of water and strument, the disclosed hematoxylin composition is par- ethylene glycol comprises from 10- 40%by volume eth- 40 ticularly useful when incorporated into the automated ylene glycol and from 60- 90%water. In another aspect, H&E staining process developed for use in the high vol- the present invention relates to an automated method for ume slide processing system that is described in U.S. histochemical staining of a biological sample, which Patent Application Publication Nos. 20040002163 and method includes contacting the biological sample with a 20050186114. Briefly, the automated slide processing disclosed hematoxylin composition and can further in- 45 system that is described in the aforementioned applica- clude contacting the sample with a counterstain. In some tions is a high-volume slide processing system that shut- embodiments, contacting the sample with a counterstain tles trays holding a plurality of slides in substantially hor- comprises contacting the sample with one or more of izontal positions (to minimize cross-contamination) be- eosin Y, orange G, light green SF yellowish, Bismark tween workstations that perform various slide processing Brown, fast green FCF, OA-6, EA25, EA36, EA50 and 50 operations on the slides. Fresh reagents can be applied EA65. The formulas and methods of making such coun- to each slide during processing, and cross-contamination terstains can be found, for example, in the StainsFile (an of slides with reagents can be substantially eliminated internet resource for histotechnologists maintained by because the slides are treated separately in spaced- Bryan Llewellyn); Kiernan, "Histological and Histochem- apart fashion in the tray. In one configuration, the system ical methods: Theory and Practice," 3rd Ed. Butterworth 55 includes a radiant heater, a combined de-paraffiniz- Heinemann, Oxford, UK; and in Horobin and Kiernan, er/stainer/solvent exchanger workstation, a convection "Conn’s biological stains: a handbook of dyes, stains and oven and a coverslipper. A tray of slides bearing paraffin- fluorochromes for us in biology and medicine," 10th ed., embedded tissue samples can be heated under the ra-

5 9 EP 2 130 023 B1 10 diant heater of the system to spread the paraffin in the 2) Hematoxylin dye (Dudley Chemical Corp, Lake- samples for easier removal and also to adhere the sam- wood, NJ), in the concentrations indicated in FIGS. ples to the slides. The tray can then be transported to 2 and 3, was then added to the solvent to form a the multifunctional de-paraffinizer/stainer/solvent ex- hematoxylin solution. changer workstation, where slides can be de-paraffin- 5 3) Sodium iodate (Sigma-Aldrich, St. Louis, MO) was ized, stained, and solvent exchanged. A tray of stained added in the concentrations indicated in FIGS. 2 and slides that is ready for coverslipping can then be shuttled 3 and allowed to oxidize the hematoxylin to he- to the coverslipper of the system where coverslips are matein, thereby forming a hematein solution having added to the slides. Once the slides are coverslipped, an initial molar concentration of hematein approxi- the tray can then be transported to the convection oven 10 mately equal to the molar concentration of the he- to cure the coverslips on the stained slides. Reagents for matoxylin minus the molar concentration of the so- the system can be provided in bag-in-box containers that dium iodate. are loaded into the fluidics module of the system. The 4) Aluminum sulphate octadecahydrate (JT Baker, high volume stainer just described is commercially avail- Phillipsburg, NJ) was added to the hematein solution able from Ventana Medical Systems, Inc, Tucson, AZ. 15 in the concentration indicated in FIGS. 2 and 3 to [0033] While the staining system just described can be form a hemalum solution. configured to perform any histological staining process, 5) Combinations of hydroquinone, n-propyl gallate the system was configured to perform a progressive H&E and β-cyclodextrin hydrate (all available from Sigma- stain using the disclosed hematoxylin compositions that Aldrich, St. Louis, MO) were then added in the con- are described in detail below. A schematic showing the 20 centrations indicated in FIGS. 2 and 3 to form the overall process is shown in FIG. 1, which process in- tested compositions. cludes: a baking step to adhere the samples to the slides, 6) The compositions were placed into separate bag- a de-paraffinization step to remove paraffin from paraffin- in-box containers that are used for on-board storage embedded samples, a hematoxylin staining step (that of reagents in the automated staining system de- can utilize the disclosed hematoxylin compositions), a 25 scribed above. bluing step that raises the pH and turns the hematoxylin blueto provide better contrastwith the eosin added down- [0036] No acid was added to the compositions used stream, an eosin staining step, a differentiation step that for these examples. is used to remove excess eosin and turn the eosin various [0037] FIGS. 2 and 3 summarize 8 different composi- shades of red to pink, a dehydration step to remove water 30 tions and the results of stability testing at several tem- from the sample using 100% ethanol, a step in which the peratures based upon observation of precipitates in the slides are exposed to an elevated temperature and air bag-in-box containers. In all cases, the addition of one flow to remove the ethanol, a coverslipping step in which or more antioxidants and the host compound improved limonene is dispensed to the sample, and a curing step. stability in comparison to an equivalent "unstabilized" he- [0034] Several hematoxylin compositions were inves- 35 matoxylin solution without an added antioxidant and/or tigated in an effort to provide a stable composition that host compound, which unstabilized hematoxylin exhibits also provided for a darker nuclear stain (by virtue of hav- precipitates throughout the container after one week at ing a higher effective initial hematein concentration). Tra- 2-8°C, after 4 weeks at ambient temperature and at 30 ditionally, solutions that have higher concentrations of °C, and after 2 weeks at 45 °C. hematein and that as a result can stain nuclei more darkly 40 [0038] Long term stability testing that included use of are made up and used within a few days because such stored compositions for manual staining of multi-tissue solutions will form copious amounts of precipitate. Water- slides also was performed. Two lots of an aqueous he- soluble antioxidants (in this example, hydroquinone and matoxylin solution including 25% ethylene glycol (v/v), n-propyl gallate) were added to a variety of hematoxylin 20 mM hematoxylin, 3.3 mM sodium iodate, 20 mM alu- formulations, singly or in combination, to determine45 minum sulfate octadecahydrate, 85 mM hydroquinone whether the antioxidants could stabilize the hematein and 10 mM β-cyclodextrin hydrate having a pH of about against oxidative degradation and precipitation, and β- 2.6 were each packed into multiple bag-in-box contain- cyclodextrin was used to determine if addition of a host ers. One container from each lot was left in ambient con- compound could further slow the natural oxidation of he- ditions, one container from each lot was subjected to matein and resulting precipitate formation. 50 freeze-thaw cycling, one container from each lot was sub- [0035] In all instances, the hematoxylin formulations jected to 45 degrees C to ambient ship stress conditions, were prepared as follows: and one container from each lot was subjected to 2-8 degrees C to ambient ship stress conditions. At monthly 1) Deionized water and either ethylene glycol (25% intervals, each of the containers was inspected for the by volume; Sigma-Aldrich, St. Louis, MO) or propyl- 55 presence of precipitates and an aliquot was removed and ene glycol(23% by volume; Sigma-Aldrich,St. Louis, checked for pH. The aliquot was then used to manually MO) were mixed together to form a solvent for the stain a microscope slide bearing multiple tissue sections composition. (liver, kidney, colon, skin, and one of tonsil, lymph node

6 11 EP 2 130 023 B1 12 or spleen). After a total of 13 months of monthly testing, wherein a molar ratio of hematoxylin to mordant in the solutions in all of the different containers consistently the composition is between 2:1 and 1:100. did not exhibit precipitates, the pH of each of the solutions in the different containers consistently remained stable, 9. A method for making a stabilized hematoxylin com- and the hematoxylin solutions in the different containers 5 position for histochemical staining of a tissue or cy- consistently provided acceptable nuclear staining of the tology sample, comprising: tissue sections following the manual staining procedure. forming a hematein solution; adding a mordant to the hematein solution to Claims 10 form a staining solution; and adding both of cyclodextrin and hydroquinone 1. A stabilized hematoxylin composition for staining of to the staining solution to form the stabilized he- a tissue or cytology sample, comprising: matoxylin composition, wherein forming the hematein solution compris- a solvent; 15 es hematoxylin; dissolving hematoxylin in an solvent, and an amount of a chemical oxidant sufficient to adding an amount of a chemical oxidant suffi- convert at least a portion of the hematoxylin to cient to convert at least a portion of the hema- hematein; toxylin to hematein. a mordant; and 20 both of cyclodextrin and hydroquinone. 10. The method of claim 9, wherein the solvent comprises water and a polyol, 2. The composition of claim 1, wherein the solvent com- wherein the polyol is one or more of glycerol, ethyl- prises one or more of water, a lower alkanol, and a ene glycol, and propylene glycol, and polyol. 25 wherein one or more of ethylene glycol and propylene glycol comprise 10 to 30% by volume of an aque- 3. The composition of claim 2, wherein the polyol com- ous solvent composition. prises one or more of glycerol, ethylene glycol, propylene glycol, poly(ethylene glycol), and poly(propyl- 11. The method of claim 9 or 10, wherein the mordant ene glycol). 30 comprises one or more of an aluminum mordant, an iron mordant, a bismuth mordant, a copper mordant, 4. The composition of claim 3, wherein one or more of a molybdenum mordant, a vanadium mordant, and ethylene glycol and propylene glycol comprise 10 to a zirconium mordant, wherein the aluminum mordant 30% by volume of an aqueous solvent composition. is alum or aluminum sulphate. 35 5. The composition of any one of claims 1 to 4, wherein 12. An automated method for histochemical staining of the chemical oxidant is one or more of sodium iodate, a tissue or cytology sample, comprising contacting mercuric oxide, potassium permanganate, potassi- the sample with a hematoxylin composition recited um periodate, and hydrogen peroxide. in any of claims 1 to 8, wherein the sample is sup- 40 ported on a substrate. 6. The composition of any one of claims 1 to 5, wherein the mordant comprises one or more of an aluminum 13. The method of claim 12, further comprising contact- mordant, an iron mordant, a bismuth mordant, a cop- ing the sample with a counterstain, wherein per mordant, a molybdenum mordant, a vanadium contacting the sample with a hematoxylin composi- mordant, and a zirconium mordant, wherein the alu- 45 tion comprises a progressive or a regressive hema- minum mordant is alum or aluminum sulphate. toxylin staining protocol, the substrate comprises a microscope slide, and 7. The composition of any one of claims 1 to 6, further the counterstain comprises one or more of eosin Y, comprising an acid, wherein the acid comprises ace- orange G, light green SF yellowish, Bismarck Brown, tic acid. 50 fast green FCF, OA-6, EA25, EA36, EA50, and EA65. 8. The composition of any one of claims 1 to 7, comprising a mixture of water and ethylene glycol as the solvent, Patentansprüche sodium iodate as the oxidant, and 55 aluminum sulphate as the mordant, 1. Stabilisierte Hämatoxylinzusammensetzung für das wherein a molar ratio of hematoxylin to oxidant in Färben einer Gewebe- oder Zytologieprobe, umfas- the composition is between 6:1 and 1:1, and send:

7 13 EP 2 130 023 B1 14

ein Lösungsmittel, Bilden einer Hämateinlösung, Hämatoxylin, Zugeben eines Beizmittels zu der Hämateinlö- eine Menge eines chemischen Oxidationsmit- sung, um eine Färbelösung zu bilden und tels, die ausreicht, mindestens einen Teil des Zugeben von sowohl Cyclodextrin als auch Hy- Hämatoxylins zu Hämatein umzuwandeln, 5 drochinon zu der Färbelösung, um die stabili- ein Beizmittel und sierte Hämatoxylinzusammensetzung zu bil- sowohl Cyclodextrin als auch Hydrochinon. den, wobei das Bilden der Hämateinlösung das 2. Zusammensetzung nach Anspruch 1, wobei das Lö- Lösen von Hämatoxylin in einem Lösungsmittel sungsmittel eines oder mehrere aus Wasser, einem 10 und niederen Alkanol und einem Polyol umfaßt. Zugeben einer Menge eines chemischen Oxi- dationsmittels, die ausreicht, mindestens einen 3. Zusammensetzung nach Anspruch 2, wobei das Po- Teil des Hämatoxylins zu Hämatein umzuwan- lyol eines oder mehrere aus Glycerin, Ethylenglycol, deln, Propylenglycol, Poly(ethylenglycol) und Poly(propy- 15 umfaßt. lenglycol) umfaßt. 10. Verfahren nach Anspruch 9, 4. Zusammensetzung nach Anspruch 3, wobei eines wobei das Lösungsmittel Wasser und ein Polyol um- oder mehrere aus Ethylenglycol und Propylenglycol faßt, 10 bis 30 Vol.-% einer wässrigen Lösungsmittel-zu- 20 wobei das Polyol eines oder mehrere aus Glycerin, sammensetzung umfassen. Ethylenglycol und Propylenglycol ist und wobei eines oder mehrere aus Ethylenglycol und 5. Zusammensetzung nach einem der Ansprüche 1 bis Propylenglycol 10 bis 30 Vol.-% einer wässrigen Lö- 4, wobei das chemische Oxidationsmittel eines oder sungsmittelzusammensetzung umfassen. mehrere aus Natriumiodat, Quecksilberoxid, Kali- 25 umpermanganat, Kaliumperiodat und Wasserstoff- 11. Verfahren nach Anspruch 9 oder 10, wobei das Beiz- peroxid ist. mittel Beizmittel eines oder mehrerer aus einem Alu- miniumbeizmittel, einem Eisenbeizmittel, einem 6. Zusammensetzung nach einem der Ansprüche 1 bis Wismutbeizmittel, einem Kupferbeizmittel, einem 5, wobei das Beizmittel eines oder mehrerer aus ei- 30 Molybdänbeizmittel, einem Vanadiumbeizmittel und nemAluminiumbeizmittel, einem Eisenbeizmittel, ei- einem Zirconiumbeizmittel umfaßt, wobei das Alu- nem Wismutbeizmittel, einem Kupferbeizmittel, ei- miniumbeizmittel Natriumalaun oder Aluminiumsul- nem Molybdänbeizmittel, einem Vanadiumbeizmit- fat ist. tel und einem Zirconiumbeizmittel umfaßt, wobei das Aluminiumbeizmittel Natriumalaun oder Aluminium- 35 12. Automatisiertes Verfahren für das histochemische sulfat ist. Färben einer Gewebe- oder Zytologieprobe, umfassend das Inkontaktbringen der Probe mit einer Hä- 7. Zusammensetzung nach einem der Ansprüche 1 bis matoxylinzusammensetzung wie in einem der An- 6, weiter umfassend eine Säure, wobei die Säure sprüche 1 bis 8 genannt, wobei die Probe von einem Essigsäure umfaßt. 40 Substrat getragen wird.

8. Zusammensetzung nach einem der Ansprüche 1 bis 13. Verfahren nach Anspruch 12, weiter umfassend das 7, umfassend eine Mischung aus Wasser und Ethy- Inkontaktbringen der Probe mit einer Gegenfärbung, lenglycol als das Lösungsmittel, Natriumiodat als wobei das Oxidationsmittel und 45 das Inkontaktbringen der Probe mit einer Hämato- Aluminiumsulfat als das Beizmittel, xylinzusammensetzung ein progressives oder re- wobei ein molares Verhältnis von Hämatoxylin zu gressives Hämatoxylinfärbeprotokoll umfaßt, Oxidationsmittel in der Zusammensetzung zwischen das Substrat einen Mikroskopobjektträger umfaßt 6:1 und 1:1 ist und und wobei ein molares Verhältnis von Hämatoxylin zu 50 die Gegenfärbung eines oder mehrere aus Eosin Y, Beizmittel in der Zusammensetzung zwischen 2:1 Orange G, Lichtgrün SF, Bismarckbraun, Fast und 1:100 ist. Green FCF, OA-6, EA25, EA36, EA50 und EA65 um- faßt. 9. Verfahren zur Herstellung einer stabilisierten Häma- toxylinzusammensetzung für das histochemische 55 Färben einer Gewebe- oder Zytologieprobe, umfas- Revendications send: 1. Composition d’hématoxyline stabilisée servant à co-

8 15 EP 2 130 023 B1 16

lorer un échantillon cytologique ou de tissu,9. Procédé de préparation d’une composition d’héma- comprenant : toxyline stabilisée à des fins de coloration histochimique d’un échantillon cytologique ou de tissu, un solvant ; comprenant : de l’hématoxyline ; 5 une quantité d’oxydant chimique suffisante pour la formation d’une solution d’hématéine ; convertir au moins une partie de l’hématoxyline l’ajout d’un mordant à la solution d’hématéine en hématéine ; pour former une solution de coloration ; et un mordant ; et l’ajout à la fois de cyclodextrine et d’hydroqui- à la fois de la cyclodextrine et de l’hydroquinone. 10 none à la solution de coloration pour former la composition d’hématoxyline stabilisée, 2. Composition selon la revendication 1, dans laquelle dans lequel la formation de la solution d’héma- le solvant comprend un ou plusieurs solvants choisis téine comprend parmi l’eau, un alcanol inférieur et un polyol. la dissolution d’hématoxyline dans un solvant, et 15 l’ajout d’une quantité d’oxydant chimique suffi- 3. Composition selon la revendication 2, dans laquelle sante pour convertir au moins une partie de l’hé- le polyol comprend un ou plusieurs polyols choisis matoxyline en hématéine. parmi le glycérol, l’éthylène glycol, le propylène glycol, le poly(éthylène glycol) et le poly(propylène gly- 10. Procédé selon la revendication 9, col). 20 danslequel le solvant comprend de l’eau et un polyol, dans lequel le polyol est un ou plusieurs polyols choi- 4. Composition selon la revendication 3, dans laquelle sis parmi le glycérol, l’éthylène glycol et le propylène l’éthylène glycol et/ou le propylène glycol compren- glycol, et nent 10 % à 30 % en volume d’une composition danslequel l’éthylène glycol et/oule propylène glycol aqueuse de solvant. 25 comprennent 10 à 30 % en volume d’une composition aqueuse de solvant. 5. Composition selon l’une quelconque des revendications 1 à 4, dans laquelle l’oxydant chimique est un 11. Procédé selon la revendication 9 ou 10, dans lequel ou plusieurs oxydants chimiques choisis parmi l’io- le mordant comprend un ou plusieurs mordants choi- date de sodium, l’oxyde mercurique, le permanga- 30 sis parmi un mordant à base d’aluminium, un mor- nate de potassium, le periodate de potassium et le dant à base de fer, un mordant à base de bismuth, peroxyde d’hydrogène. un mordant à base de cuivre, un mordant à base de molybdène, un mordant à base de vanadium et un 6. Composition selon l’une quelconque des revendica- mordant à base de zirconium, dans lequel le mordant tions 1 à 5, dans laquelle le mordant comprend un 35 à base d’aluminium est le sulfate d’alun ou d’alumi- ou plusieurs mordants choisis parmi un mordant à nium. base d’aluminium, un mordant à base de fer, un mordant à base de bismuth, un mordant à base de cuivre, 12. Procédé automatisé de coloration histochimique unmordant à base de molybdène, un mordantà base d’un échantillon cytologique ou de tissu, comprenant de vanadium et un mordant à base de zirconium, 40 la mise en contact de l’échantillon avec une compo- dans lequel le mordant à base d’aluminium est le sition d’hématoxyline selon l’une quelconque des re- sulfate d’alun ou d’aluminium. vendications 1 à 8, dans lequel l’échantillon est sup- porté sur un substrat. 7. Composition selon l’une quelconque des revendications 1 à 6, comprenant en outre un acide, dans le- 45 13. Procédé selon la revendication 12, comprenant en quel l’acide comprend l’acide acétique. outre la mise en contact de l’échantillon avec un contre-colorant, dans lequel 8. Composition selon l’une quelconque des revendica- la mise en contact de l’échantillon avec une compo- tions 1 à 7, comprenant sition d’hématoxyline comprend un protocole de coun mélange d’eau et d’éthylène glycol en tant que 50 loration progressive ou régressive à l’hématoxyline, solvant, le substrat comprend une lame pour microscope, et de l’iodate de sodium en tant qu’oxydant, et le contre-colorant comprend un ou plusieurs contre- du sulfate d’aluminium en tant que mordant, colorants choisis parmi l’éosine Y, l’orange G, le vert danslequel un rapportmolaire hématoxyline sur oxy- clair SF jaunâtre, le marron Bismarck, le verre solide dant dans la composition est situé entre 6/1 et 1/1, et 55 FCF, l’OA-6, l’EA25, l’EA36, l’EA50 et l’EA65. dans lequel un rapport molaire hématoxyline sur mordant dans la composition est situé entre 2/1 et 1/100.

9 EP 2 130 023 B1

10 EP 2 130 023 B1

11 EP 2 130 023 B1

12 EP 2 130 023 B1

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 20040002163 A [0032] • US 20050186114 A [0032]

Non-patent literature cited in the description

• CROFT ; BARTSCH. Synthesis of Chemically Mod- • HOROBIN ; KIERNAN. Conn’s biological stains: a ified Cyclodextrins. Tetrahedron, 1983, vol. 39, handbook of dyes, stains and fluorochromes for us 1417-1474 [0017] in biology and medicine. BIOS, 2002 [0030] • KIERNAN. Histological and Histochemical methods: Theory and Practice. Butterworth Heinemann [0030]