(12) United States Patent (10) Patent No.: US 7,186,522 B2 Lapen Et Al

US007 186522B2

(12) United States Patent (10) Patent No.: US 7,186,522 B2 Lapen et al. (45) Date of Patent: Mar. 6, 2007

(54) PAPANICOLAU STAINING PROCESS (56) References Cited (75) Inventors: Daniel Lapen, Lancaster, MA (US); U.S. PATENT DOCUMENTS Norman Soule, Brockton, MA (US); 6,143,512 A 11/2000 Markovic et al. Somthouk Lim, Farmingham, MA (US) 2002fOO19001 A1 2/2002 Light OTHER PUBLICATIONS (73) Assignee: CYTYC Corporation, Marlborough, MA (US) PCT International Search Report for PCT/US2004/09437, Appli cant: Cytyc Corporation, Forms PCT/ISA/210 and 220, dated Mar. 24, 2005 (4 pages). (*) Notice: Subject to any disclaimer, the term of this PCT Written Opinion of the International Search Authority for patent is extended or adjusted under 35 PCT/US2004/09437, Applicant: Cytyc Corporation, Form PCT/ U.S.C. 154(b) by 458 days. ISA/237, dated Mar. 24, 2005 (5 pages). (21) Appl. No.: 10/404.879 Primary Examiner Ralph Gitomer Assistant Examiner Kailash C. Srivastava (22) Filed: Mar. 31, 2003 (74) Attorney, Agent, or Firm Vista IP Law Group LLP (65) Prior Publication Data (57) ABSTRACT US 2004/O19 1854 A1 Sep. 30, 2004 A method for treating a biological sample with a Papanico laou staining process is provided. The method comprises (51) Int. C. incorporating a detergent treatment into the staining process GOIN L/30 (2006.01) at any of various steps. The method has been found to GOIN 33/48 (2006.01) advantageously reduce the number of artifacts produced (52) U.S. Cl...... 435/405 during Papanicolaou staining. Also provided is a sample (58) Field of Classification Search ...... 435/40.5; stained by Such a process. 51Of 406 See application file for complete search history. 18 Claims, 1 Drawing Sheet U.S. Patent Mar. 6, 2007 US 7,186,522 B2

US 7,186,522 B2 1. 2 PAPANICOLAU STAINING PROCESS and improved ability to detect cellular, nuclear and chroma tin morphology. Additionally, the removal of excess stain TECHNICAL FIELD from the cytoplasm may lead to an increased ability to detect hyperchromasia associated with abnormality. This invention relates to methods, articles and composi The term "Papanicolaou staining process,” “pap stain.” tions useful in staining biological samples. “pap test” and the like refer to a modified or unmodified Papanicolaou staining procedure which differentially stains BACKGROUND OF THE INVENTION cell nuclei and cytoplasm in a sample, for example a sample obtained during a gynecological screening examination Such Medical diagnostic testing methods are critical screening 10 as a pap Smear. tools for the early detection of pathological conditions. Early The test relies on the ability to distinguish the staining detection permits the identification of Such conditions at a pattern, staining intensity and the size and shape of the stage when Successful treatment is more likely. Early treat nucleus and cytoplasm of different cell types within the ment also frequently involves less damaging or less invasive sample. The Papanicolaou staining process advantageously treatment methods and decreases the impact on the patient. 15 provides dark stained nuclei and transparent cytoplasms, In addition to routine Screening, diagnostic testing is also which is particularly useful in detecting abnormal cells used in a variety of other applications, including biopsy within multi-layered samples provided by certain sampling analysis and monitoring the results of ongoing medical techniques including pap Smears. treatment. One particularly useful tool in diagnostic testing is the A usable test depends on the ability to read a significant Papanicolaou staining process. This process was first devel percentage of cells in the sample, otherwise test accuracy oped for the staining of gynecological specimens, and has may be comprised and samples may be rendered unusable, led to a dramatic decrease in the fatality rate from cervical leading to additional rescreening costs and increased patient cancer. Papanicolaou staining is now used in diagnosing a anxiety. variety of pathological conditions from many different tis 25 Consequently, staining artifacts which reduce the ability Sues and organs. to discern the true staining pattern of cells in a sample can However, Papanicolaou staining is subject to artifacts that dramatically impair the usefulness of a pap test. Artifacts can can adversely affect the ability of a technologist or machine reduce the ability to differentiate between the staining pat to Successfully read a processed sample. These artifacts tern of the nucleus and the cytoplasm, can increase the include spurious non-specific cytoplasmic staining, artifacts 30 percentage of cells or sample area which are unreadable, and Such as cytoplasmic cracking, cornflaking, Smudged nuclear can increase cytoplasmic staining thereby diminishing the detail, hypochromatic staining and hyperchromatic staining. transparency that is one of the primary advantages of the pap Such artifacts, at a minimum, reduce the fraction of the test, thus making it more difficult to distinguish abnormal sample which can be evaluated, can produce false staining cells within the sample. patterns that interfere with accurate diagnosis, and may 35 Before the present invention is described in further detail, render the sample unreadable. Difficulties in analyzing Such it is to be understood that this invention is not limited to the samples leads to an increase in patient anxiety, rescreening particular methodology, Solutions or apparatuses described, costs, delays in diagnosis and, more importantly, potential as such methods, Solutions or apparatuses can, of course, misdiagnosis. vary. It is also to be understood that the terminology used There is a need in the art for improved procedures for 40 herein is for the purpose of describing particular embodi staining diagnostic specimens, and for compositions and ments only, and is not intended to limit the scope of the articles of manufacture useful in Such methods. present invention. Use of the singular forms “a,” “an,” and “the include SUMMARY OF THE INVENTION plural references unless the context clearly dictates other 45 wise. Thus, for example, reference to “a sample” includes a An improved method for treating a biological sample with plurality of samples, reference to “a detergent” includes a a Papanicolaou staining process is provided. The method plurality of such detergents, reference to “a counterstain comprises incorporating a detergent treatment into the stain includes a plurality of counterstains, and the like. Addition ing process at any of various steps. The method has been ally, use of specific plural references, such as “two,” “three.” found to advantageously reduce the number of artifacts 50 etc., read on larger numbers of the same Subject unless the produced during Papanicolaou staining. Also provided is a context clearly dictates otherwise. sample stained by Such a process. Terms such as “connected,” “attached, and “linked' are used interchangeably herein and encompass direct as well as BRIEF DESCRIPTION OF THE DRAWINGS indirect connection, attachment, linkage or conjugation 55 unless the context clearly dictates otherwise. Where a range FIG. 1 shows a sample stained with a Papanicolaou of values is recited, it is to be understood that each inter staining process. vening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifi DETAILED DESCRIPTION OF THE cally disclosed, along with each Subrange between Such INVENTION 60 values. The upper and lower limits of any range can inde pendently be included in or excluded from the range, and The inventors have advantageously discovered that incor each range where either, neither or both limits are included poration of a detergent treatment into a Papanicolaou stain is also encompassed within the invention. Where a value ing procedure reduces the occurrence of artifacts to which being discussed has inherent limits, for example where a the procedure is susceptible. Use of a detergent has been 65 component can be present at a concentration of from 0 to found to clear excess stain from the cytoplasm of cells 100%, or where the pH of an aqueous solution can range stained by this procedure, allowing for increased contrast from 1 to 14, those inherent limits are specifically disclosed. US 7,186,522 B2 3 4 Where a value is explicitly recited, it is to be understood that color using an agent as known in the art (e.g., Scott's tap values which are about the same quantity or amount as the water Substitute, ammonia water, lithium carbonate). The recited value are also within the scope of the invention, as bluing solution can be incorporated without a differentiation are ranges based thereon. Where a combination is disclosed, step. each subcombination of the elements of that combination is Multiple cytoplasmic counterstains are used in Papanico also specifically disclosed and is within the scope of the laou staining to allow identification of the various cell types invention. Conversely, where different elements or groups of in the sample, their morphology and their frequency. elements are disclosed, combinations thereof are also dis The cytoplasmic stains Orange G and Eosin Y—Light closed. Where any element of an invention is disclosed as Green/Fast Green are prepared in alcohol. Orange G is a having a plurality of alternatives, examples of that invention 10 monochromatic stain that stains cell cytoplasms yellow or in which each alternative is excluded singly or in any orange if keratin is present, allowing for the distinction of combination with the other alternatives are also hereby mature or maturing keratin-producing malignant cells from disclosed; more than one element of an invention can have the less conspicuous benign cells. Orange G can be used in Such exclusions, and all combinations of elements having the form of a solution known in the art of cell staining, for Such exclusions are hereby disclosed. 15 example as OG-6. Unless defined otherwise or the context clearly dictates Traditionally, Eosin Y—Light Green/Fast Green is pre otherwise, all technical and scientific terms used herein have pared as a mixture of three stains, Eosin Y. Light Green SF the same meaning as commonly understood by one of and/or Fast Green FCF, and Bismarck Brown. Exemplary ordinary skill in the art to which this invention belongs. mixtures include EA36, EA50, and EA65. Eosin Y produces Although any methods and materials similar or equivalent to a pink color in nucleoli, cilia, red blood cells and the those described herein can be used in the practice or testing cytoplasm of mature squamous cells. Light Green SF Yel of the invention, the preferred methods and materials are lowish and Fast Green FCF produce a blue/green color in the now described. cytoplasm of metabolically active parabasal squamous cells, All publications mentioned herein are hereby incorpo intermediate Squamous cells and columnar cells. Bismarck rated by reference for the purpose of disclosing and describ 25 Brown does not produce a useable staining pattern in pap ing the particular materials and methodologies for which the staining, and can be excluded in variations of the method. reference was cited. The publications discussed herein are Substitutes for Light Green SF Yellowish may also be used, provided solely for their disclosure prior to the filing date of e.g. Fast Green FCF. Cytoplasmic counterstains can also be the present application. Nothing herein is to be construed as a mixture of OG and Eosin Y. Light Green/Fast Green an admission that the invention is not entitled to antedate 30 resulting in a single solution for use as a counterstain. such disclosure by virtue of prior invention. The alcohol in which these cytoplasmic counterstains are The term “optional' or “optionally” means that the sub prepared results in the transparent cytoplasms so useful in sequently described event or circumstance may or may not analyzing samples stained by the Papanicolaou process. occur, and includes instances where the event or circum Wash steps are incorporated into the staining procedure as stance occurs and instances in which it does not. 35 appropriate, as are hydration/dehydration series when trans ferring between aqueous and nonaqueous solutions. Upon Papanicolaou Staining completion of the staining process, the samples can be A Papanicolaou staining process comprises at least a cleared and mounted, as is known in the art. Additional hematoxylin staining step, an Orange G cytoplasmic coun procedures may be performed on the samples prior or terstaining step, and an Eosin Y-light green/fast green coun 40 Subsequent to the Papanicolaou procedure. The samples can terstaining step. The process may incorporate a “bluing step then be analyzed manually and/or by a Suitable apparatus, Pap staining advantageously produces a sample with trans for example an automated imaging system. Exemplary auto parent cell cytoplasms which is particulary useful for analy mated imaging systems include Cytyc Corporation's Thin sis of cells covering multiple layers such as those obtained Prep R. Imaging System, the TriPath FocalPointTM Profiler, from a pap Smear. 45 the ChromaVision Acis(R) System, the CompuCyt iCyte Prior to commencing a Papanicolaou staining process, the Imaging System, the Applied Imaging CytoVisionTM Sys sample must be fixed. Any fixation method producing sat tem, and the Veracel Verasys Imaging System. isfactory results may be used. Typically an alcoholic fixation A variety of modifications to the Papanicolaou staining is used with papstaining, e.g. 95% ethanol, 100% methanol, procedure may be made, and are known in the art. For 80% propanol, or 95% ethanol/ether (1:1). Commercial 50 example, the process may be modified as described in U.S. spray fixatives may also be used. Pat. Nos. 5,168,066 and 6,348,325 (issued Dec. 1, 1992 and A typical Papanicolaou staining process comprises a Feb. 19, 2002, respectively, and assigned to Cytyc Corp.) to hematoxylin staining step in combination with a metal incorporate the use of thionin. Other thiazin dyes and mordant to stain the chromatin of cells, and may be done triarylmethane dyes can be used. progressively or regressively. The chromatin staining is a 55 critical portion of this test, as the size, staining intensity and The Sample staining pattern of the chromatin is a critical element in the The portion of the sample to be analyzed can be any analysis of abnormal cells within the sample. Any form of source of biological material that can be obtained from a hematoxylin may be used, including Harris's, Gill’s, living organism directly or indirectly, including cells, tissue Delafields, Ehrlich's or Mayer's hematoxylin. Substitutes 60 or fluid. Nonlimiting examples of the sample include blood, for hematoxylin utilizing dyes e.g. Thionin, Victoria Blue B, urine, semen, milk, sputum, mucus, plueral fluid, pelvic AZure B, etc. may also be used. fluid, sinovial fluid, ascites fluid, body cavity washes, eye If done regressively, the sample is overstained and then brushing, skin scrapings, a buccal Swab, a vaginal Swab, a differentiated with an agent (e.g., dilute HCl) which allows pap Smear, a rectal Swab, an aspirate, a needle biopsy, a the staining intensity to be reduced to the desired level. This 65 section of tissue obtained for example by Surgery or autopsy, causes the staining color to shift from blue/purple to Salmon plasma, serum, spinal fluid, lymph fluid, the external secre pink/red. A “bluing step may be used to reestablish the blue tions of the skin, respiratory, intestinal, and genitourinary US 7,186,522 B2 5 6 tracts, tears, saliva, tumors, organs, and samples of in vitro tylphenyl-polyethylene glycol), Igepal(R) CA-210 (polyoxy cell culture constituents. Typically the sample will be a pap ethylene(2) isooctylphenyl ether), Igepal(R) CA-520 Smear as is routinely obtained during a gynecological exami (polyoxyethylene(5) isooctylphenyl ether), Igepal(R) CO-630 nation or a biopsy Suspected of containing abnormal cells. (polyoxyethylene(9)nonylphenyl ether), Igepal(R) CO-720 The sample can be a positive control sample which is (polyoxyethylene(12) nonylphenyl ether), Igepal(R) CO-890 known to contain abnormal cells. A negative control sample (polyoxyethylene(40) nonylphenyl ether), Igepal(R) CO-990 can also be used which is used to determine whether a given (polyoxyethylene(100) nonylphenyl ether), Igepal(R) set of staining conditions produces false positives (a positive DM-970 (polyoxyethylene(150) dinonylphenyl ether), signal even in the absence of abnormal cells in the sample). Methyl-6-O-(N-heptylcarbamoyl)-O-D-glucopyranoside, Typically the sample is provided for staining on a slide or 10 Nonaethylene glycol monododecyl ether, N-Nonanoyl-N- similar glass Surface, for example a vial, cover slip, or other methylglucamine, Octaethylene glycol monodecyl ether, Surface that is transparent to the incident light used for its Octaethylene glycol monododecyl ether, Octaethylene gly analysis. col monohexadecyl ether, Octaethylene glycol monooctade cyl ether, Octaethylene glycol monotetradecyl ether, Octyl The Detergent 15 B-D-glucopyranoside, Pentaethylene glycol monodecyl Any detergent which detectably reduces artifacts pro ether, Pentaethylene glycol monododecyl ether, Pentaethyl duced during a Papanicolaou staining procedure can be ene glycol monohexadecyl ether, Pentaethylene glycol used. The detergent is used in an amount and for a time monohexyl ether, Pentaethylene glycol monooctadecyl suitable to detectably reduce such artifacts. The percentage ether, Pentaethylene glycol monooctyl ether, Polyethylene of at least one such artifact can desirably be reduced at least glycol diglycidyl ether, Polyethylene glycol ether W-1, about 10%, 20%, 30%, 40%, 50%, 70%, 90%, 95%, 98% or Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 more by incorporating a detergent wash as described herein. stearate, Polyoxyethylene 20 isohexadecyl ether, Polyoxy When used in imaging systems which select certain areas of ethylene 20 oleyl ether, Polyoxyethylene 40 stearate, Poly the slide (“fields of interest”) to be analyzed based on any of oxyethylene 50 stearate, Polyoxyethylene 8 stearate, Poly a variety of parameters as is known in the art, the invention 25 oxyethylene bis(imidazolyl carbonyl), Polyoxyethylene 25 desirably decreases the number of falsely selected fields propylene glycol stearate, Saponin, SpanR 20 (Sorbitan (which are falsely selected for analysis based on staining monolaurate), SpanR 40 (Sorbitan monopalmitate), SpanR) artifacts that meet the selection criteria of the imaging 60 (Sorbitan monostearate), SpanR 65 (Sorbitan tristearate), system) at least about 10%, 20%, 30%, 40%, 50%, 70%, SpanR 80 (Sorbitan monooleate), SpanR 85 (Sorbitan tri 90%. 95%, 98% or more. This increases the percentage of 30 oleate), Tergitol in any form (including Types 15-S-5, 15-S- useful fields selected for analysis. 7, 15-S-9, 15-S-12, 15-S-30, NP-4, NP-7, NP-9, NP-10, The detergent may be non-ionic, cationic, anionic or NP-40, NPX (Imbentin-N/63), TMN-3 (Polyethylene glycol Zwitterionic. Mixtures of detergents may also be used. trimethylnonyl ether), TMN-6 (Polyethylene glycol trimeth Exemplary classes of detergents include alcohol ether Sul ylnonyl ether), TMN-10 (Polyethylene glycol trimethyl fates, alcohol Sulfates, alkanolamides, alkyl Sulfonates, 35 nonyl ether), MIN FOAM 1x, and MIN FOAM 2x), Tet amine oxides, amphoteric detergents, anionic detergents, radecyl-B-D-maltoside, Tetraethylene glycol monodecyl betaine derivatives, cationic detergents, disulfonates, dode ether, Tetraethylene glycol monododecyl ether, Tetraethyl cylbenzene sulfonic acid, ethoxylated alcohols, ethoxylated ene glycol monotetradecyl ether, Triethylene glycol mono alkyl phenols, ethoxylated fatty acids, glycerol esters hydro decyl ether, Triethylene glycol monododecyl ether, Trieth tropes, lauryl Sulfates, mono and diglycerides, non-ionic 40 ylene glycol monohexadecyl ether, Triethylene glycol detergents, phosphate esters, quaternary detergents, and Sor monooctyl ether, Triethylene glycol monotetradecyl ether, bitan derivatives. Triton(R) CF-21, Triton(R) CF-32, Triton R. DF-12, Triton(R) Exemplary non-ionic detergents include BigCHAP(N.N- DF-16, Triton R GR-5M, Triton(R) N-101 (Polyoxyethylene Bis3-(D-gluconamido)propylcholamide), Bis(polyethyl branched nonylphenyl ether), Triton R. QS-15, Triton R ene glycol bisimidazoyl carbonyl), BrijR 30 (Polyoxyeth 45 QS-44, Triton R. RW-75 (Polyethylene glycol 260 mono ylene 4 lauryl ether) BrijR 35 (Polyoxyethylene 23 lauryl (hexadecyl/octadecyl) ether and 1-Octadecanol), Tritone(R) ether), Brij(R) 52 (Polyoxyethylene 2 cetyl ether), Brij.R. 56 X-100 (Polyethylene glycol tert-octylphenyl ether), Triton R (Polyoxyethylene 10 cetyl ether), BrijR) 58 (Polyoxyethyl X-102, Triton(R) X-15, Triton(R) X-151, Triton(R) X-200, Tri ene 20 cetyl ether), BrijR 72 (Polyoxyethylene 2 stearyl ton(R) X-207, Triton(R) X-114, Triton R. X-165, Triton(R) ether), BrijR 76 (Polyoxyethylene 10 stearyl ether), Brij(R) 50 X-305, Triton RX-405 (polyoxyethylene(40) isooctylphenyl 78 (Polyoxyethylene 20 stearyl ether), BrijR 92 (Polyoxy ether), Triton RX-405 reduced (polyoxyethylene(40) isooc ethylene 2 oleyl ether), Brij(R 97 (Polyoxyethylene 10 oleyl tylcyclohexyl ether), Triton(R) X-45 (Polyethylene glycol ether), Brij.R. 98 (Polyoxyethylene 20 oleyl ether), BrijR 700 4-tert-octylphenyl ether), Triton(R) X-705-70, TWEENR) in (Polyoxyethylene 100 stearyl ether), Cremophor R EL (cas any form (including TWEENR 20 (Polyoxyethylenesorbi tor oil/ethylene oxide polyether), Decaethylene glycol mon 55 tan monolaurate), TWEENR 21 (Polyoxyethylenesorbitan ododecyl ether, octanoyl-N-methylglucamide (MECA-8), monolaurate), TWEENR 40 (polyoxyethylene(20) sorbitan decanoyl-N-methylglucamide (MECA-10), n-octylgluco monopalmitate), TWEENR 60 (Polyethylene glycol sorbi side, n-dodecylglucoside, isotridecyl-poly(ethyleneglyco tan monostearate), TWEENR 61 (Polyethylene glycol sor lether), N-Decanoyl-N-methylglucamine, n-Decyl C-D- bitan monostearate), TWEENR) 65 (Polyoxyethylenesorbi glucopyranoside, Decyl B-D-maltopyranoside, 60 tan Tristearate), TWEENR 80 (Polyoxyethylenesorbitan n-Dodecanoyl-N-methylglucamide, n-Dodecyl C-D-malto monooleate), TWEENR 81 (Polyoxyethylenesorbitan side, n-Dodecyl B-D-maltoside, Heptaethylene glycol monooleate), and TWEENR 85 (polyoxyethylene(20) sor monodecyl ether, Heptaethylene glycol monotetradecyl bitan trioleate)), Tyloxapol (4-(1,1,3,3-tetramethylbutyl) ether, n-Hexadecyl B-D-maltoside, Hexaethylene glycol phenol polymer with formaldehyde and oxirane), and n-Un monododecyl ether, Hexaethylene glycol monohexadecyl 65 decyl B-D-glucopyranoside. ether, Hexaethylene glycol monooctadecyl ether, Hexaeth Exemplary anionic detergents include Chenodeoxycholic ylene glycol monotetradecyl ether, Igepal CA-630 (Oc acid, Cholic acid, Dehydrocholic acid, Deoxycholic acid, US 7,186,522 B2 7 8 Digitonin, Digitoxigenin, N,N-Dimethyldodecylamine less, about 0.5% or less, about 0.2% or less, about 0.1% or N-oxide, Sodium docusate, Sodium glycochenodeoxycho less, about 0.05% or less of the solution, or any range or late, Glycocholic acid, Glycodeoxycholic acid, Glycolitho subrange therebetween. cholic acid 3-sulfate disodium salt, Glycolithocholic acid The detergent treatment may be performed at any pH ethyl ester, N-Lauroylsarcosine, Lithium dodecyl sulfate, which permits the reduction of artifacts while not adversely Lugol (Iodine Potassium Iodide), Niaproof (2-Ethylhexyl affecting the ability to analyze the sample. Thus the deter sulfate sodium salt), Niaproof 4 (7-Ethyl-2-methyl-4-unde gent treatment may be performed at about pH 1, 2, 3, 4, 5, cyl sulfate Sodium salt), optionally Substituted alkylsul 6, 7, 8, 9, 10, 11, 12, 13 or within any range or subrange fonate salts (including salts of 1-butanesulfonate, pentane therebetween. Typically the treatment will be performed at 10 a pH of about 2 to about 10. Sulfonate, hexanesulfonate, 1-Octanesulfonate, The detergent may be used at any point in a Papanicolaou 1-decanesulfonate, 1-dodecanesulfonate, 1-heptane staining process. For example, the detergent may be used Sulfonate, 1-heptanesulfonate, 1-nonanesulfonate, 1-pro prior to the hematoxylin stain, mixed with the hematoxylin panesulfonate, and 2-bromoethanesulfonate, especially the stain, after the hematoxylin stain, combined with a bluing Sodium salts), Sodium cholate, Sodium deoxycholate, 15 solution, after the bluing solution, or combined with the optionally substituted Sodium dodecyl sulfate, Sodium octyl Orange G. Eosin Y. Light Green/Fast Green or other coun Sulfate, Sodium taurocholate, Sodium taurochenodeoxycho terstain Solutions. late, Sodium taurohyodeoxycholate, Taurolithocholic acid 3-sulfate disodium salt, TaurourSodeoxycholic acid sodium EXAMPLES salt, Trizma(R) dodecyl sulfate, Ursodeoxycholic acid. The anionic detergent can be provided in acid or salt form, or The following examples are set forth so as to provide both. those of ordinary skill in the art with a complete description Exemplary cationic detergents include Alkyltrimethylam of how to make and use the present invention, and are not monium bromide, Benzalkonium chloride, Benzyldimethyl intended to limit the scope of what is regarded as the hexadecylammonium chloride, Benzyldimethyltetradecy 25 invention. Efforts have been made to ensure accuracy with lammonium chloride, BenZyldodecyldimethylammonium respect to numbers used (e.g., amounts, temperature, etc.) bromide, Benzyltrimethylammonium tetrachloroiodate, but some experimental error and deviation should be Dimethyldioctadecylammonium bromide, Dodecyleth accounted for. Unless otherwise indicated, parts are parts by yldimethylammonium bromide, Dodecyltrimethylammo weight, temperature is degree centigrade and pressure is at nium bromide, Ethylhexadecyldimethylammonium bro 30 or near atmospheric, and all materials are commercially mide, Girard's reagent T. Hexadecyltrimethylammonium available. bromide, N,N',N'-Polyoxyethylene(10)-N-tallow-1,3-diami nopropane, Thonzonium bromide, and Trimethyl(tetradecyl) Example 1 ammonium bromide. Exemplary Zwitterionic detergents include CHAPS (3- 35 Cytoplasmic cracking was found to be very visible on pap (3-cholamidopropyl)-dimethylammonio)-1-propane-Sul Smear slides processed under test Papanicolaou staining fonate), CHAPSO (3-(3-cholamidopropyl)dimethyl-am conditions. To test methods for reducing cytoplasmic crack monio)-2-hydroxy-1-propane-Sulfonate), ing, 4 different sample pap Smears were obtained, slides 3-(Decyldimethylammonio)propanesulfonate, 3-(Dode were made from the samples, fixed, allowed to dry for one cyldimethylammonio)propanesulfonate, 3-(N,N-Dimeth 40 day, and then stained with a modified Pap stain according to ylmyristylammonio)propanesulfonate, 3-(N,N-Dimethyloc the procedure below: tadecylammonio)propanesulfonate, 3-(N.N- Steps: 1 Start Station Dimethyloctylammonio)propanesulfonate, and 3-(N.N- 2 Test wash step Dimethylpalmitylammonio)propanesulfonate. 35 min 50% Reagent alcohol 45 45 min Salt Solution (10% NaCl in 50% alcohol) The detergent includes those known or discoverable in the 55 min Salt Solution (10% NaCl in 50% alcohol art. The detergent can be synthesized or can be obtained 61 min 95% Reagent alcohol commercially from any of a variety of vendors (e.g., Sigma 71 min 95% Reagent alcohol Aldrich Corp., St. Louis, Mo., USA, www.sigrnaaldrich 8–22 1 min Nuclear stain (15 min) .com). Benzalkonium chloride and CHAPS have been found 50 23 20 sec Isopropyl alcohol to be particularly useful in reducing artifacts during Papa 24 20 sec Isopropyl alcohol nicolaou staining. 25 30 sec Isopropyl alcohol A given detergent may be tested for its ability to reduce 26 20 sec Isopropyl alcohol artifacts by incorporation into a wash step in a Papanicolaou 27–34 1 min Counterstain (mixture of Orange G and eosin staining process, which may be performed under conditions 55 Fast Green/Light Green) (8 min) known to produce staining artifacts. The sample treated with 35 30 sec 100% Reagent alcohol test detergent may then be evaluated against a sample 363 min Isopropyl alcohol processed without a detergent treatment, and against a 373 min Isopropyl alcohol sample processed with a benzalkonium chloride treatment or 38 1 min Xylene other positive control treatment known to reduce staining 60 393 min Xylene artifacts. 40 End Xylene Typically the amount of detergent used will be at least Remove slides to final xylene about 0.0001% and up to about 10% of the solution in which Coverslip manually using Permount it is to be used, and may be at least about 0.0005%, 0.001%, The four samples produced cytoplasmic cracking and 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.5% or 1% or more of 65 were subjected to test wash steps to determine conditions the solution; the amount of the detergent may be about 5% which might reduce the artifact. Ten slides were prepared or less, about 2.5% or less, about 2% or less, about 1% or from each sample. Five and fifteen minute water washes as US 7,186,522 B2 9 10 well as fifteen minute washes with FC 120 surfactant (3M 3. The method of claim 1, wherein the sample is contacted Corporation) and FC 170 surfactant (3M Corporation) were with the detergent in a solution at a concentration of at least separately performed on the ten slides from each sample. about 0.0001% and less than about 10% of the solution. Neither of these wash conditions was found to dramatically 4. The method of claim 3, wherein the detergent is at least reduce the staining artifact of cytoplasmic cracking. about 0.01% of the solution. Ten additional slides were prepared from each of the four 5. The method of claim 3, wherein the detergent is at least samples and Subjected to further test wash steps, including about 0.1% of the solution. fifteen minute washes in either FC 1700.1% pH 6.9, Tween(R) 6. The method of claim 3, wherein the detergent is at least 80 0.1% pH 6.9, Igepal CA-630 0.1% pH 7.0, CHAPS 0.1% about 0.5% of the solution. pH 7.7, ZEPHIRANR) 0.1% pH 7.2, water, water with no 10 7. The method of claim 1, wherein the detergent is less 50% alcohol step before the salt solution step, water with no than about 1% of the solution. salt solution step, and water with no 50% alcohol step before 8. The method of claim 1, wherein the detergent is the salt Solution step and no salt solution steps. selected from the group consisting of alcohol ether Sulfates, ZEPHIRANR) (benzalkonium chloride) dramatically alcohol Sulfates, alkanolamides, alkyl Sulfonates, amine reduced spurious blue cytoplasmic staining and reduced 15 oxides, amphoteric detergents, anionic detergents, betaine cytoplasmic cracking. CHAPSR also reduced both artifacts derivatives, cationic detergents, disulfonates, dodecylben to a certain degree at this concentration. The other treat Zene Sulfonic acid, ethoxylated alcohols, ethoxylated alkyl ments at the indicated concentrations had little effect on phenols, ethoxylated fatty acids, glycerol esters hydrotropes, these staining artifacts. lauryl Sulfates, mono and diglycerides, non-ionic detergents, phosphate esters, quaternary detergents, and Sorbitan deriva Example 2 tives. 9. The method of claim 1, wherein the detergent is a An example of a Papanicolaou staining protocol incorpo nonionic detergent. rating a detergent wash step is given below: 10. The method of claim 9, wherein the non-ionic deter 25 gent is selected from the group consisting of Big-CHAP (N, N-Bis3-(D-gluconamido) propylcholamide), Bis (polyeth ylene glycol bis imidazoyl carbonyl), Brij 30 (Polyoxy Steps Reagent Time ethylene 4 lauryl ether) Brij 35 (Polyoxyethylene 23 lauryl 1 Distilled water 5 min ether), Brij 52 (Polyoxyethylene 2 cetyl ether), Brij 56 2 Nuclearstain 5 min 30 (Polyoxyethylene 10 cetyl ether), Brij 58 (Polyoxyethylene 3 Distilled Water dH2O10 Sec 4 Rinse solution 20 sec 20 cetyl ether), Brij 72 (Polyoxyethylene 2 stearyl ether), (detergent) Brij 76 (Polyoxyethylene 10 stearyl ether), Brij 78 (Poly 5 Distilled water 30 sec oxyethylene 20 stearyl ether), Brij 92 (Polyoxyethylene 2 6 Bluing agent 30 sec oleyl ether), Brij97 (Polyoxyethylene 10 oleyl ether), Brij 7 Distilled water 30 sec 35 98 (Polyoxyethylene 20 oleyl ether), Brij 700 (Polyoxyeth 8 50% Reagent alcohol 30 sec 9 95% Reagent alcohol 30 sec ylene 100 stearyl ether), Cremophor EL (castor oil/ethylene 10 Orange G 1 min oxide polyether), Decaethylene glycol monododecyl ether, 11 95% Reagent alcohol 15 sec octanoyl-N-methylglucamide (MECA-8), decanoyl-N-me 12 95% Reagent alcohol 15 sec 13 EA Solution 4 min thylglucamide (MECA-10), n-octylglucoside, n-dodecylglu 14 95% Reagent alcohol 1 min 40 coside, isotridecyl-poly(ethyleneglycolether), N-Decanoyl 15 95% Reagent alcohol 1 min N-methylglucamine, n-Decyl al-D-glucopyranoside, Decyl 16 100% Reagent alcohol 30 sec B-D-maltopyranoside, n-Dodecanoyl-N-methylglucamide, 17 100% Reagent alcohol 30 sec 18 100% Reagent alcohol 30 sec n-Dodecyl C-D-maltoside, n-Dodecyl B-D-maltoside, Hep 19 Xylene 1 min taethylene glycol monodecyl ether, Heptaethylene glycol 2O Xylene 3 min 45 monotetradecyl ether, n-Hexadecyl B-D-maltoside, Hexa 21 Xylene End ethylene glycol monododecyl ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol monooctadecyl Although the invention has been described in some detail ether, Hexaethylene glycol monotetradecyl ether, Igepal with reference to the preferred embodiments, those of skill CA-630 (Octylphenyl-polyethylene glycol), Igepal CA-210 in the art will realize, in light of the teachings herein, that 50 (polyoxyethylene(2) isooctylphenyl ether), Igepal CA-520 certain changes and modifications can be made without (polyoxyethylene(5) isooctylphenyl ether), Igepal CO-630 departing from the spirit and scope of the invention. Accord (polyoxyethylene(9)nonylphenyl ether), Igepal CO-720 (polyoxyethylene(12) nonylphenyl ether), Igepal CO-890 ingly, the invention is limited only by the claims. (polyoxyethylene(40) nonylphenyl ether), Igepal CO-990 What is claimed is: 55 (polyoxyethylene(100) nonylphenyl ether), Igepal DM-970 1. A process of staining a fixed biological sample, com (polyoxyethylene(150) dinonylphenyl ether), Methyl-6-O- prising in the following order: (N-heptylcarbamoyl-)-C-D-glucopyranoside. Nonaethylene (a) contacting the sample with a hematoxylin stain, glycol monododecyl ether, N-Nonanoyl-N-methylglucam (b) contacting the sample with a detergent of a type and ine, Octaethylene glycol monodecyl ether, Octaethylene in an amount Sufficient to reduce a staining artifact, 60 glycol monododecyl ether, Octaethylene glycol monohexa (c) contacting the sample with an Orange G counterstain, decyl ether, Octaethylene glycol monooctadecyl ether, Octa and ethylene glycol monotetradecyl ether, Octyl-B-D-glucopy (d) contacting the sample with an Eosin Y- Light Green/ ranoside, Pentaethylene glycol monodecyl ether, Fast Green counterstain. Pentaethylene glycol monododecyl ether, Pentaethylene gly 2. The method of claim 1, comprising contacting the 65 col monohexadecyl ether, Pentaethylene glycol monohexyl sample with the detergent in combination with a bluing ether, Pentaethylene glycol monooctadecyl ether, Pentaeth agent. ylene glycol monooctyl ether, Polyethylene glycol digly US 7,186,522 B2 11 12 cidyl ether, Polyethylene glycol ether W-1, Polyoxyethylene Lugol (Iodine Potassium Iodide), Niaproof (2-Ethylhexyl 10 tridecyl ether, Polyoxyethylene 100 stearate, Polyoxy sulfate sodium salt), Niaproof 4 (7-Ethyl-2-methyl-4-unde ethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl cyl sulfate Sodium salt), optionally Substituted alkylsul ether, Polyoxyethylene 40 stearate, Polyoxyethylene 50 fonate salts (including salts of 1-butanesulfonate, pentane stearate, Polyoxyethylene 8 stearate, Polyoxyethylene bis Sulfonate, hexanesulfonate, 1-Octanesulfonate, (imidazolyl carbonyl), Polyoxyethylene 25 propylene glycol 1-decanesulfonate, 1-dodecanesulfonate, 1-heptane stearate, Saponin, Span 20 (Sorbitan monolaurate), Span 40 Sulfonate, 1-heptanesulfonate, 1-nonanesulfonate, 1-pro (Sorbitan monopalmitate), Span 60 (Sorbitan monostearate), panesulfonate, and 2-bromoethanesulfonate, especially the Span 65 (Sorbitan tristearate), Span 80 (Sorbitan Sodium salts), Sodium cholate, Sodium deoxycholate, monooleate), Span 85 (Sorbitan trioleate), a Tergitol, Tet 10 optionally substituted Sodium dodecyl sulfate, Sodium octyl radecyl-B-D-maltoside, Tetraethylene glycol monodecyl Sulfate, Sodium taurocholate, Sodium taurochenodeoxycho ether, Tetraethylene glycol monododecyl ether, Tetraethyl late, Sodium taurohyodeoxycholate, Taurolithocholic acid ene glycol monotetradecyl ether, Triethylene glycol mono 3-sulfate disodium salt, TaurourSodeoxycholic acid sodium decyl ether, Triethylene glycol monododecyl ether, Trieth salt, Trizma dodecyl sulfate, Ursodeoxycholic acid, a salt of ylene glycol monohexadecyl ether, Triethylene glycol 15 any thereof, and an acid of any thereof. monooctyl ether, Triethylene glycol monotetradecyl ether, 13. The method of claim 1, wherein the detergent is a Triton CF-21, Triton CF-32, Triton DF-12, Triton DF-16, cationic detergent. Triton GR-SM, Triton N-101 (Polyoxyethylene branched 14. The method of claim 13, wherein the cationic deter nonylphenyl ether), Triton QS-15, Triton QS-44, Triton gent is selected from the group consisting of Alkyltrimethy RW-75 (Polyethylene glycol 260 monoChexadecyl/octade lammonium bromide, Benzalkonium chloride, Benzyldim cyl) ether and 1-Octadecanol), Triton X-100 (Polyethylene ethylhexadecylammonium chloride, glycol tert-octylphenyl ether), Triton X-102, Triton X-15, Benzyldimethyltetradecylammonium chloride, Benzyldode Triton X-151, Triton X-200, Triton X-207, Triton X-114, cyldimethylammonium bromide, Benzyltrimethylammo Triton X-165, Triton X-305, Triton X-405 (polyoxyethylene nium tetrachioroiodate, Dimethyldioctadecylammonium (40) isooctylphenyl ether), Triton X-405 reduced (polyoxy 25 bromide, Dodecylethyldimethylammonium bromide, Dode ethylene(40) isooctylcyclohexyl ether), Triton X-45 (Poly cyltrimethylammonium bromide, Ethyihexadecyldimethy ethylene glycol 4-tert-octylphenyl ether), Triton X-705-70, lammonium bromide, Girard’s reagent T. Hexadecyltrim TWEEN in any form including: TWEEN 20 (Polyoxyeth ethylammonium bromide, N,N',N'-Polyoxyethylene(10)-N- ylene sorbitan monolaurate), TWEEN 21 (Polyoxyethylene tallow- 1,3-diaminopropane, Thonzonium bromide, and sorbitan monolaurate), TWEEN 40 (polyoxyethylene(20) 30 Trimethyl(tetradecyl)ammonium bromide. sorbitan monopalmitate), TWEEN 60 (Polyethylene glycol 15. The method of claim 14, wherein the cationic deter sorbitan monostearate), TWEEN 61 (Polyethylene glycol gent is benzalkonium chloride. sorbitan monostearate), TWEEN 65 (Polyoxyethylene sor 16. The method of claim 1, wherein the detergent is a bitan Tristearate), TWEEN 80 (Polyoxyethylene sorbitan Zwitterionic detergent. monooleate), TWEEN 81 (Polyoxyethylene sorbitan 35 17. The method of claim 16, wherein the Zwitterionic monooleate), TWEEN 85 (polyoxyethylene(20) sorbitan detergent is selected from the group consisting of CHAPS trioleate), Tyloxapol (4-(1,1,3,3-tetramethylbutyl)phenol (3-(3-cholamidopropyl)-dimethylammonio)-1-propane polymer with formaldehyde and oxirane), and n-Undecyl sulfonate), CHAPSO (3-(3-chotamidopropyl) dimethyl B-D-glucopyranoside. ammonio)-2-hydroxy-1-propane-Sulfonate), 3-(Decyldim 11. The method of claim 1, wherein the detergent is an 40 ethylammonio) propanesulfonate, anionic detergent. 3-(Dodecyldimethylammonio) propanesulfonate, 3-(N, 12. The method of claim 11, wherein the anionic detergent N-Dimethylmyristylammoni- o) propanesulfonate, 3-(N, is selected from the group consisting of Chenodeoxycholic N-Dimethyloctadecylammonio) propanesulfonate, 3-(N, acid, Cholic acid, Dehydrocholic acid, Deoxycholic acid, N-Dimethyloctylammonio) propanesulfonate, and 3-(N.N- Digitonin, Digitoxigenin, N, N-Dimethyldodecylamine 45 Dimethylpalmityl-ammonio)propanesulfonate. N-oxide, Sodium docusate, Sodium glycochenodeoxycho 18. The method of claim 17, wherein the Zwitterionic late, Glycocholic acid, Glycodeoxycholic acid, Glycolitho detergent is CHAPS. cholic acid 3-sulfate disodium salt, Glycolithocholic acid ethyl ester, N-Lauroylsarcosine, Lithium dodecyl sulfate,