MXPA05010599A - Papanicolau staining process - Google Patents

Abstract

A method for treating a biological sample with a Papanicolaou staining process is provided. The method comprises incorporating a detergent treatment into the staining process at any of various steps. The method has been found to advantageously reduce the number of artifacts produced during Papanicolaou staining. Also provided is a sample stained by such a process.

Description

PAPINICOLAOU STAINING PROCESS TECHNICAL FIELD This invention relates to methods, articles and compositions useful for the staining of biological samples.

BACKGROUND OF THE INVENTION The methods for medical diagnostic tests are critical exploration tools for the early detection of pathological conditions. Early detection allows the identification of these conditions at a stage where successful treatment is most likely. Early treatment also often involves less harmful or less invasive treatment methods, and decreases the impact on the patient. In addition to routine screening, diagnostic tests are also used in a variety of different applications, including biopsy analysis and monitoring of the results of ongoing medical treatment. A particularly useful tool in diagnostic tests is the Papanicolaou staining process. This process was initially developed for the staining of gynecological samples, and has led to a dramatic decrease in the mortality rate from cervical cancer. Papanicolaou staining is now used in the diagnosis of a variety of pathological conditions of many different tissues and organs. However, Papanicolaou staining is subject to image defects that may adversely affect the ability of a technologist or machine to successfully read a processed sample. These image defects include spurious non-specific cytoplasmic staining, defects such as cytoplasmic cracking, flake appearance, fuzzy nuclear detail, hypochromatic staining and hyperchromatic staining. These image defects, at a minimum, reduce the fraction of the sample that can be evaluated, can produce false staining patterns that interfere with accurate diagnosis, and can render the sample illegible. The difficulties in analyzing these samples lead to an increase in patient anxiety, reexploration costs, delays in diagnosis, and more importantly, in potential misdiagnosis. There is a need in the art for improved methods for diagnosing samples by staining, and for compositions and articles of manufacture useful in these methods.

BRIEF DESCRIPTION OF THE INVENTION An improved method for treating a biological sample with a Papanicolaou staining process is provided. The method includes incorporating a detergent treatment in the staining process in any of several steps. It has been found that the method advantageously reduces the amount of image defects produced during Papanicolaou staining. A stained sample is also provided by this process.

DETAILED DESCRIPTION OF THE INVENTION The inventors have advantageously discovered that the incorporation of a detergent treatment in a Papanicolaou staining procedure reduces the appearance of image defects to which the method is susceptible. It has been found that the use of a detergent cleans excess cytoplasm staining of stained cells by this procedure, allowing increased contrast and improved ability to detect cellular, nuclear chromatin morphology. Additionally, removal of excess cytoplasm staining can lead to an increased ability to detect hyperchromasia associated with abnormality. The terms "pap smear process", "pap stain", "pap test" and the like, refer to a modified or unmodified Papanicolaou staining procedure, which differentially stains the nuclei and cytoplasm of cells in a sample. , for example a sample obtained during a gynecological examination such as a pap smear.

The test is based on the ability to distinguish the staining pattern, the intensity of staining and the size and shape of the nucleus and the cytoplasm of different types of cells in the sample. The Papanicolaou staining process advantageously provides dark colored stained nuclei, and transparent cytoplasms, which is particularly useful for detecting abnormal cells in multiple layer samples provided by certain sampling techniques, including pap smears. A useful test depends on the ability to read a significant percentage of cells in the sample, otherwise the accuracy of the test may be compromised, and the samples may be rendered useless, which leads to additional costs of reexploration and increased anxiety of the patient. patient. Consequently, defects in the staining image, which reduce the ability to discern the true staining pattern of cells in a sample, can dramatically impair the usefulness of a pap test. Defects in the image may reduce the ability to differentiate between the staining pattern of the nucleus and the cytoplasm, may increase the percentage of cells or the area of the sample that is illegible, and may increase cytoplasmic staining, thereby decreasing the transparency, which is one of the primary advantages of pap testing, making it more difficult to distinguish abnormal cells in the sample. Before describing the present invention in greater detail, it should be understood that this invention is not limited to the particular methodology, solutions or apparatus described, since such methods, solutions or apparatuses may vary, of course. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. The use of the singular forms "a", "an", "the" or "the" include plural references unless the context clearly dictates otherwise. Thus, for example, the reference to "a sample" includes a plurality of samples, the reference to "a detergent" includes a plurality of these detergents, the reference to "a counterstain" includes a plurality of counterstains, and the like. Additionally, the use of specific plural references, such as "two", "three", etc., informs about larger amounts of the same subject unless the context clearly dictates otherwise. Terms such as "connected", "joined", and "linked" are used interchangeably herein and include connection, link, direct link and also indirect, unless the context clearly dictates otherwise. Where a range of values is listed, it should be understood that each integral value that intervenes, and each fraction thereof, between the upper and lower bounds listed in this range, is also specifically described, together with each sub-range between said values. The upper and lower limits of any range may be included or excluded independently of the range, and each range where one, none or both limits are included is also included in the invention. Where a value that is being discussed has inherent limits, for example where a component can be present at a concentration from 0 to 100%, or where the pH of an aqueous solution can range from 1 to 14, those inherent limits are specifically described. Where a value is explicitly listed, it should be understood that values that are of approximately the same amount or number as the described value, are also within the scope of the invention, as are the ranges based thereon. where a combination is described, each sub-combination of the elements of that combination is also specifically described and is within the scope of the invention. On the contrary, where elements or groups of different elements are described, combinations of them are also described. Where any element of an invention is described as having a plurality of alternatives, the examples of that invention in which each alternative is excluded alone or in any combination with the other alternatives, is also described herein; more than one element of an invention may have these exclusions, and all combinations of elements having exclusions are described by this means. Unless otherwise defined or the context clearly dictates otherwise, all the technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described in the present application can be used for the practice or testing of the invention, preferred methods and materials are now described. The term "optional" or "optionally" means that the event or circumstance described after it may or may not occur, and includes instances where the event or circumstance occurs, and instances in which it does not.

STAINING PAPANICULAOU A Papanicolaou staining process comprises at least one staining step with hematoxylin, one step of cytoplasmic counterstaining with Orange G, and one step of counterstaining with Eosin Y-Light Green / Fast Green. The process may incorporate a "bluish" pap staining step that advantageously produces a sample with transparent cell cytoplasms, which is particularly useful for the analysis of cells covering multiple layers, such as those obtained from a pap smear. Before starting a staining process Papanicolaou, the sample should be fixed. Any method of fixation that produces satisfactory results may be used. Typically an alcoholic fixation is used with pap staining, eg, 95% ethanol, 100% methanol, 80% propanol, or 95% ethanol / ether (1: 1). Commercial fixers can also be used. A typical pap smear process comprises a step of staining with hematoxylin in combination with a mordant metal to stain the chromatin of the cells, and can be done progressively or regressively. Chromatin staining is a critical part of this test, since the size, intensity of staining and chromatin staining pattern is a critical element in the analysis of abnormal cells in the sample. Any form of hematoxylin may be used, including Harris's, Gill's, Delafield's, Ehrlich's or Mayer's hematoxylin. You can also use hematoxylin substitutes that use dyes, for example, Thionin, Victoria Blue B, Azure B, etc. If it is done regressively, the sample is superimposed, and then differentiated with an agent (for example diluted HCl), which allows the intensity of staining to be reduced to the desired level. This causes the color change from blue / purple to salmon pink / red. A "blueing" step may be used to restore the blue color using an agent known in the art (e.g., Scott jet water substitute, water with ammonium, lithium carbonate). The bluing solution can be incorporated without a differentiation step. In the Papanicolaou stain, multiple cytoplasmic countercurrents are used to allow the identification of the various typical cells in the sample, their morphology and their frequency.

The cytoplasmic stains Orange G and Eosin Y-Light Green / Fast Green are prepared in alcohol. Orange G is a monochromatic dye that stains the cytoplasms of yellow or orange cells if keratin is present, allowing the distinction of mature cells or malignant cells that are producing keratin and maturing, of the less conspicuous benign cells. Orange G can be used in the form of a solution known in the art for staining cells, for example OG-6. Traditionally, Eosin Y-Light Green / Fast Green is prepared as a mixture of three dyes, Eosin Y, Light Green SF and / or Fast Green FCF, and Bismarck Brown. Example mixtures include EA36, EA50 and EA65. Eosin Y produces a pink color in the nucleoli, cilia, red blood cells and the cytoplasm of mature squamous cells. Light Green SF Yellowish and Fast Green FCF produce a blue / green color in the cytoplasm of metabolically active parabasal squamous cells, intermediate squamous cells and columnar cells. Bismarck Brown does not produce a useful dyeing pattern in pap staining, and can be excluded in variations of the method. You can also use substitutes for Light Green SF Yellowish, for example Fast Green FCF. The cytoplasmic countercurrents can also be a mixture of OG and Eosin Y-Light Green / Fast Green, resulting in a single solution for use as a countercurrent. The alcohol in which these cytoplasmic countercurrents are prepared results in transparent cytoplasms which are useful for the analysis of samples stained by the Papanicolaou staining process. The washing steps are incorporated into the staining procedure when appropriate, as are hydration / dehydration series when transferred between aqueous and non-aqueous solutions. Upon completion of the staining process, the samples can be cleaned and assembled, as is known in the art. Additional procedures may be performed in the samples before or after the Papanicolaou procedure. The samples can then be analyzed manually and / or by an appropriate apparatus, for example an automated imaging system. Automated imaging systems include ThinPrep®l mag ing System from Cytyc Corporation, the TriPath Focal Point ™ Profiler, I Chroma Vision Acis® System, and the Veracel Verasys Imaging System. A variety of modifications to the Papanicolaou staining procedure can be made, and are known in the art. For example, the process can be modified as described in U.S. Patent Nos. 5, 168,066 and 6,348,325 (issued December 1, 1992 and February 19, 2002, respectively, and assigned to Cytyc Corp.) to incorporate the use of thionin. Other thiazine dyes and triarylmethane dyes may also be used.

THE SAMPLE The portion of the sample that is to be analyzed can be any source of biological material that can be obtained from a living organism directly or indirectly, including cells, tissue or fluid. Non-limiting examples of the sample include blood, urine, semen, milk, sputum, mucus, pleural fluid, pelvic fluid, synovial fluid, ascites fluid, body cavity washings, eye brushing, skin scrapes, a buccal swab, a swab vaginal, a pap smear, a rectal swab, an aspirate, a needle biopsy, a section of tissue obtained for example by surgery or by autopsy, plasma, serum, medullary fluid, lymphatic fluid, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, tumors, organs and samples of cell culture constituents in vitro. Typically the sample will be a Pap smear as routinely obtained during a gynecological examination or a biopsy suspected of containing abnormal cells. The sample can be a positive control sample that is known to contain abnormal cells. A negative control sample can also be used to determine whether a given set of dyeing conditions produces false positives (a positive signal even in the absence of abnormal cells in the sample). Commonly, the sample is provided for staining on a glass slide or similar surface, for example, a flask, a coverslip, or other surface that is transparent to the incident light used for analysis.

THE DETERGENT Any detergent that detectably reduces the image defects produced during a Papanicolaou staining procedure can be used. The detergent is used in an amount and for a suitable time to detectably reduce said image defects. The percentage of at least one of these image defects can desirably be reduced by at least about 10%, 20%, 30%, 40%, 50%, 70%, 90%, 95%, 98%, or more, incorporating a detergent washing as described here. When used in imaging systems, which select certain areas of the slide ("fields of interest") to be analyzed based on any of a variety of parameters, as is known in the art, the invention desirably decreases the amount of falsely selected fields (which are falsely selected by analysis based on image defects in the staining that meet the selection criteria of the imaging system) at least about 10%, 20%, 30%, 40%, 50% , 70%, 90%, 95%, 98% or more. This increases the percentage of useful fields selected for the analysis. The detergent can be non-ionic, cationic, anionic or zwitterionic. You can also use mixtures of detergents. Classes of detergents, for example, include alcohol ether sulfates, alcohol sulfates, alkanolamides, alkyl sulfonates, amine oxides, amphoteric detergents, anionic detergents, betaine derivatives, cationic detergents, disulfonates, dodecylbenzenesulfonic acid, ethoxylated alcohols, alkyl phenols. ethoxylates, ethoxylated fatty acids, hydrotropes of glycerol esters, lauryl sulfates, mono and di glycerides, nonionic detergents, phosphate esters, quaternary detergents, and sorbitan derivatives. Nonionic detergents, as an example, include BigCHAP (N, N-bis [3- (D-gIuconamido) propyl] coamide), Bis (polyethylene glycol bis [imidazoyl carbonyl]), Brij®30 (polyoxyethylene 4 lauryl ether), Brij®35 (lauryl ether of polyoxyethylene 23), Brij®52 (cetyl ether of polyoxyethylene 2), Brij®56 (cetyl ether of polyoxyethylene 10), Brij®58 (cetyl ether of polyoxyethylene 20), Brij®72 (stearyl ether of polyoxyethylene 2), Brij ®76 (polyoxyethylene stearyl ether 10), Brij®78 (polyoxyethylene stearyl ether 20), Brij®92 (polyoxyethylene oleyl ether 2), Brij®97 (polyoxyethylene oleyl ether 10), Brij®98 (polyoxyethylene 20 oleyl ether), Brij®700 (polyoxyethylene stearyl ether 100), Cremophor® EL (castor oil / ethylene oxide polyether), decaethylene glycol monododecyl ether, octanoyl-N-methylglucamide (MECA -8), decanoyl-N-methylglucamide (MECA-10), n-octylglucoside, n-dodecylglucoside, poly (ethylene glycol ether) n isotridecyl, N-decanoyl-N-methylglucamine, n-decyl-D-glucopyranoside, Decil ß -D-maltopyranoside, n-dodecanoyl-N-methylglucamide, n-dodecyl-D-maltoside, n-dodecyl-β-D-maltoside, monodecyl ether of heptaethylene glycol, monotetradecyl ether of heptaethylene glycol, n-hexadecyl-D-maltoside, monododecyl ether of hexaethylene glycol, monohexadecyl ether of hexaethylene glycol, monohectadecyl ether of hexaethylene glycol, monotetradecyl ether of hexaethylene glycol, Igepal® CA-630 (octylphenyl-polyethylene glycol), lgepal® CA-210 (polyoxyethylene isooctylphenyl ether (2)), Igepal® CA-520 ( polyoxy isooctylphenyl ether ethylene (5)), Igepal® CO-630 (polyoxyethylene nonylphenyl ether (9)), Igepal® CO-720 (polyoxyethylene nonylphenyl ether (12)), Igepal® CO-890 (polyoxyethylene nonylphenyl ether (40)) , Igepal® CO-990 (polyoxyethylene nonylphenyl ether (100)), Igepal® DM-970 (polyoxyethylene dinonylphenyl ether (150)), methyl-6-O- (N-heptylcarbamoii) -aD-glucopyranoside, monododecyl ether nonaethylene glycol, N-nonanoyl-N-methylglucamine, octaethylene glycol monodecyl ether, octaethylene glycol monododecyl ether, octaethylene glycol monohexadecyl ether, octaethylene glycol monooctadecyl ether, octaethylene glycol monotetradecyl ether, octyl-β-D-glucopyranoside, pentaethylene glycol monodecyl ether, monohexyl ether Pentaethylene glycol, pentaethylene glycol monooctadecyl ether, pentaethylene glycol monooctyl ether, polyethylene glycol diglycidyl ether, polyethylene glycol ether W-1, polyoxyethylene tridecyl ether 10, stearate polyoxyethylene 100, polyoxyethylene isohexadecyl ether 20, polyoxyethylene oleyl ether 20, polyoxyethylene stearate 40, polyoxyethylene stearate 50, polyoxyethylene 8 stearate, polyoxyethylene bis (imidazolyl carbonyl), polyoxyethylene propylene glycol stearate 25, Saponin, Span® 20 (monolaurate Sorbitan), Span® 40 (Sorbitan monopalmitate), Span® 60 (Sorbitan monostearate), Span® 65 (Sorbitan tristearate), Span® 80 (Sorbitan monooleate), Span® 85 (Sorbitan trioleate), Tergitol in any form (including types 15-S-5, 15-D-7, 15-S-9, 15-S-12, 15-S-30, NP-4. NP-7. NP-9. NP-10. NP-40, NPX (imbentin-N / 63), T N-3 (polyethylene glycol trimethylnonyl ether), TMN-6 (polyethylene glycol trimethylnonyl ether), TMN-10 (polyethylene glycol trimethylnonyl ether), MIN FOAM 1x and MIN FOAM 2x), tetradecyl-β-D-maltoside, tetraethylene glycol monodecyl ether, tetraethylene glycol monododecyl ether, tetraethylene glycol monotetradecyl ether, triethylene glycol monodecyl ether, triethylene glycol monododecyl ether, triethylene glycol monohexadecyl ether, triethylene glycol monooctyl ether, ether Triethylene glycol monotetradecyl, Triton® CF-21, Triton® CF-32, Triton® DF-12, Triton® DF-16, Triton® GR-5M, Triton® N-101 (branched polyoxyethylene nonylpentyl ether), Triton® QS -15, Triton® QS-44, Triton®RW-75 (mono (hexadecyl / octadecyl) ether of polyethylene glycol 260 and 1-octadecanol), Triton® X-100 (tert-octylphenyl ether of polyethylene glycol), Triton® X-102 , Triton®X-15, Triton® X-151, Triton® X -200, Triton® X-207, Triton® X-114, Triton® X-165, Triton® X-305, Triton® X-405 (isooctylphenyl ether of polyoxyethylene (40)), Triton® X-405 (isooctylcyclohexyl ether) polyoxyethylene (40)), Triton® X-45 (4-tert-octylphenyl polyethylene glycol ether), Triton® X-705-70, TWEEN® in any form (including TWEEN®20 (polyoxyethylene sorbitan monolaurate), TWEEN® 21 (polyoxyethylene sorbitan monolautate), TWEEN® 40 (polyoxyethylene (20) sorbitan monopalmitate), TWEEN® 60 (polyethylene glycol monostearate), TWEEN® 61 (polyethylene glycol sorbitan monostearate), TWEEN® 65 (polyoxyethylene sorbitan tristearate), TWEEN® 80 (polyoxyethylene sorbitan monooleate), TWEEN® 81 (polyoxyethylene sorbitan monooleate), and TWEEN® 85 (polyoxyethylene (20) sorbitan trioleate) )), Tyloxapol (polymer of 4- (1, 1,3,3-tetramethylbutyl) phenol with formaldehyde and oxirane), and n-undecyl-β-D-glucopyranoside. Examples of anionic detergents include chenodeoxycholic acid, cholic acid, dehydrocholic acid, deoxylic acid, digitonin, digitoxigenin, N, N-dimethyldodecylamine N-oxide, sodium docusate, sodium glycoquenodeoxycholate, glycolic acid, glycodeoxycholic acid, disodium salt of glycolytocholic acid 3-sulfate, glycolytocholic acid ethyl ester, N-lauroyl sarcosine, lithium dodecyl sulfate, Lugol (potassium iodine iodide), Niaproof (sodium salt of 2-ethylhexyl sulfate), Niaproof 4 (sodium sulfate salt of 7-ethyl-2-methyl-4-undecyl), optionally substituted alkylsulfonate salts (including salts of 1-butanesulfomate, pentanesulfonate, hexanesulfonate, 1-octanesulfonate, 1-decanesulfonate, 1-dodecanesulfonate, 1-heptanesulfonate, 1-heptanesulfonate, 1-nonanesulfonate, 1-propanesulfonate, and 2-bromoethanesulfonate, especially sodium salts), sodium cholate, sodium deoxycholate, optional substituted sodium dodecylisulfate Sodium octylsulfate, sodium taurocholate, sodium taurokenedeoxycholate, sodium tauroyodeoxycholate, disodium salt of taurolitocholic acid 3-sulfate, sodium salt of tauroursodeoxycholic acid, Trizma® dodecylisulfate, ursodeoxycholic acid. The anionic detergent can be provided in the form of acid or salt, or both. Cationic detergents example include alkyltrimethylammonium bromide, benzalkonium chloride, benzyldimethylhexadecylammonium chloride, benzyldimethyltetradecylammonium chloride, bromide bencildodecildimetilamonio, tetracloroyodato benzyltrimethylammonium bromide, dimethyldioctadecylammonium bromide dideciletildimetilamonio, dodecyltrimethylammonium bromide, bromide etilhexadecildimetilamonio, reagent Girard, hexadecyltri methyl ammonium bromide, N, N ', N'-polyoxyethylene (10) -N-tallow-1,3-diaminopropane, tonzonium bromide, and trimethyl (tetradecyl) ammonium bromide. Hybrid ion detergents as an example include CHAPS (3- (3-colamidoproyl) -dimethylammonium-1-propanesulfonate.), CHAPSO (2-hydroxy-3. (3-colamidoproyl) dimethyl-ammonium-propanesulfonate.), 3- (decyldimethylammonium) propanesulfonate, 3- (dodecyldimethylammonium) propanesulfonate, 3- (N, N-dimethyl-myristylammonium) propanesulfonate, 3- (N, N-dimethyloctanedylammonium) propanesulfonate, 3- (N, N-dimethyloctylammonium propanesulfonate) ), and 3- (N, N-dimethylpalmitylammonium) propanesulfonate. The detergent includes those that are known or that can be discovered in the art. The detergent can be synthesized or can be obtained commercially from any of a variety of vendors (eg, Sigma-Aldrich Corp., St. Louis, MO, USA, www.sigmaaldrich.com). It has been found that benzalkonium chloride and CHAPS are particularly useful for reducing image defects during Papanicolaou staining. A given detergent can be tested with respect to its ability to reduce image defects by incorporation in a wash step in a Papanicolaou staining process, which can be performed under conditions known to produce image defects in the skin. staining The sample treated with test detergent can then be evaluated against a processed sample without a detergent treatment, and against a sample processed with a benzalkonium chloride treatment in another positive control treatment that is known to reduce image defects in staining . Typically, the amount of detergent used will be at least 0.0001% and up to about 10% of the solution in which it is to be used, and can be at least about 0.0005%, 0.001%, 0.005%, 0.01%, 0.05% , 0.1%, 0.2%, 0.5%, or 1% or more of the solution; the amount of detergent can be about 5% or less, about 2.5% or less, about 2% or less, about 1% or less, about 0.5% or less, about 0.2% or less, about 0.1% or less, of approximately 0.05% or less of the solution, or any rank or sub-rank between them. The detergent treatment can be carried out at any pH that allows the reduction of image defects, provided that it does not adversely affect the ability to analyze the sample. Thus, the detergent treatment can be performed at approximately a pH of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or within any range or sub-range between them. Typically, the treatment will be carried out at a pH of about 2 to about 10. The detergent can be used at any point in a Papanicolaou staining process. For example, detergent can be used before staining with hematoxylin, mixed with hematoxylin stain, after staining with hematoxylin, combined with a bluing solution, after the bluing solution, or combined with the solutions of Orange G, Eosin Y - Light Green / Fast Green and other counterstain solutions.

EXAMPLES The following examples are set forth to provide those skilled in the art with a complete description of how to make and use the present invention, and are not intended to limit the scope of what is contemplated as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (eg, quantities, temperature, etc.), but some experimental error and deviation should be taken into account. Unless otherwise indicated, the parts are parts by weight, the temperature is expressed in degrees centigrade and the pressure is atmospheric or close to it, and all materials are commercially available.

EXAMPLE 1 Very visible cytoplasmic cracking was found on slides with pap smears processed under test pap smear conditions. For the test methods in order to reduce cytoplasmic cracking, 4 different samples of pap smear were obtained, slides were made with the samples, fixed, allowed to dry for a day, and then stained with a modified Pap stain. according to the following procedure: Steps: 1 Start station 2 Wash test step 3 5 min 50% alcoholic reagent 4 5 min saline solution (10% NaCl in 50% alcohol) 5 min saline solution (10% NaCl in 50% alcohol) 6 1 min 95% alcoholic reagent 7 1 min 95% alcoholic reagent 8-22 1 min nuclear stain (15 min) 23 20 sec isopropyl alcohol 24 20 sec isopropyl alcohol 25 30 sec isopropyl alcohol 26 20 sec isopropyl alcohol 27-34 1 min Contratinción (mix of Orange G and Eosin Fast Green / Light Green) (8 min) 35 30 sec 100% alcoholic reagent 36 3 min isoporopyl alcohol 37 3 min isopropyl alcohol 38 1 min Xylene 39 1 min Xylene 40 Fin Xylene Remove the final xylene slides Cover the slide manually using Permount All four samples produced cytoplasmic cracking and were subjected to wash test steps to determine conditions that could reduce image defects. 10 slides of each sample were prepared. Washes were made separately with water of five and fifteen minutes, as well as also fifteen minute washes with surfactant FC120 (3M Corporation) and surfactant FC170 (3M Corporation), on the ten slides of each sample. it was found that none of these washing conditions dramatically reduced the stained image defects corresponding to cytoplasmic cracking. Ten additional slides were prepared from each of the four samples, and subjected to additional washing test steps, including fifteen minute washes in 0.1% FC170, pH 6.9; Tween® 80 0.1%, pH 6.9; Igepal CA-630 0.1%, pH 7.0, CHAPS 0.1%, pH 7.7, ZEPHIRAN® 0.1%, pH 7.2, water, water without the passage of alcohol at 50% before the passage of saline, water without the passage of saline , and water without the passage of alcohol at 50% before the passage of saline and without saline passages. ZEPHIRAN® dramatically reduced spurious cytoplasmic staining and reduced cytoplasmic cracking. CHAPS® also reduced both image defects to a certain degree in this concentration. The other treatments at the indicated concentrations had little effect on these image defects by staining.

EXAMPLE 2 An example of a Papanicolaou staining protocol that incorporates a detergent wash step is given below:

Claims (31)

1. CLAIMS 1. A method for staining a fixed biological sample, comprising: contacting the sample with a haematoxylin dye; contact the sample with an Orange G counterstain; to put the sample in contact with a counterpart Eosin Y -Light Green / Fast Green; contact the sample with a detergent of a type and in an amount sufficient to reduce an image defect in the staining. The method of claim 1, further characterized in that the detergent is selected from the group consisting of alcohol ether sulfates, alcohol sulfates, alkanolamides, alkyl sulfonates, amine oxides, amphoteric detergents, anionic detergents, betaine derivatives, detergents cationics, disulfonates, dodecylbenzenesulfonic acid, ethoxylated alcohols, ethoxylated alkyl phenols, ethoxylated fatty acids, hydrotropes of glycerol esters, lauryl sulfates, mono and di glycerides, nonionic detergents, phosphate esters, quaternary detergents, and sorbitan derivatives. 3. The method of claim 1, further characterized in that the detergent is a non-ionic detergent. 4. The method of claim 3, further characterized in that the detergent is selected from the group consisting of BigCHAP (N, N-bis [3- (D-gluconamido) propyl] coamid), Bis (polyethylene glycol bis [imidazoyl carbonyl]] ), Brij®30 (polyoxyethylene 4 lauryl ether), Brij®35 (polyoxyethylene 23 lauryl ether), Brij®52 (polyoxyethylene 2 cetyl ether), Brij®56 (polyoxyethylene 10 cetyl ether), Brij®58 ( polyoxyethylene cetyl ether 20), Brij®72 (polyoxyethylene stearyl ether 2), Brij®76 (polyoxyethylene stearyl ether 10), Brij®78 (polyoxyethylene stearyl ether 20), Brij®92 (polyoxyethylene oleyl ether 2) , Brij®97 (polyoxyethylene oleyl ether 10), Brij®98 (polyoxyethylene 20 oleyl ether), Brij®700 (polyoxyethylene stearyl ether 100), Cremophor® EL (castor oil / ethylene oxide polyether), ether Decanoethylene glycol monododecyl, octanoyl-N-methylglucamide (MECA-8), decanoyl-N-methylglucamide (MECA- 10), n-octylglucoside, n-dodecylglucoside, poly (ethylene glycol ether) n of isotridecyl, N-decanoyl-N-methylglucamine, n-decyl aD-glucopyranoside, Decyl-D-maltopyranoside, n-dodecanoyl-N-methylglucamide, n-dodecyl aD-maltoside, n-dodecyl β-D-maltoside, monodecyl ether of heptaethylene glycol, monotetradecyl ether of heptaethylene glycol, n-hexadecyl β-D-maltoside, monododecyl ether of hexaethylene glycol, monohexadecyl ether of hexaethylene glycol, monooctadecyl ether of hexaethylene glycol, monotetradecyl ether of hexaethylene glycol, Igepal® CA-630 (octylphenyl-polyethylene glycol), Igepal® CA-210 (isooctylphenyl ether of polyoxyethylene (2)), Igepal® CA-520 (isooctylphenyl ether of polyoxyethylene (5)), Igepal® CO- 630 (polyoxyethylene nonylphenyl ether (9)), Igepal® CO-720 (polyoxyethylene nonylphenyl ether (12)), Igepal® CO-890 (polyoxyethylene nonylphenyl ether (40)), Igepal® CO-990 (nonylphenyl ether polyoxyethylene (100)), Igepal® DM-970 ( polyoxyethylene (150) dinonylphenyl ester, methyl-6-O- (N-heptylcarbamoyl) -aD-glucopyranoside, nonaethylene glycol monododecyl ether, N-nonanoyl-N-methylglucamine, octaethylene glycol monodecyl ether, octaethylene glycol monododecyl ether, monohexadecyl ether octaethylene glycol, octaethylene glycol monooctadecyl ether, octaethylene glycol monotetradecyl ether, octyl-β-D-glucopyranoside, pentaethylene glycol monodecyl ether, pentaethylene glycol monohexyl ether, pentaethylene glycol monooctadecyl ether, pentaethylene glycol monooctyl ether, polyethylene glycol diglycidyl ether, polyethylene glycol ether W- 1, polyoxyethylene 10 tridecyl ether, polyoxyethylene 100 stearate, polyoxyethylene 20 isohexadecyl ether, polyoxyethylene 20 oleyl ether, polyoxyethylene 40 stearate, polyoxyethylene stearate 50, polyoxyethylene 8 stearate, polyoxyethylene bis (imidazolyl carbonyl), polyoxyethylene propylene glycol stearate 25, Saponin, Span® 20 (Sorbitan monolaurate), Span® 40 (Sorbitan monopalmitate), Span® 60 (Sorbitan monostearate), Span® 65 (Sorbitan tristearate), Span® 80 (monooleate of Sorbitol), Span® 85 (Sorbitan trioleate), Tergitol in any form (including types 15-S-5, 15-D-7, 15-S-9, 15-S-12, 15-S-30 , NP-4, NP-7, NP-9, NP-10, NP-40, NPX (Imbentin-N / 63), TMN-3 (polyethylene glycol trimethylnonlyl ether), TMN-6 (polyethylene glycol trimethylnonyl ether) , TMN-10 (polyethylene glycol trimethylnonlyl ether), MIN FOAM 1x and MIN FOAM 2x), tetradecyl-β-D-maltoside, tetraethylene glycol monodecyl ether, tetraethylene glycol monododecyl ether, tetraethylene glycol monotetradecyl ether, triethylene glycol monodecyl ether, ether Triethylene glycol monododecyl, triethylene glycol monohexadecyl ether, triethylene glycol monooctyl ether, triethylene glycol monotetradecyl ether, T ritón® CF-21, Triton® CF-32, Triton® DF-12, Triton® DF-16, Triton® GR-5M, Triton® N-101 (branched polyoxyethylene nonylpentyl ether), Triton® QS-15, Triton ® QS-44, Triton®RW-75 (mono (hexadecyl / octadecyl) ether of polyethylene glycol 260 and 1-octadecanol), Triton® X-100 (tert-octylphenyl ether of polyethylene glycol), Triton® X-102, Triton®X -15, Triton® X-151, Triton® X-200, Triton® X-207, Triton® X-114, Triton® X-165, Triton® X-305, Triton® X-405 (polyoxyethylene isooctylphenyl ether ( 40)), Triton® X-405 (polyoxyethylene isooctylcyclohexyl ether (40)), Triton® X-45 (polyethylene glycol 4-tert-octylphenyl ether), Triton® X-705-70, TWEEN® in any form (including TWEEN®20 (polyoxyethylene sorbitan monolaurate), TWEEN® 21 (polyoxyethylene sorbitan monolautate), TWEEN® 40 (polyoxyethylene (20) sorbitan monopalmitate), TWEEN® 60 (polyethylene glycol monostearate), TWEEN® 61 (polyethylene glycol monostearate) l sorbitan), TWEEN® 65 (polyoxyethylene sorbitan tristearate), TWEEN® 80 (polyoxyethylene sorbitan monooleate), TWEEN® 81 (polyoxyethylene sorbitan monooleate), and TWEEN® 85 (polyoxyethylene (20) sorbitan trioleate), Tyloxapol (polymer of 4- (1, 1,3,3-tetramethylbutyl) phenol with formaldehyde and oxirane), and n-undecyl-β-D-glucopyranoside. The method of claim 1, further characterized in that the detergent is an anionic detergent. The method of claim 5, further characterized in that the detergent is selected from the group consisting of chenodeoxycholic acid, cholic acid, dehydrocholic acid, deoxycholic acid, digitonin, digitoxigenin, N, N-dimethyldodecylamine N-oxide, sodium docusate , sodium glycokenedeoxycholate, glycolic acid, glycodeoxycholic acid, disodium salt of glycolytocholic acid 3-sulfate, glycolytocholic acid ethyl ester, N-lauroyl sarcosine, lithium dodecylisphate, Lugol (potassium iodine iodide), Niaproof (sodium sulphate salt 2-ethylhexyl), Niaproof 4 (sodium salt of 7-ethyl-2-methyl-4-undecyl sulfate), optionally substituted alkylsulfonate salts (including salts of 1-butanesulfomate, pentanesulfonate, hexanesulfonate, 1-octanesulfonate, 1-decanesulfonate , 1-dodecanesulfonate, 1-heptanesulfonate, 1-heptanesulfomate, 1-nonanesulfonate, 1-propanesulfonate, and 2-bromoethanesulfonate, especially sodium salts), cholate sodium, sodium deoxycholate, optionally substituted sodium dodecylsulfate, sodium octyl sulfate, sodium taurocholate, sodium taurokenedeoxycholate, sodium tauroyodeoxycholate, taurolithocholic acid 3-sulfate disodium salt, sodium salt of tauroursodeoxycholic acid, Trizma® dodecylisulfate, acid ursodeoxycholic, a salt of any of them and an acid of any of them. The method of claim 1, further characterized in that the detergent is a cationic detergent. The method of claim 7, further characterized in that the detergent is selected from the group consisting of alkyltrimethylammonium bromide, benzalkonium chloride, Benzyldimethylhexadecylammonium chloride, benzyldimethyltetradecylammonium chloride, bromide bencildodecildimetilamonio, tetracloroyodato benzyltrimethylammonium bromide dimethyldioctadecylammonium bromide dideciletildimetilamonio, dodecyltrimethylammonium bromide, etilhexadecildimetilamonio bromide, Girard reagent, hexadecyltrimethylammonium bromide, N, N ', N'-polyoxyethylene (10) -N-tallow-1,3-diaminopropane, tonzonium bromide, and trimethyl (tetradecyl) ammonium bromide. The method of claim 8, further characterized in that the detergent is benzalkonium chloride. 10. The method of claim 1, further characterized in that the detergent is a hybrid ion detergent. The method of claim 10, further characterized in that the detergent is selected from the group consisting of CHAPS (3- (3-colamidoproyl) -dimethylammonium 1-propanesulfonate.), CHAPSO (1-propanesulfonate 2). -hydroxy-3. (3-cholamidoproyl) dimethylammonium), 3- (decyldimethylammonium) propanesulfonate, 3- (dodecyldimethylammonium) propanesulfonate, 3- (N, N-dimethyl-myristylammonium) propanesulfonate) , 3- (N, N-dimethyloctanedylammonium) propanesulfonate, 3- (N, N-dimethyloctylammonium) propanesulfonate, and 3- (N, N-dimethylpalmitylammonium) propanesulfonate. 12. The method of claim 11, further characterized in that the detergent is CHAPS. The method of claim 1, further characterized in that a mixture of detergents is used. The method of claim 1, further characterized in that the sample is contacted with the detergent before hematoxylin staining. 15. The method of claim 1, further characterized in that the detergent is combined with the hematoxylin dye. 16. The method of claim 1, further characterized in that the sample is contacted with the detergent after hematoxylin staining. 17. The method of claim 1, including contacting the sample with the detergent in combination with a bluing solution. 18. The method of claim 1, further characterized in that the sample is contacted with the detergent prior to counterstaining with Orange G. 19. The method of claim 1, further characterized in that the sample is contacted with the detergent prior to counterstaining with Eosin Y-Light Green / Fast Green. 20. The method of claim 1, further characterized in that the sample is contacted with the detergent in combination with a counterint. The method of claim 20, further characterized in that the contratinte is Orange G. 22. The method of claim 20, further characterized in that the contratinte is Eosin Y-Light Green / Fast Green. 23. The method of claim 1, further characterized in that the method is automated. 24. The method of claim 1, further characterized in that the method is performed manually. 25. The method of claim 1, further characterized in that the sample is contacted with the detergent in a solution at a concentration of at least about 0.0001% and less than about 10% of the solution. 26. The method of claim 25, further characterized in that the detergent is at least about 0.01% of the solution. 27. The method of claim 25, further characterized in that the detergent is at least about 0.1% of the solution. The method of claim 25, further characterized in that the detergent is at least about 0.5% of the solution. 29. The method of claim 25, further characterized in that the detergent is at least about 1% of the solution. 30. A fixed biological sample, which has a reduced level of image defects in the staining, said sample contains cells treated with hematoxylin, Orange G and Eosin Y - Light Green / Fast Green, further characterized because the sample has been put in contact with a detergent that reduced the amount of image defects in the sample staining. 31. Use of a detergent for the reduction of image defects in a fixed biological sample stained by a Papanicolaou staining process. SUMMARY A method is provided for treating a biological sample with a Papanicolaou staining process. The method includes incorporating a detergent treatment in the staining process in any of several steps. It has been found that the method advantageously reduces the amount of image defects produced during Papanicolaou staining. A stained sample is also provided by this process.