US 20130164760A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0164760 A1 Fischer et al. (43) Pub. Date: Jun. 27, 2013

(54) METHODS AND COMPOSITIONS FOR Publication Classi?cation PREPARING SAMPLES FOR IMMUNOSTAINING (51) Int- Cl G01N 1/30 (2006.01) (71) Applicant: TriPath Imaging, Inc., Burlington, NC (52) US, Cl, (Us) CPC ...... G01N 1/30 (2013.01) USPC ...... 435/7 .1 (72) Inventors: Timothy J. Fischer, Raleigh, NC (US); Ramona R. Nelson, Durham, NC (US); Adriann J. Taylor, Durham, NC (US); (57) ABSTRACT Clark M. Whitehead, Cary, NC (U S) (73) Assignee: TriPath Imaging, Inc., Burlington, NC COmPPSiIiOn§ and methods for Preparing a_ S_amP1_e for 1m?‘ (Us) nological staimng are provided. Compos1t1ons 1nclude kits comprising a ?rst solution comprising a surfactant and a (21) Appl. No.: 13/667,297 second solution comprising a chaotropic agent. Methods _ comprise contacting a sample, such as cells or tissues, With a (22) Flled? NOV- 2’ 2012 ?rst solution comprising a surfactant and then contacting the Related U's' Apphcatlon. . Data Thepmethodsam 1e With a seconddoes not solution require com egtremge risin a heatchaotro fof icantigen a ent. (60) Provisional application No. 61/555,139, ?led on Nov. retrieval and therefore, maintains the cellular morphology of 3,2011. the sample. Patent Application Publication Jun. 27, 2013 Sheet 1 0f 4 US 2013/0164760 A1 Patent Application Publication Jun. 27, 2013 Sheet 2 0f 4 US 2013/0164760 A1 Patent Application Publication Jun. 27, 2013 Sheet 3 0f 4 US 2013/0164760 A1

FIG. 6 Patent Application Publication Jun. 27, 2013 Sheet 4 0f 4 US 2013/0164760 A1

FIG. 8 US 2013/0164760 A1 Jun. 27, 2013

METHODS AND COMPOSITIONS FOR The exposed antigen is then available for binding to an anti PREPARING SAMPLES FOR body, Which can be detected using any method knoWn in the IMMUNOSTAINING art. The presently disclosed methods and compositions alloW for antigen retrieval in the absence of extreme heat, thus CROSS-REFERENCE TO RELATED maintaining cellular morphology. APPLICATIONS [0007] The folloWing embodiments are encompassed by [0001] This application claims the bene?t of US. Provi the present invention: sional Application No. 61/555,139, ?led Nov. 3, 2011, Which [0008] l.A kit comprising: is herein incorporated by reference in its entirety. [0009] a) a ?rst solution comprising a surfactant; and [0010] b) a second solution comprising a chaotropic FIELD OF THE INVENTION agent. [0002] The present invention relates to methods and com [0011] 2. The kit of embodiment 1, Wherein said surfactant positions to enhance the detection of proteins by making is an anionic surfactant. epitopes more accessible for antibody binding. [0012] 3. The kit of embodiment 2, Wherein said anionic surfactant is sodium dodecyl sulfate (SDS). BACKGROUND OF THE INVENTION [0013] 4. The kit of embodiment 3, Wherein said ?rst solu tion comprises about 0.01% to about 1% SDS. [0003] Fixation of tissue and cellular samples often makes [0014] 5. The kit of embodiment 4, Wherein said ?rst solu protein-based epitopes inactive, or inaccessible for immun ostaining due to protein cross-linking. Antigen retrieval (AR) tion comprises about 0.05% to about 0.5% SDS. is the process by Which target epitopes are made accessible [0015] 6. The kit of embodiment 5, Wherein said ?rst solu for immunostaining. Overcoming ?xation-induced cross tion comprises about 0.1% SDS. linking alloWs for target epitopes buried Within the tertiary [0016] 7. The kit of any one of embodiments 1-6, Wherein structure of proteins to become accessible for binding With said ?rst solution is an aqueous solution. primary antibodies. Antigen retrieval also advantageously [0017] 8. The kit of any one of embodiments 1-7, Wherein loWers the threshold for detection of antigens, thereby reduc said chaotropic agent is a chaotropic salt. ing the amount of antibody needed for detection, reducing [0018] 9. The kit of embodiment 8, Wherein said chaotropic background staining, and minimiZing the occurrence of false salt is a thiocyanate or perchlorate. negative results. Therefore, immunohistochemistry (IHC) [0019] 10. The kit of embodiment 9, Wherein said thiocy and immunocytochemistry (ICC) protocols often include a anate is guanidine thiocyanate. pretreatment step to increase the intensity of immuno staining [0020] 11. The kit of embodiment 10, Wherein said second and to retrieve the antigen of interest. solution comprises about 0.3M to about 30M guanidine thio [0004] Many existing pretreatment steps for immunostain cyanate. ing involve the incubation of the cellular or tissue sample of [0021] 12. The kit of embodiment 11, Wherein said second interest at high temperatures of about 800 C. or greater in solution comprises about 1M to about 10M guanidine thio various solutions (e.g., buffers, EDTA, acids, bases, surfac cyanate. tants) to prepare the sample for immunostaining. These types [0022] 13. The kit of embodiment 12, Wherein said second of sample pretreatment methods Were largely developed and solution comprises about 3M guanidine thiocyanate. optimiZed for processing tissue samples. Tissues can With [0023] 14. The kit of embodiment 9, Wherein said perchlo stand the high temperature of processing and maintain mor rate is lithium perchlorate. phology because these tissues are often ?xed in formalin and then embedded in para?in prior to sectioning and IHC pro [0024] 15. The kit of embodiment 14, Wherein said second solution comprises about 0.3M to about 30M lithium perchlo cessing, and because the tissue sections maintain the support of the surrounding stromal tissue architecture. Cytology rate. specimens are not ?xed to the same degree as tissue samples, [0025] 16. The kit of embodiment 15, Wherein said second are not normally embedded in paraf?n, and do not contain solution comprises about 1M to about 10M lithium perchlo stromal support material to maintain cellular morphology rate. during high heat pretreatment. Thus, high heat pretreatment [0026] 17. The kit of embodiment 16, Wherein said second methods that serve to increase epitope exposure and accessi solution comprises about 3M lithium perchlorate. bility to the primary antibody degrade the cellular morphol [0027] 18. The kit of any one of embodiments 1-17, ogy of cytology specimens. Wherein said second solution further comprises a Weak sur [0005] Therefore, pretreatment methods and compositions factant. that are effective in antigen retrieval, yet maintain cellular [0028] 19. The kit of embodiment 18, Wherein said Weak morphology, are needed for the processing of cytology surfactant is a nonionic surfactant. samples, in particular, in preparation for immunostaining. [0029] 20. The kit of embodiment 19, Wherein said Weak surfactant is nonyl phenoxypolyethoxylethanol (N P-40). BRIEF SUMMARY OF THE INVENTION [0030] 21. The kit of embodiment 20, Wherein said second [0006] Compositions and methods for preparing samples solution comprises about 0.01% to about 1% NP-40. for immunostaining are provided herein. Compositions dis [0031] 22. The kit of embodiment 21, Wherein said second closed herein include kits comprising a ?rst solution compris solution comprises about 0.05% to about 0.5% NP-40. ing a surfactant and a second solution comprising a chaotro [0032] 23. The kit of embodiment 22, Wherein said second pic agent. The tWo solutions ?nd use in retrieving protein solution comprises about 0.1% NP-40. epitopes by sequentially contacting a sample, such as cells or [0033] 24. The kit of any one of embodiments 1-23, tissues, With the ?rst solution and then the second solution. Wherein said second solution is an aqueous solution. US 2013/0164760 A1 Jun. 27, 2013

[0034] 25. The kit of any one of embodiments 1-24, [0060] 49. The method of embodiment 48, Wherein said Wherein said kit further comprises an antibody that speci? second solution comprises about 0.3M to about 30M lithium cally binds an antigen. perchlorate. [0035] 26. The kit of embodiment 25, Wherein said antigen [0061] 50. The method of embodiment 49, Wherein said is a nuclear antigen. second solution comprises about 1M to about 10M lithium [0036] 27. The kit of embodiment 25, Wherein said antigen perchlorate. is selected from the group consisting of MCM2, MCM7, p16, [0062] 51. The method of embodiment 50, Wherein said and Ki67. second solution comprises about 3M lithium perchlorate. [0037] 28. The kit of any one of embodiments 25-27, [0063] 52. The method of any one of embodiments 35-51, Wherein said kit further comprises a peroxidase blocking Wherein said second solution further comprises a Weak sur reagent, a protein blocking reagent, chemicals for the detec factant. tion of antibody binding to said antigen, a counterstain, a [0064] 53. The method of embodiment 52, Wherein said bluing agent, and instructions for use. Weak surfactant is a nonionic surfactant. [0038] 29. The kit of embodiment 28, Wherein said chemi [0065] 54. The method of embodiment 53, Wherein said cals for the detection of antibody binding comprise a chro Weak surfactant is nonyl phenoxypolyethoxylethanol (NP mogen and a secondary antibody conjugated to a labeled 40). polymer, Wherein the chromogen comprises 3',3'-diami [0066] 55. The method of embodiment 54, Wherein said nobenZidine, and Wherein the labeled polymer comprises second solution comprises about 0.01% to about 1% NP-40. horseradish peroxidase conjugated to a dextran polymer. [0067] 56. The method of embodiment 55, Wherein said [0039] 30. The kit of embodiment 28 or 29, Wherein said second solution comprises about 0.05% to about 0.5% NP-40. counterstain comprises hematoxylin. [0068] 57. The method of embodiment 56, Wherein said [0040] 31. The kit of any one of embodiments 28-30, second solution comprises about 0.1% NP-40. Wherein said bluing agent comprises a solution comprising [0069] 58. The method of any one of embodiments 35-57, Tris buffered saline, pH 7.4, TWeen-20, and sodium aZide. Wherein said second solution is an aqueous solution. [0041] 32. The kit of any one of embodiments 25-31, fur [0070] 59. The method of any one of embodiments 35-58, ther comprising a positive control sample. Wherein said sample is incubated With said ?rst solution for at [0042] 33. The kit of any one of embodiments 1-32, further least one minute. comprising reagents for Papanicolaou (Pap) staining. [0071] 60. The method of embodiment 59, Wherein said [0043] 34. The kit of embodiment 33, Wherein the reagents sample is incubated With said ?rst solution for about 1 minute for Pap staining comprise EA50 and Orange G. to about 120 minutes. [0044] 35. A method for preparing a sample for immuno [0072] 61. The method of embodiment 60, Wherein said logical staining, said method comprising: sample is incubated With said ?rst solution for about 10 min [0045] a) contacting the sample With a ?rst solution com utes to about 60 minutes. prising a surfactant; and [0073] 62. The method of embodiment 61, Where said [0046] b) contacting the sample With a second solution sample is incubated With said ?rst solution for about 19 min comprising a chaotropic agent. utes. [0047] 36. The method of embodiment 35, Wherein said [0074] 63. The method of any one of embodiments 35-62, surfactant is an anionic surfactant. Wherein said sample is incubated in said ?rst solution at room [0048] 37. The method of embodiment 36, Wherein said temperature. anionic surfactant is sodium dodecyl sulfate (SDS). [0075] 64. The method of any one of embodiments 35-62, [0049] 38. The method of embodiment 37, Wherein said Wherein said sample is heated With said ?rst solution. ?rst solution comprises about 0.01% to about 1% SDS. [0076] 65. The method of any one of embodiments 35-62, [0050] 39. The method of embodiment 38, Wherein said Wherein said sample is incubated With said ?rst solution at ?rst solution comprises about 0.05% to about 0.5% SDS. about 20° C. to about 60° C. [0051] 40. The method of embodiment 39, Wherein said [0077] 66. The method of embodiment 65, Wherein said ?rst solution comprises about 0.1% SDS. sample is incubated With said ?rst solution at about 37° C. to [0052] 41. The method of any one of embodiments 35-40, about 55° C. Wherein said ?rst solution is an aqueous solution. [0078] 67. The method of embodiment 66, Wherein said [0053] 42. The method of any one of embodiments 35-41, sample is incubated With said ?rst solution at about 50° C. Wherein said chaotropic agent is a chaotropic salt. [0079] 68. The method of any one of embodiments 35-67, [0054] 43. The method of embodiment 42, Wherein said Wherein said sample is incubated With said second solution chaotropic salt is a thiocyanate or perchlorate. for at least one minute. [0055] 44. The method of embodiment 43, Wherein said [0080] 69. The method of embodiment 68, Wherein said thiocyanate is guanidine thiocyanate. sample is incubated With said second solution for about 1 [0056] 45. The method of embodiment 44, Wherein said minute to about 120 minutes. second solution comprises about 0.3M to about 30M guani [0081] 70. The method of embodiment 69, Wherein said dine thiocyanate. sample is incubated With said second solution for about 10 [0057] 46. The method of embodiment 45, Wherein said minutes to about 60 minutes. second solution comprises about 1M to about 10M guanidine [0082] 71. The method of embodiment 70, Where said thiocyanate. sample is incubated With said second solution for about 19 [0058] 47. The method of embodiment 46, Wherein said minutes. second solution comprises about 3M guanidine thiocyanate. [0083] 72. The method of any one of embodiments 35-71, [0059] 48. The method of embodiment 43, Wherein said Wherein said sample is incubated With said second solution at perchlorate is lithium perchlorate. room temperature. US 2013/0164760 A1 Jun. 27, 2013

[0084] 73. The method of any one of embodiments 35-71, [0102] FIG. 4 provides an image of SiHa cells immun Wherein said sample is heated With said second solution. ostained With an anti-p16 antibody. Prior to immunostaining, [0085] 74. The method of any one of embodiments 35-71, the cells Were incubated at 50° C. for 19 minutes in 0.1% SDS, Wherein said sample is incubated With said second solution at Washed in TBS, and then incubated at 50° C. for 19 minutes about 30° C. to about 60° C. in 3M LiClO4/0.1% NP-40. The cells Were counterstained [0086] 75. The method of embodiment 74, Wherein said With Pap stain. sample is incubated With said second solution at about 37° C. [0103] FIG. 5 provides an image of a histological section of to about 55° C. paraf?n-embedded tonsil tissue immunostained With tWo [0087] 76. The method of embodiment 75, Wherein said anti-MCM2 antibodies and one anti-MCM7 antibody. The sample is incubated With said second solution at about 50° C. tonsil tissue had been ?xed in 10% formaldehyde for at least [0088] 77. The method of any one of embodiments 35-76, 24 hours and then embedded in paraf?n before sectioning. Wherein said sample is Washed prior to contacting the sample Prior to immunostaining, the section Was incubated at 50° C. With the second solution and after contacting the sample With for 19 minutes in 0.1% SDS, Washed in TBS, and then incu the ?rst solution. bated at 50° C. for 19 minutes in 3M LiClO4/0.1% NP-40. [0089] 78. The method of embodiment 77, Wherein said [0104] FIG. 6 provides an image of a histological section of sample is Washed With buffered saline. paraf?n-embedded cervical intraepithelial neoplasia 3 [0090] 79. The method of embodiment 78, Wherein said (CIN3) tissue immunostained With tWo anti-MCM2 antibod buffered saline is tris-buffered saline. ies and one anti-MCM7 antibody. The cervical tissue had [0091] 80. The method of any one of embodiments 35-79, been ?xed in 10% formaldehyde for at least 24 hours and then Wherein said method further comprises detecting an antigen embedded in paraf?n before sectioning. Prior to immun in said sample using an antibody. ostaining, the section Was incubated at 50° C. for 19 minutes [0092] 81. The method of embodiment 80, Wherein said in 0.1% SDS, Washed in TBS, and then incubated at 50° C. for antigen is a nuclear antigen. 19 minutes in 3M LiClO4/0.1% NP-40. [0093] 82. The method of embodiment 80, Wherein said [0105] FIG. 7 provides an image of a SurePath® loW-grade antigen is selected from the group consisting of MCM2, squamous intraepithelial lesion (LSIL) cervical cytology MCM7, p16, and Ki67. sample immunostained With tWo anti-MCM2 antibodies and one anti-MCM7 antibody. Prior to immunostaining, the [0094] 83. The method of any one of embodiments 35-82, Wherein said sample is a cervical sample. sample Was incubated at room temperature for 19 minutes in 0.1% SDS, Washed in TBS, and then incubated at room tem [0095] 84. The method of any one of embodiments 35-83, Wherein said sample comprises cells or tissue. perature for 19 minutes in 3M LiClO4/0.1% NP-40. The sample Was counterstained With Pap stain. [0096] 85. The method of any one of embodiments 35-84, [0106] FIG. 8 provides an image of a SurePath® high Wherein said method further comprises performing a mor phological analysis of said sample. grade squamous intraepithelial lesion (HSIL) cervical cytol ogy sample immunostained With tWo anti-MCM2 antibodies [0097] 86. The method of any one of embodiments 35-84, and one anti-MCM7 antibody. Prior to immunostaining, the Wherein said method further comprises Papanicolaou (Pap) sample Was incubated at room temperature for 19 minutes in staining of the sample. 0.1% SDS, Washed in TBS, and then incubated at room tem [0098] These and other aspects of the invention are dis perature for 19 minutes in 3M LiClO4/0.1% NP-40. The closed in more detail in the description of the invention given sample Was counterstained With Pap stain. beloW. DETAILED DESCRIPTION OF THE INVENTION BRIEF DESCRIPTION OF THE FIGURES [0107] Compositions and methods are provided that are [0099] FIG. 1 provides an image of a SurePath® (TriPath directed to preparing a sample for immunological staining by Imaging, Inc.) high-grade squamous intraepithelial lesion exposing protein epitopes. Compositions include kits com (HSIL) cervical cytology sample immunostained With an prising a ?rst solution comprising a surfactant (pretreatment anti-Ki67 antibody. Prior to immunostaining, the sample Was solution 1) and a second solution comprising a chaotropic incubated at 50° C. for 19 minutes in 0.1% sodium dodecyl agent (pretreatment solution 2). Methods include contacting a sulfate (SDS), Washed in Tris-buffered saline (TBS), and then sample With a ?rst pretreatment solution that comprises a incubated at 50° C. for 19 minutes in 3M lithium perchlorate surfactant and then a second pretreatment solution that com (LiClO4)/0. 1% nonyl phenoxypolyethoxyl -ethanol (NP-40). prises a chaotropic agent. Once the sample has been prepared The sample Was counterstained With Papanicolaou (Pap) and epitopes have been exposed, antigens can be contacted stain. With antibodies and detected using any antigen-antibody [0100] FIG. 2 provides an image of SiHa (human cervical binding detection method knoWn in the art. The tWo-solution squamous cancer) cells immunostained With an anti-Ki67 method disclosed herein alloWs for suf?cient antigen retrieval antibody. Prior to immunostaining, the cells Were incubated for immunological staining in the absence of extreme heat, at 50° C. for 19 minutes in 0.1% SDS, Washed in TBS, and While maintaining cellular morphology. This is especially then incubated at 50° C. for 19 minutes in 3M LiClO4/0.1% useful for cytology samples Which are more sensitive to the NP-40. The cells Were counterstained With Pap stain. extreme heat often necessary for antigen retrieval. Mainte [0101] FIG. 3 provides an image of a SurePath® HSIL nance of cellular morphology during the antigen retrieval cervical cytology sample immunostained With an anti-p16 process is important for samples for Which cellular morphol antibody. Prior to immunostaining, the sample Was incubated ogy is subsequently assessed. For example, the presently at 50° C. for 19 minutes in 0.1% SDS, Washed in TBS, and disclosed compositions and methods can be used to detect then incubated at 50° C. for 19 minutes in 3M LiClO4/0.1% antigen(s) in a cervical cytology sample in combination With NP-40. The sample Was counterstained With Pap stain. standard Papinicolau counterstaining. US 2013/0164760 A1 Jun. 27, 2013

[0108] The presently disclosed compositions and methods persed or dissolved in Water, Which may or may not comprise are directed to preparing a sample for immunological stain additional components. In other embodiments, the surfactant ing. Immunological staining or immunostaining refers to the or chaotropic agent, or both are dispersed or dissolved in a process by Which a sample is contacted With at least one polar liquid other than Water. antibody and the binding of the antibody to its corresponding [0111] The presently disclosed compositions and methods antigen Within the sample is detected using any method involve a ?rst solution that comprises a surfactant (pretreat knoWn in the art for detecting antigen-antibody binding. Non ment solution 1) and in some embodiments, the second solu limiting examples of immunological staining include immu tion (pretreatment solution 2) also comprises a surfactant. As nohistochemistry, Wherein an antigen Within a tissue sample used herein, the terms “surfactant,” “surface-active agent,” is detected, and immunocytochemistry, Wherein an antigen and “detergent” can be used interchangeably herein and refer Within a cellular sample is detected. The compositions and to molecules that can reduce the surface tension of a liquid. methods are effective in preparing samples for immunologi Surfactants have both hydrophilic and hydrophobic proper cal staining, Which refers to modi?cations of the sample to ties, and thus, canbe solubiliZed to some extent in either Water alloW for access of the antibody used in the staining process to or nonpolar solvents. Surfactants are classi?ed into four pri its antigen. Such modi?cations include permeabiliZation of mary groups: cationic, anionic, non-ionic, and ZWitterionic. the cytoplasmic membrane, and in some instances, the Without being bound by any theory or mechanism of action, nuclear membrane, reversal of protein crosslinks induced by it is believed that the presence of a surfactant in the ?rst ?xatives (e.g., methylene bridges caused by aldehyde ?xa pretreatment solution, and in some embodiments in the sec tion; see, for example, French and Edsall (1 945) Adv Protein ond pretreatment solution, contributes to the permeabiliZa Chem 2:277; Pearse (1980) Histochemistry: Theoretical and tion of the cytoplasmic and in some instances, nuclear mem Applied vol. 1; Fox et al. (1985) .1. Hislochem. Cylochem. branes. Further, surfactants, such as sodium dodecyl sulfate 33:845), and denaturation of the protein antigens. The pro (SDS), also penetrate the hydrophobic interior of proteins and cess of preparing samples for immunological staining that equilibrate the charge distribution of proteins, thereby dena alloWs for access of the antibody to the antigen is also referred turing proteins and increasing epitope exposure and accessi to herein as antigen retrieval or pretreatment. bility to the primary antibody. [0109] As used herein, the term “antigen” refers to a [0112] In some embodiments of the presently disclosed polypeptide having antigenic activity. “Antigenic activity” methods and compositions, the ?rst pretreatment solution refers to the ability of a polypeptide to be used in the produc comprises an anionic surfactant. Anionic surfactants are those tion of antibodies. The presently disclosed methods and com surfactants that have a net negative charge When dissolved or positions can be used to prepare samples for the detection of dispersed in aqueous solutions. Representative, non-limiting any type of antigen, Whether it be nuclear, cytoplasmic, examples of anionic surfactants include alkyl sulfates, such as expressed on the cell surface, or extracellular. In some ammonium lauryl sulfate and sodium dodecyl sulfate (SDS); embodiments, the presently disclosed compositions and alkyl ether sulfates, such as sodium laureth sulfate and methods can be used to enhance the immunological staining sodium myreth sulfate; docusates, such as dioctyl sodium of nuclear antigens and are thus capable of permeabiliZing sulfosuccinate; sulfonate ?uorosurfactants, such as per?uo both the cytoplasmic and nuclear membranes. Non-limiting rooctanesulfonate and per?uorobutanesulfonate; alkyl ben examples of nuclear antigens that can be immunostained Zene sulfonates; alkyl aryl ether phosphate; alkyl ether phos using the presently disclosed methods include the minichro phate; alkyl carboxylates, such as fatty acid salts, and sodium mosome maintenance (MCM) proteins, such as MCM2, stearate; sodium lauroyl sarcosinate; carboxylate ?uorosur MCM3, MCM4, MCM5, MCM6, MCM7, and MCM10, and factants, such as per?uorononanoate and per?uorooctanoate; topoisomerase II. Non-limiting additional examples of anti alkyl sulfate esters, such as sodium cetyl sulfate; alkyl sul gens that can be immunostained using the presently disclosed fonates, such as sodium dodecyl sulfonate and alkyl allyl methods include the estrogen receptor (ER), the progesterone sulfonates; sodium stearate; sodium deoxycholate; and receptor (PR), and p53. In some of these embodiments, the sodium lauroyl sarcosinate. presently disclosed methods can be used to immunostain a [0113] In other embodiments, the surfactant in the ?rst panel of antigens including ER, PR, p53, and Ki67. In other pretreatment solution is cationic (i.e., has a net positive embodiments, the presently disclosed compositions and charge When dissolved or dispersed in aqueous solutions), methods are effective in enhancing the immunological stain nonionic (i.e., has no charge When dissolved or dispersed in ing ofpl6 or Ki67. aqueous solutions), or ZWitterionic (i.e., has a net neutral [0110] The presently disclosed compositions and methods charge When dissolved or dispersed in aqueous solutions, but utiliZe tWo solutions to prepare a sample for immunological has both a negative and positive electrical charge at different staining. As used herein, the term “solution” refers to a mix locations Within the surfactant molecule). ture of at least tWo substances. The term “solution” is not [0114] In certain embodiments, the ?rst pretreatment solu limited to a homogenous mixture, but as used herein, refers to tion comprises sodium dodecyl sulfate (SDS), Which is also mixtures that comprise an ordered phase as Well as those that referred to as sodium laurilsulfate or sodium lauryl sulfate comprise a more disordered phase. For example, solutions (SLS). SDS consists of a tWelve carbon tail attached to a comprising a surfactant in Water or another polar liquid may sulfate group and has the molecular formula NaCl2H25SO4. contain an ordered phase of micelles or a disordered phase of In some of these embodiments, the ?rst pretreatment solution free surfactant molecules or ions in the solution, or a combi comprises about 0.001% to about 10% SDS, including but not nation thereof. In some embodiments, the ?rst or the second limited to about 0.001%, about 0.05%, about 0.01%, about pretreatment solution or both pretreatment solutions are 0.02%, about 0.03%, about 0.04%, about 0.05%, about aqueous solutions. As used herein, the term “aqueous solu 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, tion” refers to a mixture comprising Water. In these embodi about 0.2%, about 0.3%, about 0.4%, about 0.5%, about ments, the surfactant or chaotropic agent, or both are dis 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about US 2013/0164760 A1 Jun. 27, 2013

2%, about 3%, about 4%, about 5%, about 6%, about 7%, [0118] In some of those embodiments Wherein the second about 8%, about 9%, about 10%, and other such values pretreatment solution comprises lithium perchlorate, lithium between about 0.001% and about 10% SDS. In certain perchlorate is present Within the second pretreatment solution embodiments, the ?rst pretreatment solution comprises about at a concentration of about 0.3M to about 30M, including but 0.01% to about 1% SDS. In other embodiments, the ?rst not limited to about 0.3M, about 0.4M, about 0.5M, about pretreatment solution comprises about 0.05% to about 0.5% 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1M, about SDS. In some of these embodiments, the ?rst pretreatment 2M, about 3M, about 4M, about 5M, about 6M, about 7M, solution comprises about 0.1% SDS. about 8M, about 9M, about 10M, about 11M, about 12M, about 13M, about 14M, about 15M, about 16M, about 17M, [0115] The second pretreatment solution utiliZed in the about 18M, about 19M, about 20M, about 21M, about 22M, presently disclosed compositions and methods comprises a about 23M, about 24M, about 25M, about 26M, about 27M, chaotropic agent. As used herein, the term “chaotropic agent” about 28M, about 29M, and about 30M. In certain embodi refers to a sub stance that has the ability to destabiliZe intramo ments, the second pretreatment solution comprises about 1M lecular interactions mediated by non-covalent forces, such as to about 10M lithium perchlorate. In some of these embodi hydrogen bonds, van der Waals forces, and hydrophobic ments, the second pretreatment solution comprises about 3M interactions, Which alloWs for nonpolar compounds, such as lithium perchlorate. proteins, to dissolve more readily in aqueous solutions. With out being bound by any theory or mechanism of action, it is [0119] In other embodiments, the chaotropic salt present in believed that the chaotropic agent in the second pretreatment the second pretreatment solution is a thiocyanate. Non-limit solution contributes to the dissolution of biological mem ing examples of thiocyanates suitable for use in the presently branes and the denaturation of proteins by alloWing Water disclosed methods and compositions include potassium thio molecules to penetrate into the interior of proteins and solvate cyanate (KSCN), sodium thiocyanate (NaSCN), ammonium nonpolar side chains, thereby disrupting the hydrophobic thiocyanate (NH4SCN), and guanidine thiocyanate interactions that normally stabiliZe the native conformation. (C2H6N4S; also referred to as guanidinium thiocyanate). In Non-limiting examples of chaotropic agents suitable for use certain embodiments, the chaotropic salt is guanidine thiocy in the presently disclosed methods and compositions include anate, Which is comprised of the guanidinium cation chaotropic salts, urea, and thiourea. (CH6N3+) and the thiocyanate anion (SCN‘). [0120] In some of those embodiments Wherein the second [0116] In some embodiments, the second pretreatment pretreatment solution comprises guanidine thiocyanate, solution comprises a chaotropic salt. As used herein, the term guanidine thiocyanate is present Within the second pretreat “chaotropic salt” refers to an ionic compound comprised of ment solution at a concentration of about 0.3M to about 30M, cations and anions that can function as a chaotropic agent as including but not limited to about 0.3M, about 0.4M, about de?ned elseWhere herein. In general, it is the anion of the salt 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, that contributes to the chaotropic properties of a chaotropic about 1M, about 2M, about 3M, about 4M, about 5M, about salt. The position of an ion in the Hofmeister series (see, for 6M, about 7M, about 8M, about 9M, about 10M, about 11M, example, Hofmeister (1888) Arch. Exp. Pathol. Pharmacol. about 12M, about 13M, about 14M, about 15M, about 16M, 24:247-260; Zhang and Cremer (2006) Curr Opin Chem Biol about 17M, about 18M, about 19M, about 20M, about 21M, 10:658-663, each of Which are incorporated herein in its about 22M, about 23M, about 24M, about 25M, about 26M, entirety), Which orders ions based on their ability to solvate about 27M, about 28M, about 29M, about 30M, and other proteins, can be used to select a chaotropic salt for use in the such values betWeen about 0.3M and about 30M. In certain presently disclosed compositions and methods. Ions that embodiments, the second pretreatment solution comprises appear late in the Hofmeister series, such as SCN', C104“, I“, about 1M to about 10M guanidine thiocyanate. In some of C103‘, and Br‘, Would be expected to have greater chaotropic these embodiments, the second pretreatment solution com properties. Thus, in some embodiments, the chaotropic salt prises about 3M guanidine thiocyanate. comprises an anion selected from the group consisting of [0121] In certain embodiments, in addition to the chaotro SCN' (thiocyanate), CNS“, C103“, C104“ (perchlorate), I“, pic agent, the second pretreatment solution comprises a Weak Br“, N03“, Cl“, CH3CO2_ (acetate). Representative non-lim surfactant. As used herein, the term “Weak surfactant” refers iting examples of chaotropic salts suitable for use in the to a surfactant or concentration of a surfactant that is capable presently disclosed methods and compositions include guani of lysing mammalian cells, but maintains or has a minimal dine hydrochloride, guanidine thiocyanate, and lithium per effect on cellular morphology. Non-limiting examples of chlorate. Weak surfactants include nonionic or ZWitterionic surfactants. [0117] In particular embodiments, the chaotropic salt is a Alternatively, a Weak surfactant can be a surfactant molecule thiocyanate or perchlorate, Which refers to a salt comprising that is capable of changing cellular morphology at some cations and anions, Wherein the anion is thiocyanate (SCN) or concentrations (e.g., anionic surfactant), but is present in the perchlorate (ClO4'). In some of these embodiments, the second pretreatment solution at a loW enough concentration chaotropic salt is a perchlorate. Non-limiting examples of such that the surfactant has minimal effect on cellular mor perchlorates suitable for use in the presently disclosed meth phology. ods and compositions include ammonium perchlorate [0122] ZWitterionic surfactants are those surfactants that (NH4ClO4), cesium perchlorate (CsClO4), lithium perchlor have a net neutral charge When dissolved or dispersed in ate (LiClO4), magnesium perchlorate (Mg(ClO4)2, potas aqueous solutions, but have both a negative and positive elec sium perchlorate (KClO4), rubidium perchlorate (RbClO4), trical charge at different locations Within the surfactant mol silver perchlorate (AgClO4), perchloric acid (HClO4), cal ecule. Representative non-limiting examples of ZWitterionic cium perchlorate (Ca(ClO4)2), and sodium perchlorate (Na surfactants suitable for use in the presently disclosed methods ClO4). In certain embodiments, the chaotropic salt in the and compositions include those that are based on primary, second pretreatment solution is lithium perchlorate (LiClO4). secondary, or tertiary amines or quaternary ammonium cat US 2013/0164760 A1 Jun. 27, 2013

ions paired With sulfonates, such as 3-[(3-cholamidopropyl) using a needle to aspirate bodily ?uids. Methods for collect dimethylammonio]-1-propane sulfonate (CHAPS); carboxy ing various biological samples are Well knoWn in the art. lates, such as betaines and amino acids; or phosphates, such as [0127] The sample may be un?xed or ?xed using any lecithin. method or ?xative knoWn in the art. Non-limiting examples of [0123] Non-ionic surfactants are those surfactants that have ?xatives suitable for use in the presently disclosed composi no charge When dissolved or dispersed in aqueous solutions. tions and methods include cross-linking ?xatives, such as Representative non-limiting examples of non-ionic surfac aldehydes (e.g., formaldehyde, glutaraldehyde, formalin), tants suitable for use in the presently disclosed methods and Which create covalent chemical bonds betWeen proteins compositions include polysorbates, including but not limited through the formation of methylene bridges (see, for to, polyethoxylated sorbitan fatty acid esters (e.g., TWeen® example, French and Edsall (1 945) Adv Protein Chem 2:277; compounds), such as polyoxyethylene (POE) sorbitan Pearse (1980) Histochemistry: Theoretical and Applied vol. monooleate (Tween@ 80), POE sorbitan monostearate 1; Fox et al. (1985) J. Histochem. Cylochem. 33:845); pre (Tween@ 60), POE sorbitan monolaurate (Tween@ 20), and cipitating ?xatives, such as alcohols (e.g., ethanol, methanol) POE sorbitan monopalmitate (Tween@ 40); sorbitan deriva and acetone; oxidiZing agents, such as osmium tetroxide, tives (e.g., Span® compounds); ethylene oxide/propylene potassium dichromate, chromic acid, and potassium perman oxide copolymers (e.g., Pluronic® compounds, Which are ganate; mercurial, such as B-5 and Zenker’s; and picrates. also knoWn as poloxamers); polyoxyethylene ether com [0128] In some embodiments, the sample comprises a tis pounds, such as those of the Brij® family, including but not sue sample. Tissue samples canbe prepared using any method limited to polyoxyethylene stearyl ether (also knoWn as poly knoWn in the art, including freeZing or embedding the tissue oxyethylene (100) stearyl ether and by the trade name Brij® in para?in or an epoxy or acrylic resin prior to sectioning. In 700); polyoxyethylene glycol octylphenol ethers, such as certain embodiments, the tissue sample is ?xed in a solution polyoxyethylene p-t-octyl phenol (Triton X-1000®); polyox comprising about 10% formaldehyde for about 24 hours prior ypropylene glycol alkyl ethers; glucoside alkyl ethers, such as to sectioning. octyl glucoside; glycerol alkyl ethers; polyoxyethylene gly [0129] In particular embodiments, the sample comprises col alkylphenol ethers; nonyl phenoxylpolyethoxylethanol cervical cells, as cervical tissue samples or as cervical cells in (NP-40; also knoWn as Tergitol® type NP-40); and ethers of suspension, particularly in a liquid-based preparation. In fatty alcohols. some embodiments, cervical samples are collected according [0124] In some embodiments, the second pretreatment to liquid-based cytology specimen preparation guidelines solution comprises nonylphenol ethoxylate (NP-40). In some such as, for example, the SurePath® (TriPath Imaging, Inc.) of tho se embodiments Wherein the second pretreatment solu or the ThinPrep® preparation (CYTYC, Inc.). Cervical tion comprises NP-40, NP-40 is present Within the second samples may be transferred to a glass slide for vieWing under pretreatment solution at a concentration of about 0.001% to magni?cation. In some of these embodiments, a patient cer about 10%, including but not limited to about 0.001%, about vical sample is collected into a liquid medium, such as, for 0.05%, about 0.01%, about 0.02%, about 0.03%, about example, in a SurePathTM collection vial (TriPath Imaging, 0.04%, about 0.05%, about 0.06%, about 0.07%, about Inc.). An automated processor such as the PrepStainTM sys 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, tem (TriPath Imaging, Inc.) is used to collect cells from the about 0.4%, about 0.5%, about 0.6%, about 0.7%, about liquid medium and to deposit them in a monolayer on a glass 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, slide for further analysis. about 5%, about 6%, about 7%, about 8%, about 9%, about [0130] In one embodiment the cervical sample Will be col 10%, and other such values betWeen about 0.001% and about lected and processed to provide a monolayer sample, as set 10%. In certain embodiments, the second pretreatment solu forth in Us. Pat. No. 5,346,831, herein incorporated by ref tion comprises about 0.01% to about 1% NP-40. In other erence. The monolayer method relates to a method for pro embodiments, the second pretreatment solution comprises ducing a monolayer of cytological material on a cationically about 0.05% to about 0.5% NP-40. In some ofthese embodi charged substrate. The method comprises the steps of ments, the second pretreatment solution comprises about separating the cytological material by centrifugation over a 0.1% NP-40. density gradient, producing a packed pellet of the cytological [0125] Thus, in some embodiments, the second pretreat material, mixing the pellet of the cytological material, With ment solution comprises an aqueous solution of about 3 M draWing an aliquot of a predetermined volume from the lithium perchlorate or guanidine thiocyanate and about 0.1% mixed pellet, depositing the aliquot and a predetermined vol NP-40. In some of these embodiments, this second pretreat ume of Water into a sedimentation vessel, Which is removably ment solution is used in combination With a ?rst pretreatment secured to the cationically-charged substrate, alloWing the solution that comprises an aqueous solution of about 0.1% cytological material to settle onto the substrate under the SDS for preparing samples for immunological staining. force of gravity, and after settlement of the cytological mate [0126] The tWo presently disclosed pretreatment solutions rial, removing the Water from the sedimentation vessel. For ?nd use in preparing samples for immunological staining. As automated analysis, the sedimentation vessel may be used herein, the term “sample” refers to a biological sample detached from the substrate. Disaggregation may be by any or a sample obtained from a biological material in Which method known in the art, such as syringing, trypsiniZing, expression of a protein can be detected. Non-limiting ultrasonication, shaking, vortexing, or by use of the device examples of biological samples include cells (including described in Us. Pat. No. 5,316,814, the contents of Which cytology samples and cultured cells), tissues, biopsies, are herein incorporated by reference. smears, and bodily ?uids, such as blood, lymph, urine, saliva, [0131] Slide specimens may be ?xed or un?xed and may be and gynecological ?uids. Biological samples may be analyZed immediately folloWing preparation or may be stored obtained from a patient by a variety of techniques including, for later analysis. In some embodiments, prepared slides are for example, by lavage, scraping or sWabbing an area or by stored in about 95% ethanol for a minimum of 24 hours. US 2013/0164760 A1 Jun. 27, 2013

Alternatively, in other embodiments, slides are stored in the [0137] Following the incubation of the sample in the ?rst ?rst pretreatment solution disclosed herein (i.e., a solution pretreatment solution that comprises a surfactant, the sample comprising a surfactant). is contacted With a second pretreatment solution that com [0132] According to the presently disclosed methods for prises a chaotropic agent, and in some embodiments, a Weak preparing a sample for immunological staining, the sample is surfactant. This can be accomplished by removing the sample ?rst contacted With the ?rst pretreatment solution that com from the ?rst pretreatment solution or removing the solution prises a surfactant, and then With the second pretreatment from the sample and then transferring the sample to the sec solution that comprises a chaotropic agent, and in some ond pretreatment solution or applying the second solution to embodiments, a Weak surfactant. The sample can be con the sample. Following removal of the ?rst pretreatment solu tacted With the presently disclosed pretreatment solutions tion from the sample or the sample from the ?rst pretreatment using any method that results in the sample coming into solution, the sample can be Washed to remove the residual contact With the solution. Therefore, in some embodiments, ?rst pretreatment solution prior to contacting the sample With the pretreatment solution can be added to the sample in such the second pretreatment solution. a manner as to cover the sample With the solution. Alterna [0138] As used herein, the term “Wash” as it relates to a tively, the sample can be added to the pretreatment solution, sample refers to transiently contacting the sample With and in some embodiments, the sample can be submersed in another solution, other than the ?rst or second pretreatment the solution. solutions to remove traces of the active component of the [0133] In some embodiments, the sample is incubated With pretreatment solution (e.g., surfactant, chaotropic agent). In the ?rst pretreatment solution for at least one minute. In other some embodiments, the Wash solution is Water or a buffered embodiments, the sample is incubated With the ?rst pretreat saline. Various buffered salines used for cellular and molecu ment solution for about 1 minute to about 120 minutes, lar biological methods are knoWn in the art and include, but including but not limited to, about 1, about 2, about 3, about are not limited to, phosphate-buffered saline (PBS), HEPES 4, about 5, about 6, about 7, about 8, about 9, about 10, about buffered saline, and tris-buffered saline (TBS). In some 11, about 12, about 13, about 14, about 15, about 16, about 17, embodiments, the sample is Washed With tris-buffered saline about 18, about 19, about 20, about 21, about 22, about 23, after removal of the ?rst pretreatment solution and prior to about 24, about 25, about 30, about 40, about 50, about 60, contacting the sample With the second pretreatment solution. about 70, about 80, about 90, about 100, about 1 10, about 120 The sample may be Washed one or more times With the Wash minutes, and other such values betWeen about 1 minute and solution. about 120 minutes. In particular embodiments, the sample is incubated With the ?rst pretreatment solution for about 10 [0139] The sample is then contacted With the second pre minutes to about 60 minutes. In some of these embodiments, treatment solution that comprises a chaotropic agent and in the sample is incubated With the ?rst pretreatment solution some embodiments, a Weak surfactant. In some embodi comprising a surfactant for about 19 minutes. ments, the sample is incubated With the second pretreatment [0134] In other embodiments, the ?rst pretreatment solu solution for at least one minute. In other embodiments, the tion may also serve as a storage buffer for a sample, particu solution is incubated With the second pretreatment solution larly a sample that has been ?xed, Wherein the sample is for about 1 minute to about 120 minutes, including but not stored in the ?rst pretreatment solution for at least about 24 limited to, about 1, about 2, about 3, about 4, about 5, about 6, hours, about 48 hours, about 72 hours, about 4 days, about 5 about 7, about 8, about 9, about 10, about 11, about 12, about days, about 7 days, about 2 Weeks, about a month, about 1 13, about 14, about 15, about 16, about 17, about 18, about 19, year, or greater. about 20, about 21, about 22, about 23, about 24, about 25, [0135] The sample can be incubated in the ?rst pretreat about 30, about 40, about 50, about 60, about 70, about 80, ment solution at room temperature or heat can be applied. about 90, about 100, about 1 10, about 120 minutes, and other Thus, in some embodiments, the incubation temperature of such values betWeen about 1 minute and about 120 minutes. the sample in the ?rst pretreatment solution is about 20° C. to In particular embodiments, the sample is incubated With the about 60° C., including but not limited to about 20° C., about second pretreatment solution for about 10 minutes to about 60 21° C., about 22° C., about 23° C., about 24° C., about 25° C., minutes. In some of these embodiments, the sample is incu about 26° C., about 27° C., about 28° C., about 29° C., about bated With the second pretreatment solution for about 19 30° C., about 31° C., about 32° C., about 33° C., about 34° C., minutes. about 35° C., about 36° C., about 37° C., about 38° C., about [0140] The sample can be incubated in the second pretreat 39° C., about 40° C., about 41° C., about 42° C., about 43° C., ment solution at room temperature or heat can be applied. about 44° C., about 45° C., about 46° C., about 47° C., about Thus, in some embodiments, the incubation temperature of 48° C., about 49° C., about 50° C., about 51° C., about 52° C., the sample in the second pretreatment solution is about 20° C. about 53° C., about 54° C., about 55° C., about 56° C., about to about 60° C., including but not limited to about 20° C., 57° C., about 58° C., about 59° C., about 60° C., and other about 21° C., about 22° C., about 23° C., about 24° C., about such values betWeen about 20° C. and about 60° C. In certain 25° C., about 26° C., about 27° C., about 28° C., about 29° C., embodiments, the sample is incubated With the ?rst pretreat about 30° C., about 31° C., about 32° C., about 33° C., about ment solution at a temperature of about 37° C. to about 55° C. 34° C., about 35° C., about 36° C., about 37° C., about 38° C., In some of these embodiments, the incubation temperature is about 39° C., about 40° C., about 41° C., about 42° C., about about 50° C. 43° C., about 44° C., about 45° C., about 46° C., about 47° C., [0136] In particular embodiments, the sample is incubated about 48° C., about 49° C., about 50° C., about 51° C., about With the ?rst pretreatment solution at about 50° C. for about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., 19 minutes. In some of these embodiments, the ?rst pretreat about 57° C., about 58° C., about 59° C., about 60° C., and ment solution is an aqueous solution comprising about 0.1% other such values betWeen about 20° C. and about 60° C. In SDS. certain embodiments, the sample is incubated With the second US 2013/0164760 A1 Jun. 27, 2013

pretreatment solution at a temperature of about 370 C. to yields an F(ab')2 fragment that has tWo antigen-combining about 55° C. In some of these embodiments, the incubation sites and is still capable of cross-linking antigen. temperature is about 50° C. [0148] “Fv” is the minimum antibody fragment that con [0141] In particular embodiments, the sample is incubated tains a complete antigen recognition and binding site. In a With the second pretreatment solution at about 500 C. for tWo-chain Fv species, this region consists of a dimer of one about 19 minutes. In some of these embodiments, the sample heavy- and one light-chain variable domain in tight, non has been incubated With the ?rst pretreatment solution at covalent association. In a single-chain Fv species, one heavy about 500 C. for about 19 minutes prior to incubation With the and one light-chain variable domain can be covalently linked second pretreatment solution. by ?exible peptide linker such that the light and heavy chains [0142] One of skill in the art Will recognize that the incu can associate in a “dimeric” structure analogous to that in a bation temperature and time period of the incubation With the tWo-chain Fv species. It is in this con?guration that the three ?rst, second, or ?rst and second pretreatment solutions Will CDRs of each variable domain interact to de?ne an antigen vary depending upon the type of sample being prepared for binding site on the surface of the VH-VL dimer. Collectively, immuno staining and the extent to Which the sample has been the six CDRs confer antigen-binding speci?city to the anti ?xed. For example, those samples that have been ?xed With a body. HoWever, even a single variable domain (or half of an cross-linking ?xative might require a higher temperature or Fv comprising only three CDRs speci?c for an antigen) has longer incubation period With the ?rst, second, or ?rst and the ability to recognize and bind antigen, although at a loWer second pretreatment solutions When compared to a sample a?inity than the entire binding site. that has been ?xed With alcohol. Further, a paraf?n-embedded [0149] The Fab fragment also contains the constant domain tissue sample might be able to Withstand greater incubation of the light chain and the ?rst constant domain (C H1) of the temperatures and longer incubation periods With the ?rst, heavy chain. Fab fragments differ from Fab‘ fragments by the second, or ?rst and second pretreatment solutions than a addition of a feW residues at the carboxy terminus of the cytology sample, Without affecting cellular morphology. heavy-chain CH1 domain including one or more cysteines [0143] Once the sample has been prepared by contacting from the antibody hinge region. Fab'-SH is the designation the sample With the ?rst and second pretreatment solutions, herein for Fab' in Which the cysteine residue(s) of the constant the method can further comprise using any method knoWn in domains bear a free thiol group. F(ab')2 antibody fragments the art to detect an antigen in the prepared sample using an originally Were produced as pairs of Fab' fragments that have antibody. The terms “antibody” and “antibodies” broadly hinge cysteines betWeen them. encompass naturally occurring forms of antibodies and [0150] Polyclonal antibodies can be prepared by immuniz recombinant antibodies such as single-chain antibodies, chi ing a suitable subject (e.g., rabbit, goat, mouse, or other meric and humanized antibodies and multi-speci?c antibod mammal) With a protein immunogen. The antibody titer in the ies as Well as fragments and derivatives of all of the foregoing, immunized subject can be monitored over time by standard Which fragments and derivatives have at least an antigenic techniques, such as With an enzyme linked immunosorbent binding site. Antibody derivatives may comprise a protein or assay (ELISA) using immobilized protein. At an appropriate chemical moiety conjugated to the antibody. time after immunization, e.g., When the antibody titers are [0144] “Antibodies” and “immunoglobulins” (Igs) are gly highest, antibody-producing cells can be obtained from the coproteins having the same structural characteristics. While subject and used to prepare monoclonal antibodies by stan antibodies exhibit binding speci?city to an antigen, immuno dard techniques, such as the hybridoma technique originally globulins include both antibodies and other antibody-like described by Kohler and Milstein (1975) Nature 2561495 molecules that lack antigen speci?city. Polypeptides of the 497, the human B cell hybridoma technique (Kozbor et al. latter kind are, for example, produced at loW levels by the (1983) Immunol. Today 4:72), the EBV-hybridoma technique lymph system and at increased levels by myelomas. (Cole et al. (1985) in Monoclonal Antibodies and Cancer [0145] The term “antibody” is used in the broadest sense Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., NeW York, and covers fully assembled antibodies, antibody fragments N.Y.), pp. 77-96) or trioma techniques. The technology for that can bind antigen (e.g., Fab‘, F'(ab)2, Fv, single chain producing hybridomas is Well knoWn (see generally Coligan antibodies, diabodies), and recombinant peptides comprising et al., eds. (1994) Current Protocols in Immunology (John the foregoing. Wiley & Sons, Inc., NeW York, NY); Galfre et al. (1977) [0146] The term “monoclonal antibody” as used herein Nature 266:550-52; Kenneth (l 980) in MonoclonalAntibod refers to an antibody obtained from a population of substan ies: A New Dimension In BiologicalAnalyses (Plenum Pub tially homogeneous antibodies, i.e., the individual antibodies lishing Corp., NY); and Lerner (1981) Yale J. Biol. Meal, comprising the population are identical except for possible 54:387-402). naturally-occurring mutations that may be present in minor [0151] As an alternative to preparing monoclonal antibody amounts. secreting hybridomas, a monoclonal antibody can be identi [0147] “Antibody fragments” comprise a portion of an ?ed and isolated by screening a recombinant combinatorial intact antibody, preferably the antigen-binding or variable immunoglobulin library (e.g., an antibody phage display region of the intact antibody. Examples of antibody fragments library) With a protein of interest to thereby isolate immuno include Fab, Fab‘, F(ab')2, and Fv fragments; diabodies; lin globulin library members that bind to the protein of interest. ear antibodies (Zapata et al. (1995) Protein Eng. 8(l0):l057 Kits for generating and screening phage display libraries are 1062); single-chain antibody molecules; and multispeci?c commercially available (e.g., the Pharmacia Recombinant antibodies formed from antibody fragments. Papain digestion Phage Antibody System, Catalog No. 27-9400-01; and the of antibodies produces tWo identical antigen-binding frag Stratagene SurfZAP 0 Phage Display Kit, Catalog No. ments, called “Fab” fragments, each With a single antigen 240612). Additionally, examples of methods and reagents binding site, and a residual “Fc” fragment, Whose name particularly amenable for use in generating and screening re?ects its ability to crystallize 35 readily. Pepsin treatment antibody display library can be found in, for example, US. US 2013/0164760 A1 Jun. 27, 2013

Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619; stained With hematoxylin and, optionally, a bluing agent such WO 91/17271; WO 92/20791; WO 92/15679; 93/01288; WO as ammonium hydroxide or TBS/TWeen-20. In some aspects 92/01047; 92/09690; and 90/02809; Fuchs et al. (1991) Bio/ of the invention, samples are revieWed microscopically by a Technology 911370-1372; Hay et al. (1992) Hum. Anlibod. cytotechnologist and/or a pathologist to assess cell staining. Hybridomas 3:81-85; Huse et al. (1989) Science 24611275 Alternatively, samples may be revieWed via automated 1281; Grif?ths et al. (1993) EMBOJ. 12:725-734. microscopy or by personnel With the assistance of computer [0152] Following incubation of the sample With the ?rst softWare that facilitates the identi?cation of positive staining and second pretreatment solutions and prior to incubation cells. With an antibody, samples can be blocked using an appropri [0157] In regard to detection of antibody staining, there ate blocking agent, e.g., a peroxidase blocking reagent such also exist in the art, video-microscopy and software methods as hydrogen peroxide. In some embodiments, the samples are for the quantitative determination of an amount of multiple blocked using a protein blocking reagent to prevent non molecular species in a biological sample Wherein each speci?c binding of the antibody. The protein blocking reagent molecular species present is indicated by a representative dye may comprise, for example, puri?ed casein. marker having a speci?c color. Such methods are also knoWn [0153] An antibody, particularly a monoclonal antibody, in the art as a colorimetric analysis methods. In these meth directed to an antigen of interest is then incubated With the ods, video-microscopy is used to provide an image of the sample. More than one antibody may be used in the immun sample after it has been stained to visually indicate the pres ostaining procedure. Where more than one antibody is used, ence of a particular antigen of interest. Some of these meth these antibodies may be added to a single sample sequentially ods, such as those disclosed in Us. patent application Ser. as individual antibody reagents or simultaneously as an anti No. 09/957,446 to Marcelpoil et al. and Us. patent applica body cocktail. Alternatively, each individual antibody may be tion Ser. No. 10/057,729 to Marcelpoil et al., incorporated added to a separate sample from the same patient, and the herein by reference, disclose the use of an imaging system resulting data pooled. and associated softWare to determine the relative amounts of [0154] Techniques for detecting antibody binding are Well each molecular species present based on the presence of knoWn in the art. Antibody binding to an antigen of interest representative color dye markers as indicated by those color may be detected through the use of chemical reagents that dye markers’ optical density or transmittance value, respec generate a detectable signal that corresponds to the level of tively, as determined by an imaging system and associated antibody binding and, accordingly, to the level of expression softWare. These techniques provide quantitative determina of the antigen. Non-limiting examples of detectable sub tions of the relative amounts of each molecular species in a stances that can be used to detect antigen-antibody binding stained biological sample using a single video image that is include various enzymes, prosthetic groups, ?uorescent “deconstructed” into its component color parts. materials, luminescent materials, bioluminescent materials, [0158] Furthermore, the location of antigens Within the cell and radioactive materials. Examples of suitable enzymes is also an important consideration in immunological staining include horseradish peroxidase, alkaline phosphatase, [3-ga methods. Proteins that display nuclear, cytoplasmic, or mem lactosidase, or acetylcholinesterase; examples of suitable brane staining patterns can be con?rmed morphologically prosthetic group complexes include streptavidin/biotin and and are appropriate for immunohistochemistry methods. avidin/biotin; examples of suitable ?uorescent materials Cytoplasmic and membrane staining, hoWever, make it dif? include umbelliferone, ?uorescein, ?uorescein isothiocyan cult to identify critical morphological characteristics of cer ate, rhodamine, dichlorotriazinylamine ?uorescein, dansyl vical disease (e.g., nuclear to cytoplasmic ratio) in immuno chloride or phycoerythrin; an example of a luminescent mate cytochemistry assays. In contrast, proteins that are expressed rial includes luminol; examples of bioluminescent materials in the nucleus and shoW a nuclear staining pattern facilitate include luciferase, luciferin, and aequorin; and examples of detection of antibody staining and also permit morphological suitable radioactive material include 125I, 131I, 35S, or 3H. analysis. Thus, in some embodiments, only proteins that are [0155] In some embodiments, antibody binding is detected selectively expressed in the nucleus are detected using the through the use of a secondary antibody that is conjugated to presently disclosed pretreatment and immuno staining proce a labeled polymer. Examples of labeled polymers include but dures. are not limited to polymer-enzyme conjugates. The enzymes [0159] One of skill in the art Will recognize that optimiza in these complexes are typically used to catalyze the deposi tion of antibody titer and detection chemistry is needed to tion of a chromogen at the antigen-antibody binding site, maximize the signal to noise ratio for a particular antibody. thereby resulting in cell staining that corresponds to expres Antibody concentrations that maximize speci?c binding to an sion level of the antigen of interest. Enzymes of particular antigen of interest and minimize non-speci?c binding (or interest include horseradish peroxidase (HRP) and alkaline “background”) Will be determined. The design of assays to phosphatase (AP). Commercial antibody detection systems, optimize antibody titer and detection conditions is standard such as, for example the Dako Envision+ system and Biocare and Well Within the routine capabilities of those of ordinary Medical’s Mach 3 system, may be used to practice the present skill in the art. invention. [0160] Furthermore, one of skill in the art Will recognize [0156] In particular embodiments, antibody binding to an that the concentration of a particular antibody used to practice antigen is detected through the use of an HRP-labeled poly the presently disclosed methods Will vary depending on such mer that is conjugated to a secondary antibody. Antibody factors as time for binding and the level of speci?city of the binding can also be detected through the use of a mouse probe antibody for its antigen. Moreover, When multiple antibodies reagent, Which binds to mouse monoclonal antibodies, and a are used, the required concentration may be affected by the polymer conjugated to HRP, Which binds to the mouse probe order in Which the antibodies are applied to the sample, i.e., reagent. Samples are stained for antibody binding using the simultaneously as a cocktail or sequentially as individual chromogen 3,3-diaminobenzidine (DAB) and then counter antibody reagents. Furthermore, the detection chemistry used US 2013/0164760 A1 Jun. 27, 2013

to visualiZe antibody binding to an antigen of interest must an HRP-labeled secondary antibody) and solutions, such as also be optimized to produce the desired signal to noise ratio. hydrogen peroxide, for blocking non-speci?c staining may be [0161] The presently disclosed pretreatment methods alloW further provided. In other embodiments, antibody binding to for the maintenance of cellular morphology of the samples. an antigen is detected through the use of a mouse probe Therefore, in some embodiments, the morphological charac reagent that binds to mouse monoclonal antibodies, folloWed teristics of the sample can be assessed. For example, immu by addition of a dextran polymer conjugated With HRP that nostaining can be combined With the conventional Pap stain binds to the mouse probe reagent. Such detection reagents are so that all the morphological information from the conven commercially available from, for example, Biocare Medical. tional method is conserved. In this manner, the detection of [0167] The kits of the present invention may further com speci?c biomarkers such as those disclosed in US. Pat. No. prise a peroxidase blocking reagent (e.g., hydrogen perox 7,510,838, Which is herein incorporated by reference in its ide), a protein blocking reagent (e.g., puri?ed casein), and a entirety, can reduce the high false-negative rate of the Pap counterstain (e.g., hematoxylin). A bluing agent (e.g., ammo smear test and may facilitate mass automated screening. In nium hydroxide or TBS, pH 7.4, With Tween-20 and sodium some embodiments, the immunostaining procedure is com aZide) may be further provided in the kit to facilitate detection bined With the conventional Pap stain in a single method. A of positive staining cells. combined immunocytochemistry and Pap staining method [0168] Positive and/ or negative controls may be included in permits visualiZation of both biomarkers that are selectively the kits to validate the activity and correct usage of reagents overexpressed in high-grade cervical disease and cell mor employed in accordance With the invention. Controls may phology in a single sample (e.g., a microscope slide compris include samples, such as tissue sections, cells ?xed on glass ing a monolayer of cervical cells). The combined immuno slides, etc., knoWn to be either positive or negative for the cytochemistry and Pap staining method may permit the more presence of the antigen of interest. In a particular embodi accurate identi?cation and diagnosis of high-grade cervical ment, the positive control comprises SiHa cells. This is a disease, particularly in cases mistakenly classi?ed as normal, human cervical squamous cancer cell line that is hypertrip LSIL, or ASCUS by conventional Pap testing. loid and positive for HPV-16 infection and, therefore, serves [0162] One of skill in the art Will recogniZe that the staining as a positive control for the overexpression of biomarkers in parameters (e.g., incubation times, Wash conditions, chro high-grade cervical disease states. SiHa control cells may be mogen/ stain concentrations, etc.) for this combined method provided in the presently disclosed kits as prepared slides or ology Will need to be optimiZed such that a su?icient contrast as a cell suspension that is compatible With slide preparation. betWeen the immunostaining output (e.g., chromogen stain The design and use of controls is standard and Well Within the ing) and the Pap stain is obtained. The design of assays to routine capabilities of those of ordinary skill in the art. optimiZe staining parameters is standard and Well Within the [0169] Kits for performing the presently disclosed pretreat routine capabilities of those of ordinary skill in the art. ment methods and the combined immunostaining and Pap [0163] One of skill in the art Will further appreciate that any staining method are also encompassed by the present inven or all steps in the methods of the invention could be imple tion. Such kits comprise the reagents needed for the pretreat mented by personnel or, alternatively, performed in an auto ment step, the immunostaining procedure, as described mated fashion using, for example, the Autostainer Universal herein above, and the reagents for conventional Pap staining, Staining System (Dako) or the Biocare Nemesis Autostainer particularly EA50 and Orange G. (Biocare). Thus, the steps of sample preparation, sample [0170] Presently disclosed kits are compatible With both staining, and detection of antigen expression may be auto manual and automated pretreatment and immunostaining mated. techniques. Any or all of the kit reagents may be provided [0164] The presently disclosed compositions include kits Within containers that protect them from the external envi for practicing the presently disclosed immunostaining pre ronment, such as in sealed containers. treatment methods. These kits comprise a ?rst solution that [0171] It is to be noted that the term “a” or “an” entity refers comprises a surfactant (Which in some embodiments is an to one or more of that entity; for example, “an antibody” is anionic surfactant, such as SDS), and a second solution that understood to represent one or more antibodies. As such, the comprises a chaotropic agent (Which in some embodiments is terms “a” (or “an”), “one or more,” and “at least one” can be a thiocyanate, such as guanidine thiocyanate, or a perchlorate, used interchangeably herein. such as lithium perchlorate) and in some embodiments, a [0172] Throughout this speci?cation and the claims, the Weak surfactant (e.g., NP-40). Words “comprise,” “comprises,” and “comprising” are used in [0165] In some embodiments, the kits further comprise an a non-exclusive sense, except Where the context requires oth antibody that speci?cally binds an antigen. In some of these erWise. embodiments, the kits further comprise more than one anti [0173] As used herein, the term “about,” When referring to body that speci?cally detects the expression of at least tWo a value is meant to encompass variations of, in some embodi distinct antigens. Each antibody may be provided in the kit as ments 150%, in some embodiments 120%, in some embodi an individual reagent or, alternatively, as an antibody cocktail ments 110%, in some embodiments 15%, in some embodi comprising all of the antibodies directed to the different anti ments 11%, in some embodiments 10.5%, and in some gens of interest. embodiments 10.1% from the speci?ed amount, as such [0166] The kit can further comprise chemicals for the variations are appropriate to perform the disclosed methods detection of antibody binding to the antigen, and in some or employ the disclosed compositions. embodiments, a counterstain, and, optionally, a bluing agent [0174] Further, When an amount, concentration, or other to facilitate identi?cation of positive staining cells. In one value or parameter is given as either a range, preferred range, embodiment, the kit comprises a secondary antibody that is or a list of upper preferable values and loWer preferable val conjugated to an HRP-labeled polymer. Chromogens com ues, this is to be understood as speci?cally disclosing all patible With the conjugated enzyme (e.g., DAB in the case of ranges formed from any pair of any upper range limit or US 2013/0164760 A1 Jun. 27, 2013

preferred value and any loWer range limit or preferred value, ment process that resulted in optimal MCM2/MCM7 immu regardless of Whether ranges are separately disclosed. Where nostaining and cellular morphology for the SurePath® a range of numerical values is recited herein, unless otherwise samples involves a tWo-step procedure, Wherein the cells stated, the range is intended to include the endpoints thereof, Were ?rst treated With 0.1% SDS, folloWed by an incubation and all integers and fractions Within the range. It is not With 3M LiClO4/0.1% NP-40. This tWo-step process lever intended that the scope of the presently disclosed subject ages tWo distinct mechanisms of antigen retrieval to boost matter be limited to the speci?c values recited When de?ning immunoreactivity. Both SDS and NP-40 are surfactants and a range. While not being bound by any theory or mechanism of action, [0175] Unless de?ned otherWise, all technical and scien it is believed the primary mechanism of action of SDS and ti?c terms used herein have the same meaning as commonly NP-40 is to increase permeability of both the cytoplasmic understood by one of skill in the art to Which the invention membrane and the nuclear envelope. The protein targets of pertains. Although any methods and materials similar herein the triple antibody cocktail (MCM2 and MCM7) reside can be used in the practice or testing of the present invention, Within the cell nucleus; therefore the primary antibody needs the preferred methods and materials are described herein. to cross both the cytoplasmic membrane and nuclear enve [0176] The folloWing examples are offered by Way of illus lope. Increasing the permeability of these cellular structures tration and not by Way of limitation. aids in the ability of the primary antibody to penetrate both barriers and gain access to the target antigens. While not EXPERIMENTAL being bound by any theory or mechanism of action, it is believed the second mechanism of action of the tWo-solution Example 1 pretreatment method is the denaturing of proteins through the use of strong protein denaturants, speci?cally chaotropic Analysis of Antigen Retrieval Solutions and salts. Chaotropic salts exert this action by disrupting internal Incubation Temperatures protein bonds, thus opening up protein tertiary structure and [0177] Historically, most tissue-based antigen retrieval increasing the accessibility of masked target epitopes. (AR) techniques have utiliZed high heat, ~100° C. and/or [0185] In the remaining experiments described hereinbe treatment With various solutions. The AR procedure for the loW in Example 1, the antigen retrieval process utiliZed Pre detection of the antigens MCM2 and MCM7 in SurePath® treatment Solution 1 (0.1% SDS) and Pretreatment Solution 2 (TriPath Imaging, Inc.) cervical cytology samples using a (3M LiClO4/0.1% NP-40). The optimal incubation tempera cocktail of three antibodies disclosed in U.S. Pat. Nos. 7,595, ture and time period for the samples With each solution Was 380 (27C5 .6 and 26H6.19 anti-MCM2 antibodies) and 7,632, then determined. 498 (2E6.2 anti-MCM7 antibody), Which are herein incorpo [0186] High temperatures (greater than or equal to 80° C.) rated by reference in their entirety, Was optimiZed as are common in most antigen retrieval procedures for tissue described hereinbeloW to maintain cell morphology, While based immunohistochemistry (IHC). The heat aids in break ensuring appropriate immunostaining of cytology specimens ing the cross-linking bonds caused during tissue ?xation. by investigating multiple AR solutions and incubation tem Current methods of heating in most IHC procedures include peratures. Water baths, microWave ovens and/or pressure cookers. Tis [0178] A broad range of potential AR (pretreatment) solu sues are able to Withstand these harsh AR treatments because tions (n:43) Were tested on SurePath® cytology specimens to they are ?xed using solutions With formaldehyde and because determine Which ones Would have a positive effect on immu tissues comprise supporting stromal cellular material that nostaining With a triple antibody cocktail (tWo anti-MCM2 aids in the preservation of tissue architecture and cellular antibodies and one anti-MCM7 antibody) that detects high morphology. In contrast, cytology specimens are generally grade cervical disease. Test solutions and initial observations not ?xed or lightly ?xed and are composed of discrete cells are grouped beloW: that have no accompanying stromal support, making such [0179] EnZymes (e.g. pepsin, trypsin) (n:6)idegraded samples more sensitive to standard high-temperature AR cellular morphology With no substantial gain in immu treatments. A range of AR temperatures and incubation times noreactivity. With AR solutions Were tested to determine the temperature [0180] Acids & bases (n:4)ino substantial immunore and time period that generates appropriate immunostaining activity observed. While maintaining optimal cellular morphology. [0181] Organic solvents/miscellaneous (n:9)ino sub [0187] A range ofAR temperatures Were investigated using stantial immunoreactivity observed the triple antibody cocktail (27C5.6 and 26H6.19 anti-MCM2 [0182] Buffers (e.g. EDTA, citrate) and chaotropic salts antibodies and 2E6.2 anti-MCM7 antibody) on SurePath® (n:15)istrong chaotropic salts, especially lithium per cervical cytology samples. An analysis of 90 cervical cytol chlorate (LiClO4) alone, and in combination With some ogy samples incubated sequentially With Pretreatment Solu surfactants, preserved morphology and exhibited strong tion 1 and Pretreatment Solution 2 for time periods ranging immunoreactivity. Others demonstrated limited gains in from 10 to 30 minutes, and over a temperature range of 30° C. immunoreactivity. to 80° C. Was carried out. All slides Were evaluated by certi [0183] Surfactants (e.g. TWeen 20, Brij 35) (n:9)imost ?ed cytotechnologists. The percentage of abnormal cervical shoWed no substantial increase in immunostaining cells displaying immunostaining Was recorded as Well as the Sodium dodecyl sulfate (SDS) and nonyl phenoxypoly cellular morphology of each sample. The morphology Was ethoxylethanol (N P-40) demonstrated a moderate recorded on a scale of 1-3, With 1 being unacceptable (mor increase in immunostaining alone, and in combination phological degradation present) and 3 being optimal (no dif With the activity of some chaotropic salt solutions. ference from standard AR methods). The standard AR meth [0184] Combinations of pretreatment solutions that ods that Were used as a comparison in each of the shoWed promising activity Were tested further. The pretreat experimental examples presented herein, and Were also US 2013/0164760 A1 Jun. 27, 2013

referred to herein as the “steamer method”, consisted of the incubated at either 37° C. or 42° C. for the same time periods following steps: 1) heating a Coplin jar comprising 0.5% had no morphologic degradation (see Table 3). These data Sandopan LS (sodium laureth-13-carboxylate) in a steamer support a critical design constraint of maintaining an AR or Water bath to greater than 95° C.; 2) submersing the slide in temperature beloW 60° C. in order to ensure optimal cellular the heated 0.5% Sandopan LS solution for 20 minutes; and 3) morphology that is equivalent to a standard liquid-based removing the Coplin jar from the heat source and alloWing the SurePath® Pap. jar to cool for 10 minutes prior to proceeding With the immu no staining procedure. TABLE 3 [0188] The data indicated that all cervical samples treated With an antigen retrieval temperature of 55° C. or loWer Morphology of high-grade cervical pools incubated at various retained optimal preservation of normal cervical cellular mor temperatures and time periods With pretreatment solutions. phology, While an antigen retrieval temperature of 80° C. Incubation Time AR Temperature Cellular resulted in degraded cervical cellular morphology (see Table (min) (° C.) Morphology 1). 15 37 Optimal 15 42 Optimal TABLE 1 15 60 Cytoplasmic Curling 30 37 Optimal Assessment ofcervical cell morphology following 30 42 Optimal incubation With Pretreatment Solution 1 and Pretreatment 30 60 Cytoplasmic Curling Solution 2 at various temperatures. 60 37 Optimal 60 42 Optimal Temperature 60 60 Cytoplasmic Curling

Grading Scale 30° C. 55° C. 80° C. [0191] The AR studies demonstrate that cellular morphol 1 (unacceptable 0 0 30 morphology) ogy can be maintained betWeen the temperature range of 42° 2 (acceptable 0 0 0 C. and 55° C. For additional studies using the triple antibody morphology cocktail of tWo anti-MCM2 antibodies and one anti-MCM7 3 (optimal 30 30 0 antibody on SurePath® samples, anAR temperature of 50° C. morphology) Was selected as it resulted in optimal immunostaining and Was Total cases 30 30 30 Well beloW the upper temperature limit of 60° C.

[0189] Additional antigen retrieval temperature studies Example 2 Were carried out to investigate a focused AR temperature range from 37° C. to 60° C. These studies indicated that subtle Comparison of the Effects of the TWo-Step cellular features, such as slight curling of the periphery of Pretreatment Method With a High-Heat Pretreatment squamous cells, become apparent at an incubation tempera Method on Immunostaining and Morphology in ture of 60° C. Table 2 contains the data from 12 matched Cervical Cytology Samples cervical cytology specimens that Were treated With AR tem [0192] This study compared the tWo-step pretreatment peratures of either 37° C. or 60° C. for one hour total. After method using tWo different chaotropic salt solutions for the incubation of the samples for 30 minutes at 60° C. in AR second incubation step to each other and to a pretreatment Solution 1 and 30 minutes at 60° C. in AR Solution 2, cyto method utiliZing high heat (i.e., the steamer method described plasmic folding becomes evident. Therefore, 60° C. Was in Example 1). determined to be the upper temperature limit for incubation With antigen retrieval solutions for SurePath® samples. [0193] In these experiments, SurePath® cervical cytology specimens Were processed using the internally developed PrepStain Plus® Instrument (Tripath Imaging, Inc.) and TABLE 2 deposited for staining. For those samples treated With the Antigen retrieval temperature comparison tWo-step pretreatment method, the slides Were heated to 50° using matched cytology specimens. C. on specially-designed slide heating trays and Pretreatment Solution 1 (0.1% SDS) Was applied and the slides Were incu Temperature bated at 50° C. for 19 minutes. The cells Were Washed With a Morphology 37° C. 60° C. standard Tris-buffered saline (TBS) solution. Pretreatment Solution 2 (either 3M LiClO4/ 0.1% NP-40 or 3M guanidine Optimal morphology 1 1* 6 * Cytoplasmic curling 1 6 thiocyanate/0.1% NP-40) Was then added and the cells Were observed incubated an additional 19 minutes at 50° C. Pretreatment Solution 2 (or those samples that Were incubated With Pre Total 12 12 treatment Solution 2) comprising 3M LiClO4/0.1% NP-40

*One case exhibited degraded White blood cells at both temperatures due to poor sample are referred to herein as LiClO4 and Pretreatment Solution 2 preservation. This Was con?rmed by the presence of degraded White blood cells on the (or those samples that Were incubated With Solution 2) com standard SurePath ® Pap for this sample. prising 3M guanidine thiocyanate/0. 1% NP-40 are referred to [0190] The upper AR temperature limit of 60° C. is not herein as GT. FolloWing this incubation, the slides Were dependent upon the length of the incubation. High-grade Washed again With TBS and processed for immunostaining pooled cervical specimens incubated at 60° C. for 15, 30 or 60 With the MCM2/MCM7 triple antibody combination (27C5 .6 minutes in each of Pretreatment Solution 1 and Pretreatment and 26H6.19 anti-MCM2 antibodies and 2E6.2 anti-MCM7 Solution 2 exhibited cytoplasmic curling, While specimens antibody) and PAP counterstaining on the PrepStain Plus® US 2013/0164760 A1 Jun. 27, 2013

instrument. The slides Were then coverslipped and examined TABLE 6 by a cytologist and/ or cytopathologist. Percentage of abnormal cells immunostaining With the MCM2/MCM7 [0194] Table 4 provides the characteristics of the 481 cer triple antibody combination on CIN2+ cervical cytology samples prepared vical cytology samples analyzed in these studies. using the steamer method or treated With a tWo-step pretreatment method comprising granidine thiocyanate (GT).

TABLE 4 STEAMER GT

Characteristics of cervical cytology samples used in these studies. Frequency N/A <25% 25%—50% 50%-75% >75% Total

N/A 13 1 4 3 0 21 Biopsy <25% 2 6 4 0 0 12 25 %-5 0% 4 1 1 13 4 0 3 2 SP PAP CIN 1— CIN 2+ Total 50%-75% 0 1 25 4 61 >75% 2 4 3 5 2 16 NILM 160 0 160 ASCUS 53 12 65 Total 21 42 3 6 3 7 6 142 ASCH/AGC 18 11 29 LSIL 57 36 93 HSIL 50 84 134 Total 33 8 143 481 TABLE 7 Percentage of abnormal cells immunostaining With the MCM2/MCM7 SP PAP: SurePath ® Pap; NILM: negative for intraepithelial lesion or malignancy; triple antibody combination on CIN2+ cervical cytology samples prepared ASCUS: atypical squamous cells ofunknoWn signi?cance; using the steamer method or treated With a tWo-step pretreatment method ASCH/AGC: atypical squamous cells - cannot exclude high-grade squamous intraepithelial comprising lithium perchlorate (LiClOA). lesion/atypical glandular cells; LSIL: low-grade squamous intraepithelial lesion; STEAMER LiClOA HSlIL: high-grade squamous intraepithelial lesion; CIN 1—: cervical intraepithelial neoplasia 1 or less; Frequency N/A <25% 25%—50% 50%-75% >75% Total CTN 2+: cervical intraepithelial neoplasia 2 or greater N/A 14 3 2 2 0 21 <25% 2 5 1 0 12 [0195] There Was no statistically signi?cant difference in 25%-50% 5 10 11 6 0 32 50%-75% 2 18 21 18 2 61 terms of sensitivity or speci?city or background for immune >75% 2 3 4 5 2 16 response betWeen the LiClO4 or the GT Solution 2 in samples treated using the tWo-step antigen retrieval method. Samples Total 25 39 42 32 4 142 treated With guanidine thiocyanate had a 2.11% increase in sensitivity as compared to lithium perchlorate, Whereas the tWo treatment methods resulted in the same speci?city (see [0197] There Was no statistically signi?cant difference Table 5). More guanidine thiocyanate-treated samples had a betWeen guanidine thiocyanate-treated samples and lithium slight background staining than those samples that had been perchlorate-treated samples in terms of distribution of per treated With lithium perchlorate. cent cell immunopositivity. There Were more cases (121 vs. 117) With greater than 25 percent positive cells in guanidine TABLE 5 thiocyanate-treated samples than lithium perchlorate-treated samples for CIN2+ cases (see Table 8). There Was an increase Sensitivity and speci?city of combination of tWo anti-MCM2 in cases With >50% of the abnormal cells immunostaining in antibodies and one anti-MCM7 antibody on cervical cytology the guanidine thiocyanate-treated samples than the lithium samples treated With a tWo-step pretreatment method comprising guanidine thiocyanate (GT) or lithium perchlorate (LiClOA). perchlorate-treated samples (28 vs. 23) for HSlL/CIN2+ cases (see Table 9). In comparison, 14% (12/84) of HSIL/ Di1f(GT CIN2+ cases had >75% positivity When prepared using the 11 GT LiClO4 LiClO4) 95% c1 steamer method (see Table 10). Sensitivity 142 84.51% 82.39% 2.11% (-2.72%, 6.94%) TABLE 8 Speci?city 320 64.39% 64.39% 0% (—2.62%, 2.62%) Percentage of abnormal cells immunostaining With the MCM2/MCM7 triple antibody combination on CIN2+ cervical cytology samples prepared Diff: difference; using the tWo-step pretreatment method comprising either lithium C1: con?dence interval perchlorate (LiClO4) or gianidine thiocyanate (GT). [0196] When comparing those samples that had been pre LiClOA pared using the steamer method versus the tWo-step pretreat GT N/A <25% 25 %—50% 50%-75% >75% Total ment method, there Was no statistical difference in those samples that Were positive or negative for immunostaining on N/A 20 2 0 0 0 22 <25% 4 20 15 3 0 42 CIN2+ cases. The data, hoWever, shoWed a statistically sig 25%—50% 2 12 15 6 1 36 ni?cant difference in terms of distribution of percent cell 50%-75% 0 11 22 0 37 immunopositivity. Speci?cally, there Was an increase in cases >75% 0 1 1 1 3 6 With greater than 50% of the abnormal cells immunostaining Total 26 39 42 32 4 143 With the steamer method versus GT (77 vs. 43) and With the steamer method vs. LiClO4 (77 vs. 36) (see Tables 6 and 7). US 2013/0164760 A1 Jun. 27, 2013 14

TABLE 9 TABLE 12

Percentage of abnormal cells immunostaining With the MCM2/MCM7 Classi?cation of cervical cytology samples upon morphological analysis triple antibody combination on HSIL/C1N2+ cervical cytology samples for samples prepared using the steamer method or the tWo-step prepared using the tWo-step pretreatment method comprising either lithium pretreatment method comprising lithium perchlorate (LiClO4). perchlorate (LiClO4) or guanidine thiocyanate (GT). LiClO4 Licio4 Steamer ASCH/ GT N/A <25% 25%—50% 50%—75% >75% Total Frequency NILM AS CUS AGC LSIL HSIL Total N/A 6 O O O O 6 NILM 12 8 24 2 3 3 1 6O <25% 3 11 14 1 O 29 25%—50% O 8 8 4 1 21 ASCUS 2 34 21 4 65 50%—75% O 2 9 14 O 25 ASCH/AGC O 4 1 1 2 12 29 >75% 0 O O O 3 3 LSIL O 2 49 3 8 92 HSIL O 2 3 O 129 134 Total 9 21 31 19 4 84 Total 130 67 22 75 186 480

TABLE 10 *1 unsatisfactory sample in LiClO4

Percentage of abnormal cells immunostaining With the MCM2/MCM7 triple antibody combination TABLE 13 on HSIL/CIN2+ cervical cytology samples prepared using the steamer method. Classi?cation of CIN2+ cervical cytology samples upon morphological analysis for samples prepared using the steamer method or the tWo-step Steamer Frequency pretreatment method comprising guanidine thiocyanate (GT) (p = 0.0270).

N/A 5 Steamer GT <25% 6 25%—50% 2O Frequency ASCUS ASCH/AGC LSIL HSIL Total 50%—75% 41 >75% 12 ASCUS 10 1 1 0 12 ASCH/AGC 1 4 0 6 11 Total 84 LSIL 1 0 19 15 35 HSIL 0 2 1 81 84

[0198] When examining the morphology of cervical cytol Total 12 7 21 102 142 ogy samples prepared using the steamer method or the tWo step pretreatment method disclosed herein, the results showed there Was a statistical difference (p<0.0001) in terms TABLE 14 of the distribution of the cytology classi?cation. Guanidine Classi?cation of CIN2+ cervical cytology samples upon morphological thiocyanate treatment classi?ed 21 more ASCUS+ cases and analysis for samples prepared using the steamer method or the 39 more HSIL cases than the steamer method and the lithium tWo-step pretreatment method comprising lithium perchlorate perchlorate treatment classi?ed 30 more ASCUS+ cases and (LiClOA) (p = 0.0004). 52 more HSIL cases than the steamer method (see Tables Steamer LiClOA 1 1 -14). Frequency ASCUS ASCH/AGC LSIL HSIL Total

TABLE 11 ASCUS 9 1 2 0 12 ASCH/AGC 1 4 0 6 11 Classi?cation of cervical cytology samples upon morphological analysis LSIL 2 1 13 19 35 for samples prepared using the steamer method or the tWo-step HSIL 1 1 0 82 84 pretreatment method comprising guanidine thiocyanate GT . Total 13 7 15 107 142 GT

Steamer ASCH/ Frequency NILM ASCUS AGC LSIL HSIL Total [0199] Lithium perchlorate treatment identi?ed eight more cytologically abnormal cases than guanidine thiocyanate (32 NILM 13 6 19 2 1 2 1 60 ASCUS 3 41 12 4 65 vs. 24) in 160 cases that Were classi?ed as NILM by the ASCH/AGC 0 5 1 1 2 1 1 29 steamer method (see Table 15). Guanidine thiocyanate treat LSIL 0 3 0 5 8 3 0 91 ment classi?ed six more ASCUS than lithium perchlorate HSIL 0 0 6 2 12 6 134 treatment (49 vs. 43) in 319 cases that Were classi?ed as Total 139 68 24 75 173 479 ASCUS+ by the steamer method (see Table 16). Further, among 134 cases that Were classi?ed as HSIL+ by the steamer *2 unsatisfactory samples in GT (i.e., insuf?cient number ofcells present on slide to make a morphological determination) method, lithium perchlorate treatment classi?ed three more HSIL than guanidine thiocyanate (129 vs. 126). US 2013/0164760 A1 Jun. 27, 2013 15

TABLE 15 staining on the PrepStain Plus® instrument. The slides were then coverslipped and examined by a cytologist and/ or cyto Classi?cation ofcervical cytology samples classi?ed as NILM by the pathologist. Representative results of cervical cytology steamer method using the two-step pretreatment method comprising samples immunostained with an anti-Ki67 or anti-p16 anti guanidine thiocyanate (GT) or lithium perchlorate (LiClOA). body are shown in FIGS. 1 and 3, respectively. SiHa cells LiClOA were treated similarly and immunostained with an anti-Ki67 (FIG. 2) or anti-p16 antibody (FIG. 4). GT ASCH/ Frequency NILM ASCUS AGC LSIL HSIL Total Example 4 NILM 12 6 9 0 1 0 13 6 ASCUS 2 15 0 2 0 19 Detection of MCM2 and MCM7 in Tonsil and ASCH/AGC 0 0 2 0 0 2 LSIL 0 0 0 0 1 1 Cervical Tissues Using the Two-Step Pretreatment HSIL 0 0 0 0 2 2 Process

Total 12 8 24 2 3 3 1 60 [0202] Tonsil and cervical tissue samples were ?xed in 10% formaldehyde for at least 24 hours and then embedded in paraf?n before sectioning. Tissue sections were incubated with Solution 1 (0.1% SDS) at 500 C. for 19 minutes. The TABLE 16 sections were washed with TBS and then incubated at 500 C. Classi?cation ofcervical cytology samples classi?ed as ASCUS+ by the for 19 minutes in Solution 2 (3M LiClO4/0.1% NP-40). Fol steamer method using the two-step pretreatment method comprising lowing this incubation, the tissue sections were washed again guanidine thiocyanate GT or lithium perchlorate LiClO4. with TBS and processed for immunostaining with a triple antibody cocktail (27C5.6 and 26H6.19 anti-MCM2 antibod LiClOA ies and 2E6.2 anti-MCM7 antibody) and PAP counterstaining GT ASCH/ on the PrepStain Plus® Instrument. Representative immun Frequency NILM ASCUS AGC LSIL HSIL Total ostained tonsil and cervical tissues are shown in FIGS. 5 and NILM 2 1 0 0 0 3 6, respectively. These results demonstrate that the two-step ASCUS 0 38 2 9 0 49 pretreatment process disclosed herein can effectively retrieve ASCH/AGC 0 2 17 2 1 22 antigens from histological samples, resulting in ef?cient LSIL 0 2 1 58 13 74 immunostaining. HSIL 0 0 0 2 169 171

Total 2 43 20 71 183 319 Example 5

*2 unsatisfactory samples in GT Detection of Nuclear Antigens in Cervical Cells [0200] Overall, guandine thiocyanate and lithium perchlo Using the Two-Step Pretreatment Process in the rate treatment did not degrade cellular morphology as com Absence of Heat pared to the steamer method. Further, glandular cells and [0203] SurePath® low-grade squamous intraepithelial atrophic samples treated with the two-step pretreatment lesion (LSIL) and high-grade squamous intraepithelial lesion method exhibit less immunostaining than those processed (HSIL) cervical cytology specimens were processed as using the steamer method. The two-step antigen retrieval described in Example 3 for antigen retrieval by incubating the method did not reduce immune/clinical performance as com samples in Pretreatment Solution 1 (0.1% SDS) for 19 min pared to the steamer method, although there was a reduction utes, followed by washing with TBS, and incubation in Pre observed in the percentage of cells that were immunopositive treatment Solution 2 (3M LiClO4/0.1% SDS) for 19 minutes. compared to those samples processed with the steamer In contrast to the experiments described in Example 3, how method. For N1LM and CIN2 con?rmed cases, the two-step ever, no exogenous heat was applied to the samples during the pretreatment method upgraded the cytological classi?cation. incubation steps with the two pretreatment solutions. There fore, the incubation steps with Pretreatment Solutions 1 and 2 Example 3 were performed at room temperature. [0204] Following pretreatment, the samples were immun Detection of Antigens Ki67 and p16 in Cervical ostained with a triple antibody cocktail (27C5 .6 and 26H6. 19 Cytology Samples Using the Two-Step Pretreatment anti-MCM2 antibodies and 2E6.2 anti-MCM7 antibody) and Process PAP counterstained. Representative samples are shown in [0201] In these experiments, SurePath® cervical cytology FIG. 7 (LSIL) and FIG. 8 (HSIL). These data demonstrate the specimens were processed using the internally developed unexpected effect of the presently disclosed two-step pre PrepStain Plus® Instrument (Tripath Imaging, Inc.) and treatment method to effectively expose epitopes while main deposited for staining. Following cell deposition, the slides taining cellular morphology of cytology samples, even in the were heated to 50° C. on specially-designed slide heating absence of applied heat. trays and AR Solution 1 (0.1% SDS) was applied and the [0205] All publications and patent applications mentioned slides were incubated at 500 C. for 19 minutes. The cells were in the speci?cation are indicative of the level of those skilled washed with a standard tris-buffered saline (TBS) solution. in the art to which this invention pertains. All publications and AR Solution 2 (3M LiClO4/0.1% NP-40) was then added and patent applications are herein incorporated by reference to the the cells were incubated an additional 19 minutes at 500 C. same extent as if each individual publication or patent appli Following this incubation, the slides were washed again with cation was speci?cally and individually indicated to be incor TBS and processed for immunostaining and PAP counter porated by reference.