Solvent Free Environmentally Friendly IHC

Transcription

1 Solvent Free Environmentally Friendly IHC Furler Chase¹, Wall Carolyn¹, Henry Marianne¹, Henry Jim ¹ and Heras Alfonso¹ Bio SB Inc., 69 Santa Felicia Dr., Santa Barbara, CA, INTRODUCTION Paraffin has long been used as an embedding medium in the preparation of tissue specimens for histological studies. In some instances, plastic resins have also been used as embedding medium. Such embedding processes generally include the steps of specimen fixation (e.g., formalin fixation), dehydration, clearing, paraffin infiltration or impregnation, blocking or embedding in a block of paraffin, slicing the block and specimen into thin sections, mounting the sections on slides, removing the paraffin and solvents employed for this purpose (i.e., dewaxing or deparaffinizing), and staining and mounting the sections prior to microscopic analysis. The most commonly employed method for deparaffinization involves the dissolution of the embedding paraffin in organic solvents. For example, xylene, a flammable, volatile and a toxic organic solvent, is currently commonly used in protocols to solubilize paraffin for dewaxing of specimen sections. The simple technique of boiling formalin-fixed paraffin-embedded (FFPE) tissue sections in buffers, commonly known as Antigen Retrieval (AR) or Heat-Induced Epitope Retrieval (HIER) has played a major role in extending the reach and use of immunohistochemistry (IHC) on FFPE tissues (Shi et al. 1991; Gown et al. 1993; Taylor and Cote 2005). One notable result is the effective division of all publications with respect to IHC for FFPE tissue sections into two eras: pre-ar and post-ar (Gown 2004; Taylor 2001), indicating AR as a milestone (Jagirdar 2008). Another method commonly employed for deparaffinization and HIER involves the melting and removal of embedding paraffin in a heated bath containing an aqueous buffer solution. The use of a heated buffer solution has the additional advantage of simultaneously allowing HIER procedures to improve staining by heatinduced modification of the molecular conformation of target proteins contained in slide-mounted specimen material. Existing buffer formulations that are configured to simultaneously deparaffinize slides and perform HIER prior to IHC or in situ hybridization (ISH) utilize small concentrations of surfactants and other emulsifiers to break up paraffin. The HIER procedure places slides at an elevated temperature (e.g., from 65 to 121 C), allowing paraffin to melt and surfactants to gently melt the paraffin to the buffer surface. However, commercially available buffer formulations and methods have limited performance, sometimes creating paraffin streaking, slide recoating, and inconsistent staining results due to micro-paraffin residues, which need to be eliminated with solvent-based alcohol and xylenes before mounting. We report the use of a new generation of heat assisted 3-in-1 one-step deparaffinization, retrieval and hydration solutions and their protocols that can effectively remove paraffin from specimens prior to immunohistochemical or other diagnostic analyses, while minimizing organic solvent exposure to users, allowing compatibility with automated systems, and maintaining compatibility with downstream analyses.

2 Additionally, we report the use of a biodegradable, non-toxic stabilizing polymer solution applied prior to mounting to eliminate micro-paraffin residues and protect substrate-chromogens that are soluble or fade with solvents (prior to mounting with toxic, flammable mounting media), which allows them to be permanently mounted with non-solvent and non-flammable media, thus allowing for a solvent-free environment when conducting IHC or CISH/FISH procedures. These environmentally friendly deparaffinization, retrieval and mounting solutions and methods, produce no or minimal odors, reduce the quantity of toxic solvents used, minimize hazardous waste, and/or decrease corrosiveness and flammability plus eliminate steps, save time and money. DESIGN 1. OVERVIEW OF PROTOCOL DESIGN 1.1. This study compared the following experimental heat assisted 3-in-1 one-step deparaffinization and retrieval IHC protocols to the established solvent-based method of using xylenes and alcohol for deparaffinization and permanent mounting, which was used as control: Heat-assisted 3-in-1 one-step Deparaffinization, Retrieval and Hydration Solutions TintoDeparaffinator Citrate TintoDeparaffinator EDTA Trilogy Dewax and HIER Buffer Low Dewax and HIER Buffer High Manufacturer Bio SB, Inc, Santa Barbara, CA, USA Bio SB, Inc, Santa Barbara, CA, USA Cell Marque Corporation, Rocklin, CA, USA ThermoFisher, Runcorn, UK ThermoFisher, Runcorn, UK These 3-in-1 one-step deparaffinized specimens were mounted with AquaMounter (Bio SB), an aqueous mounting media used to evaluate paraffin residues, and the ChromoProtector, a non-toxic stabilizing polymer solution applied prior to mounting to eliminate micro-paraffin residues (without xylenes and/or alcohol treatments) Additionally, the TintoDeparaffinator Citrate and TintoDeparaffinator EDTA were evaluated and compared to fresh controls for their effectiveness to deparaffinize, retrieve antigens and produce acceptable IHC results for up to 4 repeated uses The ChromoProtector, which is used after the IHC procedure to mount tissue specimens, is a biodegradable, non-toxic stabilizing polymer solution applied prior to mounting to eliminate micro-paraffin residues and protect substrate-chromogens that are soluble or fade with solvents prior to mounting, was compared to the traditional solvent-based method of using xylenes and alcohol for permanent mounting. Additionally, different HRP and AP substrate-chromogens were mounted with either AquaMounter, PermaMounter (Bio SB), a flammable permanent mounting media containing toluene, or treated with ChromoProtector then mounted with PermaMounter or XyGreen PermaMounter (Bio SB), a biodegradable, non-toxic and non-flammable permanent mounting solution.

3 1.4. The XyGreen PermaMounter is a natural biosolvent, which is a biodegradable, non-toxic and nonflammable permanent mounting solution, was compared to an aqueous (AquaMounter) and a traditional solvent and flammable toluene-containing permanent mounting media (PermaMounter) Tissue Micro Array (TMA) and single FFPE tissues and antibodies were representative selections of the most widely used antibodies for IVD IHC: mouse and rabbit monoclonal antibodies were selected from antibodies with different isotypes and specificities for different cellular compartments: cytoplasmic, membranous, and nuclear expression. 2. MATERIALS AND EQUIPMENT 2.1 Antibodies used to test the different deparaffinization and/or mounting solutions for IHC are considered in Table 1. TABLE 1. Antibodies and Tissues used for the Different One-Step Deparaffinization, Retrieval and Mounting Solutions and Mounting Media used for IHC Antibody Clone Species Isotype Expected Signal Tissue 1 Tissue 2 CD 20 L26 Mouse Monoclonal IgG2a/K Membranous 23-core NH TMA 7-core LTMA CK AE1/AE3 AE1/AE3 Mouse Monoclonal IgG1 Cytoplasmic 23-core NH TMA Colon Carcinoma PR RBT-22 Rabbit Monoclonal IgG Nuclear 23-core NH TMA Breast Carcinoma 2.2 FFPE tissues used and TMA maps are considered in Table 2. TABLE 2. Tissue Maps of the 23- and 7-core TMA s used for the Different Immunohistochemistry Studies 23-Core Normal Human Tissue Microarray 7-Core Human Lymphoid Tissue Microarray Placenta Blank Breast Uterus Cervix Fallopian T Brain Pituitary Adrenal Pancreas Salivary Colon Liver Kidney Thyroid Lung Skin Bladder Tonsil Blank Lymph Node Spleen Tonsil Thymus Lymph Node Spleen Testis Prostate Spleen Tonsil Bone M. Thymus

4 2.3 The different experimental heat-assisted 3-in-1 one-step deparaffinization, mounting solutions and mounting media, are listed in Table 3 TABLE 3. Different Experimental Heat Assisted 3-in-1 One-Step Deparaffinization, Retrieval and Mounting Solutions and Mounting Media used for IHC Control and 3-in-1 One-Step HIER Mounting Mounting Media Deparaffinization and Solution Retrieval 1. Xylenes / Alcohol ImmunoDNA Retriever Citrate OH/Xylenes PermaMounter Toluene-based 2. TintoDeparaffinator Citrate N/A ChromoProtector XyGreen PermaMounter 3. TintoDeparaffinator EDTA N/A ChromoProtector XyGreen PermaMounter 4. Trilogy N/A OH/Xylenes PermaMounter Toluene-based 5. Dewax and HIER Buffer Low N/A OH/Xylenes PermaMounter Toluene-based 6. Dewax and HIER Buffer High N/A OH/Xylenes PermaMounter Toluene-based 2.4 Heat-induced epitope retrieval was performed using a pressure cooker (TintoRetriever Pressure Cooker with Thermometer, Bio SB) for 15 min at 121 C or a PT Module (TintoRetriever PT Module, Bio SB) at 98 C for 30 min. The Immunohistochemical stainings were carried out using the TintoStainer (Bio SB), an automated open system. TintoRetriever Pressure Cooker TintoRetriever PT Module TintoStainer Automated System

5 2.5 The IHC protocol used for HIER and Detection is shown in Table 4. TABLE 4. Immunohistochemical Protocol for the 3-in-1 One-Step Deparaffinization and Retrieval Solutions using the M/R Fab PolyDetector Plus HRP/DAB and TintoStainer Open Automated System Step Xylenes/Alcohol TintoDeparaffinator Citrate or EDTA Trilogy Dewax/HIER Low or High Deparaffinization Xylenes 3 x 5 = 15 min N/A N/A N/A Dehydration Alcohols 3 x 5 = 15 min N/A N/A N/A Hydration Buffer 1 x 5 = 5 min N/A N/A N/A HIER Citrate PC 1 x 15 min PC 1 x 15 min PC 1 x 15 min PC 1 x 15 min Citrate PT 1 x 30 min PT 1 x 30 min PT 1 x 30 min PT 1 x 30 min Cool to RT 1 x 15 min 1 x 15 min 1 x 15 min 1 x 15 min Peroxidase Block 1 x 5 min 1 x 5 min 1 x 5 min 1 x 5 min Primary Antibody 1 x 30 min 1 x 30 min 1 x 30 min 1 x 30 min M/R Link 1 x 15 min 1 x 15 min 1 x 15 min 1 x 15 min Fab Micropolymer HRP 1 x 15 min 1 x 15 min 1 x 15 min 1 x 15 min DAB 1 x 5 min 1 x 5 min 1 x 5 min 1 x 5 min Hematoxylin 1 x 0.5 min 1 x 0.5 min 1 x 0.5 min 1 x 0.5 min Mounting Xylenes 15 min ChromoProtector Xylenes 15 min Xylenes 15 min Alcohol 15 min 10 min Alcohol 15 min Alcohol 15 min Mounting Media PermaMounter XyGreen PermaMounter PermaMounter PermaMounter Total Protocol PC = min PC = min PC = min PC = min Time in Minutes PT = min PT = min PT = min PT = min Abbreviations: PC: Pressure Cooker at 121 C PT: PT Module at 98 C 3. ACCEPTANCE CRITERIA 3.1 The IHC specific signals and background were scored by qualified professionals using a scale of 0 to 4. For TMA s the signal had to be cell compartment specific and tissue specific. Tissue cores from the TMA s that are known to lack the antigen for a specific antibody, had to be negative.

6 3.2 Paraffin removal was graded by the amount of residual paraffin after IHC using the following scoring: +, ++, +++ and Results were evaluated using a paired T-test to determine equivalency or statistically significant differences of the different experimental heat-assisted 3-in-1 one-step deparaffinization, retrieval and mounting solutions compared to the established solvent-based method of using xylenes and alcohols to deparaffinize, retrieve or to mount tissues after the IHC procedure. RESULTS All 3-in-1 one-step deparaffinization and retrieval solutions produced similar IHC specificity and sensitivity and no statistically significant differences were observed. The TintoDeparaffinator EDTA had the best ability to remove paraffin with significantly less micro-paraffin residue of smaller size than other pre-treatments, followed by the TintoDeparaffinator Citrate. Trilogy, Dewax/HIER Low and Dewax/HIER High left considerable amounts of larger paraffin residues when sections were mounted with AquaMounter but the specificity and sensitivity of the IHC signals were not affected. The treatment with ChromoProtector before tissue mounting with AquaMounter for all 3-in-1 one-step deparaffinization and retrieval solutions from different vendors, completely eliminated paraffin or microparaffin residues in tissues without affecting the specificity and sensitivity of the IHC signals. TABLE 5. Average IHC Signal and Paraffin Removal of the 3-in-1 One-Step Deparaffinization and Retrieval Solutions from Different Vendors using Heat Retrieval at 121 C for 15 min and Mounted With or Without ChromoProtector and AquaMounter Deparaffinization, Retrieval and Mounting Solutions IHC Signal Residual Paraffin CD20 CK AE1/AE3 PR AVG. Xylenes & Alcohol/ HIER w/citrate TDP Citrate TDP Citrate /ChromoProtector TDP EDTA TDP EDTA/ ChromoProtector Trilogy Trilogy/ ChromoProtector Dewax/HIER Low Dewax/HIER Low/ ChromoProtector Dewax/HIER High Dewax/HIER Low/ ChromoProtector

7 FIGURE 1. Paraffin Residue of the Different Deparaffinization Solutions using CD20 and CK AE1/AE3 on FFPE Breast Tissues Mounted with Aqueous Mounting or ChromoProtector + Aqueous Mounting Ab/Mounting Xylenes Alcohol TDP Citrate TDP EDTA Trilogy Dewax/HIER High CD20 Aqueous Mounting CK AE1/AE3 Chromo Protector Aqueous Mounting TABLE 6. Average IHC Signal and Background for Repeated Use of the TintoDeparaffinator Citrate and EDTA (1X, 2X, 3X, and 4X) using Heat Retrieval at 121 C for 15 min Number of Uses of the TDP Solutions CD20 CK AE1/AE3 PR Average Solution IHC/ BCKGND IHC/ BCKGND IHC/ BCKGND IHC TDP Citrate 1X 3.5/ / / TDP EDTA 1X 3.5/ / / TDP Citrate 2X 3.5/ / / TDP EDTA 2X 3.5/ / / TDP Citrate 3X 3.5/ / / TDP EDTA 3X 3.5/ / / TDP Citrate 4X 3.5/ / / TDP EDTA 4X 3.5/ / /

8 Both the TintoDeparaffinator Citrate and EDTA each re-used up to 4 times, produced similar IHC specificity and sensitivity and no statistically significant differences were observed. Overall the TintoDeparaffinator EDTA had stronger signals than the TintoDeparaffinator Citrate. FIGURE 2. Average IHC Signal for Repeated Use of the TintoDeparaffinator Citrate (1X, 2X, 3X, and 4X) using Heat Retrieval at 121 ºC for 15 min IHC Signal for Four Times Reused Solutions TDP Citrate CD20 CK AE1/AE3 PR No statistically significant differences were observed on the IHC signal intensity for up to 4 times repeated use of the TintoDeparaffinator Citrate Solution showing good reproducible results. FIGURE 3. Difference of the Average IHC Signal for Repeated Use of the TintoDeparaffinator Citrate (1X, 2X, 3X, and 4X) Compared to the Fresh Control using Heat Retrieval at 121 ºC for 15 min 0.4 Signal Difference between Four Times Reused and Fresh Solutions TDP Citrate CD20 CK AE1/AE3 PR No statistically significant differences were observed on the IHC signal intensity for up to 4 times repeated use of the TintoDeparaffinator Citrate Solution compared to its freshly prepared TintoDeparaffinator Citrate Control. Note the graphic scale is magnified 5 times compared to Figure 2.

9 FIGURE 4. IHC of HRP Substrate-Chromogens Soluble in Organic Solvents: AEC, HRP Blue and HRP Green Mounted with ChromoProtector and PermaMounter or XyGreen PermaMounter Permanent IHC of PD-L1 on a Hodgkin s IHC of SATB2 on a Colon Ca IHC of ALK-1 on a FFPE Mounting Lymphoma Tissue with AEC Tissue with HRP Green Anaplastic Large Cell Media Lymphoma with HRP Blue ChromoProtector PermaMounter ChromoProtector XyGreen PermaMounter FIGURE 5. Optical Clarity of Progesterone Receptor IHC using AquaMounter, PermaMounter or XyGreen PermaMounter Mounting Media AquaMounter PermaMounter XyGreen PermaMounter XyGreen PermaMounter had the same optical clarity than the AquaMounter and PermaMounter controls and did not affect the IHC specificity and sensitivity.

10 CONCLUSIONS All 3-in-1 one-step deparaffinization and retrieval solutions produced similar IHC specificity and sensitivity and no statistically significant differences were observed. The Bio SB TintoDeparaffinator EDTA had the best ability to remove paraffin with significantly less micro-paraffin residue than other pre-treatments, followed by the TintoDeparaffinator Citrate. Trilogy, Dewax/HIER Low and Dewax/HIER High left considerable amounts of larger paraffin residues when sections were mounted with AquaMounter but the specificity and sensitivity of the IHC signals were not affected. The treatment with ChromoProtector prior to tissue mounting with AquaMounter for all 3-in-1 one-step deparaffinization and retrieval solutions, completely eliminated paraffin or micro-paraffin residues in tissues without affecting the specificity and sensitivity of the IHC signals. Additionally, the use of the ChromoProtector, a biodegradable, non-toxic stabilizing polymer solution applied prior to mounting to eliminate micro-paraffin residue, also protects substrate-chromogens that are soluble or fade with solvents prior to mounting with solvent mounting media and thus allows for a solvent-free environment when conducting IHC procedures. The elimination of xylenes and alcohol for the deparaffinization and mounting steps reduced the IHC procedure time by an average of 1 hour. IHC procedures that are performed using the 3-in-1 One-Step TintoDeparaffinator Citrate or EDTA to deparaffinize, retrieve and rehydrate FFPE tissues, the ChromoProtector to preserve stains and the non-toxic, biodegradable XyGreen PermaMounter to permanent mount tissues, allow for a solvent-free environment when conducting immunohistochemical procedures. These solutions are a safe, efficient and economical alternative to traditional deparaffinization and mounting of tissues with solvent containing solutions or solvent containing permanent mounting media. An additional benefit is the reduction of cost, time and number of steps in the deparaffinization, epitope retrieval and hydration steps and exposure to toxic solvents like xylene, toluene and alcohol when handling FFPE tissues for IHC.

11 REFRENCES 1. Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991; 39: Cattoretti G, Becker MH, Key G, et al. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol. 1992; 168: Gown AM, de Wever N, Battifora H Microwave-based antigenic unmasking: a revolutionary new technique for routine immunohistochemistry. Appl Immunohistochem. 1: Taylor CR. Immunohistochemistry for the age of molecular morphology. Appl Immunohistochem Mol Morphol.2001; 9: Gown AM. Unmasking the mysteries of antigen or epitope retrieval and formalin fixation. Am J Clin Pathol. 2004; 121: Taylor CR, Cote RJ Immunomicroscopy: a diagnostic tool for the surgical pathologist. Philadelphia: Elsevier Saunders. 7. Shi SR, Liu C, Taylor CR. Standardization of Immunohistochemistry for Formalin-fixed, Paraffin-embedded Tissue Sections Based on the Antigen Retrieval Technique: From Experiments to Hypothesis. J Histochem Cytochem. 2006; 39: Jagirdar J. Immunohistochemistry, then and now. Arch Pathol Lab Med. 2008; 132: Taylor CR, Shi S-R, Barr NJ Techniques of Immunohistochemistry: principles, pitfalls, and standardization. In: Dabbs DJ, editor., editor. Diagnostic immunohistochemistry: theranostic and genomic applications. 3rd ed. Philadelphia: Saunders-Elsevier; p Shan-Rong Shi, Yan Shi, and Clive R. Taylor. Antigen Retrieval Immunohistochemistry Review and Future Prospects in Research and Diagnosis over Two Decades. J Histochem Cytochem Jan; 59 (1):