Solvent Free Environmentally Friendly IHC Furler Chase¹, Wall Carolyn¹, Henry Marianne¹, Henry Jim ¹ and Heras Alfonso¹

Bio SB Inc., www.biosb.com 69 Santa Felicia Dr., Santa Barbara, CA, 93117

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 heat-induced 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.

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

Manufacturer

TintoDeparaffinator Citrate Bio SB, Inc, Santa Barbara, CA, USA

TintoDeparaffinator EDTA Bio SB, Inc, Santa Barbara, CA, USA

Trilogy Cell Marque Corporation, Rocklin, CA, USA

Dewax and HIER Buffer Low ThermoFisher, Runcorn, UK

Dewax and HIER Buffer High 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). 1.2. 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. 1.3. 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.

1.4. The XyGreen PermaMounter is a natural biosolvent, which is a biodegradable, non-toxic and non-flammable permanent mounting solution, was compared to an aqueous (AquaMounter) and a traditional solvent and flammable toluene-containing permanent mounting media (PermaMounter). 1.5. 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

Placenta Blank Breast Uterus Cervix Fallopian T

Brain Pituitary Adrenal Pancreas Salivary Colon

Liver Kidney Thyroid Lung Skin Bladder

Testis Prostate Spleen Tonsil Bone M. Thymus

7-Core Human Lymphoid Tissue Microarray

Tonsil Blank Lymph Node Spleen

Tonsil Thymus Lymph Node Spleen

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

Deparaffinization and

Retrieval

HIER Mounting

Solution

Mounting Media

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

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

Citrate PT 1 x 30 min

PC 1 x 15 min

PT 1 x 30 min

PC 1 x 15 min

PT 1 x 30 min

PC 1 x 15 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

Alcohol 15 min

ChromoProtector

10 min

Xylenes 15 min

Alcohol 15 min

Xylenes 15 min

Alcohol 15 min

Mounting Media PermaMounter XyGreen PermaMounter PermaMounter PermaMounter

Total Protocol

Time in Minutes

PC = 165.5 min

PT = 180.5 min

PC = 110.5 min

PT = 125.5 min

PC = 130.5 min

PT = 145.5 min

PC = 130.5 min

PT = 145.5 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.

3.2 Paraffin removal was graded by the amount of residual paraffin after IHC using the following scoring: +, ++, +++ and ++++. 3.3 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 micro-paraffin 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 3.5 3.5 3.5 0

TDP Citrate 3.5 3.3 3.5 ++

TDP Citrate /ChromoProtector 3.5 3.5 3.5 0

TDP EDTA 3.5 3.5 3.5 +

TDP EDTA/ ChromoProtector 3.5 3.5 3.5 0

Trilogy 3.5 3.5 3.5 +++

Trilogy/ ChromoProtector 3.5 3.5 3.5 0

Dewax/HIER Low 3.5 3.2 3.5 +++

Dewax/HIER Low/ ChromoProtector 3.3 3.5 3.2 0

Dewax/HIER High 3.5 3.5 3.5 +++

Dewax/HIER Low/ ChromoProtector 3.3 3.5 3.5 0

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 3.5/0.25 3.3/0.25 3.3/0.25 3.42

TDP EDTA 1X 3.5/0.25 3.5/0.25 3.5/0.25 3.50

TDP Citrate 2X 3.5/0.25 3.5/0.25 3.5/0.25 3.50

TDP EDTA 2X 3.5/0.25 3.6/0.25 3.5/0.25 3.56

TDP Citrate 3X 3.5/0.25 3.3/0.25 3.5/0.25 3.46

TDP EDTA 3X 3.5/0.25 3.7/0.25 3.7/0.25 3.67

TDP Citrate 4X 3.5/0.25 3.3/0.25 3.3/0.25 3.46

TDP EDTA 4X 3.5/0.25 3.5/0.25 3.3/0.25 3.46

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

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

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.

0.000

0.500

1.000

1.500

2.000

2.500

3.000

3.500

4.000

1 2 3 4

CD20

CK AE1/AE3

PR

IHC Signal for Four Times Reused Solutions

TDP Citrate

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

1 2 3 4

CD20

CK AE1/AE3

PR

TDP Citrate

Signal Difference between Four Times Reused and Fresh Solutions

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

Mounting

Media

IHC of PD-L1 on a Hodgkin’s

Lymphoma Tissue with AEC

IHC of SATB2 on a Colon Ca

Tissue with HRP Green

IHC of ALK-1 on a FFPE

Anaplastic Large Cell

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.

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.

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