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Sodium Hypochlorite Soak for Freshly Excised Skin Tissue Prior to Grossing and Frozen Sectioning

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Sodium Hypochlorite Soak for Freshly Excised Skin Tissue Prior to Grossing and Frozen Sectioning Vol. XXXIX, No. 1 May 2006 Managing Editor, Gilles Lefebvre Scientific Editor, Vinnie Della Speranza, MS, HTL(ASCP) HT, MT Sodium Hypochlorite Soak for Freshly Excised Skin Tissue Prior to Grossing and Frozen Sectioning Mechanism of Action as an Antiviral and Antimicrobial Agent and its Effect on Tissue Components and Staining Marie Tudisco, HT(ASCP) Barbara Strippoli, HT(ASCP) Memorial Sloan Kettering Cancer Center New York, NY [email protected] Fig. 1. 1999 H&E section pretreated for 15 minutes with 0.5% sodium hypochlorite immersion. 40X Abstract Histology and Mohs surgery laboratories frequently work with fresh unfixed patient samples that may pose a significant infectious risk to laboratory personnel. While fixation of tissues is thought to render them noninfectious, some IN THIS ISSUE Sodium Hypochlorite Soak for Freshly Excised Skin Tissue Prior to Grossing and Frozen Sectioning. 1 Laser Microdissection for the Histology Technician. 5 Stained Slides vs Noninvasive Imaging: Strange Bedfellows in Bone Research?. 10 Orthochromatic and Metachromatic Action of Toluidine Blue: Application in the Staining of Mohs Micrographic Frozen Sections for the Detection of Basal Cell Carcinoma . 15 Fig. 2. 2000 H&E section pretreated for 15 minutes with 0.5% sodium hypochlorite immersion. 100X The Historical Evolution of Hematoxylin and Basic Fuchsin Staining of Biological Samples. 19 Facts About Alcohol Dehydration of Tissue Samples. 23 Mark Your Calendar for 2006 . 24 1 Similarly, we have adopted a common practice of Results wetting or soaking fresh, known infectious specimens Randomly selected slides from 1999 to 2005, prepared for frozen sectioning in a 1:10 aqueous dilution of from excised human skin that had been soaked in a household bleach for a short duration prior to grossing solution of 0.5% sodium hypochlorite for 5 to and cutting. All other specimens are soaked for 15 minutes prior to frozen section processing, were 5 minutes. The known infectious samples, such as reexamined. There appeared to be comparable clarity hepatitis B and C, HIV, Kaposi’s sarcoma, and genital of stain over a 6-year period for slides having been herpes and warts, are soaked for 15 minutes. When pretreated with 0.5% sodium hypochlorite. Tissue observed microscopically, no adverse effects on tissue components were easily identified and H&E and components, frozen preparation, or staining were noted. toluidine blue stains retained their original hues (Figs. 1-8). Materials and Methods Standard methods of fixing small tissues (5 mm to 1 cm) Advantages facilitate penetration. We know that matrix hardening fixation for paraffin blocks requires 1 hour per 1. 5.25% sodium hypochlorite (household bleach) is millimeter of tissue thickness. Traditionally, frozen inexpensive and readily available (it also comes in a fresh scent). Fig. 3. 2000 Toluidine blue section pretreated for 15 minutes with 0.5% Fig. 4. 2001 H&E section pretreated for 15 minutes with 0.5% sodium sections cut on a freezing microtome are fixed in sodium hypochlorite immersion. 100X hypochlorite immersion. 100X 10% formalin for only 1 to 2 minutes before 2. Sodium hypochlorite suppresses, destroys, and 8 sectioning. The volume of fixative should be 20 times prevents transmission of infectious agents. laboratory techniques require that the samples remain Although little or no data are available on the the volume of tissue.9 unfixed for testing. While laboratory personnel prophylactic use of sodium hypochlorite as an antiviral 3. At low concentrations, for short-term exposure, understand the risks associated with working with or antimicrobial agent for fresh tissue prior to frozen Surgically excised Mohs tissue is received in the lab. sodium hypochlorite is biologically compatible with fresh unfixed samples, there is still heightened anxiety sectioning and its effects on processing and staining, we Since the tissue can begin to float in the pan when frozen section microtomy. that can be a source of distraction, possibly leading to believe reasonable conclusions can be made from data immersed, the superior 12 o’clock surface is first an accidental exposure. We report a technique for relating to its mechanism of action. marked with gentian violet to preserve orientation. 4. Its suggested use as a prophylactic against infectious organisms in the laboratory may lessen fear of adopting the prophylactic use of a 1:10 aqueous Known or suspected infectious tissue is immersed in a dilution of household bleach (5.25% sodium Sodium hypochlorite can be used in the following injury during the handling and preparation of fresh scenarios. pan containing freshly prepared 0.5% sodium tissues. hypochlorite) as an antimicrobial and antiviral agent hypochlorite (one part household bleach to nine parts prior to the preparation of frozen sections, to reduce or a) As a decontaminant of transmissible diseases such distilled water); it is placed under a fume hood for Disadvantages eliminate risk to laboratory personnel. We also as HIV, sodium hypochlorite can be used on work 15 minutes. All other specimens that come into the lab demonstrate the effects of sodium hypochlorite on surfaces and instruments. Instruments are are routinely flooded with the solution for 5 minutes 1. Sodium hypochlorite is highly corrosive and breaks fresh tissue for frozen sectioning from randomly decontaminated in 15 minutes; work surfaces using a squeeze bottle. Tissue is then blotted dry, dyed, down rapidly when diluted. A 1:10 dilution must be selected stained slides collected over a 6-year period. require 30 minutes of contact with the solution.3 grossed, and processed by frozen section method, made fresh daily. retrieved onto coated slides, and stained. Standard Introduction 2. Sodium hypochlorite can be harmful if used by b) Sodium hypochlorite is the most widely used irrigating precautions are observed during processing. In 1984, the CDC and NIH jointly published a set of solution in endodontics. It is both an antimicrobial and those with a hypersensitivity to chlorine bleach. guidelines for the safe handling of blood-borne tissue dissolving agent used in root canal procedures pathogens.1 Hepatitis B (HBV), hepatitis C (HCV), 3. Tissue will dissolve completely in the solution if left to dissolve pulp. Bovine pulp dissolves in 5.25% for long periods. and human immunodeficiency virus (HIV) are among sodium hypochlorite, or household bleach, in as little some of the transmissible agents that have been as 20 minutes and up to 2 hours.4 identified as posing a risk to laboratory personnel. While the CDC discourages the preparation of frozen c) Human brains infected with Creutzfeldt-Jakob sections on fresh frozen tissues from HIV patients, it disease (CJD) are immersed in formic acid and recognizes that this testing may be necessary in some sodium hypochlorite solutions greater than 2%. circumstances and has established decontamination Two to three days may be needed to deactivate guidelines for both the cryostat and laboratory prions that occupy the entire brain surface. Sodium countertops.2 The virus can be introduced into any hypochlorite can eliminate prions on work surfaces break in a staff member’s skin during trimming of if left undisturbed for 15 minutes.5 tissue for histological processing. To prevent infection in laboratory workers, standard precautions d) In marine biology tests, sea cucumbers were promulgated by CDC and OSHA must be observed dissolved in a test tube filled with sodium when working with fresh tissue samples. hypochlorite during a 24-hour immersion.6 Sodium hypochlorite is an important reagent used in e) Tobacco seeds, treated with 0.5% sodium many disciplines including being universally employed as hypochlorite are sterilized in 5 minutes.7 a decontaminant for work surfaces; an antimicrobial and antiviral agent in dentistry, veterinary medicine, and f) Dakin’s solution (another name for 0.5% wound management; and as a fixative in human hypochlorite with a boric acid buffer) has long been pathology, including tissue procurement; and marine and used to kill germs and prevent germ growth in plant biology. Its mechanism of action will be explored. wounds. It is still used but is no longer recommended Fig. 5. 2002 H&E section pretreated for 15 minutes with 0.5% sodium Fig. 6. 2003 H&E section pretreated for 15 minutes with 0.5% sodium because it is cytotoxic and inhibits granulation. hypochlorite immersion. 40X hypochlorite immersion. 40X 2 3 Generally, our samples are 3 mm thick. Based on the mechanism of action in regard to tissue dissolution Laser Microdissection for capacity, we believe the minimal required the Histology Technician concentration of sodium hypochlorite for use as an antimicrobial and antiviral agent can be effective in Pat Stockton, HT helping prevent the spread of infectious disease in the Laboratory of Experimental Pathology event of injury to laboratory personnel during National Institute of Environmental Health Sciences processing of frozen sections. Short-term fixation in Research Triangle Park, NC 0.5% sodium hypochlorite is biologically compatible [email protected] with the process and presents low toxicity to host cells. The integrity of the stain is preserved and remains The practice of isolating and analyzing a single cell or vivid after 5 years. The result is a permanent slide with cell type from stained tissue on a slide has come into no fading. We find that by lowering the pH through mainstream use through the technology known as laser dilution (1:10), the working solution becomes less microdissection (LM), which provides molecular alkaline, resulting in a vivid, long-lasting stain while information about DNA, RNA, and proteins in 13 preserving the integrity of the tissue. targeted tissue samples.1-4 Histology technicians (HTs) Fig. 7. 2004 H&E section pretreated for 15 minutes with 0.5% sodium Fig. 8. 2005 H&E section pretreated for 5 minutes with 0.5% sodium hypochlorite immersion. 100X hypochlorite wet flush. 40X have been incorporated into the LM process because Based upon these observations, we believe that as a 5 fixative for frozen sectioning, sodium hypochlorite of their expertise in tissue handling.
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