Alejandro S. Mendoza, M.D., Surgical Pathology/GI Fellow
John Bishop, M.D., Health Sciences Clinical Professor, Vice Chair for Clinical Services, Department of Pathology and Laboratory Medicine, UC Davis

Introduction

Tissue staining during a frozen section is one of the most important steps in obtaining an accurate pathologic diagnosis. Visualization and discernment of microscopic details can become difficult if staining is done improperly. Here, we will review the two (H&E, Toluidine blue) common staining methods used during a frozen section, compare their advantages and limitations, and outline best lab practices in achieving optimal staining result.

H&E

H&E is the most commonly used of all the various staining methods available in frozen section. H&E is simple to perform, inexpensive and reliable. The two main dye components are hematoxylin and eosin.

Hematoxylin is a natural dye derived from the Haematoxylon campechianum logwood tree, a tree native to Campeche’s Mexican state. It is a basic dye that stains acidic cell components such as nucleic acids, glycosaminoglycans, and acid glycoproteins, into a blue-purple hue. 1,2,3 Eosin is an acidic dye and serves as an excellent counterstain to hematoxylin that targets the cytoplasm of cells, specifically mitochondria, secretory granules, and collagen.3 It gives differing shades of red and pink to the cytoplasm of different types of cells and different types of connective tissues.4

H&E can be performed in two different methods: progressive or regressive. In the progressive method, the tissue is initially stained with a hematoxylin solution that contains an excess of aluminum salts or acid, which increases nuclei affinity.5 Washing with water follows before a counterstain is applied. Variation in color is due to the selective affinity of tissue components to hematoxylin. With the regressive method, overstaining the tissue section with a neutral hematoxylin solution is the initial step. An acid alcohol is then used to remove excess stain, followed by an alkaline solution to achieve a neutralized tissue section. Stain intensity is controlled by visual examination with a microscope. The progressive method yields more reproducible results, but the regressive method offers a sharper degree of differentiation.6,7

Advantages: H&E yields excellent morphology, providing excellent contrast between cellular components.5 H&E provides better visualization of individual cell necrosis, keratinization, and keratin pearls, which are frequent histologic findings in well­differentiated squamous cell carcinoma. H&E offers stability of stained tissue lasting years without fading. H&E stain is also compatible when combined with various oxidants, mordants, and differentiating agents.8 H&E is reliable and simple to perform.

Limitations: One of the main limitations of H&E is the length of the staining procedure. It can take from 3 to 5 minutes to prepare one slide, depending on practice protocol. Due to the number of stains used, performing H&E during a frozen section can sometimes become challenging especially when multiple parts or sections are processed at once. H&E also poorly stains elastic material, reticular fibers, basement membranes, and lipids. It only provides limited insight into the specific biochemical nature of the tissue, specifically only the acidic and basic components.9

Toluidine Blue

Tolonium chloride, aka Toluidine blue (TB), is a cationic (basic) thiazine metachromatic dye which has a high affinity for acidic tissue components and turns nucleic acid blue and polysaccharides purple.10 When it binds to glycosaminoglycans, it give off a reddish purple staining called metachromasia.11 This property is particularly useful in Mohs surgery for basal cell carcinoma (BCC) because the contrast of BCC’s blue aggregates with the reddish purple metachromasia of glycosaminoglycans in the surrounding stroma helps to delineate very small nests or a few clusters of tumor cells from adnexal structures.

Advantages: Toluidine blue staining is faster and simpler than H&E and requires no counterstain.11 TB is a reliable stain and requires only one primary staining agent as opposed to H&E. Minimizing the staining time reduces patient waiting, facilitates patient flow, and frees up staff to perform other tasks.12 The metachromasia in BCCs and microcystic adnexal carcinoma(MAC) can be helpful in detecting small numbers of infiltrating tumor cells that otherwise might be missed.13,14 Mast cells and mucopolysaccharides in the stroma surrounding BCC may be highlighted in a magenta hue, prompting a diligent search for residual tumor. Mast cells have bright purple metachromatic granules on TB and are abundant in the stroma of BCCs. Abundant mast cells may be indicative of residual tumor.8

Limitations: Nuclear detail is inferior compared to H&E.11 A lot of pathologists/Mohs surgeons are not trained with TB and therefore may find it difficult to use TB at first. It may take a while for them to become familiar with the staining pattern of TB.8 As with H&E, elastic material, reticular fibers, nerve fibers, and fat are difficult to identify.

Best Lab Practices

Whatever is the preferred staining method for frozen section, the following general best lab practices helps to ensure an optimal staining result.

  • Wear appropriate personal protective equipment to avoid injury and cutaneous absorption.
  • Optimal staining is achieved with 5-6 µm thick sections.
  • Do not allow frozen sections to air-dry at any time during the staining procedure for better preservation of tissue morphology.
  • Drain slides after each step to avoid injury and cutaneous absorption.
  • Change staining solutions on a regular basis according to acceptable laboratory protocol. Consideration must be given to number of slides stained as well as to time in use.
  • Store chemicals in a cool, dry, well-ventilated area, and flammables in an approved cabinet or room.
  • Ensure adequate ventilation and regularly check fume contaminant hoods that can minimize the amount of inhaled chemical vapor.
  • Quality control should be performed daily before proceeding with patient specimens.

Common Problems with Rapid H&E Staining

  1. Weak Hematoxylin staining:
    Due to autolysis or poor fixation, over-decalcification, inadequate staining time, excessive de-staining, weak hematoxylin due to carryover, contaminants, thin sections, and inadequate removal of alcohol or insufficient pre-rinsing with water prior to staining with hematoxylin
  2. Excessive Hematoxylin staining:
    Due to drying of tissue, excessive staining times, too weak or inadequate de-staining time, thick section.
  3. Weak Eosin staining:
    Due to too high Eosin pH, contaminant in the alcohol rinse, deteriorating eosin due to excessive carryover, thin sections, inadequate staining time, over-differentiation by subsequent 95% alcohol.
  4. Excessive Eosin staining:
    Due to stronger dye solution (can be due to excessive evaporation), use of isopropyl alcohol as the rinsing agent, thick sections, excessive staining times
  5. Water beads or white haze under the cover­slip:
    Due to incomplete dehydration of the section, formed by mixture of water and the clearing agent.
  6. Eosin bleeding from the tissue section:
    Due to inadequate clearing of alcohol, which will remain within the tissue section and causes the bleeding.

Sample Rapid H&E Staining Method

  1. 95% ethyl alcohol (10 dips)
  2. Formalin 10%, Phosphate Buffered (10 dips)
  3. Distilled water (10 dips)
  4. Hematoxylin Stain, Harris Modified (30 seconds)
  5. Distilled water (10 dips)
  6. Distilled water (10 dips)
  7. 95% ethyl alcohol (10 dips)
  8. Eosin Y (15 seconds)
  9. 95% ethyl alcohol (10 dips)
  10. 95% ethyl alcohol (10 dips)
  11. 100% ethyl alcohol (10 dips)
  12. 100% ethyl alcohol (10 dips)
  13. Clearing agent (10 dips)
  14. Clearing agent (10 dips)
  15. Coverslip with compatible mounting medium

Common Problems with Toluidine Blue

  1. Weak Toluidine blue staining:
    Due to autolysis or poor fixation, over-decalcification, inadequate staining time, excessive de-staining/rinsing with water, weak solution due to water carryover, contaminants, thin sections
  2. Excessive Toluidine blue staining:
    Due to drying of tissue, excessive staining times, inadequate de-staining/rinsing with water, thick sections
  3. Water beads or white haze under the cover­slip:
    Due to incomplete dehydration of the section, formed by mixture of water and the clearing agent

Sample Toluidine Blue Staining Method

  1. Alcoholic formalin (10 dips)
  2. Tap water (10 dips)
  3. Tap water (10 dips)
  4. 1% T-blue (40 seconds)
  5. Tap water (10 dips)
  6. 70% ethanol (10 dips)
  7. 95% ethanol (10 dips)
  8. 100% ethanol (10 dips)
  9. 100% ethanol (10 dips)
  10. Clearing agent (10 dips)
  11. Clearing agent (10 dips)
  12. Coverslip with compatible mounting medium

Conclusion

Preference for staining method in frozen section is usually based on individual training or personal experience. The advantages and limitations of each technique should be considered, whether the preference is H&E or TB. It is important to recognize that staining characteristics, vary with technique, brand of the solution/stain, and experience. Firm understanding of the principles of the H&E or TB staining methods is crucial in troubleshooting for problems. Tissue staining should be reproducible and reliable. Excellent visualization and discrimination of histologic detail of tissue sections are crucial in a rapid and accurate pathologic diagnosis during a frozen section. Lastly, other stains such as Oil red O for fat, Hematoxylin and periodic acid Schiff (PAS) to highlight glycogen and mucoproteins, and some immunohistochemical stains are also available during a frozen section and should be considered if available.

References

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