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45 Staining Methods in Frozen Section: Best Lab Practices

Archived Past Lab Best Practice

MARCH 15, 2020

Staining Methods in Frozen Section: Best Lab Practices

By Laboratory Best Practice blog

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

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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.

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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

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30/10/23, 13.45 Staining Methods in Frozen Section: Best Lab Practices

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
1. Bancroft JD, Gamble M, editors. Theory and Practice of Histological Techniques. 6th ed. New York: Churchill
Livingstone;2008.
2. Bancroft JD, Stevens A. Histopathological Stains and Their Diagnostic Uses. New York: Churchill Livingstone; 1975.
3. Junqueira LC, Carneiro J. Basic Histology. 11th ed. New York: McGraw Hill, Inc.; 2005.
4. Ross MH, Wojciech P. Histology: A Text and Atlas. 5th ed. Baltimore: Lippincott Williams & Wilkins; 2006.
5. Larson K, Ho HH, Anumolu PL, Chen TM. Hematoxylin and eosin tissue stain in Mohs micrographic surgery: a review.
Dermatol Surg. 2011 Aug;37(8):1089-99. doi: 10.1111/j.1524-4725.2011.02051.x. Epub 2011 Jun 2. Review. PubMed PMID:
21635628.
6. Preece A. A Manual for Histologic Techniques. 3rd ed. Boston: Little, Brown, and Company; 1972.
7. Luna L, editor. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd ed. New York:
McGraw-Hill, Inc.; 1968.
8. Humphreys TR, Nemeth A, McCrevey S, Baer SC, Goldberg LH. A pilot study comparing toluidine blue and hematoxylin and
eosin staining of basal cell and squamous cell carcinoma during Mohs surgery. Dermatol Surg. 1996 Aug;22(8):693-7.
PubMed PMID: 8780761.
9. Wittekind D. Traditional staining for routine diagnostic pathology including the role of tannic acid. 1. Value and limitations of
the hematoxylin-eosin stain. Biotech Histochem 2003;78:261-70.
10. Sridharan, G; Shankar, AA (2012). Toluidine Blue: A review of its chemistry and clinical utility. J Oral Maxillofac Pathol. 16:
251–5. doi:10.4103/0973-029X.99081.
11. Aslam A, Aasi SZ. Frozen-Section Tissue Processing in Mohs Surgery. Dermatol Surg. 2019 Dec;45 Suppl 2:S57-S69. doi:
10.1097/DSS.0000000000002260. PubMed PMID: 31764292.
12. Todd MM, Lee JW, Marks VJ. Rapid toluidine blue stain for Mohs’ micrographic surgery. Dermatol Surg. 2005 Feb;31(2):244-
5. PubMed PMID: 15762224.
13. Tehrani H, May K, Morris A, Motley R. Does the dual use of toluidine blue and hematoxylin and eosin staining improve basal
cell carcinoma detection by Mohs surgery trainees? Dermatol Surg. 2013 Jul;39(7):995-1000. doi: 10.1111/dsu.12180. Epub
2013 Mar 6. PubMed PMID: 23465179.
14. Wang SQ, Goldberg LH, Nemeth A. The merits of adding toluidine blue-stained slides in Mohs surgery in the treatment of a
microcystic adnexal carcinoma. J Am Acad Dermatol. 2007 Jun;56(6):1067-9. PubMed PMID: 17504725.
15. Carson, Freida L., and Christa Hladik Cappellano. Histotechnology: A Self-instructional Text. 4th ed. Chicago: ASCP Press,
2015. 120-121.

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