The Role of Toluidine Blue in Mohs Micrographic Surgery: A Systematic Review

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The Role of Toluidine Blue in Mohs Micrographic Surgery: A Systematic Review

Toluidine blue (TB), a dye with metachromatic staining properties, was developed in 1856 by William Henry Perkin.1 Metachromasia is a perceptible change in the color of staining of living tissue due to the electrochemical properties of the tissue. Tissues that contain high concentrations of ionized sulfate and phosphate groups (high concentrations of free electronegative groups) form polymeric aggregates of the basic dye solution that alter the absorbed wavelengths of light.2 The function of this characteristic is to use a single dye to highlight different structures in tissue based on their relative chemical differences.3

Toluidine blue primarily was used within the dye industry until the 1960s, when it was first used in vital staining of the oral mucosa.2 Because of the tissue absorption potential, this technique was used to detect the location of oral malignancies.4 Since then, TB has progressively been used for staining fresh frozen sections in Mohs micrographic surgery (MMS). In a 2003 survey study (N=310), 16.8% of surgeons performing MMS reported using TB in their laboratory.5 We sought to systematically review the published literature describing the uses of TB in the setting of fresh frozen sections and MMS.

Methods

We conducted a systematic search of the PubMed and Cochrane databases for articles published before December 1, 2019, to identify any relevant studies in English. Electronic searches were performed using the terms toluidine blue and Mohs or Mohs micrographic surgery. We manually checked the bibliographies of the identified articles to further identify eligible studies.

Eligibility Criteria—The inclusion criteria were articles that (1) considered TB in the context of MMS, (2) were published in peer-reviewed journals, (3) were published in English, and (4) were available as full text. Systematic reviews were excluded.

Data Extraction and Outcomes—All relevant information regarding the study characteristics, including design, level of evidence, methodologic quality of evidence, pathology examined, and outcome measures, were collected by 2 independent reviewers (T.L. and A.D.) using a predetermined data sheet. The same 2 reviewers were used for all steps of the review process, data were independently obtained, and any discrepancy was introduced for a third opinion (D.H.) and agreed upon by the majority.

Quality Assessment—The level of evidence was evaluated based on the criteria of the Oxford Centre for Evidence-Based Medicine. Two reviewers (T.L. and A.D.) graded each article included in the review.

PRISMA (Preferred Reporting Items for Systematic Reviews and Meta Analyses) flow diagram.
FIGURE 1. PRISMA (Preferred Reporting Items for Systematic Reviews and Meta Analyses) flow diagram.

Results

A total of 25 articles were reviewed. After the titles and abstracts were screened for relevance, 12 articles remained (Figure 1). Of these, 1 compared basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), 4 were related to BCC, 3 were related to SCC, 1 was related to microcystic adnexal carcinoma (MAC), 1 was related to primary cutaneous adenoid cystic carcinoma (PCACC), and 2 were related to technical aspects of the staining process (Table 1).

Summary of Articles Published on Toluidine Blue in Mohs Micrographic Surgery

 

 

A majority of the articles included in this review were qualitative and observational in nature, describing the staining characteristics of TB. Study characteristics are summarized in Table 1.

Comment

Basal Cell Carcinoma—Toluidine blue staining characteristics help to identify BCC nests by differentiating them from hair follicles in frozen sections. The metachromatic characteristic of TB stains the inner root sheath deep blue and highlights the surrounding stromal mucin of BCC a magenta color.18,19 In hematoxylin and eosin (H&E) stains, these 2 distinct structures can be differentiated by cleft formation around tumor nests, mitotic figures, and the lack of a fibrous sheath present in BCC tumors.20 The advantages and limitations of TB staining of BCC are presented in Table 2.

Advantages and Limitations of Toluidine Blue Staining in BCC and SCC

Humphreys et al6 suggested a noticeable difference between H&E and TB in the staining of cellular and stromal components. The nuclear detail of tumor cells was subjectively sharper and clearer with TB staining. The staining of stromal components may provide the most assistance in locating BCC islands. Mucopolysaccharide staining may be absent in H&E but stain a deep magenta with TB. Although the presence of mucopolysaccharides does not specifically indicate a tumor, it may prompt further attention and provide an indicator for sparse and infiltrative tumor cells.6 The metachromatic stromal change may indicate a narrow tumor-free margin where additional deeper sections often reveal tumor that may warrant additional resection margin in more aggressive malignancies. In particular, sclerosing/morpheaform BCCs have been shown to induce glycosaminoglycan synthesis and are highlighted more readily with TB than with H&E when compared to surrounding tissue.21 This differentiation in staining has remained a popular reason to routinely incorporate TB into the staining of infiltrative and morpheaform variants of BCC. Additionally, stromal mast cells are believed to be more abundant in the stroma of BCC and are more readily visualized in tissue specimens stained with TB, appearing as bright purple metachromatic granules. These granules are larger than normal and are increased in number.6

The margin behavior of BCC stained with TB was further characterized by Goldberg et al,8 who coined the term setting sun sign, which may be present in sequential sections of a disappearing nodule of a BCC tumor. Stroma, inflammatory infiltrate, and mast cells produce a magenta glow surrounding BCC tumors that is reminiscent of a setting sun (Figure 2). Invasive BCC is considered variable in this presentation, primarily because of zones of cell-free fluid and edema or the second area of inflammatory cells. This unique sign may benefit the inspecting Mohs surgeon by providing a clue to an underlying process that may have residual BCC tumors. The setting sun sign also may assist in identifying exact surgical margins.8

Frozen sections of a basal cell carcinoma (original magnification ×100 for both). The basophilic tumor cells were surrounded by an immediate magenta zone of stroma and an inflammatory response of mast cells, lymphocytes, and fibroblasts.
FIGURE 2. A and B, Frozen sections of a basal cell carcinoma (original magnification ×100 for both). The basophilic tumor cells were surrounded by an immediate magenta zone of stroma and an inflammatory response of mast cells, lymphocytes, and fibroblasts.

The nasal surface has a predilection for BCC.22 The skin of the nose has numerous look-alike structures to consider for complete tumor removal and avoidance of unnecessary removal. One challenge is distinguishing follicular basaloid proliferations (FBP) from BCC, a scenario that is more common on the nose.22 When TB staining was used, the sensitivity for detecting FBP reached 100% in 34 cases reviewed by Donaldson and Weber.10 None of the cases examined showed TB metachromasia surrounding FBP, thus indicating that TB can dependably identify this benign entity. Conversely, 5% (N=279) of BCCs confirmed on H&E did not exhibit surrounding TB metachromasia. This finding is concerning regarding the specificity of TB staining for BCC, but the authors of this study suggested the possibility that these exceptions were benign “simulants” (ie, trichoepithelioma) of BCC.10

The use of TB also has been shown to be statistically beneficial in Mohs training. In a single-center, single-fellow experiment, the sensitivity and specificity of using TB for BCC were extrapolated.9 Using TB as an adjunct in deep sections showed superior sensitivity to H&E alone in identifying BCC, increasing sensitivity from 96.3% to 99.7%. In a cohort of 352 BCC excisions and frozen sections, only 1 BCC was not completely excised. If H&E only had been performed, the fellow would have missed 13 residual BCC tumors.9

Bennett and Taher7 described a case in which hyaluronic acid (HA) from a filler injection was confused with the HA surrounding BCC tumor nests. They found that when TB is used as an adjunct, the HA filler is easier to differentiate from the HA surrounding the BCC tumor nests. In frozen sections stained with TB, the HA filler appeared as an amorphous, metachromatic, reddish-purple, whereas the HA surrounding the BCC tumor nests appeared as a well-defined red. These findings were less obvious in the same sections stained with H&E alone.7

 

 

Squamous Cell Carcinoma—In early investigations, the utility of TB in identifying SCC in frozen sections was thought to be limited. The description by Humphreys and colleagues6 of staining characteristics in SCC suggested that the nuclear detail that H&E provides is more easily recognized. The deep aqua nuclear staining produced with TB was considered more difficult to observe than the cytoplasmic eosinophilia of pyknotic and keratinizing cells in H&E.6

Toluidine blue may be beneficial in providing unique staining characteristics to further detail tumors that are difficult to interpret, such as spindle cell SCC and perineural invasion of aggressive SCC. In H&E, squamous cells of spindle cell SCC (scSCC) blend into the background of inflammatory cells and can be perceptibly difficult to locate. A small cohort of 3 Mohs surgeons who routinely use H&E were surveyed on their ability to detect a proven scSCC in H&E or TB by photograph.12 All 3 were able to detect the scSCC in the TB photographs, but only 2 of 3 were able to detect it in H&E photographs. All 3 surgeons agreed that TB was preferable to H&E for this tumor type. These findings suggested that TB may be superior and preferred over H&E for visualizing tumor cells of scSCC.12 The TB staining characteristics of perineural invasion of aggressive SCC have been referred to as the perineural corona sign because of the bright magenta stain that forms around affected nerves.13 Drosou et al13 suggested that TB may enhance the diagnostic accuracy for perineural SCC.

Rare Tumors—The adjunctive use of TB with H&E has been examined in rare tumors. Published reports have highlighted its use in MMS for treating MAC and PCACC. Toluidine blue exhibits staining advantages for these tumors. It may render isolated nests and perineural invasion of MAC more easily visible on frozen section.15

Although PCACC is rare, the recurrence rate is high.23 Toluidine blue has been used with MMS to ensure complete removal and higher cure rates. The metachromatic nature of TB is advantageous in staining the HA present in these tumors. Those who have reported the use of TB for PCACC prefer it to H&E for frozen sections.14

Technical Aspects—The staining time for TB-treated slides is reduced compared to H&E staining; staining can be efficiently done in frozen sections in less than 2.5 minutes using the method shown in Table 3.17 In comparison, typical H&E staining takes 9 minutes, and older TB techniques take 7 minutes.6

Rapid Toluidine Blue Staining Protocol

Conclusion

Toluidine blue may play an important and helpful role in the successful diagnosis and treatment of particular cutaneous tumors by providing additional diagnostic information. Although surgeons performing MMS will continue using the staining protocols with which they are most comfortable, adjunctive use of TB over time may provide an additional benefit at low risk for disrupting practice efficiency or workflow. Many Mohs surgeons are accustomed to using this stain, even preferring to interpret only TB-stained slides for cutaneous malignancy. Most published studies on this topic have been observational in nature, and additional controlled trials may be warranted to determine the effects on outcomes in real-world practice.

References
  1. Culling CF, Allison TR. Cellular Pathology Technique. 4th ed. Butterworths; 1985.
  2. Bergeron JA, Singer M. Metachromasy: an experimental and theoretical reevaluation. J Biophys Biochem Cytol. 1958;4:433-457. doi:10.1083/jcb.4.4.433
  3. Epstein JB, Scully C, Spinelli J. Toluidine blue and Lugol’s iodine application in the assessment of oral malignant disease and lesions at risk of malignancy. J Oral Pathol Med. 1992;21:160-163. doi:10.1111/j.1600-0714.1992.tb00094.x
  4. Warnakulasuriya KA, Johnson NW. Sensitivity and specificity of OraScan (R) toluidine blue mouthrinse in the detection of oral cancer and precancer. J Oral Pathol Med. 1996;25:97-103. doi:10.1111/j.1600-0714.1996.tb00201.x
  5. Silapunt S, Peterson SR, Alcalay J, et al. Mohs tissue mapping and processing: a survey study. Dermatol Surg. 2003;29:1109-1112; discussion 1112.
  6. Humphreys TR, Nemeth A, McCrevey S, et al. A pilot study comparing toluidine blue and hematoxylin and eosin staining of basal cell and squamous cell carcinoma during Mohs surgery. Dermatol Surg. 1996;22:693-697. doi:10.1111/j.1524-4725.1996.tb00619.x
  7. Bennett R, Taher M. Restylane persistent for 23 months found during Mohs micrographic surgery: a source of confusion with hyaluronic acid surrounding basal cell carcinoma. Dermatol Surg. 2005;31:1366-1369. doi:10.1111/j.1524-4725.2005.31223
  8. Goldberg LH, Wang SQ, Kimyai-Asadi A. The setting sun sign: visualizing the margins of a basal cell carcinoma on serial frozen sections stained with toluidine blue. Dermatol Surg. 2007;33:761-763. doi:10.1111/j.1524-4725.2007.33158.x
  9. Tehrani H, May K, Morris A, et al. Does the dual use of toluidine blue and hematoxylin and eosin staining improve basal cell carcinoma detection by Mohs surgery trainees? Dermatol Surg. 2013;39:995-1000. doi:10.1111/dsu.12180
  10. Donaldson MR, Weber LA. Toluidine blue supports differentiation of folliculocentric basaloid proliferation from basal cell carcinoma on frozen sections in a small single-practice cohort. Dermatol Surg. 2017;43:1303-1306. doi:10.1097/DSS.0000000000001107
  11. Styperek AR, Goldberg LH, Goldschmidt LE, et al. Toluidine blue and hematoxylin and eosin stains are comparable in evaluating squamous cell carcinoma during Mohs. Dermatol Surg. 2016;42:1279-1284. doi:10.1097/DSS.0000000000000872
  12. Trieu D, Drosou A, Goldberg LH, et al. Detecting spindle cell squamous cell carcinomas with toluidine blue on frozen sections. Dermatol Surg. 2014;40:1259-1260. doi:10.1097/DSS.0000000000000147
  13. Drosou A, Trieu D, Goldberg LH, et al. The perineural corona sign: enhancing detection of perineural squamous cell carcinoma during Mohs micrographic surgery with toluidine blue stain. J Am Acad Dermatol. 2014;71:826-827. doi:10.1016/j.jaad.2014.04.076
  14. Chesser RS, Bertler DE, Fitzpatrick JE, et al. Primary cutaneous adenoid cystic carcinoma treated with Mohs micrographic surgery toluidine blue technique. J Dermatol Surg Oncol. 1992;18:175-176. doi:10.1111/j.1524-4725.1992.tb02794.x
  15. 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;56:1067-1069. doi:10.1016/j.jaad.2007.01.008
  16. Chen CL, Wilson S, Afzalneia R, et al. Topical aluminum chloride and Monsel’s solution block toluidine blue staining in Mohs frozen sections: mechanism and solution. Dermatol Surg. 2019;45:1019-1025. doi:10.1097/DSS.0000000000001761
  17. Todd MM, Lee JW, Marks VJ. Rapid toluidine blue stain for Mohs’ micrographic surgery. Dermatol Surg. 2005;31:244-245. doi:10.1111/j.1524-4725.2005.31053
  18. Picoto AM, Picoto A. Technical procedures for Mohs fresh tissue surgery. J Derm Surg Oncol. 1986;12:134-138. doi:10.1111/j.1524-4725.1986.tb01442.x
  19. Sperling LC, Winton GB. The transverse anatomy of androgenic alopecia. J Derm Surg Oncol. 1990;16:1127-1133. doi:10.1111/j.1524 -4725.1990.tb00024.x
  20. Smith-Zagone MJ, Schwartz MR. Frozen section of skin specimens. Arch Pathol Lab Med. 2005;129:1536-1543. doi:10.5858/2005-129-1536-FSOSS
  21. Moy RL, Potter TS, Uitto J. Increased glycosaminoglycans production in sclerosing basal cell carcinoma–derived fibroblasts and stimulation of normal skin fibroblast glycosaminoglycans production by a cytokine-derived from sclerosing basal cell carcinoma. Dermatol Surg. 2000;26:1029-1036. doi:10.1046/j.1524-4725.2000.0260111029.x
  22. Leshin B, White WL. Folliculocentric basaloid proliferation. The bulge (der Wulst) revisited. Arch Dermatol. 1990;126:900-906. doi:10.1001/archderm.126.7.900
  23. Seab JA, Graham JH. Primary cutaneous adenoid cystic carcinoma.J Am Acad Dermatol. 1987;17:113-118. doi:10.1016/s0190 -9622(87)70182-0
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Author and Disclosure Information

Dr. Long is from the Health Corporation of America and Virginia College of Osteopathic Medicine, Blacksburg. Dr. Dunn is in private practice, Tampa, Florida. Drs. Hill and Akin are from the Department of Dermatology, Texas Tech University Health Sciences Center, Lubbock. Dr. Akin also is from Midland Dermatology and Skin Cancer Center, Texas. Dr. Goldberg is from DermSurgery Associates, Houston, Texas.

The authors report no conflicts of interest.

Correspondence: Tyler Long, DO, HCA LewisGale Hospital Montgomery Medical Education, 700 S Main St, Blacksburg, VA 24060 (longjtyler@yahoo.com).

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Dr. Long is from the Health Corporation of America and Virginia College of Osteopathic Medicine, Blacksburg. Dr. Dunn is in private practice, Tampa, Florida. Drs. Hill and Akin are from the Department of Dermatology, Texas Tech University Health Sciences Center, Lubbock. Dr. Akin also is from Midland Dermatology and Skin Cancer Center, Texas. Dr. Goldberg is from DermSurgery Associates, Houston, Texas.

The authors report no conflicts of interest.

Correspondence: Tyler Long, DO, HCA LewisGale Hospital Montgomery Medical Education, 700 S Main St, Blacksburg, VA 24060 (longjtyler@yahoo.com).

Author and Disclosure Information

Dr. Long is from the Health Corporation of America and Virginia College of Osteopathic Medicine, Blacksburg. Dr. Dunn is in private practice, Tampa, Florida. Drs. Hill and Akin are from the Department of Dermatology, Texas Tech University Health Sciences Center, Lubbock. Dr. Akin also is from Midland Dermatology and Skin Cancer Center, Texas. Dr. Goldberg is from DermSurgery Associates, Houston, Texas.

The authors report no conflicts of interest.

Correspondence: Tyler Long, DO, HCA LewisGale Hospital Montgomery Medical Education, 700 S Main St, Blacksburg, VA 24060 (longjtyler@yahoo.com).

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Toluidine blue (TB), a dye with metachromatic staining properties, was developed in 1856 by William Henry Perkin.1 Metachromasia is a perceptible change in the color of staining of living tissue due to the electrochemical properties of the tissue. Tissues that contain high concentrations of ionized sulfate and phosphate groups (high concentrations of free electronegative groups) form polymeric aggregates of the basic dye solution that alter the absorbed wavelengths of light.2 The function of this characteristic is to use a single dye to highlight different structures in tissue based on their relative chemical differences.3

Toluidine blue primarily was used within the dye industry until the 1960s, when it was first used in vital staining of the oral mucosa.2 Because of the tissue absorption potential, this technique was used to detect the location of oral malignancies.4 Since then, TB has progressively been used for staining fresh frozen sections in Mohs micrographic surgery (MMS). In a 2003 survey study (N=310), 16.8% of surgeons performing MMS reported using TB in their laboratory.5 We sought to systematically review the published literature describing the uses of TB in the setting of fresh frozen sections and MMS.

Methods

We conducted a systematic search of the PubMed and Cochrane databases for articles published before December 1, 2019, to identify any relevant studies in English. Electronic searches were performed using the terms toluidine blue and Mohs or Mohs micrographic surgery. We manually checked the bibliographies of the identified articles to further identify eligible studies.

Eligibility Criteria—The inclusion criteria were articles that (1) considered TB in the context of MMS, (2) were published in peer-reviewed journals, (3) were published in English, and (4) were available as full text. Systematic reviews were excluded.

Data Extraction and Outcomes—All relevant information regarding the study characteristics, including design, level of evidence, methodologic quality of evidence, pathology examined, and outcome measures, were collected by 2 independent reviewers (T.L. and A.D.) using a predetermined data sheet. The same 2 reviewers were used for all steps of the review process, data were independently obtained, and any discrepancy was introduced for a third opinion (D.H.) and agreed upon by the majority.

Quality Assessment—The level of evidence was evaluated based on the criteria of the Oxford Centre for Evidence-Based Medicine. Two reviewers (T.L. and A.D.) graded each article included in the review.

PRISMA (Preferred Reporting Items for Systematic Reviews and Meta Analyses) flow diagram.
FIGURE 1. PRISMA (Preferred Reporting Items for Systematic Reviews and Meta Analyses) flow diagram.

Results

A total of 25 articles were reviewed. After the titles and abstracts were screened for relevance, 12 articles remained (Figure 1). Of these, 1 compared basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), 4 were related to BCC, 3 were related to SCC, 1 was related to microcystic adnexal carcinoma (MAC), 1 was related to primary cutaneous adenoid cystic carcinoma (PCACC), and 2 were related to technical aspects of the staining process (Table 1).

Summary of Articles Published on Toluidine Blue in Mohs Micrographic Surgery

 

 

A majority of the articles included in this review were qualitative and observational in nature, describing the staining characteristics of TB. Study characteristics are summarized in Table 1.

Comment

Basal Cell Carcinoma—Toluidine blue staining characteristics help to identify BCC nests by differentiating them from hair follicles in frozen sections. The metachromatic characteristic of TB stains the inner root sheath deep blue and highlights the surrounding stromal mucin of BCC a magenta color.18,19 In hematoxylin and eosin (H&E) stains, these 2 distinct structures can be differentiated by cleft formation around tumor nests, mitotic figures, and the lack of a fibrous sheath present in BCC tumors.20 The advantages and limitations of TB staining of BCC are presented in Table 2.

Advantages and Limitations of Toluidine Blue Staining in BCC and SCC

Humphreys et al6 suggested a noticeable difference between H&E and TB in the staining of cellular and stromal components. The nuclear detail of tumor cells was subjectively sharper and clearer with TB staining. The staining of stromal components may provide the most assistance in locating BCC islands. Mucopolysaccharide staining may be absent in H&E but stain a deep magenta with TB. Although the presence of mucopolysaccharides does not specifically indicate a tumor, it may prompt further attention and provide an indicator for sparse and infiltrative tumor cells.6 The metachromatic stromal change may indicate a narrow tumor-free margin where additional deeper sections often reveal tumor that may warrant additional resection margin in more aggressive malignancies. In particular, sclerosing/morpheaform BCCs have been shown to induce glycosaminoglycan synthesis and are highlighted more readily with TB than with H&E when compared to surrounding tissue.21 This differentiation in staining has remained a popular reason to routinely incorporate TB into the staining of infiltrative and morpheaform variants of BCC. Additionally, stromal mast cells are believed to be more abundant in the stroma of BCC and are more readily visualized in tissue specimens stained with TB, appearing as bright purple metachromatic granules. These granules are larger than normal and are increased in number.6

The margin behavior of BCC stained with TB was further characterized by Goldberg et al,8 who coined the term setting sun sign, which may be present in sequential sections of a disappearing nodule of a BCC tumor. Stroma, inflammatory infiltrate, and mast cells produce a magenta glow surrounding BCC tumors that is reminiscent of a setting sun (Figure 2). Invasive BCC is considered variable in this presentation, primarily because of zones of cell-free fluid and edema or the second area of inflammatory cells. This unique sign may benefit the inspecting Mohs surgeon by providing a clue to an underlying process that may have residual BCC tumors. The setting sun sign also may assist in identifying exact surgical margins.8

Frozen sections of a basal cell carcinoma (original magnification ×100 for both). The basophilic tumor cells were surrounded by an immediate magenta zone of stroma and an inflammatory response of mast cells, lymphocytes, and fibroblasts.
FIGURE 2. A and B, Frozen sections of a basal cell carcinoma (original magnification ×100 for both). The basophilic tumor cells were surrounded by an immediate magenta zone of stroma and an inflammatory response of mast cells, lymphocytes, and fibroblasts.

The nasal surface has a predilection for BCC.22 The skin of the nose has numerous look-alike structures to consider for complete tumor removal and avoidance of unnecessary removal. One challenge is distinguishing follicular basaloid proliferations (FBP) from BCC, a scenario that is more common on the nose.22 When TB staining was used, the sensitivity for detecting FBP reached 100% in 34 cases reviewed by Donaldson and Weber.10 None of the cases examined showed TB metachromasia surrounding FBP, thus indicating that TB can dependably identify this benign entity. Conversely, 5% (N=279) of BCCs confirmed on H&E did not exhibit surrounding TB metachromasia. This finding is concerning regarding the specificity of TB staining for BCC, but the authors of this study suggested the possibility that these exceptions were benign “simulants” (ie, trichoepithelioma) of BCC.10

The use of TB also has been shown to be statistically beneficial in Mohs training. In a single-center, single-fellow experiment, the sensitivity and specificity of using TB for BCC were extrapolated.9 Using TB as an adjunct in deep sections showed superior sensitivity to H&E alone in identifying BCC, increasing sensitivity from 96.3% to 99.7%. In a cohort of 352 BCC excisions and frozen sections, only 1 BCC was not completely excised. If H&E only had been performed, the fellow would have missed 13 residual BCC tumors.9

Bennett and Taher7 described a case in which hyaluronic acid (HA) from a filler injection was confused with the HA surrounding BCC tumor nests. They found that when TB is used as an adjunct, the HA filler is easier to differentiate from the HA surrounding the BCC tumor nests. In frozen sections stained with TB, the HA filler appeared as an amorphous, metachromatic, reddish-purple, whereas the HA surrounding the BCC tumor nests appeared as a well-defined red. These findings were less obvious in the same sections stained with H&E alone.7

 

 

Squamous Cell Carcinoma—In early investigations, the utility of TB in identifying SCC in frozen sections was thought to be limited. The description by Humphreys and colleagues6 of staining characteristics in SCC suggested that the nuclear detail that H&E provides is more easily recognized. The deep aqua nuclear staining produced with TB was considered more difficult to observe than the cytoplasmic eosinophilia of pyknotic and keratinizing cells in H&E.6

Toluidine blue may be beneficial in providing unique staining characteristics to further detail tumors that are difficult to interpret, such as spindle cell SCC and perineural invasion of aggressive SCC. In H&E, squamous cells of spindle cell SCC (scSCC) blend into the background of inflammatory cells and can be perceptibly difficult to locate. A small cohort of 3 Mohs surgeons who routinely use H&E were surveyed on their ability to detect a proven scSCC in H&E or TB by photograph.12 All 3 were able to detect the scSCC in the TB photographs, but only 2 of 3 were able to detect it in H&E photographs. All 3 surgeons agreed that TB was preferable to H&E for this tumor type. These findings suggested that TB may be superior and preferred over H&E for visualizing tumor cells of scSCC.12 The TB staining characteristics of perineural invasion of aggressive SCC have been referred to as the perineural corona sign because of the bright magenta stain that forms around affected nerves.13 Drosou et al13 suggested that TB may enhance the diagnostic accuracy for perineural SCC.

Rare Tumors—The adjunctive use of TB with H&E has been examined in rare tumors. Published reports have highlighted its use in MMS for treating MAC and PCACC. Toluidine blue exhibits staining advantages for these tumors. It may render isolated nests and perineural invasion of MAC more easily visible on frozen section.15

Although PCACC is rare, the recurrence rate is high.23 Toluidine blue has been used with MMS to ensure complete removal and higher cure rates. The metachromatic nature of TB is advantageous in staining the HA present in these tumors. Those who have reported the use of TB for PCACC prefer it to H&E for frozen sections.14

Technical Aspects—The staining time for TB-treated slides is reduced compared to H&E staining; staining can be efficiently done in frozen sections in less than 2.5 minutes using the method shown in Table 3.17 In comparison, typical H&E staining takes 9 minutes, and older TB techniques take 7 minutes.6

Rapid Toluidine Blue Staining Protocol

Conclusion

Toluidine blue may play an important and helpful role in the successful diagnosis and treatment of particular cutaneous tumors by providing additional diagnostic information. Although surgeons performing MMS will continue using the staining protocols with which they are most comfortable, adjunctive use of TB over time may provide an additional benefit at low risk for disrupting practice efficiency or workflow. Many Mohs surgeons are accustomed to using this stain, even preferring to interpret only TB-stained slides for cutaneous malignancy. Most published studies on this topic have been observational in nature, and additional controlled trials may be warranted to determine the effects on outcomes in real-world practice.

Toluidine blue (TB), a dye with metachromatic staining properties, was developed in 1856 by William Henry Perkin.1 Metachromasia is a perceptible change in the color of staining of living tissue due to the electrochemical properties of the tissue. Tissues that contain high concentrations of ionized sulfate and phosphate groups (high concentrations of free electronegative groups) form polymeric aggregates of the basic dye solution that alter the absorbed wavelengths of light.2 The function of this characteristic is to use a single dye to highlight different structures in tissue based on their relative chemical differences.3

Toluidine blue primarily was used within the dye industry until the 1960s, when it was first used in vital staining of the oral mucosa.2 Because of the tissue absorption potential, this technique was used to detect the location of oral malignancies.4 Since then, TB has progressively been used for staining fresh frozen sections in Mohs micrographic surgery (MMS). In a 2003 survey study (N=310), 16.8% of surgeons performing MMS reported using TB in their laboratory.5 We sought to systematically review the published literature describing the uses of TB in the setting of fresh frozen sections and MMS.

Methods

We conducted a systematic search of the PubMed and Cochrane databases for articles published before December 1, 2019, to identify any relevant studies in English. Electronic searches were performed using the terms toluidine blue and Mohs or Mohs micrographic surgery. We manually checked the bibliographies of the identified articles to further identify eligible studies.

Eligibility Criteria—The inclusion criteria were articles that (1) considered TB in the context of MMS, (2) were published in peer-reviewed journals, (3) were published in English, and (4) were available as full text. Systematic reviews were excluded.

Data Extraction and Outcomes—All relevant information regarding the study characteristics, including design, level of evidence, methodologic quality of evidence, pathology examined, and outcome measures, were collected by 2 independent reviewers (T.L. and A.D.) using a predetermined data sheet. The same 2 reviewers were used for all steps of the review process, data were independently obtained, and any discrepancy was introduced for a third opinion (D.H.) and agreed upon by the majority.

Quality Assessment—The level of evidence was evaluated based on the criteria of the Oxford Centre for Evidence-Based Medicine. Two reviewers (T.L. and A.D.) graded each article included in the review.

PRISMA (Preferred Reporting Items for Systematic Reviews and Meta Analyses) flow diagram.
FIGURE 1. PRISMA (Preferred Reporting Items for Systematic Reviews and Meta Analyses) flow diagram.

Results

A total of 25 articles were reviewed. After the titles and abstracts were screened for relevance, 12 articles remained (Figure 1). Of these, 1 compared basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), 4 were related to BCC, 3 were related to SCC, 1 was related to microcystic adnexal carcinoma (MAC), 1 was related to primary cutaneous adenoid cystic carcinoma (PCACC), and 2 were related to technical aspects of the staining process (Table 1).

Summary of Articles Published on Toluidine Blue in Mohs Micrographic Surgery

 

 

A majority of the articles included in this review were qualitative and observational in nature, describing the staining characteristics of TB. Study characteristics are summarized in Table 1.

Comment

Basal Cell Carcinoma—Toluidine blue staining characteristics help to identify BCC nests by differentiating them from hair follicles in frozen sections. The metachromatic characteristic of TB stains the inner root sheath deep blue and highlights the surrounding stromal mucin of BCC a magenta color.18,19 In hematoxylin and eosin (H&E) stains, these 2 distinct structures can be differentiated by cleft formation around tumor nests, mitotic figures, and the lack of a fibrous sheath present in BCC tumors.20 The advantages and limitations of TB staining of BCC are presented in Table 2.

Advantages and Limitations of Toluidine Blue Staining in BCC and SCC

Humphreys et al6 suggested a noticeable difference between H&E and TB in the staining of cellular and stromal components. The nuclear detail of tumor cells was subjectively sharper and clearer with TB staining. The staining of stromal components may provide the most assistance in locating BCC islands. Mucopolysaccharide staining may be absent in H&E but stain a deep magenta with TB. Although the presence of mucopolysaccharides does not specifically indicate a tumor, it may prompt further attention and provide an indicator for sparse and infiltrative tumor cells.6 The metachromatic stromal change may indicate a narrow tumor-free margin where additional deeper sections often reveal tumor that may warrant additional resection margin in more aggressive malignancies. In particular, sclerosing/morpheaform BCCs have been shown to induce glycosaminoglycan synthesis and are highlighted more readily with TB than with H&E when compared to surrounding tissue.21 This differentiation in staining has remained a popular reason to routinely incorporate TB into the staining of infiltrative and morpheaform variants of BCC. Additionally, stromal mast cells are believed to be more abundant in the stroma of BCC and are more readily visualized in tissue specimens stained with TB, appearing as bright purple metachromatic granules. These granules are larger than normal and are increased in number.6

The margin behavior of BCC stained with TB was further characterized by Goldberg et al,8 who coined the term setting sun sign, which may be present in sequential sections of a disappearing nodule of a BCC tumor. Stroma, inflammatory infiltrate, and mast cells produce a magenta glow surrounding BCC tumors that is reminiscent of a setting sun (Figure 2). Invasive BCC is considered variable in this presentation, primarily because of zones of cell-free fluid and edema or the second area of inflammatory cells. This unique sign may benefit the inspecting Mohs surgeon by providing a clue to an underlying process that may have residual BCC tumors. The setting sun sign also may assist in identifying exact surgical margins.8

Frozen sections of a basal cell carcinoma (original magnification ×100 for both). The basophilic tumor cells were surrounded by an immediate magenta zone of stroma and an inflammatory response of mast cells, lymphocytes, and fibroblasts.
FIGURE 2. A and B, Frozen sections of a basal cell carcinoma (original magnification ×100 for both). The basophilic tumor cells were surrounded by an immediate magenta zone of stroma and an inflammatory response of mast cells, lymphocytes, and fibroblasts.

The nasal surface has a predilection for BCC.22 The skin of the nose has numerous look-alike structures to consider for complete tumor removal and avoidance of unnecessary removal. One challenge is distinguishing follicular basaloid proliferations (FBP) from BCC, a scenario that is more common on the nose.22 When TB staining was used, the sensitivity for detecting FBP reached 100% in 34 cases reviewed by Donaldson and Weber.10 None of the cases examined showed TB metachromasia surrounding FBP, thus indicating that TB can dependably identify this benign entity. Conversely, 5% (N=279) of BCCs confirmed on H&E did not exhibit surrounding TB metachromasia. This finding is concerning regarding the specificity of TB staining for BCC, but the authors of this study suggested the possibility that these exceptions were benign “simulants” (ie, trichoepithelioma) of BCC.10

The use of TB also has been shown to be statistically beneficial in Mohs training. In a single-center, single-fellow experiment, the sensitivity and specificity of using TB for BCC were extrapolated.9 Using TB as an adjunct in deep sections showed superior sensitivity to H&E alone in identifying BCC, increasing sensitivity from 96.3% to 99.7%. In a cohort of 352 BCC excisions and frozen sections, only 1 BCC was not completely excised. If H&E only had been performed, the fellow would have missed 13 residual BCC tumors.9

Bennett and Taher7 described a case in which hyaluronic acid (HA) from a filler injection was confused with the HA surrounding BCC tumor nests. They found that when TB is used as an adjunct, the HA filler is easier to differentiate from the HA surrounding the BCC tumor nests. In frozen sections stained with TB, the HA filler appeared as an amorphous, metachromatic, reddish-purple, whereas the HA surrounding the BCC tumor nests appeared as a well-defined red. These findings were less obvious in the same sections stained with H&E alone.7

 

 

Squamous Cell Carcinoma—In early investigations, the utility of TB in identifying SCC in frozen sections was thought to be limited. The description by Humphreys and colleagues6 of staining characteristics in SCC suggested that the nuclear detail that H&E provides is more easily recognized. The deep aqua nuclear staining produced with TB was considered more difficult to observe than the cytoplasmic eosinophilia of pyknotic and keratinizing cells in H&E.6

Toluidine blue may be beneficial in providing unique staining characteristics to further detail tumors that are difficult to interpret, such as spindle cell SCC and perineural invasion of aggressive SCC. In H&E, squamous cells of spindle cell SCC (scSCC) blend into the background of inflammatory cells and can be perceptibly difficult to locate. A small cohort of 3 Mohs surgeons who routinely use H&E were surveyed on their ability to detect a proven scSCC in H&E or TB by photograph.12 All 3 were able to detect the scSCC in the TB photographs, but only 2 of 3 were able to detect it in H&E photographs. All 3 surgeons agreed that TB was preferable to H&E for this tumor type. These findings suggested that TB may be superior and preferred over H&E for visualizing tumor cells of scSCC.12 The TB staining characteristics of perineural invasion of aggressive SCC have been referred to as the perineural corona sign because of the bright magenta stain that forms around affected nerves.13 Drosou et al13 suggested that TB may enhance the diagnostic accuracy for perineural SCC.

Rare Tumors—The adjunctive use of TB with H&E has been examined in rare tumors. Published reports have highlighted its use in MMS for treating MAC and PCACC. Toluidine blue exhibits staining advantages for these tumors. It may render isolated nests and perineural invasion of MAC more easily visible on frozen section.15

Although PCACC is rare, the recurrence rate is high.23 Toluidine blue has been used with MMS to ensure complete removal and higher cure rates. The metachromatic nature of TB is advantageous in staining the HA present in these tumors. Those who have reported the use of TB for PCACC prefer it to H&E for frozen sections.14

Technical Aspects—The staining time for TB-treated slides is reduced compared to H&E staining; staining can be efficiently done in frozen sections in less than 2.5 minutes using the method shown in Table 3.17 In comparison, typical H&E staining takes 9 minutes, and older TB techniques take 7 minutes.6

Rapid Toluidine Blue Staining Protocol

Conclusion

Toluidine blue may play an important and helpful role in the successful diagnosis and treatment of particular cutaneous tumors by providing additional diagnostic information. Although surgeons performing MMS will continue using the staining protocols with which they are most comfortable, adjunctive use of TB over time may provide an additional benefit at low risk for disrupting practice efficiency or workflow. Many Mohs surgeons are accustomed to using this stain, even preferring to interpret only TB-stained slides for cutaneous malignancy. Most published studies on this topic have been observational in nature, and additional controlled trials may be warranted to determine the effects on outcomes in real-world practice.

References
  1. Culling CF, Allison TR. Cellular Pathology Technique. 4th ed. Butterworths; 1985.
  2. Bergeron JA, Singer M. Metachromasy: an experimental and theoretical reevaluation. J Biophys Biochem Cytol. 1958;4:433-457. doi:10.1083/jcb.4.4.433
  3. Epstein JB, Scully C, Spinelli J. Toluidine blue and Lugol’s iodine application in the assessment of oral malignant disease and lesions at risk of malignancy. J Oral Pathol Med. 1992;21:160-163. doi:10.1111/j.1600-0714.1992.tb00094.x
  4. Warnakulasuriya KA, Johnson NW. Sensitivity and specificity of OraScan (R) toluidine blue mouthrinse in the detection of oral cancer and precancer. J Oral Pathol Med. 1996;25:97-103. doi:10.1111/j.1600-0714.1996.tb00201.x
  5. Silapunt S, Peterson SR, Alcalay J, et al. Mohs tissue mapping and processing: a survey study. Dermatol Surg. 2003;29:1109-1112; discussion 1112.
  6. Humphreys TR, Nemeth A, McCrevey S, et al. A pilot study comparing toluidine blue and hematoxylin and eosin staining of basal cell and squamous cell carcinoma during Mohs surgery. Dermatol Surg. 1996;22:693-697. doi:10.1111/j.1524-4725.1996.tb00619.x
  7. Bennett R, Taher M. Restylane persistent for 23 months found during Mohs micrographic surgery: a source of confusion with hyaluronic acid surrounding basal cell carcinoma. Dermatol Surg. 2005;31:1366-1369. doi:10.1111/j.1524-4725.2005.31223
  8. Goldberg LH, Wang SQ, Kimyai-Asadi A. The setting sun sign: visualizing the margins of a basal cell carcinoma on serial frozen sections stained with toluidine blue. Dermatol Surg. 2007;33:761-763. doi:10.1111/j.1524-4725.2007.33158.x
  9. Tehrani H, May K, Morris A, et al. Does the dual use of toluidine blue and hematoxylin and eosin staining improve basal cell carcinoma detection by Mohs surgery trainees? Dermatol Surg. 2013;39:995-1000. doi:10.1111/dsu.12180
  10. Donaldson MR, Weber LA. Toluidine blue supports differentiation of folliculocentric basaloid proliferation from basal cell carcinoma on frozen sections in a small single-practice cohort. Dermatol Surg. 2017;43:1303-1306. doi:10.1097/DSS.0000000000001107
  11. Styperek AR, Goldberg LH, Goldschmidt LE, et al. Toluidine blue and hematoxylin and eosin stains are comparable in evaluating squamous cell carcinoma during Mohs. Dermatol Surg. 2016;42:1279-1284. doi:10.1097/DSS.0000000000000872
  12. Trieu D, Drosou A, Goldberg LH, et al. Detecting spindle cell squamous cell carcinomas with toluidine blue on frozen sections. Dermatol Surg. 2014;40:1259-1260. doi:10.1097/DSS.0000000000000147
  13. Drosou A, Trieu D, Goldberg LH, et al. The perineural corona sign: enhancing detection of perineural squamous cell carcinoma during Mohs micrographic surgery with toluidine blue stain. J Am Acad Dermatol. 2014;71:826-827. doi:10.1016/j.jaad.2014.04.076
  14. Chesser RS, Bertler DE, Fitzpatrick JE, et al. Primary cutaneous adenoid cystic carcinoma treated with Mohs micrographic surgery toluidine blue technique. J Dermatol Surg Oncol. 1992;18:175-176. doi:10.1111/j.1524-4725.1992.tb02794.x
  15. 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;56:1067-1069. doi:10.1016/j.jaad.2007.01.008
  16. Chen CL, Wilson S, Afzalneia R, et al. Topical aluminum chloride and Monsel’s solution block toluidine blue staining in Mohs frozen sections: mechanism and solution. Dermatol Surg. 2019;45:1019-1025. doi:10.1097/DSS.0000000000001761
  17. Todd MM, Lee JW, Marks VJ. Rapid toluidine blue stain for Mohs’ micrographic surgery. Dermatol Surg. 2005;31:244-245. doi:10.1111/j.1524-4725.2005.31053
  18. Picoto AM, Picoto A. Technical procedures for Mohs fresh tissue surgery. J Derm Surg Oncol. 1986;12:134-138. doi:10.1111/j.1524-4725.1986.tb01442.x
  19. Sperling LC, Winton GB. The transverse anatomy of androgenic alopecia. J Derm Surg Oncol. 1990;16:1127-1133. doi:10.1111/j.1524 -4725.1990.tb00024.x
  20. Smith-Zagone MJ, Schwartz MR. Frozen section of skin specimens. Arch Pathol Lab Med. 2005;129:1536-1543. doi:10.5858/2005-129-1536-FSOSS
  21. Moy RL, Potter TS, Uitto J. Increased glycosaminoglycans production in sclerosing basal cell carcinoma–derived fibroblasts and stimulation of normal skin fibroblast glycosaminoglycans production by a cytokine-derived from sclerosing basal cell carcinoma. Dermatol Surg. 2000;26:1029-1036. doi:10.1046/j.1524-4725.2000.0260111029.x
  22. Leshin B, White WL. Folliculocentric basaloid proliferation. The bulge (der Wulst) revisited. Arch Dermatol. 1990;126:900-906. doi:10.1001/archderm.126.7.900
  23. Seab JA, Graham JH. Primary cutaneous adenoid cystic carcinoma.J Am Acad Dermatol. 1987;17:113-118. doi:10.1016/s0190 -9622(87)70182-0
References
  1. Culling CF, Allison TR. Cellular Pathology Technique. 4th ed. Butterworths; 1985.
  2. Bergeron JA, Singer M. Metachromasy: an experimental and theoretical reevaluation. J Biophys Biochem Cytol. 1958;4:433-457. doi:10.1083/jcb.4.4.433
  3. Epstein JB, Scully C, Spinelli J. Toluidine blue and Lugol’s iodine application in the assessment of oral malignant disease and lesions at risk of malignancy. J Oral Pathol Med. 1992;21:160-163. doi:10.1111/j.1600-0714.1992.tb00094.x
  4. Warnakulasuriya KA, Johnson NW. Sensitivity and specificity of OraScan (R) toluidine blue mouthrinse in the detection of oral cancer and precancer. J Oral Pathol Med. 1996;25:97-103. doi:10.1111/j.1600-0714.1996.tb00201.x
  5. Silapunt S, Peterson SR, Alcalay J, et al. Mohs tissue mapping and processing: a survey study. Dermatol Surg. 2003;29:1109-1112; discussion 1112.
  6. Humphreys TR, Nemeth A, McCrevey S, et al. A pilot study comparing toluidine blue and hematoxylin and eosin staining of basal cell and squamous cell carcinoma during Mohs surgery. Dermatol Surg. 1996;22:693-697. doi:10.1111/j.1524-4725.1996.tb00619.x
  7. Bennett R, Taher M. Restylane persistent for 23 months found during Mohs micrographic surgery: a source of confusion with hyaluronic acid surrounding basal cell carcinoma. Dermatol Surg. 2005;31:1366-1369. doi:10.1111/j.1524-4725.2005.31223
  8. Goldberg LH, Wang SQ, Kimyai-Asadi A. The setting sun sign: visualizing the margins of a basal cell carcinoma on serial frozen sections stained with toluidine blue. Dermatol Surg. 2007;33:761-763. doi:10.1111/j.1524-4725.2007.33158.x
  9. Tehrani H, May K, Morris A, et al. Does the dual use of toluidine blue and hematoxylin and eosin staining improve basal cell carcinoma detection by Mohs surgery trainees? Dermatol Surg. 2013;39:995-1000. doi:10.1111/dsu.12180
  10. Donaldson MR, Weber LA. Toluidine blue supports differentiation of folliculocentric basaloid proliferation from basal cell carcinoma on frozen sections in a small single-practice cohort. Dermatol Surg. 2017;43:1303-1306. doi:10.1097/DSS.0000000000001107
  11. Styperek AR, Goldberg LH, Goldschmidt LE, et al. Toluidine blue and hematoxylin and eosin stains are comparable in evaluating squamous cell carcinoma during Mohs. Dermatol Surg. 2016;42:1279-1284. doi:10.1097/DSS.0000000000000872
  12. Trieu D, Drosou A, Goldberg LH, et al. Detecting spindle cell squamous cell carcinomas with toluidine blue on frozen sections. Dermatol Surg. 2014;40:1259-1260. doi:10.1097/DSS.0000000000000147
  13. Drosou A, Trieu D, Goldberg LH, et al. The perineural corona sign: enhancing detection of perineural squamous cell carcinoma during Mohs micrographic surgery with toluidine blue stain. J Am Acad Dermatol. 2014;71:826-827. doi:10.1016/j.jaad.2014.04.076
  14. Chesser RS, Bertler DE, Fitzpatrick JE, et al. Primary cutaneous adenoid cystic carcinoma treated with Mohs micrographic surgery toluidine blue technique. J Dermatol Surg Oncol. 1992;18:175-176. doi:10.1111/j.1524-4725.1992.tb02794.x
  15. 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;56:1067-1069. doi:10.1016/j.jaad.2007.01.008
  16. Chen CL, Wilson S, Afzalneia R, et al. Topical aluminum chloride and Monsel’s solution block toluidine blue staining in Mohs frozen sections: mechanism and solution. Dermatol Surg. 2019;45:1019-1025. doi:10.1097/DSS.0000000000001761
  17. Todd MM, Lee JW, Marks VJ. Rapid toluidine blue stain for Mohs’ micrographic surgery. Dermatol Surg. 2005;31:244-245. doi:10.1111/j.1524-4725.2005.31053
  18. Picoto AM, Picoto A. Technical procedures for Mohs fresh tissue surgery. J Derm Surg Oncol. 1986;12:134-138. doi:10.1111/j.1524-4725.1986.tb01442.x
  19. Sperling LC, Winton GB. The transverse anatomy of androgenic alopecia. J Derm Surg Oncol. 1990;16:1127-1133. doi:10.1111/j.1524 -4725.1990.tb00024.x
  20. Smith-Zagone MJ, Schwartz MR. Frozen section of skin specimens. Arch Pathol Lab Med. 2005;129:1536-1543. doi:10.5858/2005-129-1536-FSOSS
  21. Moy RL, Potter TS, Uitto J. Increased glycosaminoglycans production in sclerosing basal cell carcinoma–derived fibroblasts and stimulation of normal skin fibroblast glycosaminoglycans production by a cytokine-derived from sclerosing basal cell carcinoma. Dermatol Surg. 2000;26:1029-1036. doi:10.1046/j.1524-4725.2000.0260111029.x
  22. Leshin B, White WL. Folliculocentric basaloid proliferation. The bulge (der Wulst) revisited. Arch Dermatol. 1990;126:900-906. doi:10.1001/archderm.126.7.900
  23. Seab JA, Graham JH. Primary cutaneous adenoid cystic carcinoma.J Am Acad Dermatol. 1987;17:113-118. doi:10.1016/s0190 -9622(87)70182-0
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The Role of Toluidine Blue in Mohs Micrographic Surgery: A Systematic Review
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Practice Points

  • Toluidine blue (TB) staining can be integrated into Mohs micrographic surgery (MMS) for enhanced diagnosis of cutaneous tumors. Its metachromatic properties can aid in differentiating tumor cells from surrounding tissues, especially in basal cell carcinomas and squamous cell carcinomas.
  • It is important to develop expertise in interpreting TB-stained sections, as it may offer clearer visualization of nuclear details and stromal components, potentially leading to more accurate diagnosis and effective tumor margin identification.
  • Toluidine blue staining can be incorporated into routine MMS practice considering its quick staining process and low disruption to workflow. This can potentially improve diagnostic efficiency without significantly lengthening surgery time.
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Nodules on the Anterior Neck Following Poly-L-lactic Acid Injection

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Nodules on the Anterior Neck Following Poly-L-lactic Acid Injection

Poly-L-lactic acid (PLLA) is a synthetic biologic polymer that is suspended in solution and can be injected for soft-tissue augmentation. The stimulatory molecule functions to increase collagen synthesis as a by-product of its degradation.1 Poly-L-lactic acid measures 40 to 63 μm and is irregularly shaped, which inhibits product mobility and allows for precise tissue augmentation.2 Clinical trials of injectable PLLA have proven its safety with no reported cases of infection, allergies, or serious adverse reactions.3-5 The most common patient concerns generally are transient in nature, such as swelling, tenderness, pain, bruising, and bleeding. Persistent adverse events of PLLA primarily are papule and nodule formation.6 Clinical trials showed a variable incidence of papule/nodule formation between 6% and 44%.2 Nodule formation remains a major challenge to achieving optimal results from injectable PLLA. We present a case in which a hyperdiluted formulation of PLLA produced a relatively acute (3-week) onset of multiple nodule formations dispersed on the anterior neck. The nodules were resistant to less-invasive treatment modalities and were further requested to be surgically excised.

Case Report

A 38-year-old woman presented for soft-tissue augmentation of the anterior neck using PLLA to achieve correction of skin laxity and static rhytides. She had a history of successful PLLA injections in the temples, knees, chest, and buttocks over a 5-year period. Forty-eight hours prior to injection, 1 PLLA vial was hydrated with 7 cc bacteriostatic water by using a continuous rotation suspension method over the 48 hours. On the day of injection, the PLLA was further hyperdiluted with 2 cc of 2% lidocaine and an additional 7 cc of bacteriostatic water, for a total of 16 cc diluent. The product was injected using a cannula in the anterior and lateral neck. According to the patient, 3 weeks after the procedure she noticed that some nodules began to form at the cannula insertion sites, while others formed distant from those sites; a total of 10 nodules had formed on the anterior neck (Figure 1).

Multiple subcutaneous nodules developed on the neck 3 weeks after poly-L-lactic acid injection.
FIGURE 1. Multiple subcutaneous nodules developed on the neck 3 weeks after poly-L-lactic acid injection.

The bacteriostatic water, lidocaine, and PLLA vial were all confirmed not to be expired. The manufacturer was contacted, and no other adverse reactions have been reported with this particular lot number of PLLA. The nodules initially were treated with injections of large boluses of bacteriostatic saline, which was ineffective. Treatment was then attempted using injections of a solution containing 1.0 mL of 5-fluorouracil (5-FU) 50 mg/mL, 0.4 mL of dexamethasone 4 mg/mL, 0.1 mL of triamcinolone 10 mg/mL, and 0.3 mL hyaluronidase. A series of 4 injections was performed in 2- to 4-week intervals. Two of the nodules resolved completely with this treatment. The remaining 8 nodules subjectively improved in size and softened to palpation but did not resolve completely. At 2 of the injection sites, treatment was complicated with steroid atrophy of the overlying skin. At the patient’s request, the remaining nodules were surgically excised (Figure 2). Histopathology revealed exogenous foreign material consistent with dermal filler (Figure 3).

Surgical excision of a nodule was performed, with the nodule tightly embedded in the underlying tissue.
FIGURE 2. Surgical excision of a nodule was performed, with the nodule tightly embedded in the underlying tissue.

Comment

Causes of Nodule Formation—Two factors that could contribute to nodule formation are inadequate dispersion of molecules and an insufficient volume of dilution. One study demonstrated that hydration for at least 24 hours is required for adequate PLLA dispersion. Furthermore, sonification for 5 minutes after a 2-hour hydration disperses molecules similarly to the 48-hour hydration.7 The PLLA in the current case was hydrated for 48 hours using a continuous rotation suspension method. Therefore, this likely did not play a role in our patient’s nodule formation. The volume of dilution has been shown to impact the incidence of nodule formation.8 At present, most injectors (60.4%) reconstitute each vial of PLLA with 9 to 10 mL of diluent.9 The PLLA in our patient was reconstituted with 16 mL; therefore, we believe that the anatomic location was the main contributor of nodule formation.

Histopathology revealed exogenous foreign material consistent with dermal filler (H&E, original magnification ×10).
FIGURE 3. Histopathology revealed exogenous foreign material consistent with dermal filler (H&E, original magnification ×10).

Fillers should be injected in the subcutaneous or deep dermal plane of tissue.10 The platysma is a superficial muscle that is intimately involved with the overlying skin of the anterior neck, and injections in this area could inadvertently be intramuscular. Intramuscular injections have a higher incidence of nodule formation.1 Our patient had prior PLLA injections without adverse reactions in numerous other sites, supporting the claim that the anterior neck is prone to nodule formation from PLLA injections.

Management of Noninflammatory Nodules—Initial treatment of nodules with injections of saline was ineffective. This treatment can be used in an attempt to disperse the product. Treatment was then attempted with injections of a solution containing 5-FU, dexamethasone, triamcinolone, and hyaluronidase. Combination steroid therapy may be superior to monotherapy.11 Dexamethasone may exhibit a cytoprotective effect on cells such as fibroblasts when used in combination with triamcinolone; monotherapy steroid use with triamcinolone alone induced fibroblast apoptosis at a much higher level.12 Hyaluronidase works by breaking cross-links in hyaluronic acid, a glycosaminoglycan polysaccharide prevalent in the skin and connective tissue, which increases tissue permeability and aids in delivery of the other injected fluids.13 5-Fluorouracil is an antimetabolite that may aid in treating nodules by discouraging additional fibroblast activity and fibrosis.14

The combination of 5-FU, dexamethasone, and triamcinolone has been shown to be successful in treating noninflammatory nodules in as few as 1 treatment.14 In our patient, hyaluronidase also was used in an attempt to aid delivery of the other injected fluids. If nodules do not resolve with 1 injection, it is recommended to wait at least 8 weeks before repeating the injection to prevent steroid atrophy of the overlying skin. In our patient, the intramuscular placement of the filler contributed to the nodules being resistant to this treatment. During excision, the nodules were tightly embedded in the underlying tissue, which may have prevented the solution from being delivered to the nodule (Figure 2).

Conclusion

Injectable PLLA is approved by the US Food and Drug Administration for soft-tissue augmentation of deep nasolabial folds and facial wrinkles. Off-label use of this product may cause higher incidence of nodule formation. Injectors should be cautious of injecting into the anterior neck. If nodules do form, treatment can be attempted with injections of saline. If that treatment fails, another treatment option is injection(s) of a mixture of 5-FU, dexamethasone, triamcinolone, and hyaluronidase separated by 8-week intervals. Finally, surgical excision is a viable treatment option, as presented in our case.

References
  1. Bartus C, William HC, Daro-Kaftan E. A decade of experience with injectable poly-L-lactic acid: a focus on safety. Dermatol Surg. 2013;39:698-705.
  2. Engelhard P, Humble G, Mest D. Safety of Sculptra: a review of clinical trial data. J Cosmet Laser Ther. 2005;7:201-205.
  3. Mest DR, Humble G. Safety and efficacy of poly-L-lactic acid injections in persons with HIV-associated lipoatrophy: the US experience. Dermatol Surg. 2006;32:1336-1345.
  4. Burgess CM, Quiroga RM. Assessment of the safety and efficacy of poly-L-lactic acid for the treatment of HIV associated facial lipoatrophy. J Am Acad Dermatol. 2005;52:233-239.
  5. Cattelan AM, Bauer U, Trevenzoli M, et al. Use of polylactic acid implants to correct facial lipoatrophy in human immunodeficiency virus 1-positive individuals receiving combination antiretroviral therapy. Arch Dermatol. 2006;142:329-334.
  6. Sculptra. Package insert. sanofi-aventis U.S. LLC; 2009.
  7. Li CN, Wang CC, Huang CC, et al. A novel, optimized method to accelerate the preparation of injectable poly-L-lactic acid by sonication. J Drugs Dermatol. 2018;17:894-898.
  8. Rossner F, Rossner M, Hartmann V, et al. Decrease of reported adverse events to injectable polylactic acid after recommending an increased dilution: 8-year results from the Injectable Filler Safety study. J Cosmet Dermatol. 2009;8:14-18.
  9. Lin MJ, Dubin DP, Goldberg DJ, et al. Practices in the usage and reconstitution of poly-L-lactic acid. J Drugs Dermatol. 2019;18:880-886.
  10. Sieber DA, Scheuer JF 3rd, Villanueva NL, et al. Review of 3-dimensional facial anatomy: injecting fillers and neuromodulators. Plast Reconstr Surg Glob Open. 2016;4(12 suppl Anatomy and Safety in Cosmetic Medicine: Cosmetic Bootcamp):E1166.
  11. Syed F, Singh S, Bayat A. Superior effect of combination vs. single steroid therapy in keloid disease: a comparative in vitro analysis of glucocorticoids. Wound Repair Regen. 2013;21:88-102.
  12. Brody HJ. Use of hyaluronidase in the treatment of granulomatous hyaluronic acid reactions or unwanted hyaluronic acid misplacement. Dermatol Surg. 2005;31:893-897.
  13. Funt D, Pavicic T. Dermal fillers in aesthetics: an overview of adverse events and treatment approaches. Clin Cosm Investig Dermatol. 2013;6:295-316.
  14. Aguilera SB, Aristizabal M, Reed A. Successful treatment of calcium hydroxylapatite nodules with intralesional 5-fluorouracil, dexamethasone, and triamcinolone. J Drugs Dermatol. 2016;15:1142-1143.
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Author and Disclosure Information

Drs. Dunn and Long are from Lake Erie College of Osteopathic Medicine, Bradenton, Florida. Dr. Zarraga is from Z-ROC Dermatology, Fort Lauderdale, Florida. Dr. Aguilera is from Shino Bay Cosmetic Dermatology & Laser Institute, Fort Lauderdale.

Drs. Dunn and Long report no conflict of interest. Dr. Zarraga is a paid speaker for Galderma Laboratories and a consultant for Bioderma and Senté. Dr. Aguilera has received honoraria for speaking engagements and training from Allergan, Cynosure, Galderma Laboratories, SkinCeuticals, Solta Medical, and Valeant Pharmaceuticals, and has served as a member of speaker bureaus for Allergan, Cynosure, Galderma Laboratories, Merz, SkinCeuticals, Solta Medical, and Valeant Pharmaceuticals.

Correspondence: Austin Dunn, DO, Lake Erie College of Osteopathic Medicine, 5000 Lakewood Ranch Blvd, Bradenton, FL 34211 (adunn23317@med.lecom.edu).

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Drs. Dunn and Long are from Lake Erie College of Osteopathic Medicine, Bradenton, Florida. Dr. Zarraga is from Z-ROC Dermatology, Fort Lauderdale, Florida. Dr. Aguilera is from Shino Bay Cosmetic Dermatology & Laser Institute, Fort Lauderdale.

Drs. Dunn and Long report no conflict of interest. Dr. Zarraga is a paid speaker for Galderma Laboratories and a consultant for Bioderma and Senté. Dr. Aguilera has received honoraria for speaking engagements and training from Allergan, Cynosure, Galderma Laboratories, SkinCeuticals, Solta Medical, and Valeant Pharmaceuticals, and has served as a member of speaker bureaus for Allergan, Cynosure, Galderma Laboratories, Merz, SkinCeuticals, Solta Medical, and Valeant Pharmaceuticals.

Correspondence: Austin Dunn, DO, Lake Erie College of Osteopathic Medicine, 5000 Lakewood Ranch Blvd, Bradenton, FL 34211 (adunn23317@med.lecom.edu).

Author and Disclosure Information

Drs. Dunn and Long are from Lake Erie College of Osteopathic Medicine, Bradenton, Florida. Dr. Zarraga is from Z-ROC Dermatology, Fort Lauderdale, Florida. Dr. Aguilera is from Shino Bay Cosmetic Dermatology & Laser Institute, Fort Lauderdale.

Drs. Dunn and Long report no conflict of interest. Dr. Zarraga is a paid speaker for Galderma Laboratories and a consultant for Bioderma and Senté. Dr. Aguilera has received honoraria for speaking engagements and training from Allergan, Cynosure, Galderma Laboratories, SkinCeuticals, Solta Medical, and Valeant Pharmaceuticals, and has served as a member of speaker bureaus for Allergan, Cynosure, Galderma Laboratories, Merz, SkinCeuticals, Solta Medical, and Valeant Pharmaceuticals.

Correspondence: Austin Dunn, DO, Lake Erie College of Osteopathic Medicine, 5000 Lakewood Ranch Blvd, Bradenton, FL 34211 (adunn23317@med.lecom.edu).

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Poly-L-lactic acid (PLLA) is a synthetic biologic polymer that is suspended in solution and can be injected for soft-tissue augmentation. The stimulatory molecule functions to increase collagen synthesis as a by-product of its degradation.1 Poly-L-lactic acid measures 40 to 63 μm and is irregularly shaped, which inhibits product mobility and allows for precise tissue augmentation.2 Clinical trials of injectable PLLA have proven its safety with no reported cases of infection, allergies, or serious adverse reactions.3-5 The most common patient concerns generally are transient in nature, such as swelling, tenderness, pain, bruising, and bleeding. Persistent adverse events of PLLA primarily are papule and nodule formation.6 Clinical trials showed a variable incidence of papule/nodule formation between 6% and 44%.2 Nodule formation remains a major challenge to achieving optimal results from injectable PLLA. We present a case in which a hyperdiluted formulation of PLLA produced a relatively acute (3-week) onset of multiple nodule formations dispersed on the anterior neck. The nodules were resistant to less-invasive treatment modalities and were further requested to be surgically excised.

Case Report

A 38-year-old woman presented for soft-tissue augmentation of the anterior neck using PLLA to achieve correction of skin laxity and static rhytides. She had a history of successful PLLA injections in the temples, knees, chest, and buttocks over a 5-year period. Forty-eight hours prior to injection, 1 PLLA vial was hydrated with 7 cc bacteriostatic water by using a continuous rotation suspension method over the 48 hours. On the day of injection, the PLLA was further hyperdiluted with 2 cc of 2% lidocaine and an additional 7 cc of bacteriostatic water, for a total of 16 cc diluent. The product was injected using a cannula in the anterior and lateral neck. According to the patient, 3 weeks after the procedure she noticed that some nodules began to form at the cannula insertion sites, while others formed distant from those sites; a total of 10 nodules had formed on the anterior neck (Figure 1).

Multiple subcutaneous nodules developed on the neck 3 weeks after poly-L-lactic acid injection.
FIGURE 1. Multiple subcutaneous nodules developed on the neck 3 weeks after poly-L-lactic acid injection.

The bacteriostatic water, lidocaine, and PLLA vial were all confirmed not to be expired. The manufacturer was contacted, and no other adverse reactions have been reported with this particular lot number of PLLA. The nodules initially were treated with injections of large boluses of bacteriostatic saline, which was ineffective. Treatment was then attempted using injections of a solution containing 1.0 mL of 5-fluorouracil (5-FU) 50 mg/mL, 0.4 mL of dexamethasone 4 mg/mL, 0.1 mL of triamcinolone 10 mg/mL, and 0.3 mL hyaluronidase. A series of 4 injections was performed in 2- to 4-week intervals. Two of the nodules resolved completely with this treatment. The remaining 8 nodules subjectively improved in size and softened to palpation but did not resolve completely. At 2 of the injection sites, treatment was complicated with steroid atrophy of the overlying skin. At the patient’s request, the remaining nodules were surgically excised (Figure 2). Histopathology revealed exogenous foreign material consistent with dermal filler (Figure 3).

Surgical excision of a nodule was performed, with the nodule tightly embedded in the underlying tissue.
FIGURE 2. Surgical excision of a nodule was performed, with the nodule tightly embedded in the underlying tissue.

Comment

Causes of Nodule Formation—Two factors that could contribute to nodule formation are inadequate dispersion of molecules and an insufficient volume of dilution. One study demonstrated that hydration for at least 24 hours is required for adequate PLLA dispersion. Furthermore, sonification for 5 minutes after a 2-hour hydration disperses molecules similarly to the 48-hour hydration.7 The PLLA in the current case was hydrated for 48 hours using a continuous rotation suspension method. Therefore, this likely did not play a role in our patient’s nodule formation. The volume of dilution has been shown to impact the incidence of nodule formation.8 At present, most injectors (60.4%) reconstitute each vial of PLLA with 9 to 10 mL of diluent.9 The PLLA in our patient was reconstituted with 16 mL; therefore, we believe that the anatomic location was the main contributor of nodule formation.

Histopathology revealed exogenous foreign material consistent with dermal filler (H&E, original magnification ×10).
FIGURE 3. Histopathology revealed exogenous foreign material consistent with dermal filler (H&E, original magnification ×10).

Fillers should be injected in the subcutaneous or deep dermal plane of tissue.10 The platysma is a superficial muscle that is intimately involved with the overlying skin of the anterior neck, and injections in this area could inadvertently be intramuscular. Intramuscular injections have a higher incidence of nodule formation.1 Our patient had prior PLLA injections without adverse reactions in numerous other sites, supporting the claim that the anterior neck is prone to nodule formation from PLLA injections.

Management of Noninflammatory Nodules—Initial treatment of nodules with injections of saline was ineffective. This treatment can be used in an attempt to disperse the product. Treatment was then attempted with injections of a solution containing 5-FU, dexamethasone, triamcinolone, and hyaluronidase. Combination steroid therapy may be superior to monotherapy.11 Dexamethasone may exhibit a cytoprotective effect on cells such as fibroblasts when used in combination with triamcinolone; monotherapy steroid use with triamcinolone alone induced fibroblast apoptosis at a much higher level.12 Hyaluronidase works by breaking cross-links in hyaluronic acid, a glycosaminoglycan polysaccharide prevalent in the skin and connective tissue, which increases tissue permeability and aids in delivery of the other injected fluids.13 5-Fluorouracil is an antimetabolite that may aid in treating nodules by discouraging additional fibroblast activity and fibrosis.14

The combination of 5-FU, dexamethasone, and triamcinolone has been shown to be successful in treating noninflammatory nodules in as few as 1 treatment.14 In our patient, hyaluronidase also was used in an attempt to aid delivery of the other injected fluids. If nodules do not resolve with 1 injection, it is recommended to wait at least 8 weeks before repeating the injection to prevent steroid atrophy of the overlying skin. In our patient, the intramuscular placement of the filler contributed to the nodules being resistant to this treatment. During excision, the nodules were tightly embedded in the underlying tissue, which may have prevented the solution from being delivered to the nodule (Figure 2).

Conclusion

Injectable PLLA is approved by the US Food and Drug Administration for soft-tissue augmentation of deep nasolabial folds and facial wrinkles. Off-label use of this product may cause higher incidence of nodule formation. Injectors should be cautious of injecting into the anterior neck. If nodules do form, treatment can be attempted with injections of saline. If that treatment fails, another treatment option is injection(s) of a mixture of 5-FU, dexamethasone, triamcinolone, and hyaluronidase separated by 8-week intervals. Finally, surgical excision is a viable treatment option, as presented in our case.

Poly-L-lactic acid (PLLA) is a synthetic biologic polymer that is suspended in solution and can be injected for soft-tissue augmentation. The stimulatory molecule functions to increase collagen synthesis as a by-product of its degradation.1 Poly-L-lactic acid measures 40 to 63 μm and is irregularly shaped, which inhibits product mobility and allows for precise tissue augmentation.2 Clinical trials of injectable PLLA have proven its safety with no reported cases of infection, allergies, or serious adverse reactions.3-5 The most common patient concerns generally are transient in nature, such as swelling, tenderness, pain, bruising, and bleeding. Persistent adverse events of PLLA primarily are papule and nodule formation.6 Clinical trials showed a variable incidence of papule/nodule formation between 6% and 44%.2 Nodule formation remains a major challenge to achieving optimal results from injectable PLLA. We present a case in which a hyperdiluted formulation of PLLA produced a relatively acute (3-week) onset of multiple nodule formations dispersed on the anterior neck. The nodules were resistant to less-invasive treatment modalities and were further requested to be surgically excised.

Case Report

A 38-year-old woman presented for soft-tissue augmentation of the anterior neck using PLLA to achieve correction of skin laxity and static rhytides. She had a history of successful PLLA injections in the temples, knees, chest, and buttocks over a 5-year period. Forty-eight hours prior to injection, 1 PLLA vial was hydrated with 7 cc bacteriostatic water by using a continuous rotation suspension method over the 48 hours. On the day of injection, the PLLA was further hyperdiluted with 2 cc of 2% lidocaine and an additional 7 cc of bacteriostatic water, for a total of 16 cc diluent. The product was injected using a cannula in the anterior and lateral neck. According to the patient, 3 weeks after the procedure she noticed that some nodules began to form at the cannula insertion sites, while others formed distant from those sites; a total of 10 nodules had formed on the anterior neck (Figure 1).

Multiple subcutaneous nodules developed on the neck 3 weeks after poly-L-lactic acid injection.
FIGURE 1. Multiple subcutaneous nodules developed on the neck 3 weeks after poly-L-lactic acid injection.

The bacteriostatic water, lidocaine, and PLLA vial were all confirmed not to be expired. The manufacturer was contacted, and no other adverse reactions have been reported with this particular lot number of PLLA. The nodules initially were treated with injections of large boluses of bacteriostatic saline, which was ineffective. Treatment was then attempted using injections of a solution containing 1.0 mL of 5-fluorouracil (5-FU) 50 mg/mL, 0.4 mL of dexamethasone 4 mg/mL, 0.1 mL of triamcinolone 10 mg/mL, and 0.3 mL hyaluronidase. A series of 4 injections was performed in 2- to 4-week intervals. Two of the nodules resolved completely with this treatment. The remaining 8 nodules subjectively improved in size and softened to palpation but did not resolve completely. At 2 of the injection sites, treatment was complicated with steroid atrophy of the overlying skin. At the patient’s request, the remaining nodules were surgically excised (Figure 2). Histopathology revealed exogenous foreign material consistent with dermal filler (Figure 3).

Surgical excision of a nodule was performed, with the nodule tightly embedded in the underlying tissue.
FIGURE 2. Surgical excision of a nodule was performed, with the nodule tightly embedded in the underlying tissue.

Comment

Causes of Nodule Formation—Two factors that could contribute to nodule formation are inadequate dispersion of molecules and an insufficient volume of dilution. One study demonstrated that hydration for at least 24 hours is required for adequate PLLA dispersion. Furthermore, sonification for 5 minutes after a 2-hour hydration disperses molecules similarly to the 48-hour hydration.7 The PLLA in the current case was hydrated for 48 hours using a continuous rotation suspension method. Therefore, this likely did not play a role in our patient’s nodule formation. The volume of dilution has been shown to impact the incidence of nodule formation.8 At present, most injectors (60.4%) reconstitute each vial of PLLA with 9 to 10 mL of diluent.9 The PLLA in our patient was reconstituted with 16 mL; therefore, we believe that the anatomic location was the main contributor of nodule formation.

Histopathology revealed exogenous foreign material consistent with dermal filler (H&E, original magnification ×10).
FIGURE 3. Histopathology revealed exogenous foreign material consistent with dermal filler (H&E, original magnification ×10).

Fillers should be injected in the subcutaneous or deep dermal plane of tissue.10 The platysma is a superficial muscle that is intimately involved with the overlying skin of the anterior neck, and injections in this area could inadvertently be intramuscular. Intramuscular injections have a higher incidence of nodule formation.1 Our patient had prior PLLA injections without adverse reactions in numerous other sites, supporting the claim that the anterior neck is prone to nodule formation from PLLA injections.

Management of Noninflammatory Nodules—Initial treatment of nodules with injections of saline was ineffective. This treatment can be used in an attempt to disperse the product. Treatment was then attempted with injections of a solution containing 5-FU, dexamethasone, triamcinolone, and hyaluronidase. Combination steroid therapy may be superior to monotherapy.11 Dexamethasone may exhibit a cytoprotective effect on cells such as fibroblasts when used in combination with triamcinolone; monotherapy steroid use with triamcinolone alone induced fibroblast apoptosis at a much higher level.12 Hyaluronidase works by breaking cross-links in hyaluronic acid, a glycosaminoglycan polysaccharide prevalent in the skin and connective tissue, which increases tissue permeability and aids in delivery of the other injected fluids.13 5-Fluorouracil is an antimetabolite that may aid in treating nodules by discouraging additional fibroblast activity and fibrosis.14

The combination of 5-FU, dexamethasone, and triamcinolone has been shown to be successful in treating noninflammatory nodules in as few as 1 treatment.14 In our patient, hyaluronidase also was used in an attempt to aid delivery of the other injected fluids. If nodules do not resolve with 1 injection, it is recommended to wait at least 8 weeks before repeating the injection to prevent steroid atrophy of the overlying skin. In our patient, the intramuscular placement of the filler contributed to the nodules being resistant to this treatment. During excision, the nodules were tightly embedded in the underlying tissue, which may have prevented the solution from being delivered to the nodule (Figure 2).

Conclusion

Injectable PLLA is approved by the US Food and Drug Administration for soft-tissue augmentation of deep nasolabial folds and facial wrinkles. Off-label use of this product may cause higher incidence of nodule formation. Injectors should be cautious of injecting into the anterior neck. If nodules do form, treatment can be attempted with injections of saline. If that treatment fails, another treatment option is injection(s) of a mixture of 5-FU, dexamethasone, triamcinolone, and hyaluronidase separated by 8-week intervals. Finally, surgical excision is a viable treatment option, as presented in our case.

References
  1. Bartus C, William HC, Daro-Kaftan E. A decade of experience with injectable poly-L-lactic acid: a focus on safety. Dermatol Surg. 2013;39:698-705.
  2. Engelhard P, Humble G, Mest D. Safety of Sculptra: a review of clinical trial data. J Cosmet Laser Ther. 2005;7:201-205.
  3. Mest DR, Humble G. Safety and efficacy of poly-L-lactic acid injections in persons with HIV-associated lipoatrophy: the US experience. Dermatol Surg. 2006;32:1336-1345.
  4. Burgess CM, Quiroga RM. Assessment of the safety and efficacy of poly-L-lactic acid for the treatment of HIV associated facial lipoatrophy. J Am Acad Dermatol. 2005;52:233-239.
  5. Cattelan AM, Bauer U, Trevenzoli M, et al. Use of polylactic acid implants to correct facial lipoatrophy in human immunodeficiency virus 1-positive individuals receiving combination antiretroviral therapy. Arch Dermatol. 2006;142:329-334.
  6. Sculptra. Package insert. sanofi-aventis U.S. LLC; 2009.
  7. Li CN, Wang CC, Huang CC, et al. A novel, optimized method to accelerate the preparation of injectable poly-L-lactic acid by sonication. J Drugs Dermatol. 2018;17:894-898.
  8. Rossner F, Rossner M, Hartmann V, et al. Decrease of reported adverse events to injectable polylactic acid after recommending an increased dilution: 8-year results from the Injectable Filler Safety study. J Cosmet Dermatol. 2009;8:14-18.
  9. Lin MJ, Dubin DP, Goldberg DJ, et al. Practices in the usage and reconstitution of poly-L-lactic acid. J Drugs Dermatol. 2019;18:880-886.
  10. Sieber DA, Scheuer JF 3rd, Villanueva NL, et al. Review of 3-dimensional facial anatomy: injecting fillers and neuromodulators. Plast Reconstr Surg Glob Open. 2016;4(12 suppl Anatomy and Safety in Cosmetic Medicine: Cosmetic Bootcamp):E1166.
  11. Syed F, Singh S, Bayat A. Superior effect of combination vs. single steroid therapy in keloid disease: a comparative in vitro analysis of glucocorticoids. Wound Repair Regen. 2013;21:88-102.
  12. Brody HJ. Use of hyaluronidase in the treatment of granulomatous hyaluronic acid reactions or unwanted hyaluronic acid misplacement. Dermatol Surg. 2005;31:893-897.
  13. Funt D, Pavicic T. Dermal fillers in aesthetics: an overview of adverse events and treatment approaches. Clin Cosm Investig Dermatol. 2013;6:295-316.
  14. Aguilera SB, Aristizabal M, Reed A. Successful treatment of calcium hydroxylapatite nodules with intralesional 5-fluorouracil, dexamethasone, and triamcinolone. J Drugs Dermatol. 2016;15:1142-1143.
References
  1. Bartus C, William HC, Daro-Kaftan E. A decade of experience with injectable poly-L-lactic acid: a focus on safety. Dermatol Surg. 2013;39:698-705.
  2. Engelhard P, Humble G, Mest D. Safety of Sculptra: a review of clinical trial data. J Cosmet Laser Ther. 2005;7:201-205.
  3. Mest DR, Humble G. Safety and efficacy of poly-L-lactic acid injections in persons with HIV-associated lipoatrophy: the US experience. Dermatol Surg. 2006;32:1336-1345.
  4. Burgess CM, Quiroga RM. Assessment of the safety and efficacy of poly-L-lactic acid for the treatment of HIV associated facial lipoatrophy. J Am Acad Dermatol. 2005;52:233-239.
  5. Cattelan AM, Bauer U, Trevenzoli M, et al. Use of polylactic acid implants to correct facial lipoatrophy in human immunodeficiency virus 1-positive individuals receiving combination antiretroviral therapy. Arch Dermatol. 2006;142:329-334.
  6. Sculptra. Package insert. sanofi-aventis U.S. LLC; 2009.
  7. Li CN, Wang CC, Huang CC, et al. A novel, optimized method to accelerate the preparation of injectable poly-L-lactic acid by sonication. J Drugs Dermatol. 2018;17:894-898.
  8. Rossner F, Rossner M, Hartmann V, et al. Decrease of reported adverse events to injectable polylactic acid after recommending an increased dilution: 8-year results from the Injectable Filler Safety study. J Cosmet Dermatol. 2009;8:14-18.
  9. Lin MJ, Dubin DP, Goldberg DJ, et al. Practices in the usage and reconstitution of poly-L-lactic acid. J Drugs Dermatol. 2019;18:880-886.
  10. Sieber DA, Scheuer JF 3rd, Villanueva NL, et al. Review of 3-dimensional facial anatomy: injecting fillers and neuromodulators. Plast Reconstr Surg Glob Open. 2016;4(12 suppl Anatomy and Safety in Cosmetic Medicine: Cosmetic Bootcamp):E1166.
  11. Syed F, Singh S, Bayat A. Superior effect of combination vs. single steroid therapy in keloid disease: a comparative in vitro analysis of glucocorticoids. Wound Repair Regen. 2013;21:88-102.
  12. Brody HJ. Use of hyaluronidase in the treatment of granulomatous hyaluronic acid reactions or unwanted hyaluronic acid misplacement. Dermatol Surg. 2005;31:893-897.
  13. Funt D, Pavicic T. Dermal fillers in aesthetics: an overview of adverse events and treatment approaches. Clin Cosm Investig Dermatol. 2013;6:295-316.
  14. Aguilera SB, Aristizabal M, Reed A. Successful treatment of calcium hydroxylapatite nodules with intralesional 5-fluorouracil, dexamethasone, and triamcinolone. J Drugs Dermatol. 2016;15:1142-1143.
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Nodules on the Anterior Neck Following Poly-L-lactic Acid Injection
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Practice Points

  • Injecting poly-L-lactic acid (PLLA) into the anterior neck is an off-label procedure and may cause a higher incidence of nodule formation.
  • Most nodules from PLLA can be treated with injections of 5-fluorouracil, dexamethasone, triamcinolone, and hyaluronidase separated by 8-week intervals.
  • Treatment-resistant nodules may require surgical excision.
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