LETTERS
Ex Vivo Blister Induction: A More Sensitive Technique for Immunofluorescence Mapping Studies in Inherited Epidermolysis Bullosa To the editor: Immunofluorescence antigen mapping (IFM) is recommended as the primary laboratory approach for diagnosis in patients suspicious of inherited epidermolysis bullosa (EB).1 In this method, structural proteins in the epidermis or dermoepidermal junction are visualized by using specific monoclonal or polyclonal antibodies. The level of split formation and expression levels of structural proteins (normal, reduced, or absent) can be detected.2 The major types of EB (simplex, junctional, and dystrophic) are determined based on the level of skin cleavage. The presence of reduced or absent staining of specific proteins can then distinguish the various subtypes of EB. In patients with milder forms of the disease, such as EB simplex, dominant dystrophic EB, and limited forms of Junctional EB (non-Herlitz type or Laryngo-onychocutaneous syndrome), nonfunctional structural proteins may be expressed normally. Therefore, in these types of EB, the diagnosis would not be achieved unless the specimen contains a blister, which helps to make the diagnosis according to the cleavage plane2 (Figs. 1A, B). The most critical step of IFM is to take a skin biopsy correctly.3 The biopsy should be taken from freshly induced blisters. The biopsy should not be taken from the existing blisters because reepithelialization under the roof of the blister results in multiple cleavage planes, and serum and inflammatory Supported by the Skin Research Center, Shahid Beheshti University of Medical Sciences. The authors declare no conflicts of interest.
TO THE
EDITOR
cells can degrade protein antigens and give false positive results.3,4 For inducing a blister, we have developed a new method by which we can have blistering in all specimens taken from patients with EB of any level of severity, leading to the diagnosis in all cases and reducing the need for rebiopsy. We reasoned that if a consistent amount of negative pressure was applied to the skin biopsy once it was taken, the EB skin should blister before the normal skin and the cleavage would occur at a specific level expected for each type. In this method, a 4-mm punch biopsy is taken from an area of nonblistered skin adjacent to the site where the patient usually develops blisters. The plastic tip of a 20 mL syringe is placed on the epidermal side of the specimen and its pistol is drawn back, causing negative pressure on the epidermis (Fig. 2A) When the area of the specimen is smaller than the area of the syringe tip (for example, a 3-mm punch biopsy), suction can be done while a 22-gauge needle is attached to the tip (Fig. 2B). If not, the whole specimen will be pulled into the syringe. The syringe is then withdrawn to apply suction, and the process is continued until a macroscopic blister appears (Fig. 2C), which typically takes 1–5 minutes. It is, of note, that in patients with milder forms of EB, the time needed for inducing the blister may take more than 2 hours. In the traditional method of taking biopsies for IFM, a clinically normal area of the patient’s skin is rubbed with a vertically oriented pencil eraser in a torsional fashion at least 20 times or until the area turns red.4 Then, a 3-mm punch biopsy is taken from the rubbed area.4 In cases with less severe form of EB such as EB simplex or dominant dystrophic EB, prolonged (up to 3 hours) torsion with a pencil eraser is required for inducing a new blister. Unfortunately younger and scared children could not tolerate the long time needed for this procedure to induce blistering. If a young child is being sedated for a skin biopsy for EB, it is not safe to wait long for blistering to develop, and rubbing is not often performed before the sedation and biopsy although it should be ideally. Moreover, unwanted large areas peel off, more than required for a 4 mm
Am J Dermatopathol Volume 36, Number 12, December 2014
punch biopsy may occur occasionally, and if the biopsy is taken from a rubbed area that is red in hope of microscopic blistering, immunofluorescence staining may reveal that no blister has been induced in the specimen. For example, in a study by Yiasemides et al,5 9% of the biopsies (3 of 33), which were taken according to the aforementioned protocol for IFM or transmission electron microscopy, were inadequate for analysis because there were no blisters in the specimens. In a similar study from Stanford, 26% of the samples (20 of 77) submitted for IFM or transmission electron microscopy were suboptimal because there were no blisters in 13 specimens, the epidermis was missing in 4, and re-epithelialization or artifactual changes occurred in 3 specimens. Only 1 of the 13 specimens without the blister was informative in which the diagnosis of junctional EB was made based on markedly reduced staining for laminin 332.6 Since the establishment of the Iranian EB registry, which is based at our center, IFM has been performed for 90 patients (aged 5 days to 45 years). A panel of primary monoclonal antibodies directed against cytokeratin 5 (MAB 3224), cytokeratin 14 (MAB 3232), a6 integrin (MAB1378), b4 integrin (MAB 1964), laminin 332 (MAB 1949), collagen IV (MAB 3326), and collagen VII (MAB 1345) was used. Fluorescein-conjugated goat anti-mouse IgG (AP124F) was used as the secondary antibody for detecting the protein-bound primary antibody. All of the above mentioned antibodies were purchased from Millipore Company (Billerica, MA). For the first 43 cases, biopsies were done according to the traditional method. In 7 of the 43 specimens, the cleavage plane was not seen, and consequently, a diagnosis was not made in 4 cases. The IFM results of 39 diagnosed cases are presented in Table 1. When the new method was applied for the rest of the 47 cases, we had blister in all our specimens except for 1 case in whom the biopsy was taken from plantar hyperkeratosis; therefore, 46 cases were diagnosed (Table 1). Fisher exact test was used for comparing the efficacy of these 2 methods. In the new method, the rate of blister www.amjdermatopathology.com |
1005
Am J Dermatopathol Volume 36, Number 12, December 2014
Letters to the Editor
FIGURE 1. A, Immunofluorescence staining for collagen VII in the skin specimen of a patient affected by dystrophic EB, the normal expression of collagen VII is evident. B, The same patient sample stained for collagen IV with dilution of 1/50 revealed sublamina densa blistering. If the specimen did not contain blister, the diagnosis would not be made.
FIGURE 2. Suctioning the specimen with a plastic tip (A) or needle (B) of syringe until a macroscopic blister appears (C).
induction was statistically higher than the traditional method (98% vs. 84%, P = 0.025). Consequently, the rate of inconclusive results was lower (2% vs. 9%), although the difference was not statistically significant (P = 0.19). When the new method was applied on the normal control skin specimen, the cleavage occurred in the dermis (Fig. 3).
The lack of genetic mutation analysis, as a diagnostic gold standard, prevented us from comparing the diagnostic accuracy of the 2 methods (traditional vs. new). However, there is no rational explanation that the torsional pressure on the EB skin induces blister in different planes from negative pressure on the specimen. The detachment is expected to occur through the weakest
part of the dermoepidermal junction in both methods. In summary, the common cause of inconclusive IFM results is inappropriate skin biopsy. It is important to take the skin biopsy from a fresh blister. A fresh blister can be induced on an appropriate area of the skin either before or after taking the biopsy. Inducing the blister after taking the biopsy has less discomfort for the patients,
TABLE 1. IFM Results for 43 and 47 Patients With EB Biopsied Through Traditional and New Methods Respectively Dystrophic, n (%)
Junctional, n (%)
Simplex, n (%)
Inconclusive, n (%)
43
36 (84)
28 (65)
9 (21)
2 (5)
4 (9)
47
46 (98)
30 (64)
5 (11)
11 (23)
1 (2)
Cases
Traditional method (Rubbing intact skin before taking biopsy) New method (Suctioning the specimen after taking biopsy) P
1006
IFM Results
Sections Contain Cleavage, n (%)
Method
| www.amjdermatopathology.com
0.025
0.19
2014 Lippincott Williams & Wilkins
Am J Dermatopathol Volume 36, Number 12, December 2014
FIGURE 3. Inducing the blister in the normal control skin specimen with the new method led to the splitting of the dermis. Collagen IV staining with dilution of 1/200 revealed that the remnants of the dermis were attached to the lamina densa.
and also induction of the blister in 100% of the specimens is feasible provided that they are not taken from the palm or sole.
ACKNOWLEDGMENTS All authors thank Professor Dedee F. Murrell for her invaluable comments and inspiration. Nikoo Mozafari, MD Mohammadreza Barzegar, MD Zahra Asadi-Kani, MD Skin Research Center, Shahid Beheshti University of Medical Sciences, Tehran Iran
REFERENCES 1. Fine JD, Eady RA, Bauer EA, et al. The classification of inherited epidermolysis bullosa (EB): report of the Third International Consensus Meeting on Diagnosis and Classification of EB. J Am Acad Dermatol. 2008;58:931–950. 2. Pohla-Gubo G, Cepeda-Valdes R, Hintner H. Immunofluorescence mapping for the diagnosis of epidermolysis bullosa. Dermatol Clin. 2010; 28:201–210, vii. 3. Rao R, Mellerio J, Bhogal BS, et al. Immunofluorescence antigen mapping for hereditary epidermolysis bullosa. Indian J Dermatol Venereol Leprol. 2012;78:692–697. 4. Intong LR, Murrell DF. How to take skin biopsies for epidermolysis bullosa. Dermatol Clin. 2010;28: 197–200, vii. 5. Yiasemides E, Walton J, Marr P, et al. A comparative study between transmission electron microscopy and immunofluorescence mapping in the diagnosis of epidermolysis bullosa. Am J Dermatopathol. 2006;28:387–394. 6. Berk DR, Jazayeri L, Marinkovich MP, et al. Diagnosing epidermolysis bullosa type and subtype in infancy using immunofluorescence microscopy: the Stanford experience. Pediatr Dermatol. 2013;30:226–233.
2014 Lippincott Williams & Wilkins
Syringocystadenocarcinoma Papilliferum With Intraepidermal Pagetoid Spread on an Unusual Location To the Editor: Syringocystadenocarcinoma papilliferum (SCACP) is a rare malignant adnexal neoplasm with apocrine differentiation, regarded as the malignant counterpart of syringocystadenoma papilliferum (SCAP).1,2 Twenty-two cases have been reported since 1980, when SCACP was first described. The most commonly affected sites are the head and neck, in particular the scalp. Three cases with intraepidermal pagetoid spread have been described.2,3 Here, we report a case of infiltrative SCACP on an unusual location with intraepidermal pagetoid spread of neoplastic cells. Dr. Shan and Chen did this work when they were fellows in Ackerman Academy of Dermatopathology. Dr Y. Guo had full access to all of the data in the study and took responsibility for the integrity and accuracy of the data analysis. S-J. Shan is responsible for the study concept and design. S-J. Shan and S. Chen are responsible for the acquisition of data. The authors declare no conflicts of interest.
Letters to the Editor
A 93-year-old man presented to his dermatologist with an ulcerated nodular lesion on the right popliteal fossa. The lesion was present for more than 10 years with a sudden rapid growth, erosion, and exudation during the last 2 years. No pruritus or tenderness was noted. On physical examination, a pink 2-cm ulcerated nodule was seen. No history of trauma or cutaneous or systemic disease could be elicited from the patient. The clinical impression was squamous cell carcinoma. An excisional biopsy was performed. Histologically, the neoplasm revealed a silhouette resembling that of SCAP (Fig. 1). There was papillated epidermal hyperplasia with cystic invagination lined by atypical epithelial cells, some columnar. Papillated projections and ductal structures were present in areas of the neoplasm. Apocrine differentiation was evidenced by the presence of “decapitation” secretion (Fig. 2). In foci within the epidermis, neoplastic cells showed pagetoid spread (Fig. 3). Neoplastic aggregates were also identified in the dermis (Fig. 4). These cells have large pleomorphic and hyperchromatic nuclei. Atypical mitotic figures were easily visible. There was an associated dense inflammatory infiltrate containing many plasma cells (Fig. 5). On immunohistochemical study, neoplastic cells were positive for CAM5.2, CK903, CEA, and EMA (Figs. 6, 7). Stain for S-100 was negative. The diagnosis of SCACP was made. The margins were clear. No local recurrence or metastasis was observed at the follow up visit. SCACP is an uncommon adnexal neoplasm with apocrine differentiation. Clinically, SCACP is presented as a longstanding raised plaque or nodule with or without ulceration. SCACP has a predilection for the head and neck, but other locations such as back, chest, suprapubic area, perianal region, upper arm, and medial calf have also been reported.2–4 In our case, the lesion is presented as a long-standing nodule with ulceration and drainage located on the right popliteal fossa. For SCACP, this unusual location has not been previously reported in the medical literature. SCACP has been thought to arise from long-standing SCAP.2 In our case, the lesion demonstrated the silhouette of SCAP, which seems to support this theory. www.amjdermatopathology.com |
1007
Ex Vivo Blister Induction: A More Sensitive Technique for Immunofluorescence Mapping Studies in Inherited Epidermolysis Bullosa To the editor: Immunofluorescence antigen mapping (IFM) is recommended as the primary laboratory approach for diagnosis in patients suspicious of inherited epidermolysis bullosa (EB).1 In this method, structural proteins in the epidermis or dermoepidermal junction are visualized by using specific monoclonal or polyclonal antibodies. The level of split formation and expression levels of structural proteins (normal, reduced, or absent) can be detected.2 The major types of EB (simplex, junctional, and dystrophic) are determined based on the level of skin cleavage. The presence of reduced or absent staining of specific proteins can then distinguish the various subtypes of EB. In patients with milder forms of the disease, such as EB simplex, dominant dystrophic EB, and limited forms of Junctional EB (non-Herlitz type or Laryngo-onychocutaneous syndrome), nonfunctional structural proteins may be expressed normally. Therefore, in these types of EB, the diagnosis would not be achieved unless the specimen contains a blister, which helps to make the diagnosis according to the cleavage plane2 (Figs. 1A, B). The most critical step of IFM is to take a skin biopsy correctly.3 The biopsy should be taken from freshly induced blisters. The biopsy should not be taken from the existing blisters because reepithelialization under the roof of the blister results in multiple cleavage planes, and serum and inflammatory Supported by the Skin Research Center, Shahid Beheshti University of Medical Sciences. The authors declare no conflicts of interest.
TO THE
EDITOR
cells can degrade protein antigens and give false positive results.3,4 For inducing a blister, we have developed a new method by which we can have blistering in all specimens taken from patients with EB of any level of severity, leading to the diagnosis in all cases and reducing the need for rebiopsy. We reasoned that if a consistent amount of negative pressure was applied to the skin biopsy once it was taken, the EB skin should blister before the normal skin and the cleavage would occur at a specific level expected for each type. In this method, a 4-mm punch biopsy is taken from an area of nonblistered skin adjacent to the site where the patient usually develops blisters. The plastic tip of a 20 mL syringe is placed on the epidermal side of the specimen and its pistol is drawn back, causing negative pressure on the epidermis (Fig. 2A) When the area of the specimen is smaller than the area of the syringe tip (for example, a 3-mm punch biopsy), suction can be done while a 22-gauge needle is attached to the tip (Fig. 2B). If not, the whole specimen will be pulled into the syringe. The syringe is then withdrawn to apply suction, and the process is continued until a macroscopic blister appears (Fig. 2C), which typically takes 1–5 minutes. It is, of note, that in patients with milder forms of EB, the time needed for inducing the blister may take more than 2 hours. In the traditional method of taking biopsies for IFM, a clinically normal area of the patient’s skin is rubbed with a vertically oriented pencil eraser in a torsional fashion at least 20 times or until the area turns red.4 Then, a 3-mm punch biopsy is taken from the rubbed area.4 In cases with less severe form of EB such as EB simplex or dominant dystrophic EB, prolonged (up to 3 hours) torsion with a pencil eraser is required for inducing a new blister. Unfortunately younger and scared children could not tolerate the long time needed for this procedure to induce blistering. If a young child is being sedated for a skin biopsy for EB, it is not safe to wait long for blistering to develop, and rubbing is not often performed before the sedation and biopsy although it should be ideally. Moreover, unwanted large areas peel off, more than required for a 4 mm
Am J Dermatopathol Volume 36, Number 12, December 2014
punch biopsy may occur occasionally, and if the biopsy is taken from a rubbed area that is red in hope of microscopic blistering, immunofluorescence staining may reveal that no blister has been induced in the specimen. For example, in a study by Yiasemides et al,5 9% of the biopsies (3 of 33), which were taken according to the aforementioned protocol for IFM or transmission electron microscopy, were inadequate for analysis because there were no blisters in the specimens. In a similar study from Stanford, 26% of the samples (20 of 77) submitted for IFM or transmission electron microscopy were suboptimal because there were no blisters in 13 specimens, the epidermis was missing in 4, and re-epithelialization or artifactual changes occurred in 3 specimens. Only 1 of the 13 specimens without the blister was informative in which the diagnosis of junctional EB was made based on markedly reduced staining for laminin 332.6 Since the establishment of the Iranian EB registry, which is based at our center, IFM has been performed for 90 patients (aged 5 days to 45 years). A panel of primary monoclonal antibodies directed against cytokeratin 5 (MAB 3224), cytokeratin 14 (MAB 3232), a6 integrin (MAB1378), b4 integrin (MAB 1964), laminin 332 (MAB 1949), collagen IV (MAB 3326), and collagen VII (MAB 1345) was used. Fluorescein-conjugated goat anti-mouse IgG (AP124F) was used as the secondary antibody for detecting the protein-bound primary antibody. All of the above mentioned antibodies were purchased from Millipore Company (Billerica, MA). For the first 43 cases, biopsies were done according to the traditional method. In 7 of the 43 specimens, the cleavage plane was not seen, and consequently, a diagnosis was not made in 4 cases. The IFM results of 39 diagnosed cases are presented in Table 1. When the new method was applied for the rest of the 47 cases, we had blister in all our specimens except for 1 case in whom the biopsy was taken from plantar hyperkeratosis; therefore, 46 cases were diagnosed (Table 1). Fisher exact test was used for comparing the efficacy of these 2 methods. In the new method, the rate of blister www.amjdermatopathology.com |
1005
Am J Dermatopathol Volume 36, Number 12, December 2014
Letters to the Editor
FIGURE 1. A, Immunofluorescence staining for collagen VII in the skin specimen of a patient affected by dystrophic EB, the normal expression of collagen VII is evident. B, The same patient sample stained for collagen IV with dilution of 1/50 revealed sublamina densa blistering. If the specimen did not contain blister, the diagnosis would not be made.
FIGURE 2. Suctioning the specimen with a plastic tip (A) or needle (B) of syringe until a macroscopic blister appears (C).
induction was statistically higher than the traditional method (98% vs. 84%, P = 0.025). Consequently, the rate of inconclusive results was lower (2% vs. 9%), although the difference was not statistically significant (P = 0.19). When the new method was applied on the normal control skin specimen, the cleavage occurred in the dermis (Fig. 3).
The lack of genetic mutation analysis, as a diagnostic gold standard, prevented us from comparing the diagnostic accuracy of the 2 methods (traditional vs. new). However, there is no rational explanation that the torsional pressure on the EB skin induces blister in different planes from negative pressure on the specimen. The detachment is expected to occur through the weakest
part of the dermoepidermal junction in both methods. In summary, the common cause of inconclusive IFM results is inappropriate skin biopsy. It is important to take the skin biopsy from a fresh blister. A fresh blister can be induced on an appropriate area of the skin either before or after taking the biopsy. Inducing the blister after taking the biopsy has less discomfort for the patients,
TABLE 1. IFM Results for 43 and 47 Patients With EB Biopsied Through Traditional and New Methods Respectively Dystrophic, n (%)
Junctional, n (%)
Simplex, n (%)
Inconclusive, n (%)
43
36 (84)
28 (65)
9 (21)
2 (5)
4 (9)
47
46 (98)
30 (64)
5 (11)
11 (23)
1 (2)
Cases
Traditional method (Rubbing intact skin before taking biopsy) New method (Suctioning the specimen after taking biopsy) P
1006
IFM Results
Sections Contain Cleavage, n (%)
Method
| www.amjdermatopathology.com
0.025
0.19
2014 Lippincott Williams & Wilkins
Am J Dermatopathol Volume 36, Number 12, December 2014
FIGURE 3. Inducing the blister in the normal control skin specimen with the new method led to the splitting of the dermis. Collagen IV staining with dilution of 1/200 revealed that the remnants of the dermis were attached to the lamina densa.
and also induction of the blister in 100% of the specimens is feasible provided that they are not taken from the palm or sole.
ACKNOWLEDGMENTS All authors thank Professor Dedee F. Murrell for her invaluable comments and inspiration. Nikoo Mozafari, MD Mohammadreza Barzegar, MD Zahra Asadi-Kani, MD Skin Research Center, Shahid Beheshti University of Medical Sciences, Tehran Iran
REFERENCES 1. Fine JD, Eady RA, Bauer EA, et al. The classification of inherited epidermolysis bullosa (EB): report of the Third International Consensus Meeting on Diagnosis and Classification of EB. J Am Acad Dermatol. 2008;58:931–950. 2. Pohla-Gubo G, Cepeda-Valdes R, Hintner H. Immunofluorescence mapping for the diagnosis of epidermolysis bullosa. Dermatol Clin. 2010; 28:201–210, vii. 3. Rao R, Mellerio J, Bhogal BS, et al. Immunofluorescence antigen mapping for hereditary epidermolysis bullosa. Indian J Dermatol Venereol Leprol. 2012;78:692–697. 4. Intong LR, Murrell DF. How to take skin biopsies for epidermolysis bullosa. Dermatol Clin. 2010;28: 197–200, vii. 5. Yiasemides E, Walton J, Marr P, et al. A comparative study between transmission electron microscopy and immunofluorescence mapping in the diagnosis of epidermolysis bullosa. Am J Dermatopathol. 2006;28:387–394. 6. Berk DR, Jazayeri L, Marinkovich MP, et al. Diagnosing epidermolysis bullosa type and subtype in infancy using immunofluorescence microscopy: the Stanford experience. Pediatr Dermatol. 2013;30:226–233.
2014 Lippincott Williams & Wilkins
Syringocystadenocarcinoma Papilliferum With Intraepidermal Pagetoid Spread on an Unusual Location To the Editor: Syringocystadenocarcinoma papilliferum (SCACP) is a rare malignant adnexal neoplasm with apocrine differentiation, regarded as the malignant counterpart of syringocystadenoma papilliferum (SCAP).1,2 Twenty-two cases have been reported since 1980, when SCACP was first described. The most commonly affected sites are the head and neck, in particular the scalp. Three cases with intraepidermal pagetoid spread have been described.2,3 Here, we report a case of infiltrative SCACP on an unusual location with intraepidermal pagetoid spread of neoplastic cells. Dr. Shan and Chen did this work when they were fellows in Ackerman Academy of Dermatopathology. Dr Y. Guo had full access to all of the data in the study and took responsibility for the integrity and accuracy of the data analysis. S-J. Shan is responsible for the study concept and design. S-J. Shan and S. Chen are responsible for the acquisition of data. The authors declare no conflicts of interest.
Letters to the Editor
A 93-year-old man presented to his dermatologist with an ulcerated nodular lesion on the right popliteal fossa. The lesion was present for more than 10 years with a sudden rapid growth, erosion, and exudation during the last 2 years. No pruritus or tenderness was noted. On physical examination, a pink 2-cm ulcerated nodule was seen. No history of trauma or cutaneous or systemic disease could be elicited from the patient. The clinical impression was squamous cell carcinoma. An excisional biopsy was performed. Histologically, the neoplasm revealed a silhouette resembling that of SCAP (Fig. 1). There was papillated epidermal hyperplasia with cystic invagination lined by atypical epithelial cells, some columnar. Papillated projections and ductal structures were present in areas of the neoplasm. Apocrine differentiation was evidenced by the presence of “decapitation” secretion (Fig. 2). In foci within the epidermis, neoplastic cells showed pagetoid spread (Fig. 3). Neoplastic aggregates were also identified in the dermis (Fig. 4). These cells have large pleomorphic and hyperchromatic nuclei. Atypical mitotic figures were easily visible. There was an associated dense inflammatory infiltrate containing many plasma cells (Fig. 5). On immunohistochemical study, neoplastic cells were positive for CAM5.2, CK903, CEA, and EMA (Figs. 6, 7). Stain for S-100 was negative. The diagnosis of SCACP was made. The margins were clear. No local recurrence or metastasis was observed at the follow up visit. SCACP is an uncommon adnexal neoplasm with apocrine differentiation. Clinically, SCACP is presented as a longstanding raised plaque or nodule with or without ulceration. SCACP has a predilection for the head and neck, but other locations such as back, chest, suprapubic area, perianal region, upper arm, and medial calf have also been reported.2–4 In our case, the lesion is presented as a long-standing nodule with ulceration and drainage located on the right popliteal fossa. For SCACP, this unusual location has not been previously reported in the medical literature. SCACP has been thought to arise from long-standing SCAP.2 In our case, the lesion demonstrated the silhouette of SCAP, which seems to support this theory. www.amjdermatopathology.com |
1007
