Calcinosis cutis (CC) is caused by the deposition of insoluble calcium salts within skin and subcutaneous tissue [1]. There are five subtypes of CC: dystrophic, metastatic, idiopathic, iatrogenic, and calciphylaxis [1],[2]. The histomorphology of CC is typically characterized by fine granules or nodules of basophilic crystalline or amorphous calcium within the dermis/subcutis [3]. If performed, special staining with von Kossa and/or Alizarin red should be positive [1]. We report a case of CC, clinically diagnosed as an epidermal inclusion cyst (EIC), which initially had ambiguous histomorphology consisting of pools of pale blue-gray material by H&E stain which were negative by von Kossa stain. Upon additional review, these findings were discovered to be the result of surface decalcification of the paraffin block, something confirmed after reprocessing of the tissue.

A 27-year-old male with no significant past medical history presented to the surgery clinic and complained of an inferior right buttock mass which had been present for at least eight years. The patient had been referred by dermatology to have the mass surgically excised in 2013 but was lost to follow up. In the years that followed, the patient described increasing discomfort while sitting, as well as multiple sporadic episodes of thick, foul-smelling drainage from the mass.

Physical exam revealed a subcutaneous mass (3×1 cm) with induration, minimal tenderness, mild erythema, and no evidence of drainage or fluctuance. The clinician noted that the mass had two adjacent heads and was felt to be consistent with EIC. The physical exam was otherwise unremarkable. The most recent laboratory findings, including calcium (though measured in 2015) were unremarkable. The patient had no systemic findings to suggest connective tissue disease or metabolic disorder.

Surgical excision demonstrated two hard, pea-sized masses underlying the skin with subsequent gross pathology revealing a gray-white pasty substance within a cyst-like structure. The specimen was entirely submitted without decalcification. Histologic examination revealed multiple pools of blue-gray, acellular material within the dermis with foreign body giant cell reaction and fibrosis. Occasional focal and minute basophilic calcifications were noted on H&E (Figure 1).

Given the unusual homogenous morphology, a differential diagnosis was considered that included calcinosis cutis/tumoral calcinosis, old cyst contents, exogenous filler material, or injection/traumatic implantation to the site. Epidermal inclusion cyst or other cystic processes were not favored due to lack of an identifiable cyst wall or keratin. The absence of staining by von Kossa or Alcian blue (controls appropriately positive) also argued against calcinosis or exogenous filler material, respectively (Figure 1). Polarizable material was not present. The patient did not have a clinical history of injections or known trauma. Immunohistochemical studies were performed due to rare cells within the amorphous material. S100 (negative), AE1/AE3 (negative), and CD163 (positive) confirmed the histologic impression of reactive macrophages. The final diagnosis was descriptive of an amorphous material in the dermis with surrounding foreign body giant cell reaction and the pathology report proposed the above differential diagnosis.

Upon subsequent review of the case, it was determined that the overall histomorphologic architecture with associated focal, small calcifications likely did represent an unusual presentation of CC. Additional case tracking in the electronic health record revealed that although decalcification didn’t occur at grossing, processing issues in the histology lab led to the embedded tissue blocks being placed in a medium-strength surface decalcification solution for ≈5–10 minutes prior to sectioning. This additional information prompted our team to reprocess the block by melting the paraffin, flipping the tissue 180° to display the surface which had not been previously exposed to surface decalcification and re-embedding the block. Repeat H&E stain showed nodular collections of basophilic material characteristic of calcifications, which were also strongly positive by von Kossa stain, consistent with a diagnosis of calcinosis cutis (Figure 2). The patient had no complications at follow-up.

Calcinosis cutis was first described by Virchow in 1855 and was later subclassified into multiple categories [1],[4]. Dystrophic CC is the most common form and is caused by local collagenous, elastic, or subcutaneous tissue damage. Dystrophic CC occurs in the setting of normal serum calcium and phosphate levels. Metastatic CC is a systemic disorder caused by abnormal calcium and/or phosphate metabolism, leading to elevated serum levels that induce calcium precipitation in the skin. Idiopathic CC is the second most common form and is a diagnosis of exclusion made when clear tissue damage or metabolic disorder is not identified. Iatrogenic CC is caused by any medical intervention which leads to calcium deposition. Calciphylaxis, the most dramatic subtype of CC, is defined as the calcification of small to medium-sized vessels in the deep dermis and subcutis, possibly leading to cutaneous ulceration and necrosis. Calciphylaxis is associated with a particularly grim prognosis [1]. A list of diagnoses and pathologic processes associated with these CC subtypes is detailed elsewhere (Table 1) [1],[2],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19],[20],[21]. Though our patient did not have autoimmune titers or recent serum calcium or phosphate levels, he also did not have clinical findings which would suggest a CTD or other systemic process. He additionally did not have a history of known trauma or medical therapies/injections to the area. Given these findings, the patient was best classified as having idiopathic CC, though additional clinical and laboratory workup could have been considered.

The histopathologic differential diagnosis of CC may include gout, oxalosis, and osteoma/chondroma cutis [3]. The lack of polarization or osteocytes/chondrocytes made these diagnoses unlikely in our patient. Given the initial gray-blue, amorphous nature of the specimen, relative lack of basophilic calcifications, and negative von Kossa, diagnostic consideration had been given to an exogenous dermal filler. However, review of the literature did not describe a dermal filler that could cause the morphologic findings seen in our case prior to reprocessing of the block [22],[23],[24]. In addition, the negative clinical history of a dermal filler or injection of other exogenous material in the context of a negative Alcian blue stain also made this diagnosis unlikely.

The histologic features which were seen in this report prior to block reprocessing were unusual. Cases of CC previously reported in the literature have shown more eosinophilic and/or homogenous CC with a lack of or very little basophilic calcifications, but we were unable to find a reported case which displayed the same unique initial morphology as seen in this patient [25],[26]. Some of the literature describes proposed inactive or early stages of tumoral calcinosis, which may have an amorphous appearance [25],[26]. However, it had seemed unlikely that the patient in our case had an early stage of calcification given the presence of the mass for many years. The originally negative von Kossa was also especially perplexing at first, and we were unable to find prior literature describing diagnostic pitfalls using von Kossa in this setting or of false negative staining in general. The use of medium-strength surface decalcification (dilute formic acid and hydrochloric acid, Easy*Cut Decal Solution) on the tissue block in this case, even after only a limited amount of time in solution, almost completely demineralized the present calcifications. This drastically altered the histomorphology and led to a completely negative von Kossa stain, representing a source of diagnostic pitfall. The definitive diagnosis of calcinosis cutis was made only after melting and re-embedding the previously unexposed/non-decalcified side of the paraffin block. The patient had no relevant clinical issues upon follow-up.

This case serves to increase awareness of this potential diagnostic pitfall in CC due to an unusual histologic presentation in the context of a negative von Kossa stain following surface decalcification, while also highlighting some of the differential diagnoses associated with the various subtypes of CC.