Identifying subcutaneous tissue microcalcification by Fluo‐3 AM imaging in cutaneous calciphylaxis

Abstract

Calciphylaxis, also known as calcific uremic arteriolopathy (CUA), is typically characterized by subcutaneous tissue calcification and excruciatingly painful cutaneous lesions. Calciphylaxis is a crippling disease with a mortality rate as high as 60%-80%, which affects 1%-4% of the population with end-stage renal disease. 1 Due to the high mortality and morbidity of calciphylaxis, it is critical for dermatologists to make early diagnosis and appropriate management, yet currently only 56% of calciphylaxis cases are correctly diagnosed by conventional histological stains. 2 The pathologic diagnosis of calciphylaxis primarily depends on high-quality staining technique. Large and chunky calcification in subcutaneous vessels can be determined by conventional histological stains such as HE, Alizarin S and von Kossa. However, these imaging techniques suffer from low sensitivity in detecting subtle calcification. 3 The subtle calcium deposits including perieccrine calcification, and intravascular and extravascular microcalcification were found to be highly specific to early diagnosis of cutaneous calciphylaxis, but the detection of these calcium deposits is difficult and not precise. 4 Fine calcium deposits even were not appreciable on those stains in some cases of proven cutaneous calciphylaxis clinically. 2 Therefore, an effective diagnosis of calciphylaxis faces a great challenge and more sensitive calcification staining is in high demand.Here, we describe a rapid, sensitive and reliable staining method using Fluo-3 AM that could be a promising diagnostic tool for cutaneous calciphylaxis. The most important property of Fluo-3 AM is an absorption spectrum compatible with excitation at 488 nm by laser sources, and a very large fluorescence intensity increase in response to Ca 2+ binding. Fluo-3 AM imaging has been widely used calcium signalling examinations in flow cytometry and confocal laserscanning microscopy, 5 but no published study has applied Fluo-3 AM to calcification detection in paraffin section of skin biopsy.We have studied 41 patients with calciphylaxis that had been confirmed by skin biopsy combined with clinical findings and imaging studies. The histopathological examination of skin biopsy was performed using conventional staining (haematoxylin-eosin HE, von Kossa and Alizarin red) and Fluo-3/AM staining. For each case, the following histological stains of calcification were evaluated: chunky calcification and stippled calcification within vessels and

Neuroprotective Mechanism of Ribisin A on H2O2-induced PC12 cell injury model

“…It is incubated with the pentaacetoxymethyl ester of the dye and then sheared into Fluo-3 by intracellular esterases upon entry into the cell. Fluo-3 does not fluoresce in its free form, but when combined with Ca 2+ , it can produce a strong fluorescence, reflecting the level of Ca 2+ concentration 40,41 . The histograms of Ca 2+ fluorescence intensity are shown in Fig.5a∼e, which correspond to the quantitative results in Fig.5f.…”

Early diagnosis strategy of calciphylaxis in dialysis patients

“…The current diagnosis of the disease is mainly based on high-risk factors, characteristic skin lesions, and features of skin histopathology [ 6 ], however, this approach has limitations in diagnosing calciphylaxis at an early stage. The comprehensive use of noninvasive radiological technology has been reported to be a good method for early warning of calciphylaxis [ 7 ], while skin biopsy and special calcium staining tailored to high-risk patients could help confirm the diagnosis in early or atypical skin lesion cases [ 8 , 9 ].…”

“…Zhu et al. recently discovered that Fluo-3 AM is a rapid, sensitive, and reliable fluorescent probe for the detection of calcium deposits, and is presumably a promising diagnostic tool for calciphylaxis [ 8 ]. Although skin biopsy is an invasive procedure, it is indispensable to confirm the diagnosis in early and atypical patients with suspected calciphylaxis.…”

Neuroprotective mechanism of ribisin A on H2O2-induced PC12 cell injury model

“…It is incubated with the pentaacetoxymethyl ester of the dye and then sheared into Fluo-3 by intracellular esterases upon entry into the cell. Fluo-3 does not fluoresce in its free form, but when combined with Ca 2+ , it can produce a strong fluorescence, reflecting the level of Ca 2+ concentration 40,41 .…”