Human samples

Samples of tattooed skin areas and regional lymph nodes of five donors as well as skin and lymph node samples of two additional donors without any tattoos were taken postmortem at the Institute of Forensic Medicine at the Ludwig-Maximilians University of Munich (court-ordered autopsies with no additional cosmetic impairment to the skin). The experiments were performed according to the Helsinki Declaration of 1975. All samples were obtained anonymously without information on the donors’ disease status, causes of death or demographics. Hence, occupational or environmental exposure with high exposure with stainless steel or metallic particles is unknown. We selected specimens with tattoos other than black which are more likely to contain TiO₂ and organic pigments.

The shave biopsy of the allergic patient was taken upon informed consent due to medical indication. The patient was tattooed with a self-made mixture of black and red ink in one session to achieve a brown color in 2014. The patient experienced immediate discomfort with additional swelling and itching with some delay of several weeks. After a year, the patient sought medical advice. The prescribed betamethasone cream applied twice a day only improved itching not swelling. The tattoo was removed in 2016. Tissue culture of removed skin was negative for mycobacteria. The patient showed a Ni (+++) patch test result with no reaction toward Cr in 2016. Removed tissue was received upon informed consent. Tissue samples were stored in plastic bags at − 20 °C directly after excision and further processed for analysis within a year. Subsamples were cut using a standard scalpel and frozen in TissueTek O.C.T. matrix (Sakura Finetek, Staufen, Germany) for cryo-microtome sectioning. Sections for XRF analyses at ID16B and ID21 were performed on 12–14 μm sections between two 4 μm Ultralene window films (Spex Sample Prep, Metuchen, NJ, USA). Sections were inactivated using a 4% formaldehyde buffer for 10 min and subsequently washed with deionized water (twice, 2–5 min) before being dried at room temperature.