Introduction −1 −1 1 2 3 4 L 5 6 7 1 8 9 Fig. 1 Chemical structures of eumelanin and phaeomelanin  Both approaches were tested here, i.e., (i) Cu determination via instrumental neutron activation analysis (INAA) in liver biopsies, nail clippings, and hair samples; and (ii) X-ray spectrum acquisition after X-ray tube excitation, followed by full spectrum data (i.e. all channel contents) processing by PCA, including the often neglected peakless part of the X-ray scattering region resulting from Compton, Rayleigh and Raman effects. Experimental Liver biopsy samples, nail clippings, and hair were collected from approximately 100 dogs with chronic hepatitis as well as from healthy animals. Nail clippings were taken from phalanx I (the thumb nail), since this nail is not in contact with the ground, thus minimizing contamination problems. Nail clippings could not be collected from all animals as in some cases breeders had removed the nail permanently shortly after birth, since sometimes such a thumb nail hinders the dog in working trials. Hair was sampled from the median abdominal wall, just cranial to the belly button. The liver biopsy samples were freeze-dried at the veterinary clinic. The nail clippings and hair samples were analyzed without cleaning to prevent changes in the nail and hair morphology that might affect the scattering information from the X-ray fluorescence (XRF) analysis. It should be noted that a Cu contamination of the nail clipping and hair is highly unlikely; the nail of the thumb nail is never in contact with, e.g., soil, and external contamination of the hair was also unlikely. INAA The needle biopsies were transferred into polyethylene capsules and lyophilized, resulting in sample masses varying typically between 1.5 and 50 mg dry weight. Nail clippings had masses of 5–50 mg, and the typical mass of the hair samples was approximately 100–200 mg. 66 64 10 17 −2 −1 69m −1 11 12 66 X-ray tube excitation The measuring procedure consisted of weighing approximately 100 mg of each hair sample and 10 mg of each nail sample directly into appropriate cells and submitting them to blank rhodium X-ray tube radiation, in triplicate. A common laboratory energy dispersive XRF instrument (EDX 700, Shimadzu) was used under the same irradiation conditions: 50-kV applied voltage in the tube, 25% dead time, 10-mm beam collimation, and 100-s irradiation time. It should be noted that the methodology of using the region in the X-ray spectrum resulting from scattering implies that only the spectrum’s shape is needed for the data processing by PCA. There is no need for quantification towards Cu mass fractions, and hence no reference materials were used for quality control as was the case in the hair, nail, and liver biopsy analysis by INAA. 13 Results and discussion 66 −1 2 2 Fig. 2 Cu levels in nail clippings and hair of Labrador Retrievers as a function of the Cu levels in liver biopsies taken from the same dogs: ○ hair, ● nail; uncertainties are omitted for clarity (see text)  3 4 Fig. 3 PCA analysis of all hair samples, classified here by the hair color: □ yellow, * chocolate, ■ black  Fig. 4 top bottom  3 1 8 A s A s e A 1 Table 1 8 Color Genotype Melanin type Black A s   B   E Eumelanin Liver (chocolate) A s –  bb   E Eumelanin Yellow, liver nose B   ee Phaeomelanin Yellow, black nose bb   ee Phaeomelanin 5 6 Fig. 5 PCA analyses of all nail samples, classified by gender  3 4 5 4 14 15 16 17 18 19 6 −1 −1 −1 −1 −1 −1 Fig. 6 top −1 −1 N bottom −1 −1 N  6 37 The results of our study show that a fairly sharp differentiation can be obtained between dogs with low and high Cu liver levels if PCA is performed on a dataset obtained from all channel contents of X-ray spectra obtained by X-ray tube excitation of nail or hair samples together with the spectral information of often neglected X-ray (Rayleigh, Raman) scattered radiation. A practical application of this technique would start with construction of a dataset using samples of known origin, encompassing all varieties expected. New and unknown samples can then be projected on this model, and subsequently classified. This approach will be further validated with new samples to be collected from Labrador Retrievers still being observed and treated along medicinal and dietary approaches. Conclusions Hair and nail clippings are widely used as bioindicators for the trace element status of man and, to a much lesser extent, also for animals. The results of this study emphasize that it is quite risky to assume a priori that the trace element levels in hair and nail reflect differences between classes of individuals, e.g., “healthy” and “sick” people or animals. In this study related to chronic hepatitis, the Cu levels in liver biopsies are not reflected by Cu levels in hair and nail. The X-ray Rayleigh scattered and X-ray Raman continuum contain valuable information about the organic, low-Z elemental matrix composition of an object. In this study, first indications were obtained that the Cu accumulation possibly may have an effect on the nail and hair melanin, thus offering a potentially favorable outlook for noninvasive monitoring of the Cu liver status. An additional advantage of the approach is that a measurement by X-ray tube excitation takes only about 2 min per sample so that, once a database has been built up, the technique might be valuable for large-scale screening purposes.