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Int Arch Occup Environ Health (2008) 81:487–493 489
means of alpha-amylase detection (InWnity amylase
reagent; Sigma, Milan, Italy). Alpha-amylase was never
detected and none of the samples was excluded. The EBC
samples were stored at ¡80°C until analysis.
Detection of urine and EBC Cr
Urinary Cr (Cr-U) was measured by means of electrothermal
atomic absorption spectrometry (ETAAS) with Zeeman
eVect background correction, using a 1/10 5 dilution of
a certiWed standard control (1 g/l) in water (Fluka) for every
ten samples and conWrmed by inductively coupled plasma
mass spectrometry (ICP-MS) in a separate laboratory.
Finally, the ETAAS method was validated using a lyophilised
urine control (Clincheck®, Recipe, Germany,
Cr = 7.5 g/L). The limit of detection (LOD) was 0.05 g/l.
Cr was normalised for urinary creatinine content (all the
samples were between 30 and 300 mg% of creatinine), and
therefore expressed as g/g creatinine. No samples of
NSCLC patients were below the LOD and in the statistical
analysis no data were censored.
EBC Cr (Cr-EBC) was measured by means of ETAAS,
as previously described (Caglieri et al. 2006), and con-
Wrmed by inductively coupled plasma mass spectrometry
(ICP-MS) in a separate laboratory. The LOD was 0.05 g/l.
No samples of NSCLC patients were below the LOD and in
the statistical analysis no data were censored. Unfortunately,
in most EBC samples, Cr was too low to be speciated
with the method proposed by Goldoni et al. 2006 and
no more sensitive methods are present in literature.
Detection of tissue Cr
Three small pieces of non-cancerous lung tissue that had
not been in direct contact with the surgical instruments
were collected from each NSCLC patient. The tumour
resection involved the upper lobe in 13 patients and the
lower lobe in 7 patients. Portions of cancerous and noncancerous
lung tissue pieces derived from the parenchyma
were ablated. It is important to note that tissue pieces
weighed, in general, a few grams and cut signs were evident
on them. Therefore, small pieces where Cr was dosed
(about 30–50 mg each) were in the opposite part of the cut.
Samples were immediately stored at ¡80°C and Cr analysis
was performed a few days later. A partial conWrmation of
contamination avoidance was that Cr was similar in the
several small pieces of tissue from the same patient (data
not shown).
Samples were boiled at 90°C overnight in order to eliminate
their water content. The dry tissue was weighed and
digested for 3 h with a solution containing 1,250 l of
water and 1,250 l of nitric acid (69%) at 65°C. Cr was
measured by means of ETAAS using certiWed standard
controls (Fluka, Milan, Italy) for every ten samples after
appropriate dilution to minimise the eVect of Cr contamination
due to the use of nitric acid. The LOD was 0.05 g/l,
but the concentrations were expressed as g/g dry tissue.
The measurement was also repeated using a piece of cancerous
tissue. No samples were below the LOD and in the
statistical analysis no data were censored.
Detection of Cr in surgical instruments
Surgical clips were incubated for 3 h in an aqueous solution,
alone or attached to the plastic tube of the EBC collection
system, as were the standard linear cutter and head of
the endosurgery machine. Cr was directly measured in the
water used for washing and expressed as ng/clip for the
clips, and g/g for the other surgical instruments.
Statistical analysis
For the controls, a value of 0.025 g/l was assigned, if Cr
was below the LOD in urine or EBC. The non-parametric
Shapiro–Wilk’s statistical test was used because of the nonnormal
nature of the data, which were expressed as median
values with interquartile ranges. Paired samples were compared
using Wilcoxon’s test for repeated measures and the
Mann–Whitney test for independent samples. For the linear
regression analysis, we used Pearson’s correlation coeYcient
on a log–log scale in order to avoid heteroscedasticity
and to normalise standardised residuals. P values of 0.05 or
less were considered statistically signiWcant.
Results
As there was neither diVerence in the EBC volume between
the controls and NSCLC patients at either collection time nor
any correlation between EBC volume and the levels of the
measured transition elements in EBC (data not shown), total
EBC volume was not considered in the statistical analysis.
Moreover, no diVerences in the EBC volume were observed
before and after the surgical intervention (data not shown).
Table 2 shows the Cr-EBC and Cr-U levels. There was
no diVerence between the NSCLC patients and controls at
time 1, but there was a signiWcant increase in Cr-EBC of
the patients at time 2 in comparison with time 1 (P < 0.05)
and in comparison with the controls (P < 0.05). There was
also a similar increase in the Cr-U levels (P < 0.01 vs time
1 and controls).
The level of Cr was 0.14 (0.04–0.24) g/g in normal pulmonary
tissue and 0.14 (0.06–0.3) g/g in cancerous tissue
(P =ns).
Surgical clips released only a small amount of Cr [0.03
(0.02–0.04) ng/clip], but the linear cutter and the head of
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