Wermers, Michelle

Abstract Background A dual filtration-based method for determination of serum labile bound copper (LBC) and LBC fraction (LBC/total copper) was developed. Reduced total copper, elevated LBC, and elevated LBC fraction have been reported in Wilson disease (WD). Methods To evaluate the diagnostic performance of these markers, samples were obtained from 21 WD treatment-naïve (WD-TN, no WD treatment or <28 days of treatment) patients, 46 WD standard-of-care–treated (WD-SOC) patients, along with 246 patients representing other potential disorders of copper status. These were then compared to 213 reference interval population patients. Results Receiver operating characteristic curves for the reference population vs WD-TN yielded areas under the curve for total copper, LBC, and LBC fraction, of 0.99, 0.81, and 0.98, respectively. Using Youden cutoffs, sensitivity/specificity for WD-TN was 95%/97% for total copper, 71%/85% for LBC, and 95%/94% for LBC fraction. LBC values, but not total copper and LBC fraction, differed substantially between WD-TN and WD-SOC cohorts. We propose a dual model wherein total copper and LBC fraction results must agree to be classified as a “positive” or “negative” result for WD. This correctly classified 19/21 WD-TN patients as positive, and 194/213 reference interval patients as negative. The remaining “indeterminate” patients (representing approximately 9% of the reference and the WD-TN populations) exhibited conflicting total copper and LBC fraction results. When indeterminate results are excluded, this model exhibited apparent 100% sensitivity/specificity. Conclusions Agreement of total serum copper and LBC fraction classification may constitute an effective “rule-in” and “rule-out” assessment for WD-TN patients.

Context.— Clinical testing for Wilson disease (WD) is potentially challenging. Measuring the fraction of labile bound copper (LBC) to total copper may be a promising alternative diagnostic tool with better sensitivity and specificity than some current biomarker approaches. A dual filtration–based inductively coupled mass spectrometry (ICP-MS) assay to measure LBC in serum was developed. Objective.— To establish a reference interval for LBC and LBC to total copper (LBC fraction) in a healthy adult population, and to examine associations between total copper, LBC, and LBC fraction with age, sex, menopausal status, hormone replacement therapy, and supplement use. Design.— Serum samples were collected from healthy male (n = 110) and female (n = 104) patients between the ages of 19 and 80 years. Total copper and LBC were analyzed using ICP-MS. Results were used to calculate the LBC fraction. Reference intervals were calculated for the 2.5th and 97.5th percentiles for both LBC and LBC fraction. Results.— The reference intervals for LBC were determined to be 13 to 105 ng/mL and 12 to 107 ng/mL for female and male patients, respectively. The reference intervals for the LBC fraction were 1.0% to 8.1% and 1.2% to 10.5% for female and male patients, respectively. No significant associations were found regarding age, menopausal status, hormone replacement therapy, or vitamin and supplement use. Conclusions.— Sex-specific reference intervals have now been established for LBC and LBC fraction. These data in conjunction with further testing of WD populations can be used to assess the sensitivity and specificity of LBC fraction in screening, monitoring, and diagnosis.

Abstract Background While clinical tests are available to aid in the diagnosis of Wilson’s Disease (WD), some such as total copper determination lack sensitivity and specificity, while others such as liver tissue copper assessment are costly and invasive. WD affects copper metabolism and is known to cause low serum copper concentrations. In addition, an elevated percentage of total serum copper exists as non-protein bound or labile bound copper (LBC). Previous studies have shown that measuring the ratio of labile bound copper to total serum copper may aid in the diagnosis of WD. To support the utility of a laboratory developed test using serum LBC ratios to aid in the diagnosis of WD, serum samples from healthy donors were used to establish a reference range for LBC and the ratio of LBC to total copper in adults. Methods Serum samples were collected in trace metal free tubes from 214 donors (n = 110 male, n = 104 female) between the ages of 19–80 years old, selected against pre-determined exclusion criteria including acute hepatitis, chronic hepatitis, renal failure, biliary cirrhosis, celiac disease, and leukemia. Prior to analysis, samples went through a centrifugation-based preparation process to isolate the LBC fraction using molecular weight size exclusion filters and EDTA chelation. Samples were then analyzed via ICP-MS in kinetic energy discrimination (KED) mode to directly measure the LBC and total copper concentrations. LBC ratio values were determined from the LBC/total copper concentrations. Results Preliminary reference ranges were developed for both LBC and the LBC ratio based on the 2.5th and 97.5th percentiles with 95% confidence intervals. Established sex specific reference intervals for total copper were deemed acceptable per CLSI guidelines in document EP28-A3c, with ≤10% of results from this sample set falling outside of the established intervals. No statistically significant relationship was found in the mid-95th percentile between LBC concentration or ratio with age or sex. The reference intervals for LBC concentration were calculated as 13.41–104.66 and 11.69–106.50 ng/mL for females and males respectively. The reference intervals for LBC ratio were calculated as 1.04–8.12 and 1.17–10.48 for females and males respectively. Conclusions A reference range of LBC and LBC ratio has now been determined for the healthy adult population. Further testing can now be done on disease state populations to determine the sensitivity and specificity of LBC ratio in the detection, monitoring, or diagnosis of WD.

Abstract Background Kidney stones are a highly prevalent disease worldwide. Additionally, both environmental and occupational exposure to Pb and Cd continue to be prevalent globally and can result in renal toxicity. The objective of this study was to examine the potential presence of Pb and Cd in kidney stones, and to assess for correlation with demographic factors including smoking, gender, age, and kidney stone matrix composition. Methods Patient kidney stones (n = 96) were analyzed using Fourier transform infrared spectroscopy to identify the stone constituents. Cd and Pb concentrations (µg/g) were determined by inductively coupled plasma mass spectrometry. Cd and Pb concentrations were correlated using bivariable and multivariable statistical analysis with demographic factors (age, gender, smoking status), and kidney stone composition. Results Kidney stone Cd (median 0.092 µg/g, range 0.014 to 2.46) and Pb concentrations (median 0.95 µg/g, range 0.060 to 15.4) were moderately correlated (r = 0.56, P < 0.0001). Cd concentrations were positively associated with patient history of smoking, patient age, and calcium oxalate monohydrate levels while negatively associated with struvite and uric acid/uric acid dihydrate. Pb concentrations were positively associated with females and apatite levels while negatively associated with uric acid/uric acid dihydrate. After holding constant other stone type composition levels, smoking status, and age, both Pb and Cd were positively associated with apatite and negatively associated with uric acid/uric acid dihydrate, struvite, and calcium carbonate. Conclusions Cd and Pb kidney stone concentrations are associated with specific kidney stone types. Cd and Pb kidney stone concentrations are both associated with smoking.

Abstract Background Arsenic is a naturally occurring element with varying species and levels of toxicity. Inorganic arsenic (e.g., arsenite (AsIII) and arsenate (AsV)) are toxic, while its metabolites (e.g., monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)) are less toxic). Symptoms of exposure can include headaches, confusion, diarrhea, and drowsiness. As these symptoms overlap with many other conditions, arsenic exposure can often be overlooked as a cause. Arsenic toxicity may be treated with chelation and/or electrolyte replacement therapy. However, treatment is not without risks and is unnecessary for exposure to organic (nontoxic) forms of arsenic. This makes screening and differentiation of arsenic important for clinical testing. Method An IC-ICP-MS method was developed using a Dionex 5000 with ion exchange chromatography for separation and iCAP Q for detection. Nontoxic species are arsenobetaine and arsenocholine, and toxic species are AsIII, DMA, MMA, and AsV. Results Precision, linearity, and specificity studies produced acceptable results. For accuracy, proficiency testing and method comparison samples were analyzed and produced acceptable results. Carryover studies demonstrated single species carryover from the diluter at levels of 500 µg/L, which can be avoided by analysis rules in the standard operating procedure. Limit of detection studies yielded a lower limit of quantitation of 1 µg/L per species. Conclusions Here, we present a rapid and reliable method for quantifying and differentiating toxic and nontoxic forms of arsenic to allow for swift and appropriate management of patients with exposure.