Samples

EDTA plasma samples from stage I to IV lung cancer patients and age and gender-matched controls from the Women Epidemiology Lung Cancer (WELCA) study were collected at 12 different collection centers in France.⁴⁶ This study was approved by Institutional Review Board of the French National Institute of Health and Medical Research and by the French data Protection Authority (IRB-INSERM, No. 3888 and CNIL No. C13-52). All specimens were collected in compliance with the Declaration of Helsinki principles. Once collected, they were coded and deidentified to protect patient identities. As part of the WELCA Study and for all samples analyzed here, all-female lung cancer patients were recruited between September 2014 and November 2016 in collection sites 2 to 14, and age-matched all-female controls were recruited between June 2015 and December 2016 at collection site 18. All women living in Paris and the lle de France area, newly diagnosed with lung cancer were considered as eligible cases. Age-matched controls were randomly sampled from women without a history of cancer living in the same area.

Primary inclusion and exclusion criteria for the WELCA study in general are provided elsewhere.⁴⁶ For the purposes of this study we considered that a wide array of clinical conditions can result in altered circulating albumin concentrations, including advanced age.^47,48 With the exception of dehydration, most such conditions result in decreased albumin concentrations.⁴⁷ Abnormal changes to the ΔS-Cys-Albumin values observed in fresh P/S samples necessarily involve albumin as well as cysteine and cystine concentrations.²⁹

To make the ΔS-Cys-Albumin assay as widely applicable across as many pathological conditions as possible, our initial survey of the population reference range of ΔS-Cys-Albumin in nonacute cardiac patients excluded only patients with severely limited kidney function (i.e., eGFR <30 mL/min × 1.73 m²; which can cause extreme elevation of cysteine and cystine^49–51 as well as albumin loss,⁴⁷ potentially resulting in extraordinarily high ΔS-Cys-Albumin measurements) and/or severe hemolysis (i.e., >250 mg/dL; which can also cause abnormal ΔS-Cys-Albumin measurements²⁹). The results of this survey indicated that the range of ΔS-Cys-Albumin in these nominally unhealthy patients was in line with (although slightly more compact and lower on average than) the theoretical ΔS-Cys-Albumin range of 11%–38% that was predicted based on known reference ranges for albumin, cysteine, and cystine.²⁹ Notably, ∼50% of the patients in this population were diabetic. Given our goal of making the ΔS-Cys-Albumin assay applicable to as wide a clinical patient population as possible, we only excluded severely hemolyzed specimens (>250 mg hemoglobin/dL) from this study. The ΔS-Cys-Albumin results from freshly processed and stored sample described below showed no indication of abnormally elevated ΔS-Cys-Albumin.

All peripheral blood samples were drawn and processed following a written standardized protocol.⁴⁶ For the lung cancer patients, each hospital had their own laboratory where the samples were processed. Control specimens were collected at the homes of individual donors by a nurse who deposited the whole blood samples at a designated laboratory. Alternatively, control specimen donors had the option of traveling to the laboratory themselves to donate their samples. For both the cases and controls, whole blood that had been collected in EDTA-containing tubes was placed on ice packs at ∼4°C and then transported to the laboratory. (While these instructions were provided to the hospitals and nurses involved in specimen collection and transport, it is possible that in some cases they may not have been followed exactly and therefore the whole blood may have been exposed to room temperature conditions before processing it into plasma.)

Once at the laboratory, blood samples were spun for 15 minutes at 3000 rpm and 4°C in a standard centrifuge. In each of the 12 collection centers, the collected plasma samples were aliquoted then temporarily kept at −80°C or −20°C while they awaited shipment to the central repository (see Results section for site-specific temporary storage details). Approximately every 3 months samples were transported on dry ice to the central repository for final storage at −80°C. No freeze/thaw cycles occurred before shipment to Arizona State University (Borges Lab) for analysis. Received plasma samples were aliquoted on ice and kept frozen at −80°C before analysis. Sample handling information, including collection site, precentrifugation delay (from blood drawing to centrifugation), postcentrifugation delay (from centrifugation to storage), total prestorage delay time (from blood drawing to storage), and initial storage temperature before shipment to the central repository were gathered and tabulated from the 413 corresponding sample collection information sheets. A freshly collected and processed 300-mL EDTA plasma sample from an individual, nominally healthy donor was obtained from BioIVT and served as a QC sample to ensure batch-to-batch quantitative reproducibility.