Subjects and data collection
With the agreement of the Ethical Committee (ref. 2014/451, Chairperson: Prof. Jean-Marie Maloteaux, av. Hippocrate, 55-14, 1200 Brussels), we performed the following analyses. Blood samples for routine control between October 2011 and 2012 were identified from 413 participants of the health care prevention program for workers. Considering the analysis as retrospective, given the fact that the samples were pre-collected and treated anonymously, the Ethical Committee gave a waiver for individual consent. Consequently, no written informed consent for participation in the study was obtained from participants.
Median age was 38 years (range: 21–66 years). Typically, these subjects were in good health, permitting a full time job without restriction, and without any active disease, including cancer and infectious disease and are representative of the population as a whole. Blood samples were obtained for routine hematologic control in workers potentially exposed to X-rays or any others ionizing radiation. Samples were treated anonymously, including sex and date of birth. No information was therefore available about habits (e.g. tobacco use) and possible comorbidities (e.g. obesity or vascular disease).
Our goal is to identify the higher boundary observed in a population in good health, but not excluding non-debilitating disease, tobacco use or oral contraception. Therefore, it would be important to compare the results to those obtained in a second, and independent, sample (n = 29) of normal controls, strictly selected on the basis of perfect health, coming from an historical cohort. This cohort was used during the process of the calibration of the blood analyser. The subjects in this second cohort were in the same range of age, but, after interrogation and examination, carefully selected on the basis of the absence of any chronic disease or substance/medications (abuse, including tobacco use).
Anticoagulated whole blood from routine controls were processed on Sysmex XE2100 [TOA Medical Electronics, Kobe, Japan], Normal controls were analysed on Sysmex XN2000 (TOA Medical electronics Co, Kobe, Japan), Advia 2120 (Bayer Diagnostics, Tarritown, NY, USA), DXH800 (Beckman Coulter, Miami, FL USA), Cell-Dyn Sapphire (Abbott Diagnostics Santa Clara, CA, USA) for the determination of the complete blood cell counts and differential counts of leukocytes. The absolute neutrophil count was divided by the absolute lymphocyte count to calculate the NLR.
For the statistical analyses, laboratory parameters fit a log-normal distribution. Logarithmic conversion was therefore used for calculating means and 95% confidence limits (95% CI) are presented as arithmetic mean ± 1.96 SD (with their 95% CI). Power analysis shows that, to test the hypothesis that <2.5% of the normal controls would be out of the proposed limits, 28 subjects are needed. It is the reason why we included 29 blood samples of the historical cohort for the external validation.
All the analyses were performed with STATISTICA (data analysis software system, version 7, Statsoft Inc. 2004, Tulsa).