Annals of Clinical Biochemistry: International Journal of Laboratory Medicine

Biomarker discovery studies often claim ‘promising’ findings, motivating further studies and marketing as medical tests. Unfortunately, the patient benefits promised are often inadequately explained to guide further evaluation, and few biomarkers have translated to improved patient care. We present a practical guide for setting minimum clinical performance specifications to strengthen clinical performance study design and interpretation.

We developed a step-by-step approach using test evaluation and decision-analytic frameworks and present with illustrative examples.

We define clinical performance specifications as a set of criteria that quantify the clinical performance a new test must attain to allow better health outcomes than current practice. We classify the proposed patient benefits of a new test into three broad groups and describe how to set minimum clinical performance at the level where the potential harm of false-positive and false-negative results does not outweigh the benefits. (1) For add-on tests proposed to improve disease outcomes by improving detection, define an acceptable trade-off for false-positive versus true-positive results; (2) for triage tests proposed to reduce unnecessary tests and treatment by ruling out disease, define an acceptable risk of false-negatives as a safety threshold; (3) for replacement tests proposed to provide other benefits, or reduce costs, without compromising accuracy, use existing tests to benchmark minimum accuracy levels.

Researchers can follow these guidelines to focus their study objectives and to define statistical hypotheses and sample size requirements. This way, clinical performance studies will allow conclusions about whether test performance is sufficient for intended use.

Autoanalyser methods for the measurement of anti-Müllerian hormone have been introduced into clinical laboratories but few reports of paediatric reference intervals using these new assays have been published.

After prior evaluation of the Roche Elecsys anti-Müllerian hormone assay against the Beckman Coulter modified second generation anti-Müllerian Hormone enzyme-linked immunosorbent assay using samples from adult females, a cohort of paediatric samples which had previously been assessed using the Beckman Coulter Access anti-Müllerian hormone assay was analysed using the Roche Elecsys anti-Müllerian hormone assay.

The Roche Elecsys anti-Müllerian hormone assay measured significantly lower than the Beckman Coulter modified second generation anti-Müllerian Hormone enzyme-linked immunosorbent assay. In the paediatric cohort measured with the Roche Elecsys assay, male levels are very high from birth to puberty after which they fall towards postpubertal female levels. Male results were similar to those previously obtained using the Beckman Coulter Access anti-Müllerian hormone assay on the same cohort. Roche Elecsys anti-Müllerian hormone in the females was very low in the neonatal and prepubertal years and the postpubertal trend, with a steady rise from 15 years, was smoother than previously modelled using the Beckman Coulter Access anti-Müllerian hormone assay.

Anti-Müllerian hormone levels measured with the Roche Elecsys assay were significantly lower than the Beckman Coulter modified second generation enzyme-linked immunosorbent assay suggesting the need for new reference ranges. In the paediatric cohort, Roche Elecsys anti-Müllerian hormone levels between boys and girls showed good prepubertal delineation and small but statistically significant differences to previously measured levels using the Beckman Coulter Access anti-Müllerian hormone assay on the same sample cohort.

In a medical laboratory, changes may be made to the analytical phase of diagnostic testing whenever a new test or the issue of a ‘new generation’ kit or new diagnostic system is required. In such cases, ISO 15189:2012 accreditation can assist laboratory professionals. The aim of the present study was to propose a working pathway for introducing new examination procedures into clinical practice in accordance with the ISO 15189:2012 standard, through the exemplars of 17-hydroxy progesterone, dehydroepiandrosterone sulphate and vitamin D.

The working pathway includes the following steps: (i) analysing examination procedures under evaluation, (ii) analysing examination procedures currently in use, (iii) verifying metrological traceability, (iv) verifying examination procedures and (v) evaluating comparability of results.

The analysis of instructions for use issued by manufacturers revealed that metrological traceability was reported only for vitamin D. The imprecision verification was satisfactory, the imprecision obtained by the laboratory in terms of total imprecision always being less than the specified total imprecision. In only one case (IQC level 1, 17-hydroxy progesterone), the total upper verification limit was calculated. The trueness verification was satisfactory for all examination procedures, except for 17-hydroxy progesterone (second material). Passing–Bablok regression analyses in the comparability study demonstrated significant differences for all the examination procedures.

The working pathway described for examination procedures in routine practice is in accordance with the requirements of ISO 15189:2012 accreditation and takes feasibility into account (as its main goal), based on the cost/patient benefit ratio.

Abnormalities of iron metabolism in pregnancy pose risks for maternal and fetal health. Robust reference intervals for iron metabolism indices have not been established in a pregnant Chinese population. The purpose of this study was to derive reference intervals for indices of iron metabolism during pregnancy in a Chinese population.

A total of 360 healthy pregnant women were recruited and divided into three groups of 120 by gestational age: first trimester (1–13 weeks), second trimester (14–27 weeks) and third trimester (≥28 weeks). An additional 120 healthy non-pregnant women were recruited as the non-pregnant control group. Serum ferritin was measured by electrochemiluminescence immunoassay. Serum iron and total iron-binding capacity were measured by a direct bathophenanthroline method. Transferrin saturation value was calculated with formula TS = SI/TIBC. The reference intervals were established using a non-parametric method.

In first and second trimesters (combined), the reference intervals for serum ferritin, serum iron, total iron-binding capacity and transferrin saturation are 14.7–184.3 mg/L, 14.50–33.45 µmol/L, 36.53–68.81 µmol/L and 19.04–64.76%, respectively. In the third trimester, the reference intervals for serum ferritin, serum iron, total iron-binding capacity and transferrin saturation are 7.2–122.2 mg/L, 5.83–21.52 µmol/L, 49.40–122.76 µmol/L and 8.22–52.75%, respectively.

The reference intervals for iron metabolism indices for healthy pregnant Chinese women were established in accordance with CLSI C28-A3 guidelines. This will be a valuable tool for clinical practice and research.

Classical and 11-oxygenated androgens both contribute to the androgen pool. Regular monitoring of the androgen status is required in disorders of steroidogenesis, and multiplexing of androgens improves the diagnostic ability of an assay. Due to the cheap non-invasive collection, saliva is advantageous when multiple samples are required. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers sensitive, simultaneous quantification of steroids with short run times. Here, we have developed an LC-MS/MS assay for the simultaneous measurement of 17-hydroxyprogesterone, androstenedione, testosterone, 11β-hydroxyandrostenedione and 11-ketotestosterone in saliva.

Samples (300 μL unstimulated whole saliva) were prepared by supported liquid extraction with dichloromethane and were reconstituted in 40% methanol. After online solid phase extraction with C18 cartridges, liquid chromatography was performed on a C8 column using a water/methanol gradient containing 0.1% formic acid and 2 mmol/L ammonium acetate. A Waters TQ-S mass spectrometer was used for quantification.

Total run time was 6.4 min. For all analytes, recovery was between 89% and 109%, ion suppression between 86% and 105%. Intra- and inter-assay comparisons showed a coefficient of variation <10% and the bias between measured and nominal concentration varied between –8% and 10%. Interference with a large set of natural and synthetic steroids was excluded. The assay was applied for the measurement of the androgen profile in healthy men (n = 17) and women (n = 10) which confirmed the sensitivity of the assay to be appropriate.

We present a novel LC-MS/MS assay for the comprehensive profiling of classical and 11-oxygenated androgens with potential for routine clinical application.

Sulfatides are found in a variety of tissues and serum lipoproteins. Sulfatide is a molecular species composed of various sphingoid bases, fatty acids and sugar chains; therefore, rapid analysis of the qualitative structure is important in clinical assessment.

In this study, sulfatide-rich fractions were isolated from serum lipids, and the sulfatide species were analysed by negative ion mode using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Sulfatide species identified in human serum included two different sugar chains, eight sphingoid molecules and various fatty acid side chains including hydroxy fatty acids. In total, 64 galactosyl sulfatides (SM4s) and 49 lactosyl sulfatides (SM3) were identified. Quantitatively, the amount of SM3 was less than 1% of the amount of SM4s. The fatty acids of SM4s of healthy serum (n = 8) were predominantly C16:0 and a hydroxylation C16:0 (C16:0h), followed by very long chain fatty acids (VLCFAs) predominant species, and SM3 was a major component of VLCFAs.

This present study described a simple method of human serum sulfatide analysis using MALDI-TOF MS. This method is suitable for clinical laboratories and is likely to increase the understanding of the roles of sulfatide species in both physiological and disease states.

Non-alcoholic fatty liver disease is a frequent ailment with known complications, including those within the cardiovascular system. Associations between several indicators of high-density lipoprotein metabolism and function with clinical and laboratory parameters for the assessment of fatty liver index, a surrogate marker of non-alcoholic fatty liver disease, were evaluated.

The study comprised 130 patients classified according to fatty liver index values: fatty liver index < 30, fatty liver index 30–59 (the intermediate group) and fatty liver index ⩾ 60. Lecithin–cholesterol acyltransferase and cholesteryl ester transfer protein activities were determined. Paraoxonase 1 concentration and its activity, paraoxonase 3 concentration and high-density lipoprotein subclass distribution were assessed.

Increased lecithin–cholesterol acyltransferase activity correlated with increased fatty liver index (P < 0.001). Paraoxonase 3 concentration was lower in the fatty liver index ⩾ 60 group compared with the fatty liver index < 30 group (P < 0.05). Cholesteryl ester transfer protein activity, paraoxonase 1 concentration and its activity did not significantly differ across the fatty liver index groups. The relative proportion of small-sized high-density lipoprotein 3 subclass was higher in the fatty liver index ⩾ 60 group compared with the other two fatty liver index groups (P < 0.01). Lecithin–cholesterol acyltransferase activity positively associated with the fatty liver index ⩾ 60 group and remained significant after adjustment for other potential confounders. Only the triglyceride concentration remained significantly associated with lecithin–cholesterol acyltransferase activity when the parameters that constitute the fatty liver index equation were examined.

Higher lecithin–cholesterol acyltransferase activity is associated with elevated fatty liver index values. Significant independent association between triglycerides and lecithin–cholesterol acyltransferase activity might indicate a role of hypertriglyceridaemia in alterations of lecithin–cholesterol acyltransferase activity in individuals with elevated fatty liver index.

Inflammatory processes that occur in subjects with obstructive sleep apnoea syndrome may contribute to progressive atherosclerosis and increased cardiovascular and cerebrovascular morbidity. Meteorin-like protein, which is also known as subfatin, is transcribed similarly to meteorin protein. Meteorin-like alleviates skeletal muscle inflammation. We aimed to investigate the serum meteorin-like status of obstructive sleep apnoea syndrome subjects and determine the potential link between serum meteorin-like concentration with the presence and severity of obstructive sleep apnoea syndrome.

The obstructive sleep apnoea syndrome group was composed of 207 obstructive sleep apnoea syndrome subjects diagnosed via polysomnography. A total of 106 healthy volunteers without clinical symptoms of obstructive sleep apnoea syndrome were recruited as the control group. Blood samples were obtained from all subjects to evaluate the serum meteorin-like concentrations via enzyme-linked immunosorbent assay method.

Decreased serum meteorin-like concentration was found in obstructive sleep apnoea syndrome subjects compared with the controls. Serum meteorin-like concentration was associated with a reduced OR for having obstructive sleep apnoea syndrome (OR 0.97, 95% CI 0.961 to 0.98; P < 0.001). Severe obstructive sleep apnoea syndrome subjects showed significantly lower meteorin-like concentration compared with mild and moderate cases. Moderate subjects exhibited decreased serum meteorin-like concentration compared with mild cases. Pearson correlation analysis revealed that serum meteorin-like concentration was negatively correlated with obstructive sleep apnoea syndrome severity. Serum meteorin-like concentration negatively correlated with body mass index, low-density lipoprotein cholesterol, apnoea–hypopnea index, number of arousals, hypopnoea and apnoea in subjects with obstructive sleep apnoea syndrome.

Serum meteorin-like concentration is inversely correlated with the presence and severity of obstructive sleep apnoea syndrome.

Immunoassays are commonly used to test for drugs of abuse in patients in a variety of settings. The increasing prevalence of ‘designer’ drugs causes difficulties for the toxicology laboratory and may result in unexpected false positives and identification of unfamiliar compounds. Within the past decade, there have been a variety of ketamine and phencyclidine analogues identified, particularly as drugs of abuse.

We present a case of intoxication with a novel ketamine analogue, deschloro-N-ethyl-ketamine, causing a false positive phencyclidine immunoassay. Additionally, we performed spiking studies and 2D molecular similarity calculations for deschloro-N-ethyl-ketamine, ketamine and three other analogues on the Siemens Viva-E EMIT-II phencyclidine assay to assess their cross-reactivity.

Four of the tested compounds (deschloro-N-ethyl-ketamine, 3-methoxy-phencyclidine, 3-methoxy-eticyclidine and methoxetamine) cause false positive phencyclidine immunoassay results, while ketamine gives a negative result. The cross-reactivity data are in accord with the similarity calculations of these molecules, further validating the ability of 2D molecular similarity analysis to predict the molecular cross-reactivity in immunoassays.

The cross-reactivity data of phencyclidine and ketamine analogues presented in this study could help toxicology laboratories and clinicians in evaluating unexpected results, particularly when novel PCP and ketamine analogues are being considered.

Measurements on clinical specimens that contain no analyte, or very low amounts of analyte, unavoidably generate assay response (signal) measurements that fall on the ‘negative’ side of the fitted zero response. It is virtually universal practice to left-censor such measurements to zero and this is frequently extended by left-censoring to the assay limit of detection (LoD) value for reporting purposes. This study considers the effect of censoring on methods comparison analysis.

Paired results were randomly generated from two hypothetical assays with zero bias, firstly assuming equal uncertainty near zero and secondly with uncertainties that differed by a moderate 50% near zero. In both cases results were left-censored to zero and to LoD and further subsets were extracted representing partial and complete removal of censored results. All data sets were subjected to overall bias evaluation and Bland–Altman and Deming regression analyses.

The combination of differing uncertainties and data censoring produced spurious biases by both Bland–Altman and regression analysis, regardless of whether censored results were retained or discarded. Biases were small for data left-censored to zero but were non-trivial with LoD-censoring. Imposing a lower limit aimed at eliminating the influence of censored results did not resolve the problem.

When high proportion of clinical results are located near zero, caution is required when using censored data (and especially LoD-censored data) in methods comparison studies. Optional access to negative results would rectify the problem, but requires the cooperation of manufacturers.

Little is known of the vitamin D status of young infants and toddlers and its determinants in West Europe. The prevalence and determinants of vitamin D deficiency of children aged 6–48 months in the centre of the Netherlands (52°N) is investigated.

In a cross-sectional population study, randomly recruited infants and toddlers (n = 150) were studied using an online questionnaire and a physical examination either in late summer (n = 52) or in late winter (n = 98). Vitamin D analysis was performed by capillary blood sampling using dried bloodspots plus LC-MS/MS.

In late winter, 32% of the children were vitamin D deficient (<50 nmol/L 25OH vitamin D3) with 5% severely deficient (<25 nmol/L). In late summer, 2% were deficient. The odds of vitamin D deficiency were higher in children aged 24–48 months, for those not using formula milk and those not adhering to the supplementation guidelines.

One-third of Dutch infants and toddlers were found to be vitamin D deficient in late winter. Suggested strategies for raising the vitamin D status may include improving the adherence to supplementation, a sensible sun exposure or the use of fortified foods. Special attention is needed for the children aged 24–48 months.