Annals of Clinical Biochemistry: International Journal of Laboratory Medicine

The US Centers for Disease Control and Prevention ensured adequate performance of the routine triglycerides methods used in Japan by a chromotropic acid reference measurement procedure used by the Centers for Disease Control and Prevention lipid standardization programme as a reference point. We examined standardized data to clarify the performance of routine triglycerides methods.

The two routine triglycerides methods were the fluorometric method of Kessler and Lederer and the enzymatic method. The methods were standardized using 495 Centers for Disease Control and Prevention reference pools with 98 different concentrations ranging between 0.37 and 5.15 mmol/L in 141 survey runs. The triglycerides criteria for laboratories which perform triglycerides analyses are used: accuracy, as bias ≤5% from the Centers for Disease Control and Prevention reference value and precision, as measured by CV, ≤5%.

The correlation of the bias of both methods to the Centers for Disease Control and Prevention reference method was: y (%bias) = 0.516 × (Centers for Disease Control and Prevention reference value) −1.292 (n = 495, R²= 0.018). Triglycerides bias at medical decision points of 1.13, 1.69 and 2.26 mmol/L was −0.71%, −0.42% and −0.13%, respectively. For the combined precision, the equation y (CV) = −0.398 × (triglycerides value) + 1.797 (n = 495, R²= 0.081) was used. Precision was 1.35%, 1.12% and 0.90%, respectively. It was shown that triglycerides measurements at Osaka were stable for 36 years.

The epidemiologic laboratory in Japan met acceptable accuracy goals for 88.7% of all samples, and met acceptable precision goals for 97.8% of all samples measured through the Centers for Disease Control and Prevention lipid standardization programme and demonstrated stable results for an extended period of time.

Synthetic cannabinoids (NOIDS) are novel psychotropic drugs (NPS) currently freely sold in the United Kingdom as ‘research chemicals’. Detection of NOIDS use is not available in current routine methods. Here we describe a marker which helps determine which patients have used these substances.

In a test case, ultra-performance liquid chromatography mass spectrometry (UPLC-Tof) was used to screen the legal high Herbal Haze II, the contents of hand-rolled cigarettes and five patient samples for NOIDS and their metabolites.

Analysis of legal high Herbal Haze II and cigarettes identified the third generation adamantyl-type NOIDS N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (AKB-48), 5F-AKB-48 and N-adamantyl-1-fluoropentylindole-3-carboxamide (STS-135). Out of 18 potential metabolites, 1-adamantylamine (C₁₀H₁₇N) was detected in all five urine samples. This adamantyl-type NOID marker was incorporated into our routine LC-MS/MS urine screen. Out of 14,436 random urine samples screened over eight months, 296 (2.05%) tested positive for the adamantyl-type NOID marker.

We have discovered a urine marker for identifying patients smoking legal high products containing the third generation adamantyl-type NOIDS such as AKB-48 and its fluoropentyl analogue 5F-AKB-48, which are among the most popular NOIDS currently available in legal high products sold in UK. This marker can be incorporated into routine LC-MS/MS drug screening alongside classic drugs of abuse. Positive detection rates for this new legal high marker are greater than for established classic drugs that are routinely screened such as amphetamine. This work highlights the need for a flexible toxicology screening service capable of adapting to changes in drug use such as the growing popularity of legal highs/NPS.

Endocan (endothelial cell-specific molecule-1) is a proteoglycan and plays an important role in angiogenesis and inflammation. The aim of this study was to evaluate of serum and urinary concentrations of endothelial cell-specific molecule-1 in bladder cancer.

The study included 50 bladder cancer patients, 50 with urinary tract infection and 51 healthy volunteers. Serum and urinary endothelial cell-specific molecule-1 concentrations were measured with enzyme linked immunosorbent assay.

In bladder cancer group, serum and urinary endothelial cell-specific molecule-1 concentrations were significantly higher than in the healthy subjects (P = 0.003 and P < 0.0001). Urinary endothelial cell-specific molecule-1 concentrations in cases with urinary tract infection were higher than in healthy volunteers (P = 0.002). There were no significant differences between bladder cancer and urinary tract infection groups in terms of serum and urinary endothelial cell-specific molecule-1 concentrations. Urinary endothelial cell-specific molecule-1 concentrations were higher than those of corresponding serum endothelial cell-specific molecule-1 concentrations (P < 0.0001 for bladder cancer and urinary tract infection groups, P = 0.002 for healthy subjects). In bladder cancer group, there was a positive correlation between serum endothelial cell-specific molecule-1 and urinary endothelial cell-specific molecule-1 concentrations (r = 0.32, P = 0.002). For serum endothelial cell-specific molecule-1, sensitivity and specificity were 50%, and 77%, and for urinary endothelial cell-specific molecule-1, 62%, and 71%, respectively.

Serum and urinary endothelial cell-specific molecule-1 concentrations increase in bladder cancer. This parameter also increases in serum and urine of cases with urinary tract infection. That urinary endothelial cell-specific molecule-1 values were higher than serum endothelial cell-specific molecule-1 values in all groups may be attributed to direct exfoliation of epithelial cells in bladder to urine.

Detection of disease-associated mutations in patients with familial hypercholesterolaemia is crucial for early interventions to reduce risk of cardiovascular disease. Screening for these mutations represents a methodological challenge since more than 1200 different causal mutations in the low-density lipoprotein receptor has been identified. A number of methodological approaches have been developed for screening by clinical diagnostic laboratories.

Using primers targeting, the low-density lipoprotein receptor, apolipoprotein B, and proprotein convertase subtilisin/kexin type 9, we developed a novel Ion Torrent-based targeted re-sequencing method. We validated this in a West Midlands-UK small cohort of 58 patients screened in parallel with other mutation-targeting methods, such as multiplex polymerase chain reaction (Elucigene FH20), oligonucleotide arrays (Randox familial hypercholesterolaemia array) or the Illumina next-generation sequencing platform.

In this small cohort, the next-generation sequencing method achieved excellent analytical performance characteristics and showed 100% and 89% concordance with the Randox array and the Elucigene FH20 assay. Investigation of the discrepant results identified two cases of mutation misclassification of the Elucigene FH20 multiplex polymerase chain reaction assay. A number of novel mutations not previously reported were also identified by the next-generation sequencing method.

Ion Torrent-based next-generation sequencing can deliver a suitable alternative for the molecular investigation of familial hypercholesterolaemia patients, especially when comprehensive mutation screening for rare or unknown mutations is required.

Obesity is associated with a state of systemic inflammation, mediated by adipose tissue-derived cytokines that may also have metabolic effects, including an effect on insulin resistance. The aim of this study was to compare the serum profile of pro- and anti-inflammatory cytokines in obese and non-obese subjects.

A total of 242 subjects who were either overweight or obese (body mass index [BMI] ≥ 25 kg/m²) and non-obese subjects (body mass index <25 kg/m²), were recruited in Mashhad in northeastern Iran. The concentrations of serum interleukin-1α, -1β, -2, -4, -6, -8 and -10 (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8 and IL-10), were measured in all subjects, together with serum vascular endothelial growth factor, interferon-γ, epidermal growth factor, monocyte chemoattractant protein-1 and tumour necrosis factor-α.

The groups differed significantly with respect to measures of adiposity and fasted lipid profile. Serum pro-inflammatory cytokines interferon-γ and interleukin-1α, and anti-inflammatory cytokines, interleukin-10, and epidermal growth factor were significantly different between obese and non-obese individuals, as was serum high-sensitivity C-reactive protein. Multivariate regression showed that waist circumference was significantly and independently related to serum monocyte chemoattractant protein-1concentrations (P = 0.001).

Despite significant differences in several cytokines between the groups, only monocyte chemoattractant protein-1appeared to be independently related to a measure of adiposity in this population sample from Iran.

Lab Tests Online-UK celebrated its 10th anniversary in 2014 and to mark the occasion the first comprehensive survey of website users was undertaken.

A pop-up box with a link to Survey Monkey was used to offer website users the chance to participate in the survey, which was live from 4 March 2014 to 11 April 2014.

Six hundred and sixty-one participants started the questionnaire and 338 completed all of the demographic questions. Although the website is designed and aimed at patients and the public, a significant number of respondents were health-care professionals (47%). The majority of survey participants found the Lab Tests Online-UK website via a search engine and were visiting the site for themselves. The majority of participants found what they were looking for on the website and found the information very easy or fairly easy to understand. The patient respondents were keen to see their laboratory test results (87%), but the majority did not have access (60%) at the time of the survey.

This survey provides good evidence that the Lab Tests Online-UK website is a useful resource for patients and health-care professionals alike. It comes at a poignant time as the release of results direct to patients starts with access to their medical records. The Lab Tests Online-UK website has a key role in enabling patients to understand their lab test results, and therefore empowering them to take an interest and engage in their own healthcare.

Serum zinc, copper and selenium are measured in patients prior to commencing on parenteral nutrition; however, their interpretation can be difficult due to acute phase reactions. We assessed (i) the relationship of raised C-reactive protein with trace elements and albumin (ii) benefits of measuring trace elements when C-reactive protein is raised in patients requiring short-term parenteral nutrition.

Samples were collected for zinc, copper, selenium and albumin at baseline and then every two weeks and correlated with C-reactive protein results in patients on parenteral nutrition. Results were categorized into four groups based on the C-reactive protein concentrations: (i) <20 mg/L, (ii) 20–39 mg/L, (iii) 40–79 mg/L and (iv) ≥80 mg/L.

In 166 patients, zinc, selenium and albumin correlated (Spearman’s) negatively with C-reactive protein; r = −0.26, P < 0.001 (95% CI −0.40 to −0.11), r = −0.44, P < 0.001 (−0.56 to −0.29) and r = −0.22 P = 0.005 (−0.36 to −0.07), respectively. Copper did not correlate with C-reactive protein (r = 0.09, P = 0.25 [−0.07 to 0.25]). Comparison of trace elements between the four groups showed no difference in zinc and copper (both P > 0.05), whereas selenium and albumin were lower in the group with C-reactive protein > 40 mg/L (P < 0.05).

In patients on short-term parenteral nutrition, measurement of C-reactive protein is essential when interpreting zinc and selenium but not copper results. Routine measurement of trace elements prior to commencing parenteral nutrition has to be considered on an individual basis in patients with inflammation.

The distributions of within-subject biological variation are usually described as coefficients of variation, as are analytical performance specifications for bias, imprecision and other characteristics. Estimation of specifications required for reference change values is traditionally done using relationship between the batch-related changes during routine performance, described as Δbias, and the coefficients of variation for analytical imprecision (CV_A): the original theory is based on standard deviations or coefficients of variation calculated as if distributions were Gaussian.

The distribution of between-subject biological variation can generally be described as log-Gaussian. Moreover, recent analyses of within-subject biological variation suggest that many measurands have log-Gaussian distributions. In consequence, we generated a model for the estimation of analytical performance specifications for reference change value, with combination of Δbias and CV_A based on log-Gaussian distributions of CV_I as natural logarithms. The model was tested using plasma prolactin and glucose as examples.

Analytical performance specifications for reference change value generated using the new model based on log-Gaussian distributions were practically identical with the traditional model based on Gaussian distributions.

The traditional and simple to apply model used to generate analytical performance specifications for reference change value, based on the use of coefficients of variation and assuming Gaussian distributions for both CV_I and CV_A, is generally useful.

Reference change values provide objective tools to assess the significance of a change in two consecutive results for a biomarker from an individual. The reference change value calculation is based on the assumption that within-subject biological variation has random fluctuation around a homeostatic set point that follows a normal (Gaussian) distribution. This set point (or baseline in steady-state) should be estimated from a set of previous samples, but, in practice, decisions based on reference change value are often based on only two consecutive results. The original reference change value was based on standard deviations according to the assumption of normality, but was soon changed to coefficients of variation (CV) in the formula (reference change value = ± Z ċ 2^½ ċ CV). Z is being dependent on the desired probability of significance, which also defines the percentages of false-positive results. The aim of this study was to investigate false-positive results using five different published methods for calculation of reference change value.

The five reference change value methods were examined using normally and ln-normally distributed simulated data.

One method performed best in approaching the theoretical false-positive percentages on normally distributed data and another method performed best on ln-normally distributed data. The commonly used reference change value method based on two results (without use of estimated set point) performed worst both on normally distributed and ln-normally distributed data.

The optimal choice of method to calculate reference change value limits requires knowledge of the distribution of data (normal or ln-normal) and, if possible, knowledge of the homeostatic set point.

As plasma is the recommended sample type for Roche adrenocorticotropic hormone (ACTH) assay, we evaluated the effect of EDTA concentration on Cobas ACTH assay.

Samples containing twofold and fourfold higher concentrations of EDTA were prepared by adding plasma to empty K2EDTA tubes and by making under-filled EDTA tubes. All measurements were performed with four replicates.

Increased EDTA concentration resulted in a significant decrease in ACTH concentration. Fifty-per cent-filled EDTA tube showed 19% decrease in ACTH concentration and 25% filled EDTA tube showed 50% decrease in ACTH concentration.

We recommend that inadequately filled EDTA specimens should be rejected when using Cobas ACTH assay.

The measurement of cardiac troponin is central for the diagnosis of myocardial infarction (MI). It is recommended that a coefficient of variation of ≤10% is achieved at the diagnostic threshold and significant change between serial measurements reported. Many modern laboratories use multiple analysers linked by automation where samples are randomly assigned to an analyser. It is therefore important to consider the combined effect of all analysers on the analytical performance of troponin measurement.

The performance of a contemporary troponin-I (cTn-I) assay run on three analysers, linked by an automated track, was undertaken across a range of cTn-I concentrations. The data for the three analysers were aggregated to obtain the combined analytical coefficient of variation (CV_A) and reference change values (RCVs).

The CV_A improved with increasing concentration and calculated RCVs ranged from 67.2% (±13 ng/L) to 32% (±160 ng/L) between cTn-I values 20 ng/L and 500 ng/L. Although there were significant differences in cTn-I measurement between analysers around the diagnostic threshold (P < 0.05), the CV_A was 13.6%.

We demonstrate that there are significant differences between the performances of analysers which can impact the biochemical criteria for the diagnosis of MI. We also show that the RCV varies according to baseline cTn-I values and that reporting a single RCV across the analytical range of cTn-I may not be appropriate.