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Background Colorectal cancer (CRC) screening using faecal tests reduces disease-specific mortality. To investigate mortality and its association with sex, rates in women and men, and in different age ranges, were examined, before and after screening began in Scotland. Methods From 1990–99, no structured screening existed. Three pilots ran from 2000 to 2007 and subsequent full roll-out completed in 2009. Crude mortality rates for 1990–2020 were calculated relative to Scottish population estimates, and age–sex standardized rates calculated for all, pre-screening (74 years) age ranges. Results CRC mortality declined from 1990 to 2020, but not linearly, and differed between sexes. In women, 1990–99 showed a steady decline [average annual percentage change (AAPC): −2.1%, 95% confidence interval (CI): −2.8% to −1.4%], but a less marked decline after 2000 (AAPC: −0.7%, 95% CI: −0.9% to −0.4%). In men, no clear decline was seen from 1990 to 1999 (AAPC: −0.4%, 95% CI: −1.1% to 0.4%), but mortality declined from 2000 to 2020 (AAPC: −1.7%, 95% CI: −1.9% to −1.5%). This pattern was exaggerated in the screening age ranges. For 2000–20, the overall reduction in mortality was less in women and in the screening age range. In the post-screening age range, reductions were smaller, but an increase was seen in the pre-screening age range, greater in women. Conclusions CRC mortality fell during 1990–2020, but the decline differed markedly between sexes, indicating a larger beneficial effect of screening on CRC mortality in men compared to women: use of different thresholds for the sexes might lead to equality.

Objective How faecal haemoglobin concentrations (f-Hb) vary from one round to the next in a colorectal cancer (CRC) screening programme, and relate to colonoscopy findings, are unknown. Our aim was to use data from the first two rounds of the faecal immunochemical test (FIT) based Scottish Bowel Screening Programme (SBoSP) to explore these issues. Methods Faecal haemoglobin concentration (f-Hb) percentiles in the second round were compared with those in the first when the first round yielded a negative FIT result (Results The profiles of f-Hb in the first and second rounds differed in (a) those who had had a negative FIT result in the first round and (b) those in whom neoplastic pathology had been found. In contrast, the pattern of difference between profiles in those who had had a negative colonoscopy was very similar to that in those in whom an adenoma had been found. In addition, the risk of CRC being diagnosed in the second round after a negative colonoscopy in the first was 3.0%, not very different to that after a negative test result (4.9%). Conclusions Adenomas may be rarely the cause of a positive FIT result. An alternative explanation as to why these are detected using FIT is required. In addition, a negative colonoscopy for a positive FIT result does not rule out the finding of significant neoplastic pathology in the next round.

Background Faecal Immunochemical tests (FITs) in the assessment of patients presenting with symptoms have generally used a single sample. Little evidence pertains to the use of replicate, where a number of tests are done prior to decision-making or repeat FIT, where additional FIT are performed following clinical decision-making. Overwhelmingly, research has focussed on FIT to help identify colorectal cancer (CRC). The aim of this review is to assess the available literature concerning replicate and repeat FIT in symptomatic patients to help generate consensus and guide future research. Methods The terms ‘faecal immunochemical test’ or ‘FIT’ were combined with ‘multiple’ or ‘repeat’. EMBASE, Medline and PubMed database and other searches were conducted. All papers published in English were included with no exclusion date limits until November 2021. Results Of the 161 initial papers screened, seven were included for review. Qualitative and quantitative FIT outcomes were assessed in the studies. The primary aims of most related to whether replicate FIT increased diagnostic yield of CRC, with colonoscopy used as the reference standard. One publication assessed the impact of a new COVID-adapted pathway on CRC detection. No consensus on replicate FIT was apparent. Some concluded that FITs may help minimise missed CRC diagnoses: others showed no increase in diagnostic yield of CRC. Conclusions Current evidence on replicate and repeat FIT is both minimal and conflicting. FIT is a superb clinical tool, but significant gaps surrounding application remain. Further studies relating to replicate and repeat FIT are required.

Objectives The exploration of the metabolites in the degradation pathways of vitamin D (VTD) has gained importance in recent years and simultaneous quantitation of twenty-five-hydroxy vitamin D (25(OH)D) mass concentration together with 24,25-dihydroxyvitamin D (24,25(OH)2D) has been proposed as a newer approach to define VTD deficiency. Yet, no data are available on 24,25(OH)2D biological variation (BV). In this study, we evaluated 24,25(OH)2D’s BV on the European Biological Variation Study (EuBIVAS) cohort samples to determine if analytical performance specifications (APS) for 24,25(OH)2D could be generated. Methods Six European laboratories recruited 91 healthy participants. 25(OH)D and 24,25(OH)2D concentrations in K3-EDTA plasma were examined weekly for up to 10 weeks in duplicate with a validated LC-MS/MS method. The Vitamin D Metabolite Ratio (24,25(OH)2D divided by 25(OH)D × 100) was also calculated at each time point. Results Linear regression of the mean 24,25(OH)2D concentrations at each blood collection showed participants were not in steady state. Variations of 24,25(OH)2D over time were significantly positively associated with the slopes of 25(OH)D concentrations over time and the concentration of 25(OH)D of the participant at inclusion, and negatively associated with body mass index (BMI), but not with age, gender, or location of the participant. The variation of the 24,25(OH)2D concentration in participants over a 10 weeks period was 34.6%. Methods that would detect a significant change linked to the natural production of 24,25(OH)2D over this period at pu%) Conclusions We have defined for the first time APS for 24,25(OH)2D examinations. According to the growing interest in this metabolite, several laboratories and manufacturers might aim to develop specific methods for its determination. The results presented in this paper are thus necessary prerequisites for the validation of such methods.

Objective Guidelines on colorectal cancer (CRC) screening with guaiac faecal occult blood tests (gFOBTs) and faecal immunochemical tests (FITs) include the need for a pilot before a programme is introduced. Interval cancers (ICs), cancers arising after a negative screening test result but before the next scheduled invite, are important indicators of programme quality. Our aim was to compare IC in the gFOBT-based Scottish Bowel Screening Programme (SBoSP), a FIT-based pilot, and the FIT-based SBoSP, to assess if the pilot provided data that was reflected in the subsequent programme. Design The IC proportions (ICPs) data ([IC/(IC + screen detected CRC)] x 100) from the penultimate year of the gFOBT-based SBoSP, the 6-month pilot and the first year of the FIT-based SBoSP were compared. To ensure appropriate comparison, these data were only from the two pilot NHS Boards. Results For all participants, and females and males, the ICPs were very similar in the gFOBT-based SBoSP and the pilot. The faecal haemoglobin concentration (f-Hb) threshold for the pilot was set at ≥80 μg Hb/g faeces. However, in marked contrast, in the FIT-based SBoSP, at the same threshold, the ICPs were lower. In all three groups, the ICPs were higher in females than in males. Conclusions Data on variables in pilots, including ICP, can be informative, but only if variables such as FIT system are held consistent between pilot and programme. Lowering the f-Hb threshold for females to give the same ICP as males might be a strategy to minimise sex inequality.

Objective There is evidence that colorectal cancer screening using faecal haemoglobin is less effective in women than men. The faecal haemoglobin concentrations were therefore examined in women and men with screen-detected colorectal cancer. Setting Scottish Bowel Screening Programme, following the introduction of a faecal immunochemical test from November 2017, to March 2020. Methods Data were collated on faecal haemoglobin concentrations, pathological stage and anatomical site of the main lesion in participants who had colorectal cancer detected. The data in women and men were compared. Results For the faecal haemoglobin concentrations studied (>80 µg Hb/g faeces), the distributions indicated lower concentrations in women. Marked differences were found between women and men diagnosed with colorectal cancer. The median faecal haemoglobin concentration for women ( n = 720) was 408 µg Hb/g faeces compared to 473 µg Hb/g faeces for men ( n = 959) ( p = 0.004) and 50.6% of the results were >400 µg Hb/g faeces in women; in men, this was 57.8%. The difference in faecal haemoglobin concentrations in women and men became less statistically significant as stage advanced from stages I–IV. For right-sided, left-sided and rectal colorectal cancer, a similar gender difference persisted in all sites. Differences in faecal haemoglobin between the genders were significant for left-sided cancers and stage I and approached significance for rectal cancers and stage II, but all sites and stages showed lower median faecal haemoglobin concentrations for women. Conclusions To minimise gender inequalities, faecal immunochemical test-based colorectal cancer screening programmes should evaluate a strategy of using different faecal haemoglobin concentration thresholds in women and men.

Objectives This study aimed to develop a risk-scoring model in the Scottish Bowel Screening Programme incorporating faecal haemoglobin concentration with other risk factors for colorectal cancer. Methods Data were collected for all individuals invited to participate in the Scottish Bowel Screening Programme between November 2017 and March 2018 including faecal haemoglobin concentration, age, sex, National Health Service Board, socioeconomic status, and screening history. Linkage with The Scottish Cancer Registry identified all screening participants diagnosed with colorectal cancer. Logistic regression was performed to identify which factors demonstrated significant association with colorectal cancer and could be used in the development of a risk-scoring model. Results Of 232,076 screening participants, 427 had colorectal cancer: 286 diagnosed following a screening colonoscopy and 141 arising after a negative screening test result giving an interval cancer proportion of 33.0%. Only faecal haemoglobin concentration and age showed a statistically significant association with colorectal cancer. Interval cancer proportion increased with age and was higher in women (38.1%) than men (27.5%). If positivity in women were mirrored in men at each age quintile interval cancer proportion would still have remained higher in women (33.2%). Moreover, an additional 1201 colonoscopies would be required to detect 11 colorectal cancers. Conclusions Development of a risk scoring model using early data from the Scottish Bowel Screening Programme was not feasible due to most variables showing insignificant association with colorectal cancer. Tailoring the faecal haemoglobin concentration threshold according to age could help to diminish some of the disparity in interval cancer proportion between women and men. Strategies to achieve sex equality using faecal haemoglobin concentration thresholds depend considerably on which variable is selected for equivalency and this requires further exploration.

Background: Fecal immunochemical test (FIT) performance can be affected by post-collection variables. Collection technique might also affect fecal hemoglobin concentration (f-Hb). Variation in quantity of feces collected in samples returned in a colorectal cancer detection program, and the effects of under-sampling, were assessed. Methods: Collection devices obtained from patients undergoing FIT were assessed for the color (in five classes) of the feces in buffer, mass, and f-Hb. Associations between these were examined in an in vitro study on Hb-spiked feces. Variables possibly associated with under-sampling were investigated using multivariable logistic regression. The effect of low sample mass on clinical performance (false-negative results) was determined. Results: Of 6,898 samples collected by 3,449 individuals (46.9% male, median age: 65.3 years), the buffer was lightest in color in 362 (5.2%), and darkest in 420 (6.1%). Samples with the lightest color had a significantly lower f-Hb compared with all darker classes (P < 0.001). Mass was recorded for 650 devices: The lightest colored samples had significantly lower mass (P < 0.05). The correlation between mass and f-Hb was confirmed in vitro (r = 0.897, P < 0.001). Low mass was not associated with age, sex, or technical factors (P > 0.05). Under-sampling related to the lightest color was not associated with false-negative results for colorectal cancer and advanced adenoma, but was for all neoplasia and inflammatory bowel disease. Conclusions: Wide variation existed in the amount of feces collected. Under-sampling results in lower measured f-Hb and may increase false-negative results. Impact: Color of sample buffer could be used to identify inadequate sampling.

Background Faecal haemoglobin concentration (f-Hb), estimated using a faecal immunochemical test, can be safely implemented in primary care to assess risk of colorectal cancer (CRC). Clinical outcomes of patients presenting with symptoms of lower gastrointestinal disease were examined using an extensive range of f-Hb thresholds to decide on reassurance or referral for further investigation. Methods All patients who attended primary care and submitted a single faecal specimen faecal immunochemical test in the first year of the routine service had f-Hb estimated using HM-JACKarc: f-Hb thresholds from Results Low f-Hb thresholds of 400 µg/g, ≥200 µg/g and ≥100 µg/g. However, patients with a f-Hb concentration of Conclusions In primary care, f-Hb, in conjunction with clinical assessment, can safely and objectively determine individual risk of CRC and decide on simple reassurance or urgent, or routine referral.

Currently, women are disadvantaged compared to men in colorectal cancer (CRC) screening, particularly in programmes that use faecal immunochemical tests for haemoglobin (FIT) followed by colonoscopy. Although there is no single cause for all the known disadvantages, many can be attributed to the ubiquitous finding that women have lower faecal haemoglobin concentrations (f-Hb) than men; there are many plausible reasons for this. Generally, a single f-Hb threshold is used in CRC screening programmes, leading to lower positivity for women than men, which causes poorer outcomes for women, including lower CRC detection rate, higher interval cancer (IC) proportion, and higher CRC mortality. Many of the now widely advocated risk scoring strategies do include factors taking account of sex, but these have not been extensively piloted or introduced. Using different f-Hb thresholds for the sexes seems advantageous, but there are difficulties, including deciding which characteristic should be selected to achieve equivalency, for example, positivity, IC proportions, or specificity. Moreover, additional colonoscopy resources, often constrained, would be required. Governments and their agencies should be encouraged to prioritise the allocation of resources to put simple strategies into practice, such as different f-Hb thresholds to create equal positivity in both sexes.

Objective Roll-out of population-based colorectal cancer (CRC) screening with faecal immunochemical test (FIT) is limited by availability of further investigations, particularly colonoscopy and examination of excised lesions. Our objective was to assess whether variation in number of faecal samples and threshold adjustment can optimise resource utilisation and CRC detection rate. Methods Three different screening strategies were compared for the same FIT threshold using a quantitative FIT system: one FIT, positive when >20 µg Hb/g faeces; two FIT, positive when either was >20 µg Hb/g faeces; and two FIT, positive when the mean was >20 µg Hb/g faeces. We calculated changes in the size of population the provider could invite to screening for an equal number of screening positive results, and CRC and adenoma detected. Results In our setting, Region of Murcia, south of Spain (not fully rolled out screening programme), changing the usual strategy of two FIT, positive when either to positive when the mean was >20 µg Hb/g faeces, would increase population invited by 37.81% with the same number of positive results (which would generate a CRC detection rate of 19.2%). In a fully rolled out programme, changing the strategy from one to two FIT (positive when the mean is >20 µg Hb/g faeces), would increase CRC detection rate by 4.64% with an increase of only 13.34% in positive FIT. Conclusions In a population-based CRC screening programme, smart use of number of FITs and positivity threshold can increase population invited and CRC detection without increasing the number of colonoscopies and pathological examinations needed.

AimsThe presence of detectable faecal haemoglobin (f-Hb) has been shown to be associated with all-cause mortality and with death from a number of chronic diseases not known to cause gastrointestinal blood loss. This effect is independent of taking medicines that increase the risk of bleeding. To further investigate the association of f-Hb with chronic disease, the relationship between f-Hb and prescription of medicines for a variety of conditions was studied.MethodsAll subjects (134 192) who participated in guaiac faecal occult blood test (gFOBT) screening in Tayside, Scotland, between March 2000 and March 2016, were studied in a cross-sectional manner by linking their gFOBT result (abnormal or normal) with prescribing data at the time of the test.ResultsThe screening participants with an abnormal gFOBT result were more likely to have been being prescribed medicines for heart disease, hypertension, diabetes and depression than those with a normal test result. This association persisted after adjustment for sex, age and deprivation (OR 1.35 (95%CI 1.23 to 1.48), 1.39 (1.27 to 1.52), 1.35 (1.15 to 1.58), 1.36 (1.16 to 1.59), all pConclusionsThe results of this study confer further substantial weight to the concept that detectable f-Hb is associated with a range of common chronic conditions that have a systemic inflammatory component; we speculate that f-Hb might have potential in identifying individuals who are high risk of developing chronic conditions or are at an early stage of disease.

Background Quantitative faecal immunochemical tests measure faecal haemoglobin concentration (f-Hb), which increases in the presence of colorectal neoplasia. Objective We examined the diagnostic accuracy of faecal immunochemical test (FIT)in patients at increased risk of colorectal cancer (CRC) attending for surveillance colonoscopy as per national guidelines. Methods A total of 1103 consecutive patients were prospectively invited to complete a FIT before their scheduled colonoscopy in two university hospitals in 2014– 2016. F-Hb was analysed on an OC-Sensor io automated analyser (Eiken Chemical Co., Ltd, Tokyo, Japan) with a limit of detection of 2 µg Hb/g faeces. The diagnostic accuracy of f-Hb for CRC and higher-risk adenoma was examined. Results A total of 643 patients returned a faecal test. After excluding 4 patients with known inflammatory bowel disease, 639 (57.9%) remained in the study: age range: 25–90 years (median: 64 years, interquartile range (IQR): 55–71): 54.6% male. Of 593 patients who also completed colonoscopy, 41 (6.9%) had advanced neoplasia (4 CRC, 37 higher-risk adenoma). Of the 238 patients (40.1%) who had detectable f-Hb, 31 (13.0%) had advanced neoplasia (2 CRC, 29 higher-risk adenoma) compared with 10 (2.8%) in those with undetectable f-Hb (2 CRC, 8 higher-risk adenoma). Detectable f-Hb gave negative predictive values of 99.4% for CRC and 97.2% for CRC plus higher-risk adenoma. Conclusion In patients at increased risk of CRC under colonoscopy surveillance, a test measuring faecal haemoglobin can provide an objective estimate of the risk of advanced neoplasia, and could enable tailored scheduling of colonoscopy.

Objectives Faecal immunochemical tests for haemoglobin (FIT) are becoming widely used in colorectal cancer (CRC) screening and assessment of symptomatic patients. Faecal haemoglobin concentration (f-Hb) thresholds are used to guide subsequent investigation. We established the distributions of f-Hb in a large screening population by sex, age, deprivation and geography. Methods Single estimates of f-Hb were documented for all individuals participating in the first 18 months of the Scottish Bowel Screening Programme (SBoSP). The distributions of f-Hb were generated for all participants, all men and women, and men and women by age quintile and deprivation quintile. Distributions were also generated by geographical region for all participants, men and women, and by deprivation. Comparisons of f-Hb distributions with those found in a pilot evaluation of FIT and three other countries were performed. Results f-Hb was documented for 887,248 screening participants, 422,385 men and 464,863 women. f-Hb varied by sex, age, deprivation quintile and geographical region. The f-Hb distributions by sex and age differed between the SBoSP and the pilot evaluation and the three other countries. Conclusions f-Hb is higher in men than in women and increases with age and deprivation in both sexes. f-Hb also varies by geographical region, independently of deprivation, and by country. The f-Hb distribution estimated by pilot evaluation may not represent the population distribution. Decision limits have advantages over reference intervals. Use of partitioned f-Hb thresholds for further investigation, based on the data generated, has advantages and disadvantages, as do risk scores based on a spectrum of influencing variables.

Background Polypectomy may be performed at colonoscopy and then subsequent surveillance undertaken. It is thought that faecal haemoglobin concentration (f-Hb), estimated by quantitative faecal immunochemical tests (FIT), might be a useful tumour marker. Methods Consecutive patients enrolled in colonoscopy surveillance were approached at two hospitals. A specimen for FIT was provided before colonoscopy and, ideally after 3 weeks, a second FIT sample from those who had polypectomy. A single FIT system (OC-Sensor io, Eiken Chemical Co., Ltd) was used to generate f-Hb. Results 1103 Patients were invited; 643 returned a FIT device (uptake: 58.3%). Four patients had known inflammatory bowel disease (IBD) and were excluded, leaving 639 (57.9%) with an age range of 25–90 years (median 64 years), 54.6% male. Of 593 patients who had a f-Hb result and completed colonoscopy, advanced neoplasia was found in 41 (6.9%); four colorectal cancer (CRC): 0.7% and 37 advanced adenoma (AA): 6.3%, and a further 127 (21.4%) had non-advanced adenoma (NAA). The median f-Hb was significantly greater in AA as compared to NAA; 6.0 versus 1.0 μg Hb/g faeces, p < 0.0001.134/164 (81.7%) of invited patients returned a second FIT device: 28 were patients with AA in whom median pre-polypectomy f-Hb was 19.2, falling to 3.5 μg Hb/g faeces post-polypectomy, p = 0.01, and 106 with NAA had median pre-polypectomy f-Hb 0.8 compared to 1.0 μg Hb/g faeces post-polypectomy, p = 0.96. Conclusions Quantitative FIT could provide a good tumour marker in post-polypectomy surveillance, reduce colonoscopy requirements and minimise potential risk to patients.

Objectives Faecal immunochemical tests for haemoglobin (FIT) are used in colorectal cancer (CRC) screening programmes and to triage patients presenting with symptoms suggestive of CRC for further bowel investigations. There are a number of quantitative FIT analytical systems available. Currently, there is no harmonisation or standardisation of FIT methods. The aim of the study was to assess the comparability of numerical faecal haemoglobin concentrations (f-Hb) obtained with four quantitative FIT systems and the diagnostic accuracy at different f-Hb thresholds. Methods A subgroup of the National Institute for Health and Care Excellence (NICE) FIT study, a multicentre, prospective diagnostic accuracy study were sent four FIT specimen collection devices from four different FIT systems or two FIT devices for one FIT system. Faecal samples were examined and analysis of results carried out to assess difference between methods at thresholds of limit of detection (LoD), 10 µg haemoglobin/g faeces (µg/g) and 100 μg/g. Results 233 patients returned specimen collection devices for examination on four different systems; 189 patients returned two FIT kits for one system. At a threshold of 100 μg/g the sensitivity is the same for all methods. At lower thresholds of LoD and 10 μg/g differences were observed between systems in terms of patients who would be referred and diagnostic accuracies. Conclusions The lack of standardisation or harmonisation of FIT means that differences are observed in f-Hb generated on different systems. Further work is required to understand the clinical impact of these differences and to minimise them.

Background Current guidelines document persistent rectal bleeding as an alarm symptom in patients presenting to primary care. We studied whether a faecal immunochemical test could assist in their assessment. Methods From December 2015, faecal immunochemical tests were routinely available to primary care when assessing patients with new-onset bowel symptoms: general practitioners were encouraged to include faecal haemoglobin concentration (f-Hb) within any referral to secondary care. Results with f-Hb ≥10 μg Hb/g faeces were defined as positive. The incidence of significant bowel disease (SBD: colorectal cancer [CRC], higher-risk adenoma [HRA: any ≥1 cm, or three or more] and inflammatory bowel disease [IBD]) at subsequent colonoscopy, referred symptoms and f-Hb were recorded. Results Of 1447 patients with a faecal immunochemical test result and colonoscopy outcome, SBD was diagnosed in 296 patients (20.5%; 95 with CRC, 133 with HRA, and 68 with IBD). Four hundred and sixty-two patients (31.9%) reported rectal bleeding: 294 had f-Hb ≥10 μg Hb/g faeces. At colonoscopy, 105/294 had SBD versus 14/168 with rectal bleeding and f-Hb Conclusion Patients with rectal bleeding and f-Hb

Objectives Flexible sigmoidoscopy screening at around age 60 can reduce colorectal cancer incidence. Insufficient evidence exists on flexible sigmoidoscopy at age 60 in a population being offered biennial faecal occult blood test screening from age 50. This randomized controlled trial assessed if flexible sigmoidoscopy would be an effective adjunct to faecal occult blood test. Methods In the Scottish Bowel Screening Programme between June 2014 and December 2015, 51,769 individuals were randomized to be offered flexible sigmoidoscopy instead of faecal occult blood test at age 60 or to continue faecal occult blood test. Those not accepting flexible sigmoidoscopy and those with normal flexible sigmoidoscopy were offered faecal occult blood test. All with flexible sigmoidoscopy-detected neoplasia or a positive faecal occult blood test result were offered colonoscopy. Results Overall flexible sigmoidoscopy uptake was 17.8%, higher in men than women, and decreased with increasing deprivation (25.7% in the least to 9.2% in the most deprived quintile). In those who underwent flexible sigmoidoscopy, detection rate for colorectal cancer was 0.13%, for adenoma 7.27%, and for total neoplasia 7.40%. In those who underwent colonoscopy after a positive flexible sigmoidoscopy, detection rate for colorectal cancer was 0.28%, adenoma 8.66%, and total neoplasia 8.83%. On an intention to screen basis, there was no difference in colorectal cancer detection rate between the study and control groups. Adenoma and total neoplasia detection rate were significantly higher in the study group, with odds ratios of 5.95 (95%CI: 4.69–7.56) and 5.10 (95%CI: 4.09–6.35), respectively. Conclusions In a single screening round at age 60, there was low uptake and neoplasia detection rate. Flexible sigmoidoscopy detected significantly more neoplasia than faecal occult blood test alone.

Background Monthly medians of patient results are useful in assessment of analytical quality in medical laboratories. Separate medians by gender makes it possible to generate two independent estimates of contemporaneous errors. However, for plasma creatinine, reference intervals (RIs) are different by gender and also higher over 70 years of age. Methods Daily, weekly and monthly patient medians were calculated from the raw data of plasma creatinine concentrations for males between 18 and 70 years, males >70 years, females between 18 and 70 years and females >70 years. Results The medians of the four groups were all closely associated, with similar patterns. The mean of percentage bias from each group defined the best estimate of bias. The maximum half-range (%) of the bias evaluations provided an estimate of the uncertainty comparable to the analytical performance specifications: thus, bias estimates could be classified as optimum, desirable or minimum quality. Conclusions Medians by gender and age are useful in assessment of analytical stability for plasma creatinine concentration ranging from 60 to 90 μmol/L. The daily medians are valuable in rapid detection of large systematic errors, the weekly medians in detecting minor systematic errors and monthly medians in assessment of long-term analytical stability.

Background Reference change values are used to assess the significance of a difference in two consecutive results from an individual. Reference change value calculations provide the limits for significant differences between two results due to analytical and inherent biological variations. Often more than two serial results are available. Using the reference change value concept on more than two measurements results in an increased number of false-positive results. This problem has been solved for both uni- and bidirectional differences through use of wider limits when additional results are included. Methods Based on normally (Gaussianly) distributed simulated data, a dynamic reference change value model was developed using more than two results and total coefficients of variation. The dynamic reference change value model includes validation of a set-point as the mean of the four first serial results and additional results are assessed for compliance to the steady state with the same set-point. Furthermore, the dynamic reference change value model compensates for increasing false-positive results with subsequent results. The dynamic reference change value model was designed to calculate significant limits for bidirectional differences. Results Reference change factors were calculated for multiplication of the mean of previous results to create the limits for significant differences. The reference change factors are provided as a function of number of results and total coefficients of variation both in tables and in figures. Conclusions The dynamic reference change value model is appropriate for ongoing assessment of the steady state of a biomarker using more than two serial results.

Currently the 25-hydroxy vitamin D (25(OH)D) concentration is thought to be the best estimate of the vitamin D status of an individual. Unfortunately, its measurement remains complex, despite recent technological advances. We evaluated the biological variation (BV) of 25(OH)D in order to set analytical performance specifications (APS) for measurement uncertainty (MU). Six European laboratories recruited 91 healthy participants. The 25(OH)D concentrations in K3-EDTA plasma were examined weekly for up to 10 weeks in duplicate on a Lumipulse G1200 (Fujirebio, Tokyo, Japan). The linear regression of the mean 25(OH)D concentrations at each blood collection showed that participants were not in a steady state. The dissection of the 10-sample collection into two subsets, namely collections 1–5 and 6–10, did not allow for correction of the lack of homogeneity: estimates of the within-subject BV ranged from 5.8% to 7.1% and the between-subject BV ranged from 25.0% to 39.2%. Methods that would differentiate a difference induced by 25(OH)D supplementation at p &lt, 0.05 should have MU &lt, 13.6%, while at p &lt, 0.01, the MU should be &lt, 9.6%. The development of APS using BV assumes a steady state of patients. The findings in this study suggest that patients are not in steady state. Therefore, APS that are based on MU appear to be more appropriate.

AIM: Lower gastrointestinal (GI) symptoms are poor predictors of colorectal cancer (CRC). The aim of this study was to examine the diagnostic yield of colonoscopy by faecal haemoglobin (f-Hb) concentration in symptomatic patients assessed in primary care by faecal immunochemical testing (FIT). METHOD: In three Scottish NHS Boards, FIT kits (HM-JACKarc, Hitachi Chemical Diagnostics Systems Co., Ltd, Tokyo, Japan) were used by general practitioners to guide referrals for patients with lower GI symptoms (laboratory data studied for 12 months from December 2015 onwards in Tayside, 18 months from June 2018 onwards in Fife and 5 months from September 2018 onwards in Greater Glasgow and Clyde). Cases of CRC diagnosed at colonoscopy were ascertained from colonoscopy and pathology records. RESULTS: Four thousand eight hundred and forty one symptomatic patients who underwent colonoscopy after FIT submission were included. Of the 2166 patients (44.7%) with f-Hb

ObjectivesIn developed countries, the incidence of colorectal cancer (CRC) has declined in the over 50 years age group but increased in younger people. We studied CRC incidence by age and the influence of screening uptake.DesignAge-standardised and sex-standardised incidences for CRC from 1997 to 2017 were obtained from the Scottish Cancer Registry (SCR). In addition, linkage between the Scottish Bowel Screening Database and the SCR allowed investigation of any association between screening participation and CRC incidence.SettingScotland and the Scottish Bowel Screening Programme, in which guaiac faecal occult blood test screening was piloted from March 2000 and fully rolled by December 2009.ParticipantsFrom the introduction of screening in 2000 through to 2017, 2 395 172 were invited to participate, of whom 1 487 999 participated at least once.Main outcome measuresIncidence of CRC.ResultsIn the screening age range (50–74 years), CRC incidence peaked at 156.5 cases per 100 000 in 2010 after full roll-out of screening across Scotland but fell to 123.9 per 100 000 in 2017. However, under 50 years, there was a rise from 5.3 cases per 100 000 in 2000 to 6.8 per 100 000 in 2017. When CRC incidence was examined in those who had been offered screening, incidence fell in the participant group more than in the non-participant group after roll-out of screening was complete. Analysis of cumulative incidence demonstrated that CRC incidence in the participant group remained consistently below that of the non-participant from around 7 years of follow-up.ConclusionsThe incidence of CRC in Scotland has declined in the over 50 years age group but increased in younger people. It is likely that population screening has contributed to the reduction in CRC incidence in the over 50 years age group.

I read with interest the CMAJ article “The appropriate use of fecal immunochemical testing”[1][1] (FIT). I agree with most of the points made and firmly support the view that there is no evidence that fecal occult blood testing (FOBT) and FIT are of any value in assessment of inpatients in

Aim The delivery of the Scottish Bowel Screening Programme (SBoSP) is rooted in the provision of a high quality, effective and participant-centred service. Safe and effective colonoscopy forms an integral part of the process. Additional accreditation as part of a multi-faceted programme for participating colonoscopists, as in England, does not exist in Scotland. This study aimed to describe the quality of colonoscopy in the SBoSP and compare this to the English national screening standards. Methods Data were collected from the SBoSP between 2007 and 2014. End-points for analysis were caecal intubation, cancer, polyp and adenoma detection, and complications. Overall results were compared with 2012 published English national standards for screening and outcomes from 2006 to 2009. Results During the study period 53 332 participants attended for colonoscopy. The colonoscopy completion rate was 95.6% overall. The mean cancer detection rate was 7.1%, the polyp detection rate was 45.7% and the adenoma detection rate was 35.5%. The overall complication rate was 0.47%. Conclusion Colonoscopy quality in the SBoSP has exceeded the standard set for screening colonoscopy in England, despite not adopting a multi-faceted programme for screening colonoscopy. However, the overall adenoma detection rate in Scotland was 9.1% lower than that in England which has implications for colonoscopy quality and may have an impact on cancer prevention rates, a key aim of the SBoSP.

Background:Recently, the use of separate gender-partitioned patient medians of serum sodium has revealed potential for monitoring analytical stability within the optimum analytical performance specifications for laboratory medicine. The serum albumin concentration depends on whether a patient is sitting or recumbent during phlebotomy. We therefore investigated only examinations requested by general practitioners (GPs) to provide data from sitting patients.Methods:Weekly and monthly patient medians of serum albumin requested by GP for both male and female patients were calculated from the raw data obtained from three analysers in the hospital laboratory on examination of samples from those >18 years. The half-range of medians were applied as an estimate of the maximum bias. Further, the ratios between the two medians were calculated (females/males).Results:The medians for male and female patients were closely related despite considerable variation due to the current analytical variation. This relationship was confirmed by the calculated half-range for the monthly ratio between the genders of 0.44%, which surpasses the optimum analytical performance specification for bias of serum albumin (0.72%). The weekly ratio had a half-range of 1.83%, which surpasses the minimum analytical performance specifications of 2.15%.Conclusions:Monthly gender-partitioned patient medians of serum albumin are useful for monitoring of long-term analytical stability, where the gender medians are two independent estimates of changes in (delta) bias: only results requested by GP are of value in this application to ensure that all patients are sitting during phlebotomy.

Background Uptake of population-based screening for colorectal cancer in Scotland is around 55 per cent. Abdominal aortic aneurysm (AAA) screening has recently been introduced for men aged 65 years and the reported uptake is 78 per cent. The aim was to determine the impact of a brief intervention on bowel screening in men who attended AAA screening, but previously failed to complete bowel screening. Methods Men invited for AAA screening between September 2015 and March 2016 within NHS Tayside were included. Attendees who had not responded to their latest bowel screening invitation were seen by a colorectal cancer clinical nurse specialist. Reasons for not completing the faecal occult blood test (FOBT) were recorded; brief information on colorectal cancer screening was communicated, and participants were offered a further invitation to complete a FOBT. Those who responded positively were sent a further FOBT from the Scottish Bowel Screening Centre. Subsequent return of a completed FOBT within 6 months was recorded. Results A total of 556 men were invited for AAA screening, of whom 38·1 per cent had not completed a recent FOBT. The primary reason stated for not participating was the time taken to complete the test or forgetting it (35·1 per cent). Other reasons included: lack of motivation (23·4 per cent), confusion regarding the aim of screening (16·2 per cent), disgust (19·8 per cent), fear (6·3 per cent) and other health problems (9·9 per cent). Following discussion, 81·1 per cent agreed to complete the FOBT and 49 per cent subsequently returned the test. Conclusion A substantial proportion of previous bowel screening non-responders subsequently returned a completed FOBT following a brief intervention with a nurse specialist. Attendance at non-bowel screening appointments may provide a valuable opportunity to improve bowel screening uptake.

Background Many clinical decisions are based on comparison of patient results with reference intervals. Therefore, an estimation of the analytical performance specifications for the quality that would be required to allow sharing common reference intervals is needed. The International Federation of Clinical Chemistry (IFCC) recommended a minimum of 120 reference individuals to establish reference intervals. This number implies a certain level of quality, which could then be used for defining analytical performance specifications as the maximum combination of analytical bias and imprecision required for sharing common reference intervals, the aim of this investigation. Methods Two methods were investigated for defining the maximum combination of analytical bias and imprecision that would give the same quality of common reference intervals as the IFCC recommendation. Method 1 is based on a formula for the combination of analytical bias and imprecision and Method 2 is based on the Microsoft Excel formula NORMINV including the fractional probability of reference individuals outside each limit and the Gaussian variables of mean and standard deviation. The combinations of normalized bias and imprecision are illustrated for both methods. The formulae are identical for Gaussian and log-Gaussian distributions. Results Method 2 gives the correct results with a constant percentage of 4.4% for all combinations of bias and imprecision. Conclusion The Microsoft Excel formula NORMINV is useful for the estimation of analytical performance specifications for both Gaussian and log-Gaussian distributions of reference intervals.

Objective Age, sex, and deprivation are known factors influencing colorectal (bowel) cancer screening uptake. We investigated the influence of these factors on uptake over time. Methods Data from the Scottish Bowel Screening Programme (SBoSP) were collected between 2007 and 2014. End-points for analysis were uptake, faecal occult blood test positivity, and disease detection, adjusted for age, sex, deprivation, and year of screening. Results From 5,308,336 individual screening episodes documented, uptake gradually increased with increasing age up to 65–69 and was lower in men than women (52.4% vs. 58.7%, respectively). Deprivation had a significant effect on uptake by men and women of all age groups, with the most deprived least likely to complete a screening test. Uptake has increased with time in both sexes and across the deprivation gradient. The number needed to screen to detect significant neoplasia was significantly lower in men than women overall (170 vs. 365), and this held over all age and deprivation groups. The number needed to screen was also lower in the more deprived population. Conclusions Although lower age, male sex, and increased deprivation are associated with lower bowel cancer screening uptake in Scotland, uptake has increased since SBoSP introduction in all age groups, both sexes, and across the deprivation gradient. Despite a lower uptake, the number needed to screen to find significant disease was lower in men and in those with higher levels of deprivation.

Prediction models for colorectal cancer (CRC) detection in symptomatic patients, based on easily obtainable variables such as fecal haemoglobin concentration (f-Hb), age and sex, may simplify CRC diagnosis. We developed, and then externally validated, a multivariable prediction model, the FAST Score, with data from five diagnostic test accuracy studies that evaluated quantitative fecal immunochemical tests in symptomatic patients referred for colonoscopy. The diagnostic accuracy of the Score in derivation and validation cohorts was compared statistically with the area under the curve (AUC) and the Chi-square test. 1,572 and 3,976 patients were examined in these cohorts, respectively. For CRC, the odds ratio (OR) of the variables included in the Score were: age (years): 1.03 (95% confidence intervals (CI): 1.02–1.05), male sex: 1.6 (95% CI: 1.1–2.3) and f-Hb (0–

ObjectiveFaecal immunochemical tests (FIT) are replacing guaiac faecal occult blood tests (FOBT) in colorectal cancer (CRC) screening. Data from the first year of FIT screening were compared with those from FOBT screening and assumptions based on a pilot evaluation of FIT.DesignData on uptake, positivity, positive predictive value (PPV) for CRC and higher-risk adenoma from participants in the first year of the FIT-based Scottish Bowel Screening Programme (n=919 665), with a threshold of 80 µg Hb/g faeces, were compared with those from the penultimate year of the FOBT-based programme (n=862 165) and those from the FIT evaluation (n=66 225).ResultsOverall, uptake of FIT was 63.9% compared with 56.4% for FOBT. Positivity was 3.1% and 2.2% with FIT and FOBT; increases were seen in both sexes, and across age range and deprivation. More CRC and adenomas were detected by FIT, but the PPV for CRC was less (5.2% with FIT and 6.4% with FOBT). However, for higher-risk adenoma, PPV was greater with FIT (24.3% with FIT and 19.3% with FOBT). In the previous FIT evaluation, uptake was 58.5% with FIT compared with 54.0% with FOBT; positivity was 2.5% with FIT and 2.0% with FOBT.ConclusionTransition to FIT from FOBT produced higher uptake and positivity with lower PPV for CRC and higher PPV for adenoma. The FIT pilot evaluation underestimated uptake and positivity. Introducing FIT at the same threshold as the evaluation caused a 67.2% increase in colonoscopy demand instead of a predicted 10%.

Objectives Changes in the prevalence of faecal occult blood test positivity over time have not been previously reported, but could have important implications. This study examined the positivity of the initial guaiac faecal occult blood test, a surrogate marker for colorectal bleeding, in participants aged 50 in a national bowel screening programme. Methods Data from the Scottish Bowel Screening Programme were used to study the initial positivity of the guaiac faecal occult blood test between 2007 and 2017. Positive predictive values of the testing process for colorectal cancer and adenoma were assessed over the same time period. Results Across Scotland, the initial guaiac faecal occult blood test positivity increased from 4.1 to 10.8%. In NHS Grampian and NHS Fife, two of the three NHS Boards which began roll-out of screening in 2007, it increased from 4.0 to 10.9%. In the Scottish Bowel Screening Programme, the positive predictive value declined from 11.0 to 6.6% for colorectal cancer and increased from 31.6 to 39.8% for adenoma. Conclusions In the Scottish programme over a decade, initial guaiac faecal occult blood test positivity increased in participants aged 50. This may be associated with changes in lifestyle and might have implications for screening clinical outcomes, including positive predictive value.

ObjectiveTo determine whether a faecal immunochemical test (FIT) for faecal haemoglobin concentration (f-Hb) can be safely implemented in primary care as a rule-out test for significant bowel disease (SBD) (colorectal cancer (CRC), higher risk adenoma (HRA) and inflammatory bowel disease (IBD)) when used as an adjunct to the clinical assessment of new bowel symptoms.DesignSingle-centre prospective cohort study of all patients who attended primary care and submitted a FIT in the first calendar year of the service beginning December 2015. f-Hb was estimated using HM-JACKarc (Kyowa Medex) with a clinical cut-off of ≥10 µg Hb/g faeces. Incident cases of CRC were verified via anonymised record linkage to the Scottish Cancer Registry.Results5422 patients submitted 5660 FIT specimens, of which 5372 were analysed (positivity: 21.9%). 2848 patients were referred immediately to secondary care and three with f-Hb ConclusionIn primary care, measurement of f-Hb, in conjunction with clinical assessment, can safely and objectively determine a patient’s risk of SBD.

Background Faecal immunochemical tests for haemoglobin have been recommended to assist in assessment of patients presenting in primary care with lower bowel symptoms. The aim was to assess if, and which, additional variables might enhance this use of faecal immunochemical tests. Methods Faecal immunochemical test analysis has been a NHS Tayside investigation since December 2015. During the first year, 993 patients attending colonoscopy were invited to complete a detailed questionnaire on demographic background, symptoms, smoking status, alcohol use, dietary fibre, red and processed meat intake, physical activity, sitting time, dietary supplement use, family history of colorectal cancer, adenoma, inflammatory bowel disease and diabetes. Significant bowel disease was classified as colorectal cancer, advanced adenoma or inflammatory bowel disease. Results A total of 470 (47.3%) invitees agreed to complete the questionnaire and 408 (41.1%) did. Unadjusted odds ratios for the presence of significant bowel disease compared with undetectable faecal haemoglobin increased with increasing faecal haemoglobin and for faecal haemoglobin 10–49, 50–199, 200–399 and ⩾400 μg Hb/g faeces were 0.95 (95% CI: 0.16–5.63), 2.47 (0.55–1.03), 6.30 (1.08–36.65) and 18.90 (4.22–84.62), respectively. Rectal bleeding and family history of polyps were the only other variables with statistically significant ( P Conclusions Faecal haemoglobin is the most important factor to be considered when deciding which patients presenting in primary care with lower bowel symptoms would benefit most from referral for colonoscopy.

Background Many patients present in primary care with lower bowel symptoms, but significant bowel disease (SBD), comprising colorectal cancer (CRC), advanced adenoma (AA), or inflammatory bowel disease (IBD), is uncommon. Quantitative faecal immunochemical tests for haemoglobin (FIT), which examine faecal haemoglobin concentrations (f-Hb), assist in deciding who would benefit from colonoscopy. Incorporation of additional variables in an individual risk-score might improve this approach. We investigated if the published f-Hb, age and sex test score (FAST score) added value. Methods Data from the first year of routine use of FIT in primary care in one NHS Board in Scotland were examined: f-Hb was estimated using one HM-JACKarc FIT system (Kyowa Medex Co., Ltd., Tokyo, Japan) with a cut-off for positivity ≥10 μg Hb/g faeces. 5660 specimens were received for analysis in the first year. 4072 patients were referred to secondary care: 2881 (70.6%) of these had returned a FIT specimen. Of those referred, 1447 had colonoscopy data as well as the f-Hb result (group A): 2521 patients, also with f-Hb, were not immediately referred (group B). The FAST score was assessed in both groups. Results 1196 (41.7%) of patients who returned a specimen for FIT analysis had f-Hb ≥10 μg Hb/g faeces. In group A, 252 of 296 (85.1%) with SBD had f-Hb > 10 μg Hb/g faeces, as did 528 of 1151 (45.8%) without SBD. Using a FAST score > 2.12, which gives high clinical sensitivity for CRC, only 1143 would have been referred for colonoscopy (21.0% reduction in demand): 286 of 296 (96.6%) with SBD had a positive FAST score, as did 857 of 1151 (74.5%) without SBD. However, one CRC, five AA and four IBD would have been missed. In group B, although 95.2% had f-Hb > 2.12 was found in 13 of 15 (86.7%) with SBD. Conclusions The performance characteristics of the FAST score did not seem to enhance the utility of f-Hb alone. Locally-derived formulae might confer desired benefits.

Background: Quantitative faecal immunochemical tests (FIT) measure faecal haemoglobin concentration (f-Hb), which increases in the presence of colorectal neoplasia and is related to the severity of disease. We examined the diagnostic accuracy of FIT in patients at moderate to high risk of colorectal cancer (CRC) attending for surveillance colonoscopy as per national guidelines. Methods: We undertook a prospective study in two University hospitals, one in Scotland and one in England. Between June 2014 and September 2016, 1103 consecutive patients were invited to complete a FIT before their scheduled colonoscopy: f-Hb was analysed on an OC-Sensor io automated analyser (Eiken Chemical Co., Ltd, Tokyo, Japan)) with a limit of detection (LoD) of 2 µg Hb/g faeces. The diagnostic accuracy of f-Hb for CRC and higher-risk adenoma (HRA) was examined. Findings: 643 patients returned a FIT. Four patients with known IBD were excluded, leaving 639 (57·9%) in the study: age range: 25-90 years (median: 64 years, IQR: 55-71): 54·6% male. Of 593 patients who returned a FIT and completed colonoscopy, 41 (6·9%) had advanced neoplasia (4 CRC, 37 HRA): 238/593 patients (40·1%) had detectable f-Hb of whom 31 (13·0%) had advanced neoplasia (2 CRC, 29 HRA) compared to 10 (2·8%) in those with undetectable f-Hb (2 CRC, 8 HRA).Detectable f-Hb gave negative predictive values of 99·4% for CRC and 97·2% for CRC plus HRA. Interpretation: In patients at moderate to high risk of CRC who are under colonoscopy surveillance, a FIT at the LoD can provide an objective estimate of the risk of advanced neoplasia and could enable tailored scheduling of colonoscopy. Funding Statement: Chief Scientist Office; grant reference number CZH/4/1032. Declaration of Interests: CGF has undertaken consultancy with Kyowa-Medex Co., Ltd, Tokyo, Japan, and has received travel support from Alpha Labs Ltd, Eastleigh, UK. Other authors - none. Ethics Approval Statement: This study had ethical approval from NRES North of Scotland Ethics Committee: reference: 14/NS/0059.

AimTo compare acceptability and diagnostic accuracy of a recently available faecal immunochemical test (FIT) system (HM-JACKarc) with the FIT routinely used in an established screening programme (OC-Sensor).DesignRandomised controlled trial (ISRCTN20086618) within a population-based colorectal cancer (CRC) screening programme. Subjects eligible for invitation in the Umbria Region (Italy) programme were randomised (ratio 1:1) to be screened using one of the FIT systems.ResultsScreening uptake among the 48 888 invitees was the same for both systems among subjects invited in the first round and higher with OC-Sensor than with HM-JACKarc (relative risk (RR): 1.03; 95% CI 1.02 to 1.04) among those invited in subsequent rounds. Positivity rate (PR) was similar with OC-Sensor (6.5%) as with HM-JACKarc (6.2%) among subjects performing their first FIT screening and higher with OC-Sensor (5.6%, RR: 1.25, 95% CI 1.12 to 1.40) than with HM-JACKarc (4.4%) among those screened in previous rounds. Positive predictive value (PPV) (OC-Sensor: 25.9%, HM-JACKarc: 25.6%) and detection rate (DR) (OC-Sensor: 1.40%; HM-JACKarc: 1.42%) for advanced neoplasia (AN: CRC + advanced adenoma) were similar among subjects performing their first FIT screening. The differences in the AN PPV (OC-Sensor: 20.3%, HM-JACKarc: 22.6%) and DR (OC-Sensor: 0.96%, HM-JACKarc: 0.83%) among those screened in previous rounds were not statistically significant. The number needed to scope to detect one AN was 3.9 (95% CI 5.8 to 2.9) and 3.9 (95% CI 5.5 to 2.9) at first and 4.9 (95% CI 5.8 to 4.2) and 4.4 (95% CI 5.3 to 3.7) at subsequent screening, with OC-Sensor and HM-JACKarc, respectively.ConclusionsOur results suggest that acceptability and diagnostic performance of HM-JACKarc and of OC-Sensor systems are similar in a screening setting.Trial registration numberISRCTN20086618; Results.

Although colonoscopy, despite disadvantages, remains the “gold standard” for investigation of suspected colorectal disease, faecal immunochemical tests for haemoglobin (FIT) are now considered the best non-invasive investigation for use in asymptomatic population-based programmatic screening for colorectal neoplasia (1). FIT come in two formats, qualitative tests, based upon immunochromatography, and quantitative tests, based on immunoturbidimetry. Qualitative FIT have some advantages, including potential as point-of-care tests, but have major disadvantages (2), particularly that they have different analytical detection limits and thus give very different clinical outcomes (3). Quantitative FIT are much more suitable for larger-scale screening programmes and have many advantages (4), especially that estimates of faecal haemoglobin concertation (f-Hb) are obtained, allowing considerable flexibility in programme design.

Background 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 2½ 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. Methods The five reference change value methods were examined using normally and ln-normally distributed simulated data. Results 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. Conclusions 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.