Lone G. M. Jørgensen

Optimization of preanalytical conditions and analysis of plasma glucose. 1. Impact of the new WHO and ADA recommendations on diagnosis of diabetes mellitus

The new diagnostic criteria for type 2 diabetes from the American Diabetes Association (ADA) and World Health Organization (WHO) recommend measurements on plasma and a lowering of the glucose threshold for diabetes by 0.8mmol/L. This narrows the distance between the upper end of the reference limit and the discriminatory level to a degree where analytical quality becomes critical. The quality demands for the preanalytical and analytical phase and their consequences on diagnostic performance have to be established in the new technical system, measuring in plasma rather than in capillary whole blood. Because of the instability of glucosein blood samples it is necessary to clarify the influence of different preanalytical and analytical factors on the number of false-positive and false-negative classifications. Thus the aim of the present study was to find optimal conditions for sampling, additives, storage, transport and analysis of plasma glucose combining feasibility with an analytical bias close to zero and a within-imprecision around 1%. We have documented the analytical performance of the method itself and its traceability to an international standard. The preanalytical conditions, such as influence of antiglycolytic agent NaF, conditions for plasma separation, storage temperature and storage time before and after plasma separation were investigated. In conclusion, we recommend that blood should be drawn in tubes containing heparin and NaF and kept on ice water for not more than 1h until centrifugation at minimum 1000 g for 10min. The plasma is then stable for at least 48h at room temperature.The new diagnostic criteria for type 2 diabetes from the American Diabetes Association (ADA) and World Health Organization (WHO) recommend measurements on plasma and a lowering of the glucose threshold for diabetes by 0.8 mmol/L. This narrows the distance between the upper end of the reference limit and the discriminatory level to a degree where analytical quality becomes critical. The quality demands for the preanalytical and analytical phase and their consequences on diagnostic performance have to be established in the new technical system, measuring in plasma rather than in capillary whole blood. Because of the instability of glucose in blood samples it is necessary to clarify the influence of different preanalytical and analytical factors on the number of false-positive and false-negative classifications. Thus the aim of the present study was to find optimal conditions for sampling, additives, storage, transport and analysis of plasma glucose combining feasibility with an analytical bias close to zero and a within-imprecision around 1%. We have documented the analytical performance of the method itself and its traceability to an international standard. The preanalytical conditions, such as influence of antiglycolytic agent NaF, conditions for plasma separation, storage temperature and storage time before and after plasma separation were investigated. In conclusion, we recommend that blood should be drawn in tubes containing heparin and NaF and kept on ice water for not more than 1 h until centrifugation at minimum 1000 x g for 10 min. The plasma is then stable for at least 48 h at room temperature.

Combination of analytical quality specifications based on biological within- and between-subject variation

At a conference on ‘Strategies to Set Global Analytical Quality Specifications in Laboratory Medicine’ in Stockholm 1999, a hierarchy of models to set analytical quality specifications was decided. The consensus agreement from the conference defined the highest level as ‘evaluation of the effect of analytical performance on clinical outcomes in specific clinical settings’ and the second level as ‘data based on components of biological variation’. Here, the many proposals for analytical quality specifications based on biological variation are examined and the outcomes of the different models for maximum allowable combined analytical imprecision and bias are illustrated graphically. The following models were investigated. (1) The Cotlove et al. (1970) model defining analytical imprecision (%CVA) in relation to the within-subject biological variation (%CVw-s) as: %CVA≤ 0·5 × %CVW-S (where %CV is percentage coefficient of variation), (2) The Gowans et al. (1988) concept, which defines a functional relationship between analytical imprecision and bias for the maximum allowable combination of errors for the purpose of sharing common reference intervals. (3) The European Group for the Evaluation of Reagents and Analytical Systems in Laboratory Medicine (EGE Lab) Working Group concept, which combines the Cotlove model with the Gowans concept using the maximal acceptable bias. (4) The External Quality Assessment (EQA) Organizers Working Group concept, which is close to the EGE Lab Working Group concept, but follows the Gowans et al. concept of imprecision up to the limit defined by the model of Cotlove et al. (5) The ‘three-level’ concept classifying analytical quality into three levels: optimum, desirable and minimum. The figures created clearly demonstrated that the results obtained were determined by the basic assumptions made. When %CVW-S is small compared with the population-based coefficient of variation [%CVp = (%CV2 W-S +%CV2 B-S)1/2], the EGE Lab and EQA Organizers Working Group concepts become similar. Examples of analytical quality specifications based on biological variations are listed and an application on external quality control is illustrated for plasma creatinine.

Can capillary whole blood glucose and venous plasma glucose measurements be used interchangeably in diagnosis of diabetes mellitus

According to new proposals from the American Diabetes Association (ADA) and WHO, venous peripheral plasma is the preferred system for measuring glucose for diagnosing diabetes mellitus. Owing to the instability of glucose in plasma after blood sampling, strict well-defined and standardized preanalytical conditions are essential to ensure that glucose concentration measured in plasma reflects real blood glucose in the patient. This is in contrast to the capillary whole blood measurements, which are easy to perform and well established. We investigated whether it is possible to perform analysis on capillary whole blood but express the results as plasma glucose values and hence obtain comparable results and the same predictive values for diagnosis in the individual patient? The conclusion of our investigations is that these two systems are not interchangeable and that conversion should not be done for diagnostic purposes where plasma determinations are recommended.

Consequences Of Bias and Imprecision in Measurements of Glucose and Hba1c for the Diagnosis and Prognosis of Diabetes Mellitus

Aim: To investigate the effect of composite analytical bias and imprecision in the measurements of fasting plasma‐glucose (fPG) for diagnosis of diabetes mellitus and estimation of risk of development and progression of retinopathy using measurements of Haemoglobin A1C (HbA1C%). Materials and methods: Data on biological within‐subject variation for fPG (5.7% and 4.9%) and HbA1C% (1.9%) from literature and data on fPG for a ‘low‐risk’ population (regarding diabetes) from own investigations (ln‐values of mean=1.6781∼geometric mean population=5.36 mmol/L and standard deviation=0.0891∼CV population=8.9%). Further, guidelines for diagnosis of diabetes (two consecutive measurements of fPG above 7.0 mmol/L) were obtained from literature as also the risk of development of and progression of retinopathy using measurements of HbA1C (a change in risk of 44% for a change in HbA1C% of 10%). It was assumed that each individual had values which over a short time had a Gaussian distribution about a biological set‐point. Calculations of the effect of analytical bias and imprecision were performed by linear addition of bias and squared addition of imprecision to the squared error‐free biological distribution. Composite variations of bias and imprecision were obtained by varying assumed imprecision and calculating the maximum acceptable bias for the stated situation. Results: Two diagnostic examples are described for fPG and one for risk related to HbA1C%. Firstly, the risk of diabetes as a function of set‐point and bias and imprecision was investigated, using functions where the probability of two measurements above 7.0 mmol/L was plotted against biological set‐points, resulting in a S‐shaped curve with a 25% probability for a set‐point equal to 7.0 mmol/L. Here, a maximum 5% probability of classifying an individual with a set‐point of 6.4 mmol/L (upper reference limit for the ‘low‐risk’ population) as diabetic was used to calculate the analytical quality specifications. Comparably, the 5% probability of misclassifying a diabetic with fPG of 8.0 mmol/L was investigated, and both specifications were illustrated in an imprecision‐bias plot. Secondly, the percentage of ‘low‐risk’ individuals which would be falsely diagnosed as diabetic was calculated, and this percentage was plotted as a function of bias for different assumed values of imprecision. Thirdly, the confidence intervals for a certain risk‐difference for HbA1C% of 5% or 10% was used to draw an imprecision‐bias plot for different assumed changes and probabilities. Discussion: Analytical quality taking the demands for bias and imprecision in account are obtainable in laboratories, but may be questionable for use of capillary blood and POCT instruments with considerable consequences for the number of individuals classified as diabetics, and thereby for the economy etc. Conclusion: For clinical settings, with so clear recommendations and descriptions of risk curves as in diabetes, it is relatively easy to estimate the analytical quality specifications according to the highest level of the model hierarchy, when relevant probabilities for the events are assumed.

Omega-3 fatty acids inhibit an increase of proinflammatory cytokines in patients with active Crohn's disease compared with omega-6 fatty acids

Background : Crohns disease is a chronic inflammatory condition affecting the gastrointestinal tract. Polyunsaturated omega‐3 fatty acids given orally may reduce the secretion of proinflammatory cytokines and hereby downregulate the inflammatory process.

Should we maintain the 95 percent reference intervals in the era of wellness testing? A concept paper.

Abstract The reference interval is probably the most widely used decision-making tool in clinical practice, with a modern use aiming at identifying wellness during health check and screening. Its use as a diagnostic tool is much less recognised and may be obsolete. The present study investigates the consequences of the newpractice for the interpretation of prospective value, negative vs. positive, the probability of confirming wellness, and number of false results based on selected strategy for reference interval establishment. Calculations assumed normalised Gaussian-distributed reference intervals with analytical variation set to zero and absolute accuracy. Also assumed is the independency of tests. Probability for no values outside reference intervals in healthy subjects was calculated from the formula pno outside=(1−psingle) and according to the formula for repeated testing: pone outside=n×psingle (1−psingle)n−1 etc. Here n is the number of tests performed and psingle is the probability of one result outside reference limits with the general formula pi outside n−i=k×psinglei (1−psingle)n−i, with k being the binominal coefficient and i the number outside the reference intervals. Use of the 99.9 centile for health checks will increase the probability for no false from 60% to 99% for 10 tests, and from 46% to 98% for 15 tests. The probability for one false-positive result in 10 tests in a panel can be reduced from 32% to 1% if the 99.9% centile is substituted for the 95% centile. For two in 10 tests, the probability can be reduced from 8% to below 0.1%. In both cases, selection of the 99.9% centile improves the diagnostic accuracy. Reference intervals are needed as a “true” negative reference for absence of disease, and should cover the 99.9% centile of the reference distribution of an analyte to avoid false positives. For this new use, it is critical that reference persons are absolutely normal without clinical, genetic and biochemical signs of the condition being investigated. However, reference intervals cannot substitute clinical decision limits for diagnosis and medical intervention.

Plasma glucose reference interval in a low-risk population. 2. Impact of the new WHO and ADA recommendations on the diagnosis of diabetes mellitus.

The aim of the study was to establish a reference interval of fasting venous plasma glucose (FPG) from healthy individuals. A prospective modified cross-sectional population-based study was made with random selection of 2100 persons in age-stratified groups > or = 18 years identified from the local Personal Identification Register. The invitation was accepted by 755 persons, of which 726 aged 18-92 years were eligible. They did not have a diabetes diagnosis, were non-pregnant and capable of fasting for 8 h. All participants filled in a questionnaire on medical risk factors. Blood for the FPG and haemoglobin Alc (HbAlc) measurements was drawn in accordance with a standardized procedure. A total of 302 participants carried diabetes risk indicators and were ruled out. The FPG concentrations in the remaining low-risk population (n=424) was ln Gaussian distributed. The FPG 97.50 centile in this group was 6.4 mmol/L (95% CI: 6.3-6.5 mmol/L), in contrast to the WHO and ADA theoretical limit of 6.1 mmol/L. Their diagnostic decision limit of 7.0 mmol/L FPG corresponded to the 99.86 centile of the FPG reference distribution (95% CI: 6.8-7.1). Subclassification of the reference population showed increasing FPG with increasing BMI and age and was higher in men than in women. The study determined the FPG 95% interfractile reference interval in a healthy population. The interval in glucose concentration between the 97.5 centile of the reference interval and the ADA-WHO diagnostic limit is very narrow. The clinical application of the diagnostic discriminator and the interpretation of the WHO-ADA grey zone from 6.1 to 7.0 mmol/L FPG may consequently be biased because of poorly defined limits and influence from BMI, age and gender.Theaim ofthe studywas to establish a referenceintervaloffasting venous plasma glucose (FPG) from healthy individuals. A prospective modified cross-sectional population-based study was made with random selection of 2100 persons in agestratified groups 18 years identified from the local Personal Identification Register. The invitation was accepted by 755 persons, of which 726 aged 18-92 years were eligible. They did not have a diabetes diagnosis, were non-pregnant and capable offasting for 8h. Allparticipants filled in a questionnaireon medical risk factors. Blood for the FPG and haemoglobin A1c (HbA1c) measurements was drawn in accordance with a standardized procedure. A total of 302 participants carried diabetes risk indicators and wereruled out.TheFPG concentrations in the remaining low-risk population (n=424) was ln Gaussian distributed. The FPG 97.50 centile in this group was 6.4mmol/L (95% CI: 6.3-6.5mmol/L), in contrast to the WHO and ADA theoretical limit of 6.1mmol/L. Their diagnostic decision limit of 7.0mmol/L FPG corresponded to the 99.86 centile of the FPG reference distribution (95% CI: 6.8-7.1). Subclassification of the reference population showed increasing FPG with increasing BMI and age and was higher in men than in women. The study determined the FPG 95% interfractile reference interval in a healthy population. The interval in glucose concentration between the 97.5 centile of the reference interval and the ADA-WHO diagnostic limit is very narrow. The clinical application of the diagnostic discriminator and the interpretation of the WHO-ADA grey zone from 6.1 to 7.0mmol/L FPG may consequently be biased because of poorly defined limits and influence from BMI, age and gender.

Graphical interpretation of confidence curves in rankit plots

Abstract A well-known transformation from the bell-shaped Gaussian (normal) curve to a straight line in the rankit plot is investigated, and a tool for evaluation of the distribution of reference groups is presented. It is based on the confidence intervals for percentiles of the calculated Gaussian distribution and the percentage of cumulative points exceeding these limits. The process is to rank the reference values and plot the cumulative frequency points in a rankit plot with a logarithmic (ln=loge) transformed abscissa. If the distribution is close to ln-Gaussian the cumulative frequency points will fit to the straight line describing the calculated ln-Gaussian distribution. The quality of the fit is evaluated by adding confidence intervals (CI) to each point on the line and calculating the percentage of points outside the hyperbola-like CI-curves. The assumption was that the 95% confidence curves for percentiles would show 5% of points outside these limits. However, computer simulations disclosed that approximate 10% of the series would have 5% or more points outside the limits. This is a conservative validation, which is more demanding than the Kolmogorov-Smirnov test. The graphical presentation, however, makes it easy to disclose deviations from ln-Gaussianity, and to make other interpretations of the distributions, e.g., comparison to non-Gaussian distributions in the same plot, where the cumulative frequency percentage can be read from the ordinate. A long list of examples of ln-Gaussian distributions of subgroups of reference values from healthy individuals is presented. In addition, distributions of values from well-defined diseased individuals may showup as ln-Gaussian. It is evident from the examples that the rankit transformation and simple graphical evaluation for non-Gaussianity is a useful tool for the description of sub-groups.

Insulin-like growth factors (IGFs) and IGF binding proteins in active Crohn's disease treated with ω-3 or ω-6 fatty acids and corticosteroids

Objective. Catabolism and growth impairment are well-known complications of inflammatory bowel disease (IBD). This may be caused by the disease activity itself and/or the medical treatment, and both may lead to changes in the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. The aim of the present study was to examine the effects of enteral nutrition, Impact Powder®, as adjuvant therapy to corticosteroid treatment on changes in the GH/IGF-I axis in patients with Crohns disease (CD). Material and methods. The patients were randomized to 3-IP (ω-3-fatty acid (FA), 3 g/day) or 6-IP (ω-6-FA, 9 g/day). Changes in total IGF-I (tIGF-I) and total IGF-II (tIGF-II), free IGF-I (fIGF-I), IGF binding proteins (IGFBP-1 and IGFBP-3), IGFBP-3 protease activity and insulin levels were examined in 31 patients with active CD (CDAI: 186–603) during treatment with prednisolone (40 mg for 1 week) and tapering the dose by 5 mg/week. Clinical and biochemical markers of inflammation were studied at day 0, and after 5 and 9 weeks. Results. There were no differences at baseline between the two groups. During the treatment period, tIGF-I, fIGF-I and IGFBP-3 increased significantly in both groups compared to baseline (p<0.05) without differences between the groups. Insulin and IGFBP-1 showed no significant changes throughout the treatment period. Conclusions. There was no difference between 3-IP and 6-IP as adjuvant enteral nutrition on the GH/IGF-I axis. The changes observed in the GH/IGF-I axis are in line with previously published studies and may be explained by corticosteroid treatment; however, we cannot exclude an additional effect of ω3-/ω6 FA as adjuvant enteral nutrition.

The clinical impact of screening for gestational diabetes

Abstract Gestational diabetes mellitus (GDM) is defined as carbohydrate intolerance during pregnancy. In Denmark the health service offers selective screening for GDM, i.e., admission to an oral glucose tolerance test (OGTT) after pre-screening with interview for clinical risk factors for GDM, two capillary fasting blood glucose (cFBG) measurements and a urine test for glucosuria. The aim of the present study was to investigate the power of the pre-screening to identify GDM and the screening to predict adverse clinical outcome. A retrospective investigation of pregnant women undergoing screening during 1998 at Vejle County Hospital, Denmark was undertaken. The two most frequent pre-screening criteria for OGTT were body-mass index (BMI) ≥ 27 kg/m2 and age ≥ 35 years. The highest odds ratio (OR) of 9.07 (95% CI: 2.60 to 63.70) for GDM had glucosuria and the lowest (zero) had cFBG. The frequency of complicated delivery was similar in GDM (58%) compared to non-GDM (56%). The best predictor of complicated delivery was a BMI with OR=1.50 (95% CI: 0.87 to 2.60) for BMI ≥ 27 kg/m2 vs. < 27 kg/m2. The best predictor of adverse neonatal outcome was a capillary blood glucose 120 min after glucose load (cBG120 min) ≥9.0 mmol/l (OR=3.18, 95% CI: 1.14 to 8.89). The intermediary endpoint GDM was not superior for predicting adverse maternal and neonatal outcome. The cumulative probability distribution of cBG120 min after a 75 g glucose load was not homogeneously distributed in groups stratified according to maternal and foetal outcome. A changed slope was seen after cBG120 min 9.0 mmol/l. Screening cFBG of 4.1 mmol/l was unable to predict GDM and adverse outcome. Glucosuria was too rare to be effective as a screening tool. Pre-screening did not identify GDM. The best predictor of complicated delivery was a high BMI. The best predictor of foetal adverse outcome was cBG120 min ≥ 9.0 mmol/l after a 75 g glucose load. Identical fraction complications were present in GDM and non-GDM. A refinement of the screening procedure is highly needed, and this has been initiated in Denmark.