Stability of heparin blood samples during transport based on defined pre-analytical quality goals.

BACKGROUND: In many countries and especially in Scandinavia, blood samples drawn in primary healthcare are sent to a hospital laboratory for analysis. The samples are exposed to various conditions regarding storage time, storage temperature and transport form. As these factors can have a severe impact on the quality of results, we wanted to study which combination of transport conditions could fulfil our pre-defined goals for maximum allowable error. METHODS: Samples from 406 patients from nine general practitioners (GPs) in two Danish counties were sent to two hospitals for analyses, during two periods (winter and summer). Transport conditions (mail, courier pick-up, or brought to hospital by public coach), storage time, storage temperature and centrifugation requirements were different in the two counties. Results were tested for deviation from a "0-sample", the blood sample taken, centrifuged and separated at the doctor's office within 45-60 min. This sample was considered as the best estimate of a comparison value. RESULTS: The pre-set quality goals were fulfilled for all the investigated components for samples transported to hospital by courier either as whole blood or as "on gel" after centrifugation, as long as the samples were stored at 20-25 degrees C and centrifuged/analysed within 5-6 h. A total of 4% of the samples sent by mail had mismatched identity, probably due to plasma being transferred to a new tube. CONCLUSIONS: Samples can be sent as unprocessed anticoagulated whole blood if the above mentioned conditions are met. There is no need for centrifugation in the primary sector. Neither mailing of samples with plasma "on gel" nor public transport by coach bus fulfil our analytical goals.

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 approximately geometric mean population=5.36 mmol/L and standard deviation=0.0891 approximately 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.

Controlled storage conditions prolong stability of biochemical components in whole blood.

Blood specimens from primary care centres are normally transported to central laboratories by mail. This necessitates centrifugation and separation, especially since the potassium ion concentration in whole blood changes during storage at ambient temperature. Thus, because of the growing awareness of and concern for pre-analytical contributions to the uncertainty of measurements, we investigated 27 components and their stability under controlled temperature conditions from 17 to 23 degrees C. We found that storage of whole blood can be prolonged by up to 8-12 h for all components examined, including potassium ions, when stored at 20+/-0.2 degrees C. We conclude that this opens the possibility for establishing a pick-up service, by which whole blood specimens stored at 20-21 degrees C can be collected at the doctor's office, making centrifugation, separation and mailing superfluous. In addition, the turn-around time from sample drawing to reporting the analytical result would be shortened. After investments in thermostatted boxes and logistics, the system could reduce costs for transporting blood samples from general practice centres to central laboratories.

Creation of a low-risk reference group and reference interval of fasting venous plasma glucose.

Reference intervals are recommended for naturally occurring quantities and required in the evaluation of new components in order to provide clinically useful information. The aim of the present study is to present a method for selecting reference individuals for the determination of fasting venous plasma glucose (f-vPG) reference intervals and ways to determine if disease groups can share reference intervals with an ideal reference population. Reference subjects were randomly selected, eligibility was judged according to predetermined inclusion and exclusion criteria. Using the literature we selected risk indicators for diabetes mellitus (DM) and used these indicators to rule out high-risk individuals in order to obtain a reference distribution of f-vPG determined using individuals with low risk of DM. The distribution of f-vPG in the high-risk individuals was compared with that determined for the low-risk group. We then estimated the ability of the high-risk individuals to share the reference interval of the low-risk individuals, and calculated the fraction that was outside this interval. Distributions were also investigated for linearity in the cumulated frequency rankit distribution of In-values. The allowable difference between two reference limits could not exceed 0.375 times the population biological variation. Most risk indicators were powerful predictors of high f-vPG values. Subgroups with these risk indicators should not be included in the homogeneous In-normally distributed reference distribution. Distributions of f-vPG concentrations in individuals with risk factors were not homogeneous and varying percentages of individuals were outside the reference distribution, having f-vPG greater than 7.0 mmol/l. We conclude that randomisation is only useful to recruit candidate reference subjects. To rule out subjects according to clinical risk factors for diabetes, it is necessary to identify a reference population with low risk of exhibiting increased f-vPG concentrations. This method may be used to validate a reference interval for a particular analyte with respect to an investigated disease, and to stratify risk factors of importance.

The effect of the new ADA and WHO guidelines on the number of diagnosed cases of diabetes mellitus.

In the new lowered diagnostic discriminator for diabetes mellitus (DM) from the American Diabetes Association (ADA), fasting peripheral venous plasma glucose (f-vPG) of 7.0 mmol/l is identical to the 99.9 centile of f-vPG (7.05 mmol/l, 95%CI: 6.91-7.20 mmol/l) in a low-risk reference population. We investigated its diagnostic concordance with other diagnostic discriminators. As no index test is available for DM we used the ADA discriminator as gold standard. We isolated a low-risk reference population (n = 424) from a randomised general population (n = 726) by ruling out of all cases with clinical and biochemical risk indicators for DM. We based our analysis on measurements traceable to primary standard concentration, a bias of < 1.5% and CV% < 2.5. The distribution of the fasting capillary whole blood glucose (f-CBG; mmol/l) in the reference population was in Gaussian with the 99.9 centile of 6.62 mmol/l (95% CI 6.47-6.77 mmol/l) and the 97.5 centile of 5.92 mmol/l (5.82-6.02 mmol/l). The 6.1 mmol/l f-CBG WHO limit corresponds approximately to the 97.6 centile, and this limit is thus not traceable to the ADA discriminator, which corresponds to f-CBG of 6.4 mmol/l. This is the case in groups only, as recalculation will introduce unpredictable errors. Thus, in our general population a varying number of subjects will be at risk of DM as a mere consequence of different limits. The f-CBG limit of 6.1 mmol/l will thus lead to 2.4% false-positive diagnoses or, in EU, to around 44 x 10(6) adults being diagnosed. The number of cases at risk of DM vary from 5.4 x 10(6) to 44 x 10(6) in EU. We conclude that application of different diagnostic limits results in highly variable number of diagnosed DM cases, and therefore one diagnostic discriminator is needed to provide reproducible diagnoses.

Fasting and post-glucose load--reference limits for peripheral venous plasma glucose concentration in pregnant women.

Recently both the American Diabetes Organization (ADA) and World Health Organization (WHO) have revised the diagnostic recommendations for gestational diabetes mellitus (GDM), however, they did not not reach agreement on the criteria for diagnosis, the referral criteria for the confirmatory oral glucose tolerance test (OGTT), its standardization, and diagnostic cut-off point. The aims of this study were to investigate if the fasting venous plasma glucose mmol/l (f-vPG) and the 2-hour venous plasma glucose mmol/l (2h-vPG) after a WHO standardized 75 g oral glucose tolerance test (OGTT) in a non-risk group of pregnant women during first and third trimester of pregnancy deviated from that of risk groups, to establish a reference interval for f-vPG and 2h-vPG, and to investigate the predictive role of f-vPG for the 2h-vPG glucose concentration. This is a population-based case-control study where a consecutive number of pregnant women were invited to screening irrespective of their risk factors for GDM. All women filled in a questionnaire of the Danish national screening program on risk factors and had f-vPG and the 2h-vPG measured. By ruling out women with GDM and risk factors, we isolated a non-risk reference class. The In f-vPG parametric 97.5 centile was less than 5% higher during week 32 of pregnancy than during week 20, and therefore these groups were combined. The f-vPG 95% reference interval was from 4.01 mmol/l (95% CI: 3.96 to 4.07 mmol/l) to 5.26 mmol/l (95% CI: 5.19 to 5.34 mmol/l). "The true upper normal limit", the 99.9 centile, was 5.69 mmol/l (95% CI: 5.59 to 5.80 mmol/l). The f-vPG was 0.6 mmol/l lower over the whole range in pregnant women compared to age-matched non-pregnant women. The distribution of 2h-vPG concentrations at week 20 was non-Gaussian and therefore considered non-homogeneous, while it was Gaussian distributed and homogeneous at week 32. The 2h-vPG 95% reference interval of the combined weeks was from 2.80 mmol/l (95% CI: 2.56 to 3.04 mmol/l) to 7.58 mmol/l (95% CI: 7.34 to 7.82 mmol/l), and the upper limit of normal (99.9 centile) was 8.96 mmol/l (95% CI: 8.63 to 9.29 mmol/l). Distributions of f-vPG and 2h-vPG were distinct in our defined risk classes. In individual cases, no systematic correlation was found between the f-vPG concentration at week 20 and week 32. The f-vPG concentrations at any of the weeks did not predict the 2h-vPG level and no single clinical risk factor was decisive for the presence of GDM.

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 (%CV(W-S)) as: %CVA < or = 0.5 x %CV(W-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 %CV(W-S) is small compared with the population-based coefficient of variation [%CV(P) = (%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.