Preanalytical Sources Of Variation Research Articles

Effect of Collection Methods on Salivary Cortisol

Salivary cortisol is a useful measure of hypothalmic-pituitary axis function, but the effects of collection method, sample volume, and environmental conditions on hormone recovery are poorly described. These potential sources of pre-analytical variation were assessed using spiked pooled saliva and assay by liquid chromatography and tandem mass spectrometry (LC-MS/MS). When saliva was collected onto cotton swabs, hormone recovery was determined primarily by volume recovery, which was in turn determined by sample volume. To ensure adequate cortisol recovery, ≥2 ml of saliva must be absorbed onto swabs. This may be difficult for many children and some adults to achieve. Salivary cortisol is remarkably stable to a range of environmental conditions, including repeated freeze-thaw cycles, storage at room temperature and exposure to sunlight. This study shows that it is preferable to obtain saliva by passive drool without swabs if adequate sample volume (≥2 ml) cannot be achieved, and that saliva samples can be returned by standard post without affecting hormone recovery.

Banking of clinical samples for proteomic biomarker studies: A consideration of logistical issues with a focus on pre‐analytical variation

Biobanks with their collections of clinical samples and data are essential resources for the success of clinical proteomics in delivering and validating candidate biomarkers. Samples must be banked in a manner that allows maximum subsequent compatibility with analytical techniques and additionally many critical factors must be taken into account when establishing a biobank or selecting samples from a biobank. These include logistical, ethical, legal and security issues and, very importantly, steps to minimise any pre-analytical variability introduced through sample processing and handling (technical effects). The inherent variation present within the samples must also be taken into account. In this review, we examine the impact of these factors and issues to be faced when banking samples with a particular focus on sources of pre-analytical variation, which must be rigorously controlled and recorded. It is encouraging that several initiatives are now addressing such key issues and these are also discussed.

Biological variation and reference change values of CA 19‐9, CEA, AFP in serum of healthy individuals

Objective. The use of tumour markers in diagnosis and monitoring is very common. Tumour marker results vary – preanalytical sources of variation, total random analytical error (CVa), and within‐subject (intraindividual) normal biological variation. There are not so many studies evaluating the biological variations and reference change values (RCV) of these parameters. The aim of our study was to assess: (i) the average inherent intra‐ and inter‐individual biological variation (CVi and CVg) for CA 19‐9, CEA, AFP in a group of healthy individuals; (ii) the significance of changes in serial results of each marker; and (iii) the index of individuality. Material and methods. The study group comprised 49 healthy volunteers ranging in age between 18 and 60 years (25 M and 24 F). Four blood samples were obtained from each subject; one at each 14‐day interval. Each sample from one individual was assayed in duplicate. CA 19‐9, CEA, AFP levels were measured by an immunoluminometric assay on a random‐access analyser (Architect i2000; Abbott Diagnostics Division). The intra‐ (CVi) and inter‐individual (CVg) biological variations were estimated from the data generated. Reference change value (RCV) was calculated. Results. The intra‐individual/inter‐individual biological variations (CVs) for CA 19‐9, CEA, AFP were 27.2/64.24 %, 30.87/37.14 % and 26.67/43.65 %, respectively. The critical differences (RCVs) of CA 19‐9, CEA, AFP were 64.71 %, 72.57 % and 62.62 %, respectively (Z = 1.65 for unidirectional changes; p<0.05). Conclusions. Intra‐individual biological variation contributes to the variation in serial results and should therefore be included in the criteria for serum tumour marker assessment.

Pre-analytical variation of some haematological quantities

It is customary to assume that pre-analytical variation is negligible if all pre-analytical sources of variation have been standardised. The aim of this study was to quantify the pre-analytical variation of some haematological quantities frequently measured in clinical laboratories. The experimental design considers different sources of pre-analytical variation that usually occur day-to-day for samples from patients. The results demonstrate that for concentrations of erythrocytes, haemoglobin, leukocytes, neutrophilocytes (segmented) and lymphocytes, and for the volume fraction of erythrocytes ("packed cell volume") in blood, the pre-analytical variance is not negligible. We suggest that these variances should be taken into account when estimating the uncertainty of measurement for patient results.

Evolution of Methods for Measurement of HDL-Cholesterol: From Ultracentrifugation to Homogeneous Assays

Adoption of automated homogeneous assays for HDL-cholesterol (HDL-C) is increasing, driven by the need of clinical laboratories to cope with increasing workloads while containing costs. However, performance characteristics of homogeneous assays often differ in important aspects from those of the earlier precipitation methods. This review provides an overview of the new generation of homogeneous assays for HDL-C within the historical context of the evolution of methods and the efforts to standardize measurements of the lipoproteins. This is a narrative review based on method evaluations conducted in the laboratories of the authors as well as on relevant publications, especially comparative evaluation studies, from the literature. Publications considered here have been collected by the authors over the past 30 years of involvement as methods for HDL-C made the transition from their early use in lipid research laboratories to clinical laboratories and the recent emergence of homogeneous assays. The presentation includes descriptions of methodologies, including homogeneous, precipitation, electrophoresis, and ultracentrifugation assays. Reference methods and recommended approaches for assessing accuracy are described. Accuracy and imprecision are summarized in the context of the National Cholesterol Education Program (NCEP) standards for analytical performance. The effects of interfering substances and preanalytical sources of variation are presented. Homogeneous assays have been shown to be reasonably well suited for use in routine clinical laboratories, generally meeting the NCEP criteria for precision, accuracy, and total error. However, discrepant results compared with the reference methods have been observed with some of the assays, and the sources of discrepancies are not well characterized. Some homogeneous reagents have not been thoroughly evaluated. At least three of the reagents have experienced successive adjustments in formulation; hence, the reagents may not yet be fully optimized. For these reasons, the homogeneous assays cannot be confidently recommended for use in long-term clinical trials and other research applications without thorough validation.

Biological Variation of Free and Total Carnitine in Serum of Healthy Subjects

Determination of carnitine (l-β-hydroxy-γ-trimethylaminobutyric acid) in biological samples plays an important role in the diagnosis of diseases with carnitine deficiency (1). In the past few years, and also in this Journal, several spectrophotometric methods for assaying carnitine in serum have been described (2)(3)(4)(5)(6). To our knowledge, however, no data have been published on the biological variation of this serum analyte. For any new test, data on the biological variation generated from the healthy population may be used (a) in setting desirable performance standards or analytical goals, (b) in assessing the utility of conventional population-based reference intervals, and (c) in critically evaluating the significance of changes in serial results from an individual (7). To investigate the analytical and biological variation of free and total carnitine, as well as of the acyl/free carnitine ratio, we collected blood samples once a week for 4 weeks from each of 14 healthy subjects (7 men and 7 nonpregnant women, ages 21–23 years), students of Mathias Belius University. The subjects agreed to maintain current dietary habits, body weight, and exercise program (if any) for the duration of the study. The criteria of the hospital Ethics Committee were respected in this experiment. To minimize sources of preanalytical variation, venous blood specimens were drawn between 0800 and 0900 h after ∼12 h of fasting. The subjects …

Traditional lipoprotein profile: clinical utility, performance requirements, and standardization

The lipid and lipoprotein parameters which are predominantly measured and effectively comprise the traditional lipoprotein profile include total cholesterol, high density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol, and triglyceride. Total cholesterol is accepted as the initial entry point in a case finding approach such as that recommended by the National Cholesterol Education Program (NCEP). HDL cholesterol, known to be a strong inverse predicator of risk, is an additional measurement to total cholesterol to improve risk assessments. The evidence for triglyceride association remains mixed: although strong associations are found in some studies, the evidence as an independent risk factor is still incomplete. Triglyceride is therefore measured primarily for LDL estimation. Final classification and potential intervention is ultimately based on the measurement of LDL cholesterol. Reliability in the measurement of total cholesterol, HDL, LDL, and triglyceride is especially important if the uniform decision points established by the NCEP are to be properly implemented. Attention must be placed on controlling preanalytical sources of variation, which can account for as much as 60% of the total measurement variability. The major analytical source of error comes from matrix effects, which results in problems of proper analytical calibration. Instrument system calibration should be verified by a comparison with an accuracy base using fresh patient specimens. CDC has established a network of reference method laboratories to provide access to these lipid and lipoprotein accuracy bases.

Blood lipid measurements: Variations and practical utility

Objective. —To describe the magnitude and impact of the major biological and analytical sources of variation in serum lipid and lipoprotein levels on risk of coronary heart disease; to present a way to qualitatively estimate the total intraindividual variation; and to demonstrate how to determine the number of specimens required to estimate, with 95% confidence, the true underlying total cholesterol value in the serum of a patient. Data Sources. —Representative references on each source of variation were selected from more than 300 reviewed publications, most published within the past 5 years, to document current findings and concepts. Most articles reviewed were in English. Study Selections. —Studies on biological sources of variation were selected using the following criteria: representative of published findings, clear statement of either significant or insignificant results, and acquisition of clinical and laboratory data under standardized conditions. Representative results for special populations such as women and children are reported when results differ from those of adult men. Data Extraction. —References were selected based on acceptable experimental design and use of standardized laboratory lipid measurements. Data Synthesis. —The lipid levels considered representative for a selected source of variation arose from quantitative measurements by a suitably standardized laboratory. Statistical analysis of data was examined to assure reliability. The proposed method of estimating the biological coefficient of variation must be considered to give qualitative results, because only two or three serial specimens are collected in most cases for the estimation. Conclusions. —Concern has arisen about the magnitude, impact, and interpretation of preanalytical as well as analytical sources of variation on reported results of lipid measurements of an individual. Preanalytical sources of variation from behavioral, clinical, and sampling sources constitute about 60% of the total variation in a reported lipid measurement of an individual. A technique is presented to allow physicians to qualitatively estimate the intraindividual biological variation of a patient from the results of two or more specimens reported from a standardized laboratory and to determine whether additional specimens are needed to meet the National Cholesterol Education Program recommendation that the intraindividual serum total cholesterol coefficient of variation not exceed 5.0. A National Reference Method Network has been established to help solve analytical problems. ( JAMA . 1992;267:1652-1660)

Clinical pathology: preanalytical variation in preclinical safety assessment studies--effect on predictive value of analyte tests.

Significant differences in concentrations of analytes in samples may be introduced before samples enter analyzers. These differences are known as preanalytical variation and are part of the overall variation in analytical data. Preanalytical variation is caused by factors that operate during animal preparation prior to sampling, sample collection, sample processing, and sample storage prior to measurement. Preanalytical variation is important because it detracts from the predictive value of analyte measurements. Preanalytical variation may permanently damage data. Because its effects are difficult to quantitate it should be minimized in safety assessment studies. Sources of preanalytical variation are actions performed on animals prior to sample collection and actions performed on the specimen prior to analysis. Preanalytical variation produces a range of artefacts in experimental data. Consequences of preanalytical variation are loss of confidence in the data, obfuscation of real test article effects, false effects, and possibly the expense of repeating a study. To limit preanalytical variation, its sources must be identified, the effects documented, and measures devised to eliminate its sources. Predictive value (likelihood of actual disease) of appropriate clinical pathology tests in toxicology is inversely dependent on preanalytical variation: uncontrolled variation produces data with low predictive values, and controlled variation produces data with high predictive values.

Blood Lipid Measurements

To describe the magnitude and impact of the major biological and analytical sources of variation in serum lipid and lipoprotein levels on risk of coronary heart disease; to present a way to qualitatively estimate the total intraindividual variation; and to demonstrate how to determine the number of specimens required to estimate, with 95% confidence, the "true" underlying total cholesterol value in the serum of a patient. Representative references on each source of variation were selected from more than 300 reviewed publications, most published within the past 5 years, to document current findings and concepts. Most articles reviewed were in English. Studies on biological sources of variation were selected using the following criteria: representative of published findings, clear statement of either significant or insignificant results, and acquisition of clinical and laboratory data under standardized conditions. Representative results for special populations such as women and children are reported when results differ from those of adult men. References were selected based on acceptable experimental design and use of standardized laboratory lipid measurements. The lipid levels considered representative for a selected source of variation arose from quantitative measurements by a suitably standardized laboratory. Statistical analysis of data was examined to assure reliability. The proposed method of estimating the biological coefficient of variation must be considered to give qualitative results, because only two or three serial specimens are collected in most cases for the estimation. Concern has arisen about the magnitude, impact, and interpretation of preanalytical as well as analytical sources of variation on reported results of lipid measurements of an individual. Preanalytical sources of variation from behavioral, clinical, and sampling sources constitute about 60% of the total variation in a reported lipid measurement of an individual. A technique is presented to allow physicians to qualitatively estimate the intraindividual biological variation of a patient from the results of two or more specimens reported from a standardized laboratory and to determine whether additional specimens are needed to meet the National Cholesterol Education Program recommendation that the intraindividual serum total cholesterol coefficient of variation not exceed 5.0. A National Reference Method Network has been established to help solve analytical problems.