Significant Interlaboratory Variation Research Articles

Belgian Recommendations for Analytical Verification and Validation of Immunohistochemical Tests in Laboratories of Anatomic Pathology.

Analytical verification and validation of immunohistochemical (IHC) tests and their equipment are common practices for today's anatomic pathology laboratories. Few references or guidelines are available on how this should be performed. The study of Sciensano (the Belgian national competent authority regarding licensing of medical laboratories) performed in 2016, demonstrated a significant interlaboratory variation in validation procedures of IHC tests among Belgian laboratories. These results suggest the unavailability of practical information on the approach to the verification and validation of these tests. The existing Belgian Practice Guideline for the implementation of a quality management system in anatomic pathology laboratories has been reviewed to meet this demand and, in addition, to prepare the laboratories for the EU-IVD revised regulations (IVDR). This paper describes Belgian recommendations for the verification and validation of IHC tests before implementation, for ongoing validation, and for revalidation. For each type of test (according to the IVDR classification and the origin) and its intended use (purpose), it addresses how to perform analytical verification/validation by recommending: (1) the number of cases in the validation set, (2) the performance characteristics to be evaluated, (3) the objective acceptance criteria, (4) the evaluation method for the obtained results, and (5) how and when to revalidate. A literature study and a risk analysis taking into account the majority of variables regarding verification/validation of methods have been performed, resulting in an expert consensus recommendation that is a compromise among achievability, affordability, and patient safety. This new consensus recommendation has been incorporated in the aforementioned ISO 15189:2012-based Practice Guideline.

Assessment ofurinary 6-hydroxy-2,4-cyclohexadienyl mercapturic acid as a novel biomarker ofbenzene exposure.

Assessing benzene exposure is a public health priority due to its deleterious health effects and ubiquitous industrial and environmental sources of exposure. Phenyl mercapturic acid (PhMA) is a commonly used urinary biomarker to assess benzene exposure. However, recent work has identified significant interlaboratory variation in urinary PhMA concentrations related to methodological differences. In this study, we present urinary 6-hydroxy-2,4-cyclohexadienyl mercapturic acid (pre-PhMA), a metabolite that undergoes acid-catalyzed dehydration to form PhMA, as a novel and specific urinary biomarker for assessing benzene exposure. We developed and validated the first quantitative liquid chromatography-tandem mass spectrometry assay for measuring urinary concentrations of pre-PhMA. The pH effect on the method of ruggedness testing determined that pre-PhMA is stable across the normal human urine pH range and that neutral conditions must be maintained throughout quantification for robust and accurate measurement of urinary pre-PhMA concentrations. The method exhibited below 2 ng/mL sensitivity for pre-PhMA, linearity over three orders of magnitude, and precision and accuracy within 10%. Urinary pre-PhMA concentrations were assessed in 369 human urine samples. Smoking individuals exhibited elevated levels of pre-PhMA compared to non-smoking individuals. Furthermore, the relationship between benzene exposure and urinary pre-PhMA levels was explored by examining the correlation of pre-PhMA with 2-cyanoethyl mercapturic acid, a smoke exposure biomarker. The urinary biomarkers exhibited a positive correlation (r = 0.720), indicating that pre-PhMA levels increased with benzene exposure. The results of this study demonstrate that urinary pre-PhMA is a rugged and effective novel biomarker of benzene exposure that can be widely implemented for future biomonitoring studies.

The variability of standard artificial soils: cadmium and phenanthrene sorption measured by a batch equilibrium method

Artificial soil (AS) is used in soil ecotoxicology as a test medium or reference matrix. AS is prepared according to standard OECD/ISO protocols and components of local sources are usually used by laboratories. This may result in significant inter-laboratory variations in AS properties and, consequently, in the fate and bioavailability of tested chemicals. In order to reveal the extent and sources of variations, the batch equilibrium method was applied to measure the sorption of 2 model compounds (phenanthrene and cadmium) to 21 artificial soils from different laboratories. The distribution coefficients (Kd) of phenanthrene and cadmium varied over one order of magnitude: from 5.3 to 61.5L/kg for phenanthrene and from 17.9 to 190L/kg for cadmium. Variations in phenanthrene sorption could not be reliably explained by measured soil properties; not even by the total organic carbon (TOC) content which was expected. Cadmium logKd values significantly correlated with cation exchange capacity (CEC), pHH2O and pHKCl, with Pearson correlation coefficients of 0.62, 0.80, and 0.79, respectively. CEC and pHH2O together were able to explain 72% of cadmium logKd variability in the following model: logKd=0.29pHH2O+0.0032 CEC –0.53. Similarly, 66% of cadmium logKd variability could be explained by CEC and pHKCl in the model: logKd=0.27pHKCl+0.0028 CEC –0.23. Variable cadmium sorption in differing ASs could be partially treated with these models. However, considering the unpredictable variability of phenanthrene sorption, a more reliable solution for reducing the variability of ASs from different laboratories would be better harmonization of AS preparation and composition.

A multicenter assessment of single-cell models aligned to standard measures of cell health for prediction of acute hepatotoxicity

Assessing the potential of a new drug to cause drug-induced liver injury (DILI) is a challenge for the pharmaceutical industry. We therefore determined whether cell models currently used in safety assessment (HepG2, HepaRG, Upcyte and primary human hepatocytes in conjunction with basic but commonly used endpoints) are actually able to distinguish between novel chemical entities (NCEs) with respect to their potential to cause DILI. A panel of thirteen compounds (nine DILI implicated and four non-DILI implicated in man) were selected for our study, which was conducted, for the first time, across multiple laboratories. None of the cell models could distinguish faithfully between DILI and non-DILI compounds. Only when nominal in vitro concentrations were adjusted for in vivo exposure levels were primary human hepatocytes (PHH) found to be the most accurate cell model, closely followed by HepG2. From a practical perspective, this study revealed significant inter-laboratory variation in the response of PHH, HepG2 and Upcyte cells, but not HepaRG cells. This variation was also observed to be compound dependent. Interestingly, differences between donors (hepatocytes), clones (HepG2) and the effect of cryopreservation (HepaRG and hepatocytes) were less important than differences between the cell models per se. In summary, these results demonstrate that basic cell health endpoints will not predict hepatotoxic risk in simple hepatic cells in the absence of pharmacokinetic data and that a multicenter assessment of more sophisticated signals of molecular initiating events is required to determine whether these cells can be incorporated in early safety assessment.

Clinical application and standardization of antiphospholipid antibodies tests

According to the consensus criteria of antiphospholipid syndrome(APS), the diagnosis of APS requires the persistent presence of antiphospholipid antibodies (aPLs), indicating the critical role of aPLs in the diagnosis of APS. During the last decade, great efforts have been made to improve the laboratory detection and standardization of aPLs testing. Unfortunately, the heterogeneous nature of aPLs, lacking of standardization in aPLs test, and significant inter-laboratory variation have hampered the clinical application of aPLs test. In this commentary, the clinical application and standardization of aPLs test are focused on, and how to establish the standardization system in aPLs test in order to improve the performance of aPLs test in clinical practice are discussed.(Chin J Lab Med, 2015, 38: 793-796) Key words: Antiphospholipid syndrome; Antibodies, antiphospholipid; Reference standards

Current status and prospects of antiphospholipid antibodies tests

Antiphospholipid antibodies (APLs) are important for the diagnosis of antiphospholipid syndrome (APS), especially for predicting the risk of thrombosis and pathological pregnancy. However, the heterogeneity of antiphospholipid antibodies, lacking of standardization and significant interlaboratory variation binder the clinical application of APLs and better understanding of APS diagnosis and treatment. Therefore, it is urgent to establish a standardize system for antiphospholipid antibodies test and to improve the performance of the test and perform well-designed clinical evaluation.（Chin J Lab Mes，2014,37： 564-566） Key words: Antiphospholipid syndrome; Antibodies, antiphospholipid; Reference values

Quantifying inter-laboratory variability in stable isotope analysis of ancient skeletal remains.

Over the past forty years, stable isotope analysis of bone (and tooth) collagen and hydroxyapatite has become a mainstay of archaeological and paleoanthropological reconstructions of paleodiet and paleoenvironment. Despite this method's frequent use across anthropological subdisciplines (and beyond), the present work represents the first attempt at gauging the effects of inter-laboratory variability engendered by differences in a) sample preparation, and b) analysis (instrumentation, working standards, and data calibration). Replicate analyses of a 14C-dated ancient human bone by twenty-one archaeological and paleoecological stable isotope laboratories revealed significant inter-laboratory isotopic variation for both collagen and carbonate. For bone collagen, we found a sizeable range of 1.8‰ for δ13Ccol and 1.9‰ for δ15Ncol among laboratories, but an interpretatively insignificant average pairwise difference of 0.2‰ and 0.4‰ for δ13Ccol and δ15Ncol respectively. For bone hydroxyapatite the observed range increased to a troublingly large 3.5‰ for δ13Cap and 6.7‰ for δ18Oap, with average pairwise differences of 0.6‰ for δ13Cap and a disquieting 2.0‰ for δ18Oap. In order to assess the effects of preparation versus analysis on isotopic variability among laboratories, a subset of the samples prepared by the participating laboratories were analyzed a second time on the same instrument. Based on this duplicate analysis, it was determined that roughly half of the isotopic variability among laboratories could be attributed to differences in sample preparation, with the other half resulting from differences in analysis (instrumentation, working standards, and data calibration). These findings have serious implications for choices made in the preparation and extraction of target biomolecules, the comparison of results obtained from different laboratories, and the interpretation of small differences in bone collagen and hydroxyapatite isotope values. To address the issues arising from inter-laboratory comparisons, we devise a novel measure we term the Minimum Meaningful Difference (MMD), and demonstrate its application.

Development and testing of external quality assessment samples for Salmonella detection in poultry samples.

Salmonella-contaminated poultry house dust plus 10 g chicken faeces inoculated with Salmonella Enteritidis and then frozen for storage and transport were used as candidate external quality assurance test samples. Variations in faeces sample preparation, storage and culture were examined initially. This indicated that, within modest limits, the age of the inoculating culture and of the faeces did not affect detection, nor did swirling the pre-enrichment culture or extending its duration. Under optimal conditions of preparation and storage, Salmonella numbers of 70 colony-forming units (CFU) and above were reliably detected at the originating laboratory. A ring trial was performed, involving 13 external UK laboratories plus the originating laboratory. Faeces samples inoculated with Salmonella Enteritidis were frozen, transported on dry ice and tested by the ISO 6579:2002 (Annex D) method. Detection by the originating laboratory was consistent with the previously established lower limit for reliability of 70 CFU. However, the sensitivity of detection by the external laboratories was apparently poorer in several cases, with significant interlaboratory variation seen at the lowest inoculum level, using Fisher's exact test. Detection of Salmonella in poultry house dust appeared to be more sensitive and uniform among laboratories. Significance and impact of the study: Salmonella surveillance and control regimes in the European poultry industry and elsewhere require sensitive culture detection of Salmonella in environmental samples, including poultry faeces. A ring trial was conducted, and the results highlighted that some of the participating laboratories failed to identify Salmonella. This suggests that contaminated frozen faeces cubes could be beneficial to assess proficiency, according to the results of this preliminary study. The data obtained in this study can be used as an indication for the design of realistic external quality assurance for laboratories involved in official testing of Salmonella in poultry flocks.

Proficiency Testing Results for Heparin-Induced Thrombocytopenia in North America

Between 2010 and 2012, North American Specialized Coagulation Laboratory Association (NASCOLA) distributed five proficiency testing challenges to evaluate laboratory testing for heparin-induced thrombocytopenia (HIT). Results (n = 355) were submitted from 43 unique laboratories for 10 samples (3 positive, 2 weak positive, and 5 negative). The vast majority of results were from commercial enzyme-linked immunosorbent assay (ELISA) methods, predominantly polyvalent assays. Laboratories performed well in the classification of clear negative and positive samples. All results (100%) submitted for the five negative samples (n = 173) and 97% of immunological results submitted for the three positive samples (n = 105) were correctly classified (the incorrect responses were two borderline classifications and, from a gel-agglutination method, one negative classification). There was more difficulty in the classification of the two weak positive samples (n = 70). In one survey, 61% of results from the weak positive sample were classified as positive, while 21% were called negative, 16% were called borderline, and 2% were called inconclusive. In a second survey, 16% of results from the weak positive sample were called positive, while 56% were called negative, and 28% were called borderline. Significant interlaboratory variation was observed for ELISA results, with coefficients of variation of about 20 to 30%. We conclude that there is variability in HIT laboratory testing and that identification of weak positive samples can be challenging.

An update on international consensus guidelines for antiphospholipid antibody testing

In spite of the publication of several proposals, consensus documents and expert opinions, there still exists significant inter-assay and inter-laboratory variation in the results of both aCL and anti-b2GPI testing, which affects the consistency of the diagnosis of the antiphospholipid syndrome (APS). The USA-based but internationally-represented Clinical and Laboratory Standards Institute (CLSI) therefore convened a Document Development Committee on Antiphospholipid Antibodies in mid 2011. The overall purpose of this committee is to provide clinical laboratories, assay users and kit developers and manufacturers with recommendations and consensus guidelines that will hopefully improve the consistency, performance and reliability of aCL and anti-b2GPI immunoassays.

Lack of Impact of Semen Quality on Fertilization in Assisted Conception

Defective semen quality is one of the commonest causes of infertility. The diagnosis of male fertility depends upon a descriptive evaluation of human semen, however a normal semen analysis does not necessarily indicate satisfactory fertility potential. (i) to examine the semen quality of patients undergoing treatment by assisted conception, (ii) to explore relationships between semen quality and treatment outcomes, and (iii) to look at inter-laboratory variation in the assessment of semen quality. Semen quality in patients undergoing assisted conception treatment between 2001 and 2004 was reviewed. Data on female age, egg numbers and fertilization outcomes was obtained by case note review. The thresholds used to direct patients towards IVF or ICSI treatment were comparable with the normal values promulgated by WHO, with the exception of morphology. Semen quality was not predictive of fertilization rates. When the results of independent measurements of the same sample were compared, there was diagnostic disagreement in between 10%-29% of samples. The conventional criteria of semen quality are used to determine treatment strategy for couples undergoing assisted conception but are not reflected in fertilization rates, emphasising the limited utility of the conventional criteria of semen quality in the assessment of sperm function. There remains significant inter-laboratory variation in the results of semen analysis.

Technical External Quality Assessment for SurePath® stained liquid‐based cytology gynaecological cervical samples using control material – a novel approach

The Technical External Quality Assessment Scheme (TEQA) introduced in Wales is based on NHSCSP publication No 19 [External Quality Assessment Scheme for the Evaluation of Papanicolaou Staining in Cervical Cytology] which sets out the policies and standard operating procedures for the TEQA of Papanicolaou staining of gynaecological cervical samples. As part of a development plan for the TEQA scheme in Wales, the use of a control sample was introduced to the assessment process - a common control sample can provide a consistent assessment parameter independent of the recommended slide selection process [External Quality Assessment Scheme for the Evaluation of Papanicolaou Staining in Cervical Cytology, NHSCSP Publication No 19, February 2004] enabling direct comparison of staining standards for laboratories within the region; this counters selection variation bias, establishing a process that may be more representative of routine staining results. A cervical sample was selected in line with the criteria described in publication 19 [External Quality Assessment Scheme for the Evaluation of Papanicolaou Staining in Cervical Cytology, NHSCSP Publication No 19, February 2004]. Thirteen slides were prepared by the scheme facilitator from this anonymized sample. These control slides were subsequently 'fixed' but not stained, then distributed to the laboratories participating in the TEQA scheme. The slides were stained using their standard regime, then returned to the facilitator for assessment. The slides showed consistent staining with no significant inter-laboratory variation, however, the eosinophilic stained components exhibited an artificial colouration, which slightly altered the expected stained appearance; this was thought to be due to 'cross-reactivity' of the spray fixative with the preserving agent. To address this artefact, a further development of control procedures was devised utilizing a pooled control sample procedure. Residual material from a number of similar samples was pooled and distributed in aliquots to participant laboratories for standard processing and staining; the completed slides were returned to the scheme facilitator for assessment. The pooled sample slides were assessed at the next scheduled quarterly TEQA assessment. The overall scoring for these samples produced an acceptable level of Papanicolaou staining for 12 of the laboratories - only one laboratory produced a marginal score. The artefactual presentation of eosinophilia was not seen. This method of producing control material establishes consistency in the TEQA comparative assessment process, counters selection bias and reduces the time demands associated with slide selection. It may also prove useful in identifying technical problems within laboratories during sample preparation prior to or during staining, including equipment or process faults. This technique is now well established locally as an enhancement of the current TEQA scheme for the assessment of slide staining. We feel that this enhancement could be incorporated as a new initiative in the current National TEQA scheme as a complement to the established selection process.

Interlaboratory Comparison of Epstein-Barr Virus Viral Load Assays

To assess interlaboratory variability in qualitative and quantitative Epstein-Barr virus (EBV) viral load (VL) testing, we distributed a panel of samples to 28 laboratories in the USA, Canada and Europe who performed testing using commercially available reagents (n = 12) or laboratory-developed assays (n = 18). The panel included two negatives, seven constructed samples using Namalwa and Molt-3 cell lines diluted in plasma (1.30-5.30 log(10) copies/mL) and three clinical plasma samples. Significant interlaboratory variation was observed for both actual (range 1.30-4.30 log(10) copies/mL) and self-reported (range, 1.70-3.30 log(10) copies/mL) lower limits of detection. The variation observed in reported results on individual samples ranged from 2.28 log(10) (minimum) to 4.14 log(10) (maximum). Variation was independent of dynamic range and use of commercial versus laboratory-developed assays. Overall, only 47.0% of all results fell within acceptable standards of variation: defined as the expected result +/- 0.50 log(10). Interlaboratory variability on replicate samples was significantly greater than intralaboratory variability (p < 0.0001). Kinetics of change in VL appears more relevant than absolute values and clinicians should understand the uncertainty associated with absolute VL values at their institutions. The creation of an international reference standard for EBV VL assay calibration would be an initial important step in quality improvement of this laboratory tool.

Neutralizing and Binding Antibodies to Interferon Beta in Patients with Multiple Sclerosis: A Comparison of Assay Results from Three Italian Centres

Interferon (IFN) beta therapy for multiple sclerosis (MS) is associated with the development of binding antibodies (BAbs) and neutralizing antibodies (NAbs) in a percentage of patients. This study investigated the reproducibility of results of two different antibody detection techniques using serum from 100 patients with MS who were receiving IFN beta therapy. Fifty samples were analysed using a commercially available kit-based BAb assay and a further 50 different samples were analysed using a widely used NAb cytopathic effect assay, at three different laboratories. All three centres agreed on the BAb status of all serum samples. However, only 84% agreement was reached on serum NAb status, and there was significant inter-laboratory variation in NAb titre values. Further analysis of these data revealed a correlation between the mean NAb titre and the coefficient of variation of serum samples, indicating greater discordance with higher NAb titres. A significant interlaboratory variation in NAb titres does exist; thus, caution is required when comparing titres from different centres. It is clear that validated detection assays are needed to accurately quantify NAb titres.

Biological and Technical Variables Affecting Immunoassay Recovery of Cytokines from Human Serum and Simulated Vaginal Fluid: A Multicenter Study

The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1β and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1β and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal−Wallis analysis of variance with Dunn’s multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences (P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1β determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface.

How difficult is it to achieve compliance with the quality control requirements of the Clinical and Laboratory Standards Institute's guideline M42-A?

Data accumulated in a multi-laboratory study of disc diffusion susceptibility data for Escherichia coli ATCC 25922 was analysed with respect to the acceptable ranges for this strain specified in the Clinical and Laboratory Standards Institute's M42-A guidelines. Seven laboratories each generated 60 zone size measurements for discs containing five agents for which acceptable ranges had been published. Of the 35 data sets analysed 37% contained >10% of their measurements outside the acceptable ranges. For each agent, between one and four of the seven data sets obtained contained >10% of the zone sizes outside the acceptable ranges. Only one laboratory obtained data sets that for all agents, contained <10% of the zone sizes outside the acceptable. For the other six, non-compliant data sets were obtained for between one and four of the agents. These data suggest that the adherence to a specified test protocol cannot be assumed to result in the achievement of compliance with the quality control requirements of that protocol. They would further suggest that the need to undertake corrective actions, as specified in M42-A, would be frequently required before a laboratory could claim to be using this protocol and could legitimately apply any cut-off values or breakpoints that are or that might be, associated with it use. As laboratories involved in susceptibility testing of bacteria associated with aquaculture frequently handle only a small number of strains per year, the achievement of compliance with the quality control requirements of a standard protocol might require a disproportionate and unacceptable increase in their work load. The data obtained in this retrospective study also demonstrated that, even if all data sets containing >10% non-compliant measurements were eliminated; significant inter-laboratory variation in mean zone sizes would remain.Laboratory-independent cut-off values will be of value only if inter-laboratory variation in disc diffusion data can be reduced to a minimum. It is argued that the data presented here provides strong grounds for questioning whether, particularly in the case of laboratories with small strain throughputs, inter-laboratory variation can be appropriately resolved by rigorous specifications of test protocols and quality control criteria. Thus, they would rather support the development of cut-off values by the application of normalised resistance interpretation. As the cut-off values generated by this approach are laboratory-specific, they are not influenced by inter-laboratory variation.

Noninvasive Markers of Fibrosis for Longitudinal Assessment of Fibrosis in Chronic Liver Disease: Are They Ready for Prime Time?

Over the past decade, there has been a renewed enthusiasm to develop noninvasive serum markers or tests to assess the presence and severity of fibrosis in chronic liver disease. Although a single marker or test has lacked the necessary accuracy to predict fibrosis, different combinations of these markers or tests have shown encouraging results. However, inter-laboratory variability and inconsistent results with liver diseases of varying etiologies have made it difficult to assess the reliability of these markers in clinical practice. In this issue of the journal, Poynard et al. describe the "histological" response to lamivudine in patients with chronic Hepatitis B Virus (HBV) over a 24-month period using surrogate serum biomarkers (Fibrotest-Actitest, FT-AT) without corroborating histological data. Investigators found improvement in fibrosis and inflammation in 85% and 91%, respectively, despite the emergence of YMDD mutation in 41.5% of patients. The higher improvement rates reported in this study should be interpreted with caution for a number of reasons including the absence of data on virological response rates, corroboratory histological data, and data on the validity of FT to evaluate fibrosis in a longitudinal manner. Although FT has been studied extensively by the authors of the current study, to date there are only few independent studies. In addition to significant inter-laboratory variations, these studies have shown that significant fibrosis could be missed, or conversely significant fibrosis diagnosed in the absence of minimal or no fibrosis in about 15% to 20% of patients. We may be approaching a time when serum biomarkers may become an integral part of the assessment of patients with chronic liver disease, but published evidence suggests that these markers are not yet ready for prime time.

Noninvasive Markers of Fibrosis for Longitudinal Assessment of Fibrosis in Chronic Liver Disease: Are They Ready for Prime Time?

Over the past decade, there has been a renewed enthusiasm to develop noninvasive serum markers or tests to assess the presence and severity of fibrosis in chronic liver disease. Although a single marker or test has lacked the necessary accuracy to predict fibrosis, different combinations of these markers or tests have shown encouraging results. However, interlaboratory variability and inconsistent results with liver diseases of varying etiologies have made it difficult to assess the reliability of these markers in clinical practice. In this issue of the Journal, Poynard and colleagues describe the "histological" response to lamivudine in patients with chronic HBV over a 24-month period using surrogate serum biomarkers (FibroTest-ActiTest) without corroborating histological data. Investigators found improvement in fibrosis and inflammation in 85% and 91%, respectively, despite the emergence of YMDD mutation in 41.5% of patients. The higher improvement rates reported in this study should be interpreted with caution for a number of reasons including the absence of data on virological response rates, corroboratory histological data, and data on the validity of FibroTest to evaluate fibrosis in a longitudinal manner. Although FibroTest has been studied extensively by the authors of the current study, to date there are only few independent studies. In addition to significant interlaboratory variations, these studies have shown that significant fibrosis could be missed, or conversely significant fibrosis diagnosed in the absence of minimal or no fibrosis in about 15-20% of patients. We may be approaching a time when serum biomarkers may become an integral part of the assessment of patients with chronic liver disease, but published evidence suggests that these markers are not yet ready for prime time.

A Multilaboratory Peer Assessment Quality Assurance Program-Based Evaluation of Anticardiolipin Antibody, and beta2-Glycoprotein I Antibody Testing

We evaluated the performance of anticardiolipin (aCL) and beta2-glycoprotein I (beta2-GPI) antibody assays through a large external quality assurance program. Data from the 2002 cycle of the Royal College of Pathologists of Australasia Quality Assurance Program (RCPA QAP) were analyzed for variation in reported numerical values and semiquantitative results or interpretations according to method type or group and in conjunction with available clinical data. High interlaboratory variation in numerical results and notable method-based variation, combined with a general lack of consensus in semiquantitative reporting, continues to be observed. Numerical results from cross-laboratory testing of 12 serum samples (for immunoglobulin G [IgG]-aCL, IgM-aCL, and IgG-beta2-GPI) yielded interlaboratory coefficients of variation (CVs) that were higher than 50% in six of 12 (50%) specimens for IgG-aCL, and 12 of 12 (100%) specimens for IgM-aCL and IgG-beta2-GPI. Semiquantitative reporting also varied considerably, with total (100%) consensus occurring in only four of 36 (11%) occasions. General consensus (where > 90% of participating laboratories agreed that a given serum sample gave a result of either negative or positive) was only obtained on 13 of 36 (36%) occasions. Variation in results between different method types or groups were also present, resulting in potential biasing of the RCPA QAP-defined target results by the large number of laboratories using the dominant aCL assays. Finally, laboratory findings frequently did not agree with the available clinical information. In conclusion, in a large proportion of specimens from the 2002 RCPA QAP cycle, laboratories could not agree on whether a serum sample tested was aCL-positive or aCL-negative, or beta2-GPI-positive or beta2-GPI-negative. Despite prior attempts to improve the standardization of testing and reporting practices, laboratory testing for aCL and anti-beta2-GPI still demonstrates significant interlaboratory and intermethod variation, which needs to be taken into account for the clinical interpretation of test results, especially those from different laboratories.

Consensus guidelines for anticardiolipin antibody testing

Despite the use of standardized GPL and MPL units, significant inter-laboratory and inter-method variation in anticardiolipin antibody (aCL) testing still exists, limiting the clinical utility and inter-laboratory portability of test results. This article reviews published and unpublished guidelines (some developed using consensus procedures) that can be used to improve various aspects of: (1) specimen collection; (2) aCL assay manufacture and testing procedures; (3) quality control; and (4) interpretation (including reporting) of results; and in doing so, improve the consistency of aCL results between different laboratories, assays and runs. However, there is a still a need for consensus guidelines that combine and deal with all of the aforementioned aspects of aCL testing and reporting.