Volume 56, Issue 10 - Special Issue: Harmonization in Laboratory Medicine: the Request, the Sample, the Measurement, and the Report – an Update. Part 1 / Editors: Jillian R. Tate, MariaStella Graziani and Mario Plebani

The goal of harmonizing laboratory information is to contribute to quality in patient care, ultimately improving upon patient outcomes and safety. The main focus of harmonization and standardization initiatives has been on analytical processes within the laboratory walls, clinical chemistry tests in particular. However, two major evidences obtained in recent years show that harmonization should be promoted not only in the analytical phase but also in all steps of the testing process, encompassing the entire field of laboratory medicine, including innovative areas (e.g. “omics”) rather than just conventional clinical chemistry tests. A large body of evidence demonstrates the vulnerability of the extra-analytical phases of the testing cycle. Because only “good biological samples” can assure good analytical quality, a closer interconnection between the different phases of the cycle is needed. In order to provide reliable and accurate laboratory information, harmonization activities should cover all steps of the cycle from the “pre-pre-analytical” phase (right choice of test at right time for right patient) through the analytical steps (right results with right report) to the “post-post-analytical” steps (right and timely acknowledgment of laboratory information, right interpretation and utilization with any necessary advice as to what to do next with the information provided). In addition, modern clinical laboratories are performing a broad menu of hundreds of tests, covering both traditional and innovative subspecialties of the discipline. In addition, according to a centered viewpoint, harmonization initiatives should not be addressed exclusively to clinical chemistry tests but should also include all areas of laboratory medicine.

The article tries to reply to the following three questions: Are External Quality Assessment Schemes (EQAS) really fit for purpose? Are all schemes equivalent and sufficiently harmonized? Is the role of EQAS similar and necessary in all branches of laboratory medicine? Although the reply to the first two questions is, unfortunately, negative for several reasons (lack of commutable material with reference method values, EQAS with different scopes, etc.), the reply to the third one is positive: EQAS are a necessary source of information on trueness and accuracy and must be fully developed for all the branches of the clinical laboratory.

The European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) has initiated many harmonization activities in all phases of the examination process. The EFLM is dealing with both the scientific and the educational aspects of harmonization, with the intention of disseminating best practice in laboratory medicine throughout Europe. Priorities have been given (1) to establish a standard for conducting and assessing biological variation studies and to construct an evidence based EFLM webpage on biological variation data, (2) to harmonize preanalytical procedures by producing European guidelines, (3) to improve test ordering and interpretation, (4) to produce other common European guidelines for laboratory medicine and play an active part in development of clinical guidelines, (5) to establish a common basis for communicating laboratory results to patients, (6) to harmonize units of measurement throughout Europe, (7) to harmonize preanalytical procedures in molecular diagnostics and (8) to harmonize and optimize test evaluation procedures. The EFLM is also now launching the 5th version of the European Syllabus to help the education of European Specialists in Laboratory Medicine (EuSpLM), which is being supported by the development of e-learning courses. A register of EuSpLM is already established for members of National Societies in EU countries, and a similar register will be established for specialists in non-EU countries.

In our efforts to advance the profession and practice of clinical laboratory medicine, strong coordination and collaboration are needed more than ever before. At the dawn of the 21st century, medical laboratories are facing many unmet clinical needs, a technological revolution promising a plethora of better biomarkers, financial constraints, a growing scarcity of well-trained laboratory technicians and a sharply increasing number of International Organization for Standardization guidelines and new regulations to which medical laboratories should comply in order to guarantee safety and effectiveness of medical test results. Although this is a global trend, medical laboratories across continents and countries are in distinct phases and experience various situations. A universal underlying requirement for safe and global use of medical test results is the standardization and harmonization of test results. Since two decades and after a number of endeavors on standardization/harmonization of medical tests, it is time to reflect on the effectiveness of the approaches used. To keep laboratory medicine sustainable, viable and affordable, clarification of the promises of metrological traceability of test results for improving sick and health care, realization of formal commitment among all stakeholders of the metrological traceability chain and preparation of a joint and global plan for action are essential prerequisites. Policy makers and regulators should not only overwhelm the diagnostic sector with oversight and regulations but should also create the conditions by establishing a global professional forum for anchoring the metrological traceability concept in the medical test domain. Even so, professional societies should have a strong voice in their (inter-) national governments to negotiate long-lasting public policy commitment and funds for global standardization of medical tests.

An imbalance between bone formation and bone resorption is a factor in the development of osteoporosis. Bone turnover markers (BTM) are useful in assessing bone remodeling at the cellular level. Measurement of BTM is useful for assessing bone turnover and therefore fracture risk as well as for monitoring response to treatment response. This paper describes the steps that have been taken so far and the ongoing work to harmonize the total testing process for the assessment of bone turnover in osteoporosis internationally and collaboratively between the clinical and laboratory professions. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) in collaboration with the International Osteoporosis Foundation (IOF) as well as the National Bone Health Association (NBHA) in the US have designated procollagen type I N propeptide (s-PINP), and cross-linked β-isomerized type I collagen C-telopeptide (s-βCTX), in blood as reference standard BTM. Collaborative efforts are ongoing in order to standardize preanalytical steps as well as the analysis of s-PINP and s-CTX by standardizing or harmonizing their measurement as appropriate and reporting of values. Reporting units have been standardized. Reference interval studies and examination of appropriate decision thresholds, reference change values and treatment targets are ongoing with the aim of harmonizing the total testing process for the assessment of bone turnover in osteoporosis.

Accepting that standardizing and harmonizing laboratory practice has considerable value within laboratory medicine, including within the field of hemostasis, this paper concentrates on some recent and important initiatives in harmonizing hemostasis practice. Harmonization of hemostasis practice to improve clinical diagnosis and management is best driven by evidence and, in the absence of evidence, by consensus and expert opinion. To such end, there are various groups involved in such initiatives, and recent initiatives by these groups are highlighted in this review.

The European Autoimmunity Standardisation Initiative (EASI) has been founded in order to improve autoimmune diagnostics by stimulating the interaction between the clinicians and laboratory specialists, by standardization of autoantibody tests, and by harmonization of testing algorithms. The ultimate goal of EASI is to utilize autoimmune diagnostics in the best way in order to optimize patient care. This mini-review gives an overview of the historical perspective of EASI and summarizes the major achievements.

Clinical microbiology is a highly challenging technical discipline, which makes it difficult to harmonize processes and protocols. In addition, the lack of scientific consensus on some diagnostic algorithms and the need to address the diverse needs of different patient populations contribute to the lack of interlaboratory consistency. Laboratories utilize a number of measures and metrics, but the lack of standardized approaches and data collection means that they cannot effectively evaluate their performance against their peers. Coordinated efforts are required to develop tools that can be used across laboratories regardless of size or complexity.

Biological variation (BV) data have many applications in laboratory medicine. However, concern has been raised that some BV estimates in use today may be irrelevant or of unacceptable quality. A number of initiatives have been launched by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) and other parties to deliver a more harmonized practice in the generation, reporting and application of BV data. Resulting from a necessary focus upon the veracity of historical BV studies, critical appraisal and meta-analysis of published BV studies is possible through application of the Biological Variation Data Critical Appraisal Checklist (BIVAC), published in 2017. The BIVAC compliant large-scale European Biological Variation Study delivers updated high-quality BV data for a wide range of measurands. Other significant developments include the publication of a Medical Subject Heading term for BV and recommendations for common terminology for reporting of BV data. In the near future, global BV estimates derived from meta-analysis of BIVAC appraised publications will be accessible in a Biological Variation Database at the EFLM website. The availability of these high-quality data, which have many applications that impact on the quality and interpretation of clinical laboratory results, will afford improved patient care.

Over the years, ISO15189 has established itself as the international standard for quality management systems for all fields in laboratory medicine. As a result, also accreditation to this standard has increased in international adaptation. As a consequence, its impact grows as governments make the standard obligatory and/or national funding systems require ISO15189 accreditation for reimbursement. All these trends amplify each other. This overview aims to specify how ISO15189 has gained its current position as the harmonized quality standard that is not affected by borders between scientific (sub)fields nor between nations. From there, future challenges, opportunities and risks are addressed.

Effective management of clinical laboratories participating in external quality assessment schemes (EQAS) is of fundamental importance in ensuring reliable analytical results. The International Standard ISO 15189:2012 requires participation in interlaboratory comparison [e.g. external quality assessment (EQA)] for all tests provided by an individual laboratory. If EQAS is not commercially available, alternative approaches should be identified, although clinical laboratories may find it challenging to choose the EQAS that comply with the international standards and approved guidelines. Great competence is therefore required, as well as knowledge of the characteristics and key elements affecting the reliability of an EQAS, and the analytical quality specifications stated in approved documents. Another skill of fundamental importance is the ability to identify an alternative approach when the available EQAS are inadequate or missing. Yet the choice of the right EQA program alone does not guarantee its effectiveness. In fact, the fundamental steps of analysis of the information provided in EQA reports and the ability to identify improvement actions to be undertaken call for the involvement of all laboratory staff playing a role in the specific activity. The aim of this paper was to describe the critical aspects that EQA providers and laboratory professionals should control in order to guarantee effective EQAS management and compliance with ISO 15189 accreditation requirements.

Background: The mainstay of patient-oriented laboratory testing in emergency settings entails selecting a number and the type of tests according to valid criteria of appropriateness. Since the pattern of urgent tests requesting is variable across different institutions, we designed a joined survey between the Academy of Emergency Medicine and Care (AcEMC) and the Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC) for reaching tentative consensus about the most informative diagnostic tests in emergency settings. Methods: A survey, containing the most commonly ordered urgent laboratory tests and the relative clinical indications, was disseminated to eight relevant members of AcEMC and eight relevant members of SIBioC. All contributors were asked to provide numerical scores for the different laboratory parameters, where 1 indicated “strongly recommended”, 2 “recommended in specific circumstances”, and 3 “strongly discouraged”. The mean results of the survey were presented as the mean of responders’ values, and the parameters were finally classified as “strongly recommended” (mean value, 1.00–1.49), “weakly recommended” (mean value, 1.50–1.99), “discouraged” (mean value, 2.00–2.49) and “strongly discouraged” (mean value, 2.50–3.00). Results: The results of the survey allowed defining a hierarchy of priority, wherein 24 tests were “strongly recommended”. The use of five common tests was instead “strongly discouraged”. For 16 additional parameters in the list, the consensus ranged between “weakly recommended” and “discouraged”. Conclusions: We hope that results presented in this joint AcEMC-SIBioC consensus document may help harmonizing panel of tests and requesting patterns in emergency setting, at least at a national level.

The Working Group for the Preanalytical Phase (WG-PRE) was officially established by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) in 2013, with the aim of improving harmonization in the preanalytical phase across European member societies. Since its early birth, the WG-PRE has already completed a number of projects, including harmonizing the definition of fasting status, patient and blood tubes identification, color coding of blood collection tubes, sequence of tubes during blood drawing and participation in the development of suitable preanalytical quality indicators. The WG-PRE has also provided guidance on local validation of blood collection tubes, has performed two European surveys on blood sampling procedures and has organized four European meetings to promote the importance of quality in the preanalytical phase. The future activities entail development and validation of an external quality assessment scheme focused on preanalytical variables, development and dissemination of a survey about the local management of unsuitable samples in clinical laboratories, as well as release of EFLM phlebotomy guidelines. This article summarizes all recent achievements of the WG-PRE and illustrates future projects to promote harmonization in the preanalytical phase.

The International Consortium for Harmonization of Clinical Laboratory Results (ICHCLR) was established to fulfill recommendations identified by an international conference convened to review the available infrastructure and challenges in achieving harmonization of results among different measurement procedures. The specific objectives of the ICHCLR are to: prioritize measurands by medical importance, coordinate the work of different organizations, and stimulate development of technical and regulatory processes to achieve harmonization. Central to these objectives is the website “www.harmonization.net” developed by the ICHCLR as an information portal designed to provide a resource center for global activities to harmonize and standardize results from clinical laboratory measurement procedures. Priorities based on medical impact are provided for measurands for which harmonization is needed or work to implement harmonization is incomplete or inactive. By making information available regarding harmonization activities in progress or planned, coordination of work to harmonize laboratory measurement procedures will minimize duplication of effort and optimize the use of limited resources. A toolbox of technical procedures developed by ICHCLR to be considered when developing a process to achieve harmonization for a measurand is presented. The ICHCLR initiated a proposal to ISO Technical Committee 212 for a harmonization protocol as the basis for calibration traceability when there are no other higher order reference system components available. The ICHCLR offers a unique service to prioritize measurands in need of harmonization and to provide a centralized approach to organize global efforts to achieve harmonization of clinical laboratory test results.

Calibration 2.000 was initiated 20 years ago for standardization and harmonization of medical tests. The program also intended to evaluate adequate implementation of the In Vitro Diagnostics (IVD) 98/79/EC directive, in order to ensure that medical tests are fit-for-clinical purpose. The Calibration 2.000 initiative led to ongoing verification of test standardization and harmonization in the Netherlands using commutable external quality assessment (EQA)-tools and a type 1 EQA-design, where feasible. National support was guaranteed by involving all laboratory professionals as well as laboratory technicians responsible for EQA and quality officers. A category 1 EQA-system for general chemistry analytes, harmonizers for specific analytes like hGH and IGF-1, and commutable materials for other EQA-sections have been developed and structurally introduced in the EQA-schemes. The type 1 EQA-design facilitates the dialogue between individual specialists in laboratory medicine and the IVD-industry to reduce lot-to-lot variation and to improve standardization. In such a way, Calibration 2.000 sheds light on the metrological traceability challenges that we are facing and helps the laboratory community to get the issues on the table and resolved. The need for commutable trueness verifiers and/or harmonizers for other medical tests is now seen as paramount. Much knowledge is present in the Netherlands and for general chemistry, humoral immunology and protein chemistry, a few endocrinology tests, and various therapeutic drug monitoring (TDM) tests, commutable materials are available. Also the multi sample evaluation scoring system (MUSE) and the category 1 EQA-design offer many possibilities for permanent education of laboratory professionals to further improve the between and within laboratory variation and the test equivalence.

Background: Mass spectrometry (MS)-based 17-hydroxyprogesterone (17OHP) methods show considerable variation in results in external quality assurance (EQA) programs. An understanding of the current status of MS-based serum/plasma 17OHP quantification is important to facilitate harmonization. Methods: A 50-item e-survey related to (1) laboratory characteristics, (2) pre-analytical considerations and (3) analysis of 17OHP was developed and circulated to clinical MS laboratories via professional associations in Asia Pacific, Europe and North America. Results: Forty-four laboratories from 17 countries completed the survey. Sample preparation varied between laboratories with protein precipitation and liquid-liquid extraction being the most common processes. Analyte separation was most commonly achieved by liquid chromatography (LC) using a C18 column and mobile phases of water, methanol and formic acid. The ions selected for quantification were 331>97 m / z or 331>109 m / z . Alternative transition ions were used as qualifiers. Twenty-seven of 44 respondents reported preparing their calibrators in-house and variations in material purity and matrix were evident. Nine of 44 laboratories did not participate in an EQA program, and half did not know if their method separated out isobars. The reference intervals, and also their partitioning, reported by the laboratories were highly discrepant, in some cases, by multiple folds. Conclusions: Although MS-based methods are similar in many facets, they are highly disparate. Five recommendations have been developed as an outcome of this survey to support the continued improvement of analysis of serum/plasma 17OHP by MS.

Background: This study was designed to obtain an overview of the analytical quality of the prothrombin time, reported as international normalized ratio (INR) and to assess the variation of INR results between European laboratories, the difference between Quick-type and Owren-type methods and the effect of using local INR calibration or not. In addition, we assessed the variation in INR results obtained for a single donation in comparison with a pool of several plasmas. Methods: A set of four different lyophilized plasma samples were distributed via national EQA organizations to participating laboratories for INR measurement. Results: Between-laboratory variation was lower in the Owren group than in the Quick group (on average: 6.7% vs. 8.1%, respectively). Differences in the mean INR value between the Owren and Quick group were relatively small (<0.20 INR). Between-laboratory variation was lower after local INR calibration (CV: 6.7% vs. 8.6%). For laboratories performing local calibration, the between-laboratory variation was quite similar for the Owren and Quick group (on average: 6.5% and 6.7%, respectively). Clinically significant differences in INR results (difference in INR>0.5) were observed between different reagents. No systematic significant differences in the between-laboratory variation for a single-plasma sample and a pooled plasma sample were observed. Conclusions: The comparability for laboratories using local calibration of their thromboplastin reagent is better than for laboratories not performing local calibration. Implementing local calibration is strongly recommended for the measurement of INR.

Background: Antithrombin (AT) is a critical regulator of coagulation, and its overall activity is typically measured using functional tests. A large number of molecular forms of AT have been identified and each individual carries multiple molecular proteoforms representing variable activities. Conventional functional tests are completely blind for these proteoforms. A method that ensures properly defined measurands for AT is therefore needed. We here assess whether mass spectrometry technology, in particular multiple reaction monitoring (MRM), is suitable for the quantification of AT and the qualitative detection of its molecular proteoforms. Methods: Plasma proteins were denatured, reduced and alkylated prior to enzymatic digestion. MRM transitions were developed towards tryptic peptides and glycopeptides using AT purified from human plasma. For each peptide, three transitions were measured, and stable isotope-labeled peptides were used for quantitation. Completeness of digestion was assessed using digestion time curves. Results: MRM transitions were developed for 19 tryptic peptides and 4 glycopeptides. Two peptides, FDTISEK and FATTFYQHLADSK, were used for quantitation, and using a calibration curve of isolated AT in 40 g/L human serum albumin, CVs below 3.5% were obtained for FDTISEK, whereas CVs below 8% were obtained for FATTFYQHLADSK. Of the 26 important AT mutations, 20 can be identified using this method, while altered glycosylation profiles can also be detected. Conclusions: We here show the feasibility of the liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) technique for the quantitation of AT and the qualitative analysis of most of its molecular proteoforms. Knowing the measurands will enable standardization of AT tests by providing in-depth information on the molecular proteoforms of AT.

Background For the analysis of 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine ribonucleotides (6-MMPR), no external quality assessment scheme (EQAS) is currently available and no quality control samples can be made because of the absence of pure substances. An experimental design is tested to compare laboratory analytical results. Methods In this EQAS, participating laboratories were asked to select patient samples from their routine analysis and exchange these with a coupled laboratory. Because of large differences in results between laboratories, all standard operating procedures were reviewed, revealing that the origin of these differences could be in the method of hydrolysis and the preparation of calibrators. To investigate the contribution of the calibrators to these differences, one participating laboratory was asked to prepare a batch of calibrators to be shipped to the participating laboratories for analysis. Results Results for 6-TGN differed more between laboratories, compared with results for 6-MMPR. For 6-TGN and 6-MMPR 43% and 24% of the results, respectively, were out of the 80%–120% range. When correcting the results from the exchange of the patient samples with the results of the calibrators, the mean absolute difference for 6-TGN improved from 24.8% to 16.3% (p<0.001), while the results for 6-MMPR worsened from 17.3% to 20.0% (p=0.020). Conclusions This first EQAS for thiopurine drugs shows that there is a difference between laboratories in the analysis of 6-TGN, and to a lesser extent in the analysis of 6-MMPR. This difference for 6-TGN can partially be explained by the use of in-house-prepared calibrators that differ among the participants.

Background: Real-time PCR methods are increasingly used in routine patient care settings not only to determine the presence or absence of pathogens in patient materials, but also to obtain semiquantitative results to estimate the pathogen load. However, it is so far unknown how well these methods are harmonized among different laboratories. Methods: Sets of stool samples were distributed three to four times per year to ca. 25–40 participating laboratories within the European Union as part of an external quality assessment scheme (EQAS) for the detection of gastrointestinal protozoa. This paper presents the results obtained over a 3-year period for Entamoeba histolytica , Entamoeba dispar , Giardia lamblia , Cryptosporidium species and Dientamoeba fragilis . Results: Although both false-positive and false-negative results were reported, the overall sensitivity and specificity were high. The substantial differences in the quantitative output of the real-time PCR assays could be traced back to differences in DNA isolation procedures between different laboratories. Conclusions: Participation in an EQAS proved to be important as it provides information on how the real-time PCR methods used by the participant compares to the generally reported results and indicates how procedures could be improved. Semiquantitative results of real-time PCR methods are not exchangeable between laboratories as long as the diagnostic procedures are not harmonized. Intralaboratory comparison of semiquantitative real-time PCR results seems only possible by the use of calibration curves derived from well-validated standards in clinical material and not by spiking solutions with purified DNA.

Background: The ratio of albumin to creatinine (ACR) is an important parameter used for detection of albuminuria in patients with early kidney damage. The aim of the study was to evaluate the harmonization of ACR results among Polish participants in an international external quality assessment (EQA) program, and to evaluate the impact of albumin and creatinine analytical performance on the harmonization of ACR results. Methods: We analyzed 182 results of albumin, 202 of creatinine, and 180 of ACR obtained from Polish laboratories in an EQA program organized by Labquality. The dispersion of the results in surveys and percentage differences between the results and target values were calculated. Moreover, differences between method groups were assessed. Results: The inter-laboratory coefficient of variation (CV) for ACR was 36%. Only 74% of results of Polish laboratories were within the target limits; for 11% of the results, an incorrect albuminuria category would have been reported. The inter-laboratory CV for albumin was 20%, 2.6-fold higher than for creatinine. Significant differences between method groups for albumin determination have been observed, even when the same measurement technique was used. The greatest difference between two groups was 23%, 2.5-fold greater in comparison to creatinine. Conclusions: There is an insufficient harmonization of ACR values among Polish laboratories, caused mainly by urine albumin analytical performance. Given the important role of ACR in the classification, monitoring and treatment of kidney damage, the harmonization of albumin measurements is crucial and urgently needed.

Correct measurement of autoantibodies is essential for the diagnosis of autoimmune diseases. However, due to the variability of autoantibody results and the heterogeneity of testing, wrong diagnosis is a reality. For this and more reasons, harmonization of testing is of the outmost importance. In this review we have summarized the factors contributing to this variability. The ways with which the working group on harmonization of autoantibody testing of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has been trying to tackle the issue with the production and correct use of certified reference materials (CRMs), is discussed. Finally the advantages and the limitations of the use of CRMs are presented.

Autoantibodies are helpful tools not only for the diagnosis and the classification of systemic autoimmune rheumatic diseases (SARD) but also for sub-grouping patients and/or for monitoring disease activity or specific tissue/organ damage. Consequently, the role of the diagnostic laboratory in the management of SARD is becoming more and more important. The advent of new techniques raised the need of updating and harmonizing our use/interpretation of the assays. We discuss in this opinion paper some of these issues. Indirect immunofluorescence (IIF) was originally suggested as the reference technique for anti-nuclear antibody (ANA) detection as previous solid phase assays (SPA) displayed lower sensitivity. The new available SPA are now offering better results and can represent alternative or even complementary diagnostic tools for ANA detection. The improved sensitivity of SPA technology is also changing our interpretation of the results for other types of autoantibody assays, but we need updating their calibration and new reference materials are going to be obtained in order to harmonize the assays. There is growing evidence that the identification of autoantibody combinations or profiles is helpful in improving diagnosis, patients’ subgrouping and predictivity for disease evolution in the field of SARD. We report some explanatory examples to support the idea to make the use of these autoantibody profiles more and more popular. The technological evolution of the autoimmune assays is going to change our routine diagnostic laboratory tests for SARD and validation of new algorithms is needed in order to harmonize our approach to the issue.

Background: Accurate measurements of rheumatoid factors (RFs), autoantibodies binding IgG, are important for diagnosing rheumatoid arthritis (RA) and for predicting disease course. Worldwide, various RF assays are being used that differ in technique and target antigens. We studied whether assay choice leads to clinically important discrepancies in RF status and level. Methods: RF measurements using four commercial RF assays were compared in 32 RF + samples. Using enzyme-linked immunosorbent assays (ELISAs), the influence of the target antigen source – human IgG (hIgG) versus rabbit IgG (rIgG) – on measured RF levels was investigated in arthralgia patients and RA patients. Results: Substantial discrepancies were found between RF levels measured in the four commercial assays. Six samples (19%) with RF levels below or slightly above the cutoff in the rIgG-based Phadia assay were RF + in three assays using hIgG as the target antigen, some with very high levels. Direct ELISA comparisons of RF reactivity against hIgG and rIgG estimated that among 173 ACPA + arthralgia patients, originally RF negative in rIgG-based assays, up to 10% were single positive against hIgG. Monoclonal RFs binding to hIgG and rIgG or hIgG only supported these findings. In a cohort of 69 early RA patients, virtually all RF responses reacted with both targets, although levels were still variable. Conclusions: The use of RF assays that differ in technique and target antigen, together with the different specificities of RF responses, leads to discrepancies in RF status and levels. This has important consequences for patient care if RA diagnosis and disease progression assessments are based on RF test results.

Background: Detection of anti-neutrophil cytoplasmic antibodies (ANCA) is important for the diagnosis of the ANCA-associated vasculitides (AAV). For AAV, especially ANCA directed against myeloperoxidase (MPO) and proteinase 3 (PR3) are most relevant. ANCA with less well-defined specificities may, however, also be detected in other inflammatory and non-inflammatory conditions. Methods: A questionnaire, initiated by the European Autoimmunity Standardisation Initiative (EASI), was used to gather information on methods and testing algorithms used for ANCA in clinical laboratories of 12 European countries (EASI survey). Results: Four hundred and twenty-nine responses were included in the EASI survey analysis which revealed differences within countries and between countries. Laboratories overall were poor in adherence to international consensus on ANCA testing. Substantial variation was observed with respect to the use of ANCA indirect immunofluorescence (IIF) in the algorithm, application of distinct methods for MPO- and PR3-ANCA, the daily availability of new ANCA results, and interpretation of test results. Conclusions: Awareness of these differences may stimulate further harmonization and standardization of ANCA testing. This may be promoted by an update of the international ANCA consensus and the introduction of international standards.

Background: Early detection of antinuclear antibodies (ANA) in asymptomatic subjects is useful to predict autoimmune diseases years before diagnosis. ANA have been determined by indirect immunofluorescence (IIF) using human epithelial type 2 (HEp-2) cells, which is considered the gold standard technique. Multiplex technology (BioPlex ANA Screen) has been introduced for ANA evaluation in recent years. Nevertheless, concordance between BioPlex and IIF is low and there is no harmonization between both methods for detection of autoantibodies. This study has aimed to clarify the clinical significance of autoantibodies detected by BioPlex ANA Screen in subjects with undiagnosed clinical suspicion of autoimmune disease and to determine the predictive value of autoantibodies detected by BioPlex ANA Screen. Methods: A 3-year follow-up study was performed of 411 subjects without a clear diagnosis of autoimmune diseases in whom autoantibodies were detected by BioPlex ANA Screen that were negative by IIF on HEp-2 cells. Results: At 3 years of follow-up, 312 (76%) subjects were positive for autoantibodies by IIF and 99 subjects continued to be negative. A diagnosis of autoimmune disease was found in most of the subjects (87%). Conclusions: BioPlex ANA Screen has greater sensitivity than IIF on HEp-2 cells for autoantibodies detection. Early detection of these antibodies by BioPlex can predict possible development of autoimmune diseases.

In this review we analyze all aspects of total testing process regarding the measurement of antithyroid peroxidase, antithyroglobulin and antithyrotropin receptor autoantibodies. The main critical points related to the preanalytical, analytical and postanalytical steps of autoimmune thyroid disease diagnostics are considered, focusing on harmonization of autoimmune thyroid tests request, retesting intervals, terminology of thyroid autoantibodies, measurement units and definition of reference limits. Harmonization in thyroid autoantibody testing is a relevant example of feasible harmonization in autoimmunology.

The indirect immunofluorescence assay (IFA) on HEp-2 cells is the reference method for autoantibody screening. The HEp-2 IFA pattern provides useful information on the possible autoantibodies in the sample. The International Consensus on Antinuclear Antibody Patterns (ICAP) initiative seeks to define and harmonize the nomenclature of HEp-2 IFA patterns. The most relevant and usual patterns have been assigned an alphanumeric code from anti-cell (AC)-1 to AC-28 and were organized into a classification algorithm (www.ANApatterns.org). The systemic sclerosis-associated autoantibodies to DNA topoisomerase I (Topo I) produce a peculiar composite 5-element HEp-2 IFA pattern (Topo I-like pattern) comprising the staining of the nucleus, metaphase chromatin plate, nucleolar organizing region, cytoplasm and nucleolus. In a recent assessment of the European Consensus Finding Study Group on autoantibodies, a well-defined anti-Topo I sample was blindly analyzed and classified according to ICAP AC patterns by 43 participant laboratories across Europe. There were wide variations among these laboratories in reporting nuclear, nucleolar and cytoplasmic patterns, indicating the inadequacy of the existing AC patterns to report the Topo I-like pattern. Several ICAP member laboratories independently demonstrated the overall consistency of the HEp-2 IFA Topo I-like pattern using HEp-2 slides from different manufacturers. The ICAP committee reviewed 24 candidate images and selected the four most representative images to be available on the ICAP website. The proper recognition of the AC-29 pattern should trigger suspicion of the presence of anti-Topo I antibodies, which may engender appropriate analyte-specific reflex tests to confirm the autoantibody specificity.

Background: Anti-mitochondrial antibodies (AMA) are found in >90% of primary biliary cholangitis patients. Anti-rods/rings antibodies (anti-RR) are most commonly associated with interferon-α and ribavirin treatment in hepatitis C patients. Clinical laboratories routinely screen for AMA and anti-RR using indirect immunofluorescence on HEp-2 cells (HEp-2-IFA). Therefore, we sought to establish reference materials for use in AMA and anti-RR testing. Methods: AMA-positive and anti-RR-positive human plasma samples (AMA-REF and RR-REF), identified as potential reference materials based on preliminary data, were further validated by multiple laboratories using HEp-2-IFA, immunoprecipitation (IP), western blotting, IP-western, line immunoassay (LIA), addressable laser bead immunoassay (ALBIA) and enzyme-linked immunosorbent assay (ELISA). Results: AMA-REF showed a strong positive cytoplasmic reticular/AMA staining pattern by HEp-2-IFA to ≥1:1280 dilution and positive signal on rodent kidney/stomach/liver tissue. AMA-REF reacted with E2/E3, E3BP, E1α and E1β subunits of the pyruvate dehydrogenase complex by IP and western blotting and was positive for AMA antigens by LIA, ALBIA and ELISA. RR-REF showed a strong positive rods and rings staining pattern by HEp-2-IFA to ≥1:1280 dilution. RR-REF reacted with inosine monophosphate dehydrogenase by IP, IP-western and ALBIA. RR-REF also produced a nuclear homogenous staining pattern by HEp-2-IFA, immunoprecipitated proteins associated with anti-U1RNP antibody and reacted weakly with histones, nucleosomes, Sm and nRNP/Sm by LIA. Conclusions: AMA-REF and RR-REF are useful reference materials for academic or commercial clinical laboratories to calibrate and establish internal reference standards for immunodiagnostic assays. AMA-REF and RR-REF are now available for free distribution to qualified laboratories through Plasma Services Group.