Laboratory practice is central to earlier myeloma diagnosis: Utilizing a primary care diagnostic tool and laboratory guidelines integrated into haematology services.

Treatment advances have greatly improved survival, but myeloma is among the worst of all cancers for delayed diagnosis, causing serious morbidities and early deaths. This delay is largely because the symptom profile of myeloma has very low specificity, and in primary care, myeloma is rare. However, initiating the journey to diagnosis simply requires considering myeloma and sending blood to test for monoclonal immunoglobulin. Laboratory tests reliably detect monoclonal immunoglobulin, which is present in 99% of myeloma cases, so why do health care systems have such a problem with delayed diagnosis? The Myeloma UK early diagnosis programme has brought together diverse expertise to investigate this problem, and this article was prepared by the programme's working group for laboratory best practice. It reviews evidence for test requesting, analysis and reporting, for which there is large variation in practice across the United Kingdom. It presents a 'GP Myeloma diagnostic tool' and how it can be integrated into laboratory practice alongside a laboratory best practice tool. It proposes improved requesting and integration with haematology services for reporting and interpretation. Here the laboratory has a central role in creating efficient and cost-effective pathways for appropriate and timely bone marrow examination for myeloma diagnosis.

Development of a certified reference material for anti-ß2-glycoprotein I IgG - commutability studies.

Objectives: In this paper, we describe the steps followed for the development of a certified reference material for immunoglobulin G antibodies against ß2-glycoprotein I (also known as apolipoprotein H). These steps include processing of the material, commutability, the impact of dilution, the appropriate reconstitution conditions, homogeneity and stability during transport and storage. Methods: We analysed 69 clinical samples from patients suffering from antiphospholipid syndrome with several commercial enzyme-linked immunosorbent assays (ELISA) purchased from in vitro diagnostic manufacturers. Results: Analysis of the results indicated that the candidate reference material can be safely freeze-dried, and that the user should carefully follow the reconstitution instructions as small changes in e.g. temperature may have unwanted effects. The statistical analysis of the commutability studies indicated that the analytical response of the reference material upon dilution is similar to that of clinical samples, and that correlation between results may differ from assay to assay. Finally yet importantly, the presented and developed candidate reference material is commutable for most assays tested, homogeneous and stable. Conclusions: Immunoglobulin G antibodies against ß2-glycoprotein I are associated with a higher risk of thrombosis and pregnancy complications. Their measurement is essential for the diagnosis and monitoring of antiphospholipid syndrome. These antibodies are detected by specific immunoassays, routinely used in clinical diagnostics, but various of these methods show enormous variability, in part due to the lack of a reference material.

Certified reference material against PR3 ANCA IgG autoantibodies. From development to certification.

Background The importance of the standardisation of immunoassays for autoantibodies has been widely discussed. The appropriate use of certified reference materials (CRM) could contribute to a more accurate diagnosis and follow-up of a series of diseases such as small vessel-associated vasculitis. This is a systemic autoimmune disorder during which two autoantibodies can be present, MPO ANCA IgG and PR3 ANCA IgG. Results from different commercially available immunoassays used for PR3 ANCA IgG measurement can vary significantly. Therefore the potential for improvement using a suitable certified reference material was assessed and led to the development of a CRM. Methods Thirty clinical samples were evaluated using 10 immunoassays. The correlation between results from these assays was assessed in a pairwise manner. Feasibility studies were conducted in order to find a reference material format most suitable for the preparation of a CRM. Results The evaluation of two sets of 30 clinical samples with 10 assays showed that differences between assays can result in different interpretations for individual clinical samples. Most of the samples had the same result classification in all assays. However, six of the samples tested led to inconsistent results. Conclusions The correlation between results from clinical samples was systematically good for combinations of eight of those assays. Therefore, it should be possible to improve the comparability of results using a commutable CRM for calibration. Based on these studies, a final format for the CRM was selected and eventually produced and certified for its PR3 ANCA IgG content.

Evaluation of cryoprotein investigation using a digital external quality assurance scheme.

Background: Robust preanalytical and analytical processes are critical for the detection of cryoproteins. There is significant variation in practice in the detection, analysis and reporting. Results: A survey in 2018 of 137 laboratories participating in the UK National External Quality Assessment Service (UK NEQAS) (6) quality control program showed significant variation in the laboratory processes which highlighted the need for standardisation of the detection, analysis and reporting of cryoglobulins.Conclusion: The first available EQA scheme aiming to harmonise practice for cryoprotein testing has been developed by UK NEQAS and laboratories should participate in an appropriate EQA scheme to fulfil requirements for ISO accreditation.

The importance of commutability of reference materials used as calibrators: the example of ceruloplasmin.

BACKGROUND: Different methods for ceruloplasmin tend to give different results in external quality assessment schemes. During the production of the certified reference material ERM-DA470k/IFCC discrepant measurement results were also found for ceruloplasmin measured with different methods, and consequently the protein could not be certified in the material. METHODS: We performed a commutability study with 30 serum samples and the reference materials ERM-DA470, ERM-DA470k/IFCC, and ERM-DA472/IFCC, using 6 different methods. Data were analyzed according to the CLSI Guideline C53-A to assess whether the reference materials had the same behavior as the serum samples with respect to measurement results obtained with combinations of the methods used. RESULTS: Measurement results from different methods showed a good linear correlation for the serum samples. ERM-DA470 showed marked noncommutability for certain combinations of methods. ERM-DA470k/IFCC and ERM-DA472/IFCC were commutable for more combinations of methods. The lack of commutability of ERM-DA470 for certain combinations of methods correlates with results from the UK National External Quality Assessment Service showing discrepancies between results from these methods. For serum stored in the presence of sodium azide the results from different methods are essentially equivalent. CONCLUSIONS: Ceruloplasmin in ERM-DA470 is a fully documented example of a situation in which, due to lack of commutability, the use of a common material for calibration did not lead to harmonization .

Characterization of the new serum protein reference material ERM-DA470k/IFCC: value assignment by immunoassay.

BACKGROUND: The availability of a suitable matrix reference material is essential for standardization of the immunoassays used to measure serum proteins. The earlier serum protein reference material ERM-DA470 (previously called CRM470), certified in 1993, has led to a high degree of harmonization of the measurement results. A new serum protein material has now been prepared and its suitability in term of homogeneity and stability has been verified; after characterization, the material has been certified as ERM-DA470k/IFCC. METHODS: We characterized the candidate reference material for 14 proteins by applying a protocol that is considered to be a reference measurement procedure, by use of optimized immunoassays. ERM-DA470 was used as a calibrant. RESULTS: For 12 proteins [α(2) macroglobulin (A2M), α(1) acid glycoprotein (orosomucoid, AAG), α(1) antitrypsin (α(1)-protease inhibitor, AAT), albumin (ALB), complement 3c (C3c), complement 4 (C4), haptoglobin (HPT), IgA, IgG, IgM, transferrin (TRF), and transthyretin (TTR)], the results allowed assignment of certified values in ERM-DA470k/IFCC. For CRP, we observed a bias between the lyophilized and liquid frozen materials, and for CER, the distribution of values was too broad. Therefore, these 2 proteins were not certified in the ERM-DA470k/IFCC. Different value transfer procedures were tested (open and closed procedures) and found to provide equivalent results. CONCLUSIONS: A new serum protein reference material has been produced, and values have been successfully assigned for 12 proteins.

Standardizing plasma protein measurements worldwide: a challenging enterprise.

The need for harmonizing laboratory results is particularly intense in the field of quantitative protein assays in consideration of the clinical impact of specific protein measurements and their relevance in monitoring disease. We report the efforts made by the Committee on Plasma Proteins of the IFCC Scientific Division to achieve worldwide comparability in plasma protein results. We focus on the production of reference materials and the methods applied throughout their production process. Particularly, the recent characterization of ERM-DA470k/IFCC and ERM-DA472/IFCC has demonstrated that it is possible to reproduce the earlier established procedures and thereby maintain standardization. Plasma protein reference materials have had a substantial impact in improving the harmonization of patient protein results that should translate into better patient care.

Development and preparation of a new serum protein reference material: feasibility studies and processing.

BACKGROUND: The availability of matrix reference materials is essential for the standardisation of (immuno)assays used to measure proteins. The reference material ERM-DA470 (previously called CRM470) certified in 1993 has led to a large degree of harmonisation of these assays. A new serum protein reference material has now been produced (ERM-DA470k). It is intended to replace ERM-DA470, and will additionally be certified for beta(2)-microglobulin (B2M). METHODS: Serum from 390 healthy donors was pooled and processed so as to stabilise, delipidate and 'maturate' it. Purified C-reactive protein (CRP) and recombinant B2M were added. Pilot batches were produced to study the stability, homogeneity, and commutability of the material. On the basis of the results with the trial batches it was decided to proceed with the processing of the main batch of a candidate reference material. RESULTS: Two pilot batches were produced and the processed and spiked serum lyophilised after filling (1 mL). The B2M in the material was shown to be stable and commutable. For CRP, it was discovered that freeze-drying led to a decrease in measurable protein. The main batch of candidate reference material was produced and fulfilled the required criteria in terms of optical transparency, homogeneity and stability. CONCLUSIONS: A new serum protein reference material has been produced with the properties required for a serum protein reference material for 14 proteins. An apparent loss of CRP of approximately 20% was observed upon freeze-drying of the material.

Identification of a novel Haemophilus influenzae protein important for adhesion to epithelial cells.

Non-typable Haemophilus influenzae (NTHi) is an important human-specific respiratory pathogen colonizing the mucosa of the upper respiratory tract. The bacterium is a common cause of acute otitis media in children and exacerbations in patients with chronic obstructive pulmonary disease (COPD). An immunoglobulin (Ig) D-lambda myeloma protein was found to detect a 16 kDa surface protein that we designated protein E (PE). The pe gene was cloned using an NTHi genomic DNA library, and a truncated PE-derived protein lacking the endogenous signal peptide (PE22-160) was synthesized and produced in large amounts in Escherichia coli. Interestingly, PE was expressed at the bacterial surface of NTHi as revealed by flow cytometry using the IgD-lambda myeloma protein or PE-specific polyclonal antibodies. A PE-deficient NTHi mutant was produced and lost 50% of its adhesive capacity as compared to the wild-type counterpart when analysed for adhesion to type II lung alveolar epithelial cells. In parallel, E. coli expressing full-length PE1-160 adhered significantly more efficiently to epithelial cells as compared to wild-type E. coli. Recombinant IgD that recognized the chemical dansyl-chloride did not interact with PE indicating that the IgD-lambda myeloma protein most likely was an antibody directed against the H. influenzae surface epitope. In conclusion, we have discovered a novel NTHi outer membrane protein with adhesive properties using an IgD-myeloma protein.

Large abnormal peak on capillary zone electrophoresis due to contrast agent.

Background Some iodinated radio-contrast media absorb ultraviolet light and can therefore be detected by capillary zone electrophoresis. If seen, these peaks are typically small with 'quantifications' of below 5 g/L. Here, we describe the detection of a large peak on capillary zone electrophoresis that was due to the radio-contrast agent, Omnipaque™. Methods Serum from a patient was analysed by capillary zone electrophoresis, and the IgG, IgA, IgM and total protein concentrations were measured. The serum sample was further analysed by gel electrophoresis and immunofixation. Results Capillary zone electrophoresis results for the serum sample showed a large peak with a concentration high enough to warrant urgent investigation. However, careful interpretation alongside the serum immunoglobulin concentrations and total protein concentration showed that the abnormal peak was a pseudoparaprotein rather than a monoclonal immunoglobulin. This was confirmed by analysis with gel electrophoresis and also serum immunofixation. The patient had had a CT angiogram with the radio-contrast agent Omnipaque™; addition of Omnipaque™ to a normal serum sample gave a peak with comparable mobility to the pseudoparaprotein in the patient's serum. Conclusions Pseudoparaproteins can appear as a large band on capillary zone electrophoresis. This case highlights the importance of a laboratory process that detects significant electrophoretic abnormalities promptly and interprets them in the context of the immunoglobulin concentrations. This should avoid incorrect reporting of pseudoparaproteins which could result in the patient having unnecessary investigations.

Electrophoretic patterns post daratumumab.

Background Daratumumab (Darzalex) is a human IgG1 kappa monoclonal antibody targeting CD38 that has been recently approved for the treatment of refractory multiple myeloma. As it is a monoclonal protein, it can be detected on routine serum protein electrophoresis and by immunofixation. Methods Serum samples from four patients were analysed by serum protein electrophoresis immediately pre- and post-treatment with daratumumab. Results For all four patients, daratumumab was visible on serum protein electrophoresis as an additional small band (approximately 1 g/L) in the slow gamma region. Conclusion Diagnostic laboratories should be aware that daratumumab can be detected on routine serum protein electrophoresis of myeloma patients and should liaise closely with clinicians to ensure the presence of daratumumab is not misinterpreted as development of a new monoclonal protein.

Development of a Certified Reference Material for myeloperoxidase-anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA).

A serum Certified Reference Material (CRM) for supporting reliable autoimmune diagnostics was recently released by the Institute for Reference Materials and Measurements (IRMM) of the Joint Research Centre of the European Commission. It was produced in collaboration with a Working Group on the Harmonisation of Autoimmune Tests of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC WG-HAT). This material is aimed at facilitating the standardisation of measurements of anti-myeloperoxidase immunoglobulin G antibodies. The CRM could be used as a common calibrant by clinicians and manufacturers thereby significantly improving the comparability of results from commercial immunoassays used for IgG anti-MPO measurements. This paper provides information on the new CRM and its intended use.

Strategies for building reference standards for autoantibodies.

Producing robust, certified, traceable reference material for autoantibody testing is a vital element in maintaining the validity of results that are generated in the daily clinical laboratory routine. This is a huge challenge because of the high number of variables involved in the detection and measurement of the autoantibodies. The production of such materials is time consuming and needs rigorous attention to detail; this is best achieved by an overarching independent body who will oversee the process in a "not for profit" manner. Much effort has been made to build international standards for quantitative and qualitative assays based on monoclonal antibodies, obtained from affinity purification and plasmapheresis. The big challenge is to respect individual differences in immune response to the same antigen. A promising ongoing initiative is the construction of pools with monospecific samples from different individuals.

Laboratory testing in autoimmune rheumatic diseases.

There are a number of pathological conditions in which tissue damage occurs in association with immune activation directed against components of normal tissue. The initial damaging events usually involve cells of the immune system, the T-cells, but the cell damage releases antigens that become targets for an antibody response. The detection and quantification of autoantibodies has become an important component in the diagnosis and management of autoimmune rheumatic diseases such as rheumatoid arthritis, systemic lupus erythematosus, the systemic vasculitides and systemic sclerosis. Each of these diseases is associated with a particular autoantibody or group of autoantibodies. They are usually detected by their reaction against tissue components using subjective methods such as indirect immunofluorescence. Any positive samples are further analysed using more specific and quantitative methods for the 'quantification' of the specific autoantibody concentration. It is important that these autoantibodies are not considered to be 'gold standard' tests: they are no more than markers of the disease with significant limitations. They are best used as part of a diagnostic panel rather than as a marker indicating one particular disease. Techniques are gradually improving, giving numerical results rather than titres, but a lack of standardization makes these results extremely variable. Many of the markers show no correlation with disease activity. Their use should be restricted to the initial investigation and not repeated every time the patient is followed up. Other markers do, however, correlate with disease activity and can be used to monitor disease. When investigating patients who have symptoms associated with autoimmune rheumatic diseases, analytes such as immunoglobulins, complement components and C-reactive protein may all be measured.

Standardization of autoantibody testing: a paradigm for serology in rheumatic diseases.

Autoantibody measurement is an excellent tool to confirm the diagnosis of rheumatic autoimmune diseases. Hence, reliability and harmonization of autoantibody testing are essential, but these issues are still a matter of debate. Intrinsic variability in analytes and reagents as well as heterogeneity of the techniques are the main reasons for discrepancies in inter-laboratory variations and reporting of test results. This lack of reliability might be responsible for wrong or missed diagnoses, as well as additional costs due to assay repetition, unnecessary use of confirmatory tests and/or consequent diagnostic investigations. To overcome such issues, the standardization of autoantibody testing requires efforts on all aspects of the assays, including the definition of the analyte, the pre-analytical stages, the calibration method and the reporting of results. As part of such efforts, the availability of suitable reference materials for calibration and quality control would enable the development of a reliable reference system. Strong-positive sera from patients have been used as reference materials in most of the autoantibody assays for rheumatic diseases; however, antigen-affinity-purified immunoglobulin fractions or in some cases reliable monoclonal antibody preparations offer more adequate tools for standardization. Systematic assessments of reference materials are currently underway, and preliminary results appear to be encouraging.