An international multi-center serum protein electrophoresis accuracy and M-protein isotyping study. Part II: limit of detection and follow-up of patients with small M-proteins.

Background Electrophoretic methods to detect, characterize and quantify M-proteins play an important role in the management of patients with monoclonal gammopathies (MGs). Significant uncertainty in the quantification and limit of detection (LOD) is documented when M-proteins are <10 g/L. Using spiked sera, we aimed to assess the variability in intact M-protein quantification and LOD across 16 laboratories. Methods Sera with normal, hypo- or hyper-gammaglobulinemia were spiked with daratumumab or elotuzumab, with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Laboratories blindly analyzed samples according to their serum protein electrophoresis (SPEP)/isotyping standard operating procedures. LOD and intra-laboratory percent coefficient of variation (%CV) were calculated and further specified with regard to the method (gel/capillary electrophoresis [CZE]), gating strategy (perpendicular drop [PD]/tangent skimming [TS]), isotyping (immunofixation/immunosubtraction [ISUB]) and manufacturer (Helena/Sebia). Results All M-proteins ≥1 g/L were detected by SPEP. With isotyping the LOD was moderately more sensitive than with SPEP. The intensity of polyclonal background had the biggest negative impact on LOD. Independent of the method used, the intra-laboratory imprecision of M-protein quantification was small (mean CV = 5.0%). Low M-protein concentration and high polyclonal background had the strongest negative impact on intra-laboratory precision. All laboratories were able to follow trend of M-protein concentrations down to 1 g/L. Conclusions In this study, we describe a large variation in the reported LOD for both SPEP and isotyping; overall LOD is most affected by the polyclonal immunoglobulin background. Satisfactory intra-laboratory precision was demonstrated. This indicates that the quantification of small M-proteins to monitor patients over time is appropriate, when subsequent testing is performed within the same laboratory.

An international multi-center serum protein electrophoresis accuracy and M-protein isotyping study. Part I: factors impacting limit of quantitation of serum protein electrophoresis.

Background Serum protein electrophoresis (SPEP) is used to quantify the serum monoclonal component or M-protein, for diagnosis and monitoring of monoclonal gammopathies. Significant imprecision and inaccuracy pose challenges in reporting small M-proteins. Using therapeutic monoclonal antibody-spiked sera and a pooled beta-migrating M-protein, we aimed to assess SPEP limitations and variability across 16 laboratories in three continents. Methods Sera with normal, hypo- or hypergammaglobulinemia were spiked with daratumumab, Dara (cathodal migrating), or elotuzumab, Elo (central-gamma migrating), with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Provided with total protein (reverse biuret, Siemens), laboratories blindly analyzed samples according to their SPEP and immunofixation (IFE) or immunosubtraction (ISUB) standard operating procedures. Sixteen laboratories reported the perpendicular drop (PD) method of gating the M-protein, while 10 used tangent skimming (TS). A mean percent recovery range of 80%-120% was set as acceptable. The inter-laboratory %CV was calculated. Results Gamma globulin background, migration pattern and concentration all affect the precision and accuracy of quantifying M-proteins by SPEP. As the background increases, imprecision increases and accuracy decreases leading to overestimation of M-protein quantitation especially evident in hypergamma samples, and more prominent with PD. Cathodal migrating M-proteins were associated with less imprecision and higher accuracy compared to central-gamma migrating M-proteins, which is attributed to the increased gamma background contribution in M-proteins migrating in the middle of the gamma fraction. There is greater imprecision and loss of accuracy at lower M-protein concentrations. Conclusions This study suggests that quantifying exceedingly low concentrations of M-proteins, although possible, may not yield adequate accuracy and precision between laboratories.

How comparable are total human chorionic gonadotropin (hCGt) tumour markers assays?

Background Total human chorionic gonadotropin (hCGt) tumour marker testing is regarded as an "off label" application for most commercial methods. We compared four assays in patients with a hCGt tumour marker request. We hypothesised that regression slopes would be altered and that outliers would be more common with tumour marker than with pregnancy samples if the detection of malignancy associated hCG molecular forms differed amongst assays. Further such systematic differences would be obvious and large enough to change clinical management decisions. Results We measured hCGt in 390 samples from 137 females and 253 males with a tumour marker request and 208 pregnancy controls with the following methods: Access Total ßhCG, Architect Total-ßhCG, Cobas hCG + ß and Immulite HCG. The between method regressions determined on tumour marker and pregnancy samples were not significantly different. The outlier rates were similar for male and female tumour marker and the pregnancy groups: 1.6% (95% confidence interval [CI] 0%-3.1%), 2.2% (95% CI 0%-4.7%) and 2.9% (95% CI 0.6%-5.2%). The outliers were randomly distributed amongst the methods and we were confident that they would not adversely influence clinical decisions. Conclusions The hCGt results were clinically equivalent with no systematic difference amongst the four assays.

The Paraprotein - an Enduring Biomarker.

The 'paraprotein', also known as M-protein, monoclonal protein and monoclonal component, has stood the test of time as the key biomarker in monoclonal gammopathies. It continues to reinvent itself as new electrophoretic and immunoassay methods are developed that are analytically more sensitive. Use of the serum free light chain immunoassay in particular has led to new clinical discoveries and improvements in the diagnosis and monitoring of patients with plasma cell dyscrasia and other monoclonal gammopathies. In addition, minimal residual disease can be detected using mass spectrometry and flow cytometry methods.

Report of the Survey Conducted by RCPAQAP on Current Practice for Paraprotein and Serum Free Light Chain Measurement and Reporting: a Need for Harmonisation.

Clinical laboratory testing is vital in the diagnosis, monitoring and prognostication of monoclonal gammopathies. Although the 2012 recommendations for standardised reporting of protein electrophoresis in Australia and New Zealand aimed to harmonise the laboratory practices related to paraprotein testing, the between-laboratory variation still exists. A survey was conducted to assess the between-laboratory variation in certain aspects of laboratory testing related to monoclonal gammopathy.

Proposed Addendum to 2012 Recommendations for Standardised Reporting of Protein Electrophoresis in Australia and New Zealand.

It is apparent that there is a need for greater harmonisation of the reporting and quantification of paraproteins on protein electrophoresis with the introduction of the electronic health record and recent survey findings indicating ongoing areas of heterogeneity on serum protein electrophoresis. The proposed addendum aims to update the 2012 recommendations for standardised reporting of protein electrophoresis in Australia and New Zealand. The sections which need to be updated include those on the quantification of gamma- and non-gamma-migrating paraproteins; interpretive commenting in specimens with a paraprotein and/or small abnormal bands; the utility of serum free light chains compared with Bence Jones protein measurement; and a new table with interpretive commenting for serum free light chains. It is expected that such standardised reporting will reduce both variation between laboratories and the risk of misinterpretation of results.

Paraprotein Sample Exchange in Australia and New Zealand - 2018.

Quantification of co-migrating paraproteins in the beta-region presents an ongoing challenge for laboratories performing serum protein electrophoresis. The between-laboratory variation may impact patient care if the patient uses different pathology services during plasma cell dyscrasia monitoring. To identify the practical difficulties and determine the extent of agreement in the reporting of beta-migrating paraproteins in Australia and New Zealand (NZ), sample exchanges were conducted in five Australian states and in NZ in early 2018. This study has highlighted the variation in quantification and reporting of beta-migrating paraproteins which could potentially affect patient monitoring and management.

A critical evaluation of the Beckman Coulter Access hsTnI: Analytical performance, reference interval and concordance.

INTRODUCTION: We investigated the analytical performance, outlier rate, carryover and reference interval of the Beckman Coulter Access hsTnI in detail and compared it with historical and other commercial assays. MATERIALS AND METHODS: We compared the imprecision, detection capability, analytical sensitivity, outlier rate and carryover against two previous Access AccuTnI assay versions. We established the reference interval with stored samples from a previous study and compared the concordances and variances with the Access AccuTnI+3 as well as with two commercial assays. RESULTS: The Access hsTnI had excellent analytical sensitivity with the calibration slope 5.6 times steeper than the Access AccuTnI+3. The detection capability was markedly improved with the SD of the blank 0.18-0.20 ng/L, LoB 0.29-0.33 ng/L and LoD 0.58-0.69 ng/L. All the reference interval samples had a result above the LoB value. At a mean concentration of 2.83 ng/L the SD was 0.28 ng/L (CV 9.8%). Carryover (0.005%) and outlier (0.046%) rates were similar to the Access AccuTnI+3. The combined male and female 99th percentile reference interval was 18.2 ng/L (90% CI 13.2-21.1 ng/L). Concordance amongst the assays was poor with only 16.7%, 19.6% and 15.2% of samples identified by all 4 assays as above the 99th, 97.5th and 95th percentiles. Analytical imprecision was a minor contributor to the observed variances between assays. CONCLUSION: The Beckman Coulter Access hsTnI assay has excellent analytical sensitivity and precision characteristics close to zero. This allows cTnI measurement in all healthy individuals and the capability to identify numerically small differences between serial samples as statistically significant. Concordance in healthy individuals remains poor amongst assays.

An update report on the harmonization of adult reference intervals in Australasia.

The Australasian Association of Clinical Biochemists (AACB) has over the past 5 years been actively working to achieve harmonized reference intervals (RIs) for common clinical chemistry analytes using an evidence-based checklist approach where there is sound calibration and metrological traceability. It has now recommended harmonized RIs for 18 common clinical chemistry analytes which are performed in most routine laboratories and these have been endorsed by the Royal College of Pathologists of Australasia (RCPA). In 2017 another group of analytes including urea, albumin and arterial blood gas parameters were considered and suggested harmonized RIs proposed. This report provides an update of those harmonization efforts.

Establishing consensus-based, assay-specific 99th percentile upper reference limits to facilitate proper utilization of cardiac troponin measurements.

Implementation of the 99th percentile as the upper reference limit for cardiac troponin (cTn) assays is a seemingly lucid recommendation, but, in reality, is incredibly complex. Lack of harmonization between cTn assays diminishes the ability to have a single medical decision point across manufacturer assay/instruments. Moreover, even within a single cTn assay there are several published values corresponding to the "99th percentile". Variability in the determined value is primarily a function of population selection including: sample size, age, sex, exclusion criteria, and statistical methods. Given the complexities associated with this value, some countries have taken an expert consensus approach to endorsing harmonized, assay-specific, cTn 99th percentile values. The purpose of this manuscript is to highlight the intricacies associated with selecting a cTn 99th percentile and to review the approach that Australia used to endorse a nationwide upper reference limit for the Architect STAT hs-cTnI assay.

Harmonising Adult Reference Intervals in Australia and New Zealand - the Continuing Story.

Reference intervals (RIs) are used to help clinicians determine if a patient can be classified as being in a diseased or healthy state and there are often sound scientific and clinical reasons for differences in RIs. One of the current strategic priorities for the Australasian Association of Clinical Biochemists is to encourage and assist laboratories to achieve harmonisation of RIs for common clinical chemistry analytes where sound calibration and traceability are in place. This need is based on good laboratory practice, providing the clinician with results that allow appropriate and reliable clinical interpretation and progression further toward the national e-health framework and a single electronic health record. After reviewing and considering studies related to bias as well as both a priori and a posteriori RI studies nationally and internationally and the consideration of flagging rates and clinical relevance, an initial group of 12 harmonised RIs were endorsed by the Royal College of Pathologists of Australasia in 2014. In 2015, after further stakeholder consultation, a second group of six harmonised RIs for common chemistry analytes has been proposed for adults which includes ALT and AST where methods do not use pyridoxal-5'-phosphate as an activator and lipase excluding the Ortho Clinical Diagnostics and Siemens Dimension assays.

Opinion Paper: Deriving Harmonised Reference Intervals - Global Activities.

Harmonisation of reference intervals (RIs) refers to use of the same or common RI across different platforms and /or assays for a specified analyte. It occurs optimally for those analytes where there is sound calibration and traceability in place and evidence from a between-method comparison shows that bias would not prevent the use of a common RI. The selection of the RI will depend on various sources of information including local formal RI studies, published studies from the literature, laboratory surveys, manufacturer's product information, relevant guidelines, and mining of databases. Pre-analytical and partitioning issues, significant figures and flagging rates, are assessed for each analyte. Several countries and regions including the Nordic countries, United Kingdom, Japan, Turkey, and Australasia are using common RIs that have been determined either by direct studies or by a consensus process. In Canada, the Canadian Society of Clinical Chemists Taskforce is assessing the feasibility of establishing common reference values using the CALIPER (Canadian Laboratory Initiative on Pediatric Reference Intervals) and CHMS (The Canadian Health Measures Survey) databases as the basis. Development of platform-specific common reference values for each of the major analytical systems may be a more practical approach especially for the majority of analytes that are not standardised against a primary reference method and are not traceable to a primary or secondary reference material. We encourage laboratories to consider adopting reference intervals consistent with those used by other laboratories in your region where it is possible and appropriate for your local population. Local validation of the adopted reference interval is also recommended as per CLSI guidelines.

Is accuracy of serum free light chain measurement achievable?

The serum free light chain (FLC) assay has proven to be an important complementary test in the management of patients with monoclonal gammopathies. The serum FLC assay has value for patients with plasma cell disorders in the context of screening and diagnosis, prognostic stratification, and quantitative monitoring. Nonetheless, serum FLC measurements have analytical limitations which give rise to differences in FLC reporting depending on which FLC assay and analytical platform is used. As the FLC measurements are incorporated in the International Myeloma Working Group guidelines for the evaluation and management of plasma cell dyscrasias, this may directly affect clinical decisions. As new certified methods for serum FLC assays emerge, the need to harmonise patient FLC results becomes increasingly important. In this opinion paper we provide an overview of the current lack of accuracy and harmonisation in serum FLC measurements. The clinical consequence of non-harmonized FLC measurements is that an individual patient may or may not meet certain diagnostic, prognostic, or response criteria, depending on which FLC assay and platform is used. We further discuss whether standardisation of serum FLC measurements is feasible and provide an overview of the steps needed to be taken towards harmonisation of FLC measurements.