Abstract
Background
Serum free light chain (sFLC) measurements are increasingly important in the context of screening for monoclonal gammopathies, prognostic stratification and monitoring of therapy responses. In this study we have performed a method comparison of four sFLC assays that are currently available for routine clinical use.
Methods
In a retrospective study, sFLC analyses were performed on a cohort that included 139 patients with various monoclonal gammopathies and 54 control sera without an M-protein. Method comparisons of the following four FLC assays were performed: Freelite (Binding Site), N-Latex FLC (Siemens), Seralite (Abingdon Health) and Sebia FLC (Sebia).
Results
Bland-Altman agreement analysis showed biases varying between −0.1 and 16.2 mg/L for κFLC, −6.0 and 6.8 mg/L for λFLC and −0.04 and 0.38 for the ratio of the involved to uninvolved FLC. Strong agreements were observed for FLC-concentrations below 100 mg/L. The clinical concordance of the κ/λFLC-ratio of the four methods varied between 86% and 92%. Significant quantitative differences were observed between the different methods, mainly in sera with high FLC concentrations. Most assays consistently overestimated FLC concentrations compared to SPE.
Conclusions
Good overall clinical concordances were observed between the four sFLC assays that were compared in this study. Although good agreements were observed between the FLC assays, significant absolute differences in FLC concentrations in individual patients can be seen, particularly at higher FLC concentrations. Because of inequivalent absolute sFLC values between the methods in individual patients, none of the four sFLC assays can be used interchangeably.
Introduction
Monoclonal gammopathies including benign monoclonal gammopathies of undetermined significance (MGUS) to life-threatening diseases such as multiple myeloma (MM), are characterized by clonal proliferation of plasma cells and the production of monoclonal immunoglobulin proteins (M-proteins). Traditionally, electrophoretic methods form the cornerstone for the detection and quantification of these M-proteins [1], [2]. The introduction of highly sensitive Freelite sFLC assays by Bradwell et al. in the early 2000s strongly advanced M-protein diagnostics [3]. Ever since, serum free light chain (sFLC) analysis became an increasingly important complementary test in the management of patients with monoclonal gammopathies. The sFLC has proven clinical value in the context of screening and diagnosis, prognosis and monitoring response to therapy [4], [5]. A decade after the introduction of the Freelite assay, new sFLC assays across different analytical platforms became available for routine diagnostics [6], [7], [8].
Table 1 highlights the characteristics of the different available FLC assays. Although all four are immunoassays, they show relevant differences in the type of analytical platform and the type of antibodies that are used for FLC detection. Freelite uses polyclonal anti-human FLC antisera in automated nephelometric and turbidimetric assays. FLC value assignment was performed on purified FLC material as a calibrator [3]. Reference intervals were determined on large cohorts of healthy controls [9] and patients with impaired renal function [10]. The FLC assays that followed were all calibrated on the Freelite assay, with assay-specific reference intervals that were established on independent control cohorts. The four currently available FLC assays are strikingly different in terms of the principle of the immunoassay and the choice of the detection reagents that make use of either polyclonal or monoclonal antibodies. The N-Latex FLC nephelometric assay [6], [11] and the Seralite lateral flow assay [7], [12] both make use of monoclonal antibodies. The Sebia FLC is an automated ELISA and, similar to Freelite, makes use of polyclonal antibodies [8], [13]. Further properties of these various immunoassays and the implications for clinical use have been reviewed recently by Tate [14].
Characteristics of FLC assays.
| Freelite [3], [9], [10] | N-Latex FLC [6], [11] | Seralite [7], [12] | Sebia FLC [8], [13] | |
|---|---|---|---|---|
| Assay principle | Nephelometry/turb | Nephelometry | Lateral flow | ELISA |
| Antibodies | Polyclonal | Monoclonal | Monoclonal | Polyclonal |
| Calibrator | Polyclonal FLC | Polyclonal FLC | Monoclonal FLC | Polyclonal FLC |
| Sample volume | 20 μL | κ: 90 μL, λ: 40 μL | 100 μL | 8 μL |
| Intra-assay VC | 0.4–2.2% | 0.3–1.2% | 2.7–9.2% | 5.1–7.6% |
| Inter-assay VC | 0.7–4.1% | 0.5–1.9% | 2.7–9.6% | 1.9–7.6% |
| Reference values | κ: 3.3–19.4 mg/L | κ: 6.7–22.4 mg/L | κ: 5.2–22.7 mg/L | κ: 5.2–15.3 mg/L |
| λ: 5.7–26.3 mg/L | λ: 8.3–27.0 mg/L | λ: 4.0–25.1 mg/L | λ: 8.2–18.1 mg/L | |
| κ/λ: 0.26–1.65 | κ/λ: 0.31–1.56 | κ/λ: 0.5–2.5 | κ/λ: 0.37–1.44 | |
| Adj. FLC-ratioa | κ/λ: 0.37–3.1 | No | No | κ/λ: 0.46–2.23 |
| Company | The Binding Site | Siemens | Abingdon Health | Sebia |
aAdjusted κ/λ FLC-ratio reference values for patients with impaired renal function. FLC, free light chain; VC, variation coefficient.
All newly introduced FLC assays have been extensively compared to Freelite results. Studies comparing both assays have demonstrated acceptable concordance in their capacity to detect patients with monoclonal gammopathies. However, assay comparison studies have confirmed significant differences in absolute FLC values in individual patient samples [15]. Because the bias is not constant across the measuring range of two FLC assays, it is not possible to apply a slope-correction to harmonize the assays. Identifying which of the two FLC assays accurately measures sFLC concentration in the case of discrepancies is challenging because of the lack of an internationally accepted sFLC reference methods and reference materials.
Guidelines of FLC-thresholds for treatment decision making have been defined based on studies utilizing the Freelite sFLC assay. In order to interpret sFLC results from different vendors in a broader context, we have performed a comprehensive method comparison of all four sFLC assays that are currently available for routine clinical use.
Materials and methods
sFLC assays
Serum Freelite (The Binding Site, Birmingham, UK) and N-Latex FLC (Siemens, Marburg, Germany) analyses were performed on a BNII analyzer according to manufacturer’s guidelines. Seralite (Abingdon Health, York, UK) analyses were performed according to manufacturer’s guidelines on the ADxLR5 reader system using competitive inhibition lateral flow technology. Sebia FLC (Sebia, Lisses, France) analyses were performed according to manufacturer’s guidelines on the automated AP22 ELITE processor (DAS, Palombara Sabina, Italy). Reference values for all four sFLC assays were used as indicated in the respective kit-inserts (see Table 1).
Samples
Assay performances were analyzed by using the remaining sera that were assessed for routine diagnostics from 62 patients with MM, 36 with LCMM, 16 with MGUS, seven with smoldering MM, 13 patients with AL amyloidosis and five patients with Waldenström macroglobulinemia. Control samples were selected in which no M-protein was detected using SPE or immunofixation electrophoresis (IFE, Hydragel 4IF, Sebia). These samples were obtained from 15 blood bank donors and 39 patients without monoclonal gammopathy. Twenty-four of these patients had chronic kidney disease. Samples were either freshly measured or stored at −20 °C and thawed prior to analysis. All samples were coded and anonymized prior to analysis. The study was performed in accordance with the Helsinki guidelines and was approved by the institutional Medical Ethics Review Boards (Erasmus MC 2017-415 and Radboudumc 2018-4140).
sFLC methods comparison
Method comparisons between the four sFLC assays were performed according to the CLSI EP9 guideline by using Bland-Altman evaluation and Passing and Bablok regression analysis on log-transformed values. The 193 selected serum samples covered the entire dynamic range of FLC concentrations. All samples were used for analysis, no outliers were discarded. Concordance analysis of the FLC-ratio was performed for all assay-combinations. For this, all 193 samples were divided into below the reference range (abnormal low), within the reference range (normal) and above the reference range (abnormal high) as determined by the reference values indicated in the kit-insert. In the case of patients with renal impairment, the adjusted FLC-ratio as defined for the Freelite and Sebia-FLC assay was used. Reference values for creatinine (Roche Diagnostics, Almere, The Netherlands) are 55–90 μmol/L for females and 65–115 μmol/L for males. The reference value for eGFR is >60 mL/min/1.73m2.
sFLC methods comparison with SPE
Sera obtained from eight patients had measurable involved FLC (iFLC) peaks on SPE (Hydrasys, Sebia). iFLC concentrations measured in the four different FLC-assays were compared to iFLC peak quantification on SPE.
Statistics
Bland-Altman evaluation, Passing-Bablok regression and Pearson’s correlation coefficient (R) analysis were performed with the results for κFLC, λFLC and FLC-ratio obtained in all four FLC assays. The results were calculated for all (six) paired-assay combinations. As results were not normally distributed, non-parametric methods were used. To evaluate qualitative concordance of the four FLC assays, the Cohen kappa (κ) coefficient was calculated. Complete agreement between two FLC-assays was defined as κ coefficient =1, high agreement as 0.81≤ κ coefficient <1 and a good agreement when 0.61≤ κ coefficient <0.8. Differences between FLC-ratio in patients with renal impairment compared to controls were calculated using Mann-Whitney U-test unpaired. Statistical analysis was performed using Analyse-it for Microsoft Excel Method Comparison Edition (v30.2, Analyse-it Software Ltd., Leeds, UK).
Results
Method comparisons
κFLC and λFLC using four different sFLC-assays were measured in 139 sera from individual patients with monoclonal gammopathies and 54 control sera from individuals without an M-protein. The exact diagnosis of all included patients is specified in Table 2. The agreement of κFLC, λFLC and the ratio of the involved to the noninvolved FLC (iFLC/niFLC) were presented by Bland-Altman representations for all six possible combinations. Each FLC assay was compared to the three other FLC assays and graphs are shown in Figure 1A for κFLC, Figure 1B for λFLC and in Figure 2 for iFLC/niFLC. Bland-Altman evaluation illustrates small constant biases, ranging from −9.8 to 16.2 mg/L for κFLC, −6.0 to 6.8 mg/L for λFLC and −0.04 to 0.38 for iFLC/niFLC. In general, good agreement can be seen between methods in the absolute low concentration range (<100 mg/L), while increasing absolute differences are visible at higher concentrations. Above 100 mg/L Freelite FLC measures higher, while Sebia FLC measures lower compared to the other methods. Conversely, Seralite FLC shows in this range both higher and lower results compared to Freelite FLC and N latex FLC. The iFLC/niFLC agreement is poor above a ratio of 50. The Passing-Bablok regression analysis showed slopes varying from 0.83 to 1.02 for κFLC, from 0.80 to 1.12 for λFLC and from 0.78 to 0.99 for the iFLC/niFLC (Supplementary material, Figures 1 and 2). Pearson correlations (R) between the methods ranged from 0.88 to 0.98 for κFLC and correlations for λFLC ranged from 0.82 to 0.94. Each FLC assay was correlated to three other FLC assays. The highest average correlation for κFLC was observed in the N Latex FLC assay (mean R=0.95), followed by Freelite (R=0.94), Sebia FLC (R=0.92) and Seralite (R=0.90). For λFLC the highest average correlation was observed in the Sebia FLC assay (mean R=0.91), followed by the N Latex FLC (R=0.90), Freelite (R=0.88) and Seralite (R=0.85). For the FLC-ratio, the Pearson correlations between the methods ranged from 0.90 to 0.96. The highest average correlation for the FLC-ratio was observed in the N Latex FLC assay (mean R=0.95), followed by the Freelite and Sebia FLC-assay (R=0.94), and Seralite (R=0.91).
Patient cohort.
| Diagnosis | Patients |
|---|---|
| Monoclonal gammopathies | |
| Multiple myeloma | 62 |
| Kappa LCMM | 19 |
| Lambda LCMM | 17 |
| MGUS | 16 |
| sMM | 7 |
| AL amyloidosis | 13 |
| Waldenstrom macroglobulinemia | 5 |
| Controls | |
| Blood bank donor without M-protein | 15 |
| Patients without M-protein | 15 |
| Chronic kidney disease without M-protein | 24 |
| Total | 193 |
sMM, smouldering multiple myeloma; MGUS, monoclonal gammopathy of unknown significance; LCMM, light chain multiple myeloma.
Bland-Altman representation of FLC concentrations.
Bland-Altman graphs are presented by plotting the difference between method vs. the mean of the methods of κFLC (A) and λFLC (B) levels in 193 samples analyzed using the following methods: Freelite, N Latex FLC, Seralite, and Sebia FLC. Median, 2.5th and 97.5th percentile of the agreement is shown in each figure.
Bland-Altman representation of involved to uninvolved ratio FLC.
Bland-Altman graphs are presented by plotting the difference between method vs. the mean of the methods of the involved to uninvolved FLC-ratios in 193 samples analyzed using the following methods: Freelite, N Latex FLC, Seralite, and Sebia FLC. Median, 2.5th and 97.5th percentile of the agreement is shown in each figure.
Based on the FLC-ratio cut-off values specified in each kit-insert, all 193 samples could be classified per assay as ‘abnormal low’, ‘normal’ or ‘abnormal high’. No large differences in the concordances of the clinical interpretation of the FLC-ratio’s between the four FLC-assays were observed, they ranged from 86 to 92% (Figure 3). The highest average concordance was observed for N Latex FLC (90.2%), followed by Sebia FLC (89.8%), Freelite (88.4%) and Seralite (87.7%). This means that out of the 193 samples, the number of discrepant FLC-ratios ranges from 16 to 27 in each comparison (Figure 3). The high/good agreement in the total group of 193 samples was further shown by Cohen κ coefficients that ranged from 0.76 to 0.86. In all method comparisons, the discrepancies were observed in samples with FLC-ratio values close to the cut-off value of the reference ranges (Figure 3).
Concordance analysis.
Concordance analysis of the FLC-ratio clinical interpretation between all four FLC assays is shown. The grey boxes indicate agreement between both assays. For each analysis the % concordance and the Cohen kappa coefficient over all 193 samples is provided. On the right of each panel the samples with a discrepant FLC-ratio between both assays are illustrated by red dots. H, above reference range; L, below; N, within.
These high concordance rates for the various FLC assays are only observed when the FLC-ratio is used to differentiate between a normal and abnormal ratio, for example, as a screening tool for the presence of a monoclonal gammopathy. Table 3 indicates that the concordances are substantially lower when the FLC-ratio is used as high-risk marker for progression (iFLC/niFLC ≥8) [16], and poor when used as myeloma defining event (iFLC/niFLC-ratio ≥100) [17].
Concordances (%) at different FLC-ratio levels.
| Freelite N latex FLC | Freelite Seralite | Freelite Sebia FLC | N Latex FLC Seralite | N Latex FLC Sebia FLC | Seralite Sebia FLC | |
|---|---|---|---|---|---|---|
| Normal FLC-ratio | 91 | 86 | 89 | 88 | 92 | 89 |
| FLC-ratio ≥8a | 77 | 71 | 71 | 71 | 77 | 74 |
| FLC-ratio ≥100a | 53 | 35 | 25 | 24 | 46 | 15 |
aDefined as the involved: uninvolved FLC-ratio.
An interesting difference between the four FLC assays is that both Freelite and the Sebia FLC assay have introduced adjusted reference ranges for patients with renal impairment. We compared FLC-ratio’s in control sera (i.e. no M-protein) from individuals with normal renal function (n=30) and individuals with renal impairment (n=24). FLC-ratio’s were significantly increased in individuals with renal impairment when tested using the Freelite assay (average FLC-ratio increased from 1.2 to 1.9; p=0.0001) and Sebia FLC (average FLC-ratio increased from 0.87 to 1.14; p=0.002). FLC-ratio’s were not significantly different in patients with renal impairment when tested using the N-Latex FLC and Seralite assay (see Figure 4).
FLC-ratio in patients with renal impairment.
Box and whiskers plot for all four FLC assays in control samples (no M-protein) from individuals with normal renal function (n=30, white boxes) and individuals with renal impairment (n=24, gray boxes). Median FLC ratios are indicated by horizontal lines in each box. FLC-ratios were significantly increased in individuals with renal impairment using the Freelite assay (p=0.0001) and Sebia FLC (p=0.002). FLC-ratios were not significantly increased in patients with renal impairment using the N Latex FLC and Seralite assay.
Accuracy of FLC measurements
Absolute sFLC differences between the various methods were mainly visible at the high end of the concentration range. Up to 10-fold differences in sFLC concentration between the sFLC assays in individual sera were regularly observed in this study.
Within the entire cohort of samples, five samples had a κFLC concentration above 1000 mg/L measured in at least one of the sFLC assays (highest 5090 mg/L measured with Freelite) and 12 samples had a λFLC concentration above 1000 mg/L (highest 12,400 mg/L measured with Freelite). In all these 17 sera samples the sFLC result in the initial dilution was correctly indicated as ‘above curve’. In conclusion, antigen excess was not observed in any of the samples tested with any of the four sFLC assays.
To assess which FLC assays are most accurate in these 17 samples with relatively high monoclonal FLC concentrations, data were compared to results obtained with electrophoretic methods. In one serum sample no monoclonal λFLC could be detected even though Seralite quantified this sample as 1.980 mg/L. The other three FLC assays quantified the λFLC ranging from 76 to 265 mg/L). In eight out of 17 samples a monoclonal FLC band on IFE was observed, however, this band could not be quantified because it was either a faint band or a band that co-migrated with an intact M-protein or with other proteins in the β-region. SPE concentrations of the iFLC in the remaining eight serum samples ranged from 70 to 5300 mg/L. As shown in Figure 5A–D, the iFLC concentrations measured by the sFLC assays were significantly higher compared to SPE in a vast majority of the samples. Results in one sample clearly deviated from this trend, Figure 5E shows that Freelite strongly overestimated the monoclonal λFLC, while the other three sFLC assays strongly underestimated its concentration compared to the SPE value. The absolute differences in iFLC concentration between SPE and the sFLC assays were highest for Freelite with an average difference per sample of 2500 mg/L, followed by N Latex FLC (1300 mg/L) and Seralite/Sebia (840 mg/L).
Method comparison to electrophoresis.
(A) Quantification of eight monoclonal FLC samples measured both by Freelite (black symbols), N Latex FLC (red), Seralite (green) and Sebia FLC (blue) was compared to quantification by Serum Protein Electrophoresis (SPE). The black dotted line indicates perfect agreement between FLC quantification with SPE. Blue arrows represent four patients shown in more detail (B–E). Represented for each patient is the IFE, the SPE densitogram with the FLC clone spiked in gray, and the FLC-concentrations obtained with all four FLC assays.
Discussion
This study describes a method comparison of the four sFLC assays that are currently commercially available for routine clinical diagnostics. The sFLC assay is an important complementary test in the context of screening patients suspected of monoclonal gammopathy, prognostic stratification and monitoring of therapy [4], [5], [17].
The Freelite FLC assay was introduced as a nephelometric/turbidimetric sFLC assay in the early 2000s [3]. Meanwhile, three other sFLC immunoassays across different analytical platforms have become available for clinical laboratories [6], [7], [8]. These methods show relevant differences in the type of detection antibodies, polyclonal vs. monoclonal, that are used. Among the advantages of using polyclonal detection antibodies, is their ability to recognize a broader range of epitopes. Results reported in literature indeed suggest that assays using polyclonal reagents detect a higher proportion of monoclonal FLCs [18], [19]. It has, on the other hand, also been suggested that assays using monoclonal reagents may perform better in terms of assay reproducibility, which is important for patient monitoring [18], [20]. Non-linearity and poor accuracy are analytical limitations associated with all currently available assays, regardless if polyclonal or monoclonal reagents are used [15]. The consequence of non-harmonized sFLC measurements is that an individual patient may or may not meet certain diagnostic, prognostic or response criteria, depending on the FLC assay and platform used.
Method comparisons performed so far, have all focused on single method-to-method comparisons in which one of the newly introduced FLC-assays is compared to Freelite [15]. In these studies, it is challenging, if not impossible, to determine which assay is correct in case discrepancies are observed between both assays. In this study we compare four sFLC assays, which allows more in-depth analyses of the characteristics of these assays and occasional discrepancies in specific samples. Overall, we observed good agreements for κFLC, λFLC and FLC-ratio between all four assays. The observed differences increased with the analyte concentrations, especially above 100 mg/L. The clinical concordances of the four FLC assays was high and ranged from 86% (Freelite and Seralite) to 92% (N Latex and Sebia FLC). In this study, discrepancies were only observed in samples with FLC-ratio values relatively close to the cut-off value of the reference ranges. Of the four assays, the lowest agreements, correlations and concordances were observed for the Seralite assay. The advantage of this assay is that it generates the most rapid FLC results using a portable diagnostic device, facilitating near-patient testing and small size laboratories.
The N Latex FLC, Seralite and Sebia FLC assays have all calibrated their assays to the Freelite reference ranges in healthy controls. Highest concordance is therefore, as expected, observed in samples with FLC-ratio’s close to normal. In 2008, an iFLC/niFLC ≥8 was reported to be an independent risk factor for progression of smoldering MM [16]. And in 2014, the International Myeloma Working Group (IMWG) defined an iFLC/niFLC ≥100 as a myeloma defining event with treatment indication because it was shown that 80% of these patients progressed to MM within 2 years [17]. These IMWG recommendations are based on clinical studies that used Freelite reagents on a BNII analyzer. It is therefore important to note that concordance between the different sFLC assays is considerably lower at FLC-ratio cut-off value of 8, and extremely poor at a cut-off value of 100. This study therefore further stresses the importance for independent clinical studies for each FLC-assay to clinically validate what FLC ratio is equivalent to a Freelite ratio of 8 and 100. The first studies that addressed this point stated that an involved:uninvolved sFLC-ratio ≥100 is approximately equivalent to an N-Latex sFLC-ratio of ≥35 [21], [22] and a Sebia sFLC ratio of ≥16 [23].
Another interesting difference between the four FLC assays is that both Freelite and the Sebia FLC assay have introduced adjusted reference ranges for patients with renal impairment [10], [13]. In contrast, no significant increase of the FLC-ratio is observed when the N-Latex FLC and Seralite assay is applied to sera derived from patients with renal impairment without a monoclonal gammopathy [24], [25]. Our study verifies exactly these results in which significantly increased FLC-ratios were exclusively observed in individuals with renal impairment tested using the Freelite and Sebia FLC assays. With renal impairment, FLC clearance is increasingly accomplished via pinocytosis in the reticuloendothelial system, which is independent of protein size. This is in contrast to renal FLC clearance which more efficiently clears monomers (typically κFLC) than dimers (typically λFLC). As a result, the sFLC-ratio in patients with renal impairment better reflects the underlying FLC production rate, which is higher for κFLC. The discrepancy why an adjusted FLC-ratio reference range is introduced only for some FLC assays, might be explained by the fact that some FLC assays may preferentially recognize monomeric λFLC while other preferentially detect dimeric λFLC [26].
Previous comparative studies between the various FLC assays showed that significant absolute differences in FLC concentrations in individual patients can be seen, particularly at higher FLC concentrations [8], [15], [27], [28]. In this study we confirm these data, and demonstrate that up to 10-fold differences in sFLC concentration between the sFLC assays in individual sera were regularly observed. Because an international reference method for sFLC quantification is not available, it is challenging to assess which of the four sFLC assays is most accurate. Overall it can be concluded that the iFLC concentrations measured by the sFLC assays were significantly higher compared to SPE in a vast majority of the samples. The absolute differences in iFLC concentration between SPE and the sFLC assays were highest for Freelite. Mass spectrometry methods appear to be promising in overcoming the analytical problems of immunoassays and have the potential to improve both diagnostic accuracy and sensitivity [29], [30], [31], [32]. As such mass spectrometry could in the future be a potential candidate as FLC reference method.
Results from the Sebia FLC assay were obtained after 4 months’ storage at −20 °C, while other results were produced on fresh samples. Samples from blood bank donors were also stored for several months and thawed prior to analysis. A recent study showed that FLC in serum samples were sufficiently stable following long-term frozen storage (for a minimal period of 568 days) and therefore suitable for comparison studies [33].
Because of differences between the various FLC assays, they cannot be used interchangeably. An adequate period of parallel testing is required to enable continued accurate follow-up of individual patients in the case of changing from one FLC assay to another. Along the same line, caution is warranted when patients switch from the hospital in which they are treated and sFLC concentrations are communicated via referral letters. Our data also clearly indicate that clinical FLC thresholds published in guidelines and obtained with Freelite data, do not apply to the other FLC assays. For these assays clinical studies are needed to establish assay-specific FLC thresholds.
Acknowledgments
Reagents to perform the Seralite and Sebia FLC tests in this study were kindly provided by BMD and Sebia, respectively.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2019-0533).
©2021 Henk Russcher et al., published by De Gruyter, Berlin/Boston
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