Received: 28 October 2023 Revised: 15 March 2024 Accepted: 19 March 2024

DOI: 10.1002/jha2.886

RESEARCH ARTICLE

The choice of serum-free light chain analysis method could

potentially have clinical consequences for myeloma patients

Ljupco Veskovski1 Ingvar Jakobsson2 Per-Ola Andersson3

Therese Gustafsson2 Annelie Sedigh2 Dorota Knut-Bojanowska4
Markus Hansson3 Cecilie Hveding Blimark3 Ulf-Henrik Mellqvist1

1
Department of Research, Education and
Innovation and Department of Medicine Abstract
(Hematology Unit), Södra Älvsborg Hospital
Multiple myeloma (MM) is a disease, that at times poses diagnostic and monitoring
(SÄS) Borås, Region Västra Götaland (VGR),
Boras, Sweden challenges. Over the last decades laboratory methods have been expanded with serum
2
Laboratory for Clinical Chemistry SÄS Borås free light chain (FLC) analysis. Alerted by two index cases with clinical impact due to
and Department of Clinical Chemistry
Sahlgrenska University Hospital, Goteborg,
failure of the FLC analysis to indicate a disease progression, we aimed to identify any
Sweden clinical consequences due to known differences between FLC analysis methods. We
3
Department of Haematology, Sahlgrenska applied two FLC analysis methods (Freelite Binding Site [FBS] and N-Latex Siemens
University Hospital, Goteborg, Sweden
4
[NLS]) on all patients with MM and monoclonal gammopathy of uncertain significance
Department of Hematology, Norra Älvsborg
Hospital (NÄL), Trollhattan, Sweden diagnosed/followed up at Södra Älvsborg Hematology Unit, from April to December
2022. From a total of 123 patients with malignant plasma cell disorder, we identified
Correspondence
Ljupco Veskovski, Brämhultsvägen 53, 501 82
five cases (4.1%) where solely the FBS method, as opposed to NLS, urine and serum
Borås, Sweden. electrophoresis, could support diagnosis or detect progression. The consequences of
Email: [email protected]
this discrepancy included not only change of diagnosis or delayed therapy but also
Funding information change of treatment. Our findings indicate that a stronger awareness of the potential
The Department of Research, Education and
weaknesses of different FLC methods is needed, which calls for a closer collaboration
Innovation, Södra Älvsborg Hospital (SÄS)
Borås, Grant/Award Number: sas-981725 between clinical chemists and hematologists.

KEYWORDS
disease progression, hematology, immunoglobulin light chains, monoclonal gammopathy of
undetermined significance, multiple myeloma, paraproteinemia

1 INTRODUCTION κ-, λ-chains in urine (Bence Jones proteinuria) respectively. These

biomarkers are a diagnostic criterium for a majority of the plasma
Multiple myeloma (MM) is a malignant plasma cell disorder that cell disorders, pointing towards these diseases in a clinical investi-
can present diagnostic and monitoring challenges, even now in an gation, and may also serve as prognostic features during follow-up,
era of improved diagnostic methods [1, 2] and increasingly efficient where changes in concentrations could indicate disease progres-
therapies prolonging survival [3]. Core components in the diagnostic sion or treatment response. Although easily accessible, not all MM
and monitoring methods for MM are the serum and urine elec- patients will present with these biomarkers [1–3]. Also, over the
trophoreses that measure monoclonal immunoglobulin in serum and course of the malignant plasma cell disorder (primarily myeloma),

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2024 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.

eJHaem. 2024;5:455–461. wileyonlinelibrary.com/journal/jha2 455

 26886146, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jha2.886 by Bangladesh Hinari NPL, Wiley Online Library on [19/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
456 VESKOVSKI ET AL .

the production of M-protein may be diminished or completely TA B L E 1 Patient characteristics. One case with IgM-type
lost [4]. paraprotein had multiple plasmacytomas. Only light chain means the
patient did not have measurable levels of the four immunoglobulin
The implementation of serum-free light chain (FLC) analysis has
types, only light chains in either urine or serum or in both urine and
become a valuable addition to the clinician’s toolset. Some of the
serum. In a couple of cases, one MM and one MGUS, paraproteinemia
benefits are, 1) diagnosis and monitoring of oligo-secretory plasma was detected in the gamma region but could not be further specified,
cell disorders, 2) distinction between plasma cell disorders requir- nor could their clonal type be determined. In one case of
ing treatment (MM) and those with a watch-and-wait approach IgG-M-protein, the clonal type could not be assessed and the FLC was
(monoclonal gammopathy of uncertain significance [MGUS] and smol- normal. In another case with MM in complete remission since 1996,
neither type nor clonality of M-protein could be recovered, and the κ-,
dering myeloma [SMM]), and 3) detection of FLCs in serum with
λ-, light chains have remained normal since the introduction of
regards to their potentially debilitating effect on renal function. The FLC-measurement.
specific FLC criteria for plasma cell disorders are clarified in the
n (%)
updated guidelines of the International Myeloma Working Group
(IMWG)[5, 6]. Included patients 175

In the wake of the introduction of FLC analysis in the clinical set- Male 99 (57)
ting [7–9], the development of different (competing) methods for the Female 76 (43)
detection and quantification of FLC [10–13], revealed some common Median age years (range) 74 (32–92)
analytical challenges [14]. The most alarming was the poor correlation Plasma cell disorders 175
between different methods [15]. So far, this has led to the common rec-
MM 110 (63)
ommendation by researchers that the same method of FLC analysis
SMM 5 (2.9)
should be utilized for monitoring patients since concentration values,
Plasmacytoma 7 (4.0)
and consequently ratios and differences over time, can vary widely
Plasma cell leukemia 1 (0.6)
between assays [16, 17].
Initially, there was only one method available for measuring FLC, the MGUS 48 (27)

Serum Freelite (The Binding Site Group Ltd., Freelite Binding Site [FBS]) AL 4 (2.3)
method [7, 8]. It is widely used in Europe and the United States and is Paraprotein type 175
the method on which the IMWG has based its serum-FLC diagnostic IgA 31 [18]
and prognostic criteria. The method carried some technical challenges, IgD 2 (1.1)
primarily the issue of antigen excess [18], which required experienced
IgG 117 (67)
vigilance and time-consuming reevaluations and corrections. When
IgM 1 (0.6)
alternative methods for FLC analysis were made available, hospitals
Only light chain 21 [12]
in the Västra Götaland region (VGR) of Sweden (comprising 1.7 mil-
lion inhabitants), among other regions in Sweden, looked for a method Gamma region 2 (1.1)

with less of the issues seen with the FBS method. In 2017, Södra Älvs- Undetermined 1 (0.6)
borg Hospital, in the VGR, adopted the N-Latex (Siemens Healthineers) Clonal type 175
(NLS) method [10], for its perceived advantages when compared to the Clonal Type: kappa (κ) 106 (61)
FBS method. Clonal Type: lambda (λ) 65 (37)
In 2020, we noticed two separate clinical cases (Table 4 and
Clonal Type: undetermined 4 (2.2)
Table S1) where patients developed severe disease progression with
Abbreviations: AL, light chain amyloidosis; FLC, free light chain; IgA,
significant and debilitating clinical consequences for the patients,
immunoglobulin A; IgD, immunoglobulin D; IgG, immunoglobulin G; IgM,
without fair warning from the monitoring analyses of FLC. A great immunoglobulin M; MGUS, monoclonal gammopathy of undetermined
discrepancy was detected in the assessment of FLC with the NLS significance; MM, multiple myeloma; SMM, smoldering myeloma.
method compared to serum electrophoresis. To further evaluate this
discrepancy, samples were analyzed also with the FBS method. These suffering the same consequences as the two index cases, and to find out
analyses showed a much higher level of the involved FLC, and the if and when, one or both, FLC methods are essential for diagnosing MM
results were in line with what the serum electrophoresis had signaled. disease and assessing its response during follow-up.
One of the patients suffered severe renal failure which led to life-long
hemodialysis, whereas the other patient experienced progressive renal
failure, overall deterioration of the general physical status, and later 2 METHODS
death.
The purpose of this study was to compare two methods for FLC anal- 2.1 Patient selection
ysis, the formerly used FBS and the currently used NLS, in a real-life
setting at the hematology unit at Södra Älvsborg Hospital. With a regi- From April 21, 2022, to December 31, 2022, all referred, investi-
men of double testing for FLC, we aimed to discover patients at risk of gated, or monitored patients regarding MGUS and malignant plasma
 26886146, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jha2.886 by Bangladesh Hinari NPL, Wiley Online Library on [19/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
VESKOVSKI ET AL . 457

TA B L E 2 Outcome of Freelite Binding Site (FBS) and N-Latex 2.2 Laboratory analysis of M-protein and FLCs
Siemens (NLS) comparisons. The table demonstrates number of cases
where either of the two FLC methods, FBS and NLS detect at least a
Blood sampling and urine collection were conducted routinely as part
three times higher level than the other, in at least one of the three
applications of FLC results, 1) involved light chain concentration, 2) of clinical investigations or follow-up procedures. However, during
involved/non-involved light chain ratio, and 3) increase in difference the study period, a portion of the serum samples were fresh frozen
between involved and non-involved light chain. For five patients the ≥ at −80◦ C, reserve samples were primarily used to perform extra
3:1 difference is deemed clinically relevant because it had a significant analyses and adjustments whenever there were clear or suspected
impact on the clinical outcome for the patient. This is further clarified
signs of estimation issues, including antigen excess. Serum FLC analysis
in Table 3.
was conducted with two different assays, the polyclonal FBS and
n (%) the monoclonal NLS method. A BN Prospec Nephelometer (Siemens
Included double tested 175 (100) Healthineers) was used for both assays. All patients analyzed for FLC,
≥3:1 difference in result between FLC-methods 31 [18] were also analyzed for total serum immunoglobulin (IgG, IgA, and IgM)

MM, SMM, plasmacytoma, and plasma cell leukemia 24 [14] concentrations, using Alinity c (Abbott Laboratories). Serum/urine
protein electrophoresis (S/uPEP) and immunofixation, for assessment
FBS > NLS 16 (9.1)
of M-protein and Bence-Jones-proteinuria, were performed with
NLS > FBS 8 (4.6)
agarose gels on the Hydrasys 2 Scan (Sebia, Lisses, France) and Alinity
MGUS 7 (4.0)
c (Abbott Laboratories). Serum-M-Protein was measured in g/L. Refer-
FBS > NLS 4 (2.3)
ence values for FLC-analyses were: NLS: S-FLC κ 6.7–22.4 mg/L, S-FLC
NLS > FBS 3 (1.7) λ mg/L 8.3–27.0 mg/L, κ/λ-ratio 0.31–1.56, FBS: S-FLC κ 3.3–19 mg/L,
Clinically relevant ≥3:1 difference 5 (2.9) and S-FLC λ 5.7–26 mg/L, κ/λ-ratio 0.3–1.7, Δ κ−λ (absolute difference
MM, SMM, plasmacytoma, and plasma cell leukemia 5 (2.9) between κ and λ) mg/L. Urine protein analysis: tU-Albumin mg/24 h,
FBS 3:1 > NLS 5 (2.9) U-Albumin mg/L, tU-κ mg/24 h, U-κ mg/L, tU-λ mg/24 h, and U-λ mg/L.

NLS 3:1 > FBS 0

2.3 Data analysis
MGUS 0
FBS > NLS 0
M-protein and double-tested FLC data were registered and checked
NLS > FBS 0
against other standard laboratory data, patient chart registries, and
Abbreviations: FBS, Freelite Binding Site; FLC, free light chain; MGUS, mon- skeletal surveys when necessary. The first step of comparing FLC
oclonal gammopathy of undetermined significance; MM, multiple myeloma; data between the FBS and NLS methods aimed to determine cases
NLS, N-Latex Siemens; SMM, smoldering myeloma.
where there was a ≥3:1 difference in FLC concentration levels of
involved(inv)-FLC, or a ≥3:1 difference in FLC-ratio, or a ≥3:1 differ-
ence in increase of difference between involved and non-involved FLC,
at least once (for patients tested more than once) and the first time it is
cell disorders at the Hematology unit of Södra Älvsborg Hospital, observed (for patients tested more than once). Secondly, a patient chart
were potential study candidates. All patients, for whom the clinician review was performed to confirm diagnosis and response, according to
requested an analysis of serum FLC, were tested with two meth- the IMWG criteria [5, 6], to determine cases where diagnosis and/or
ods (see below). The aim was to perform double testing at each response assessment rested only on FLC (-concentrations, or -ratio,
requested FLC analysis. Study patients were then identified retro- or -an increase of difference), and excluding cases where FLC simply
spectively by checking all double tested against all patients that were was not necessary to take adequate clinical action (i.e. other clinical
diagnosed or followed up at the Hematology unit during the study features sufficed to assess diagnosis or response). The purpose was
period. Patients were diagnosed and/or regularly assessed during to identify cases where a difference between the two FLC methods
follow-up according to the diagnostic and prognostic criteria in the had a clinical consequence, for example, a change of diagnosis, a ther-
IMWG guidelines [5, 6]. In total 896 (768 with malignant plasma cell apeutic delay with the development of debilitating symptoms (skeletal
disorder, 99 with MGUS, and 29 with AL-amyloidosis) double tests lesions, renal failure), or the opposite, a timely therapeutic intervention
were performed on the included patients with a median of seven to prevent a clinical consequence.
tests per patient with malignant plasma cell disorder, a median of two
tests per patient with MGUS and a median of eight tests per patient
with AL-amyloidosis. 3 RESULTS
Patients where the M-protein was of an IgM-type in MGUS or indo-
lent lymphoma were excluded. This was also the case for patients with 3.1 Patient characteristics
multiple myeloma only monitored with a significant M-protein and
for those treated with bispecific antibodies where the therapy led to In total, 199 patients had an investigation, treatment, follow-up, or sec-
unmeasurable FLC. ond opinion assessment, due to diagnosed or suspected plasma cell
 26886146, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jha2.886 by Bangladesh Hinari NPL, Wiley Online Library on [19/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
458 VESKOVSKI ET AL .

TA B L E 3 Relevant cases from April to December 2022. The table summarizes the clinical consequence of the difference in measurement
between the FBS and NLS methods for the identified five cases. Only FBS results meet the diagnostic or prognostic criteria (according to
International Myeloma Working Group guidelines) to prompt adequate and timely action and to reveal a diagnostic delay.

Case Analysis Clinical consequence

1 NLS-FLC fails to signal MM disease progression Delayed therapy, fracture
2 NLS-FLC is not enough to meet MDE criteria, only diagnosed as SMM Delayed correct diagnosis (MM), exclusion from SMM
clinical study
3 NLS-FLC is not enough to meet malignant plasma cell disorder criteria, only Delayed diagnosis (plasma cell leukemia), acute renal and
diagnosed as MGUS respiratory failure
4 Only FBS-FLC supports MM diagnosis and later disease progression Timely onset of therapy and later therapy adjustment
5 Only FBS-FLC supports MM disease progression Timely onset of therapy

Abbreviations: FBS, Freelite Binding Site; FLC, free light chain; MGUS, monoclonal gammopathy of undetermined significance; MM, multiple myeloma; NLS,
N-Latex Siemens; SMM, smoldering myeloma.

disorder during the study period, and 175 met the inclusion criteria. Fif- 3.3 Case reports
teen patients were excluded due to not being double-tested with both
FLC methods along with nine double-tested. The first index case detected is described in Table 4, whereas index case
Patient characteristics are presented in Table 1. The male/female 2 and the relevant cases 1–5 detected during the study period (Table 3)
ratio was balanced, and the median age was representative of the are detailed in Tables S2–S7.
study population. The malignant plasma cell disorder patients (n = 123)
include 109 MM, five SMM, eight plasmacytoma, and one plasma
cell leukemia patient. Twenty-four (20%) of the MM-patients had 3.4 Index case 1
unmeasurable levels of whole M-protein monitored with serum elec-
trophoresis during the observation period. 3.4.1 68-year-old male with MM diagnosed in
2015 (no previous medical conditions)

3.2 Outcome of FBS and NLS comparisons At diagnosis in 2015, the patient presented with progressing fatigue,
anemia (Hb 82 g/L), acute renal failure (creatinine 511 μmol/L), S-
Thirty-one patients were identified who had a relative difference of ≥ Albumin 41 g/L, S-Calcium 2.68 mmol/L, S-IgA λ 1 g/L, S-κ 16.1 mg/L, S-
3:1 in involved FLC levels or inv/non-inv FLC ratio at diagnosis (where λ 17000 mg/L (FBS method), tU-Albumin 46 mg/24 h, tU-κ < 6.11 mg/L,
the FLC level is a myeloma defining event [MDE] criteria at diag- and tU-λ 16448 mg/24 h. Bone marrow biopsy showed an extensive
nosis)[5] or ≥ 3:1 difference in the increase of difference between plasma cell infiltration and a skeletal CT survey revealed multiple oste-
involved and non-involved chain during follow-up [6]. Twenty-four olytic lesions. The first two lines of therapy resulted in a very good
patients with a malignant plasma cell disorder (MM, SMM, plasmacy- partial response (VGPR). The renal function partially recovered (crea-
toma, and plasma cell leukemia) and seven with MGUS (Table 2). In tinine around 200 μmol/L) whereas S-κ, S-λ and κ/λ-ratio normalized.
5 verified cases (4.1% of the included patients with malignant plasma There were no other remaining signs or symptoms of MM-related
cell disorder), the ≥ 3:1 difference was clinically relevant (Table 3 and organ impairment or MDEs.
Tables S2–S7), revealing a diagnostic delay with serious clinical conse- In late February 2020, the patient was hastily admitted to the
quence (two cases), an earlier assessment of disease progression and hospital, due to frequent diarrhea, anemia, and acute deterioration
timely shift or onset of therapy (two cases), and a change of diagno- of the chronic renal insufficiency, creatinine 1300 μmol/L, and urgent
sis and follow-up routines (one case). The assessment of diagnosis, or hemodialysis was initiated. There was an increase in the involved
disease progression in these five cases rested entirely on the FLC mea- serum light chain (S-κ 21.7 mg/L and S-λ 250 mg/L with the NLS
surement from one of the two methods (FBS), that is, there were no method), however, the serum electrophoresis indicated a much higher
other clinical features to indicate the diagnosis or the disease pro- level of λ-chains. Samples were sent to neighboring NÄL Hospital
gression. Renal insufficiency as a potential consequence of myeloma utilizing the FBS method, which yielded significantly higher λ-levels
(creatinine > 177 μmol/L) was observed in five of 31 patients with a sig- (S-λ 30800 mg/L). Urine analysis revealed a Bence-Jones proteinuria
nificant difference in FLC between methods. Only the index case had (tU-Albumin 132 mg/24 h, tU-κ 20 mg/24 h, tU-λ 9018 mg/24 h, and
end stage renal insufficiency that could have affected the FLC-levels tU-Protein 280 mg/24 h). Further, a bone marrow aspirate revealed
[19], however that patient had improved FLC levels with a lesser dif- 12% plasma cells, an increased serum lactate dehydrogenase 9.5
ference between FLC methods during the observation period and was μkat/L, and the presence of +1q21 mutation in the cytogenetic FISH
hence not part of the 31 identified cases. analysis (stage III myeloma, according to R-ISS [2]). The patient was
 26886146, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jha2.886 by Bangladesh Hinari NPL, Wiley Online Library on [19/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
VESKOVSKI ET AL . 459

TA B L E 4 Index case 1. Clinical parameters monitored for Index case 1.

Feb Mar Jul Apr May Jun May Dec

Variable 2020 2020 2020 2021 2021 2021 2022 2022
M-protein 1 <1 0 5 7 0 0 0
-type IgA λ IgA λ n/a λ λ n/a n/a n/a
N-Latex Siemens
κ 21.7 31.2 68.6 63 22.3 48.1 77.3 88.6
λ 250 39.8 78.4 67 105 46.4 84.2 81.7
κ/λ 0.09 0.78 0.88 0.94 0.21 1 0.92 1.1
Δ κ−λ 228 9 10 4 83 2 13 8
Freelite Binding Site
κ 19.8 24.9 8.,2 76 n/a n/a 102 122
λ 30800 754 32.7 3470 14800 169 39 61
κ/λ <0.01 0.03 2.51 0.02 n/a n/a 2.6 2,0
Δ κ−λ 30780 729 50 3394 n/a n/a 63 61
Urine analysis
tU-Albumin 132 284 38 mg/L 51 mg/L 121 84 – –
tU-κ 20 22 15 mg/L 34 mg/L 11 57 – –
tU-λ 9018 282 6.9 mg/L 519 mg/L 9688 64 – –
Clinical Features
Hb 80 87 106 88 68 81 89 116
Leucocytes 7.7 3.29 6.73 8.84 11.1 1.62 8.9 3.5
Thrombocytes 222 33 139 272 132 116 101 63
Creatinine 1300 778 509 508 603 345 558 550
Calcium 2.31 – 2.32 2.48 2.81 2.34 2.46 2.53
Lesion n/a yes† n/a n/a n/a n/a n/a n/a
‡ ‡
Symptom yes yes no no no no no no
Therapy
-type – VTD VTD Btz/Dex – PCD PCD PCD

M-Protein g/L, -type (immunoglobulin type and clonal type κ or λ of M-protein), Serum free light chain analysis-N-Latex Siemens: κ 6.7–22.4 mg/L, λ 8.3–
27.0 mg/L, κ/λ 0.31–1.56, Δ κ−λ mg/L, -Freelite Binding-Site: κ 3.3–19 mg/L, and λ 5.7–26 mg/L, κ/λ 0.3–1.7, Δ κ−λ mg/L, Urine protein analysis: tU-Albumin,
tU-κ, and tU-λ mg/24 h. Results reported in mg/L instead of mg/24 h when insufficiently collected total-24h-urine volume, Clinical Features: Hb 117–
153 g/L, Leucocytes 3.5–8.8 × 109 /L, Thrombocytes 165–387 × 109 /L, Creatinine 60–105 mmol/L, Calcium 2.15–2.50 μmol/L, Lesion (skeletal) yes, no, or
n/a, Symptom (disease-related symptoms, primarily pain, but also fatigue, neuropathy, etc.) yes or no.
Abbreviations: BtzDex, bortezomib, dexamethasone; Hb, hemoglobin; IgA, immunoglobulin A; M-protein, monoclonal immunoglobulin, paraprotein; n/a, not
assessed; PCD, pomalidomide, cyclophosphamide, dexamethasone; tU, total-24h-urine; VTD, bortezomib, thalidomide, dexamethasone.; Δ κ−λ, absolute
difference between kappa and lambda; κ, kappa; κ/λ, kappa/lambda-ratio; λ, lambda.
†
Multiple skeletal lesions, both new and known deteriorated lesions compared to previous assessments in 2015.
‡
Diarrhea and fatigue, however no pain.

started on third-line therapy including maintenance therapy, again 4 DISCUSSION AND CONCLUSIONS
attaining VGPR, but unfortunately had to remain on permanent
hemodialysis, keeping the creatinine concentrations fluctuating Our study revealed five cases where the only means for diagnosis or
around 500 μmol/L. detecting disease progression was FLC analysis and where only one
In April 2021 serum electrophoresis revealed 5 g/L of serum-free (FBS) of the two parallel applied methods served to detect the relevant
λ chains, which did not match what the FLC-analysis (NLS) had mea- changes.
sured, so again samples were sent to NÄL lab conforming significantly The introduction of methods measuring FLCs in serum has been
higher levels of serum free λ chains (3470 mg/L) using the FBS method. important in facilitating the diagnosis and monitoring of multiple
This immediately prompted a shift in therapy which eventually proved myeloma. Even though the FBS method was the first to see a wider use
efficient against the disease, attaining VGPR for the fourth time. [7–9], to this day, there is no gold standard method for analyzing serum
 26886146, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jha2.886 by Bangladesh Hinari NPL, Wiley Online Library on [19/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
460 VESKOVSKI ET AL .

FLCs [15, 20]. Where the polyclonal FBS method had a tendency for the Department of Clinical Chemistry, Sahlgrenska University Hos-
both overestimation and underestimation, with issues such as antigen pital, Göteborg. This work was supported by The Department of
excess [18], the monoclonal methods such as NLS [10] were introduced Research, Education and Innovation, Södra Älvsborg Hospital (SÄS)
to achieve more consistent results, less sensitive to antigen excess and Borås, funded the study.
potentially superior for disease monitoring. This prompted a change of
method from FBS to NLS in several hospitals in Sweden in 2017. CONFLICT OF INTEREST STATEMENT
The discrepancies detected between FLC methods have been The authors declare no conflict of interest.
described earlier [20]. Studies have demonstrated that all available
methods display a high degree of correlation at the lower normal DATA AVAILABILITY STATEMENT
and close to normal ranges and a significant degree of discrepancy The data that supports the findings of this study are available in the
at higher concentration levels [21], highlighting the issue of non- supplementary material of this article. Additional data can be made
interchangeability between the methods, and at the same time pushing available upon request.
for further research. There are many possible reasons for the dis-
crepancy, instrument- and calibrator-associated [22], assay-related ETHICS STATEMENT
[20], difference in reference intervals, or analytical difficulties con- The study was approved by the Swedish Ethical Review Authority (Dnr
nected to the presence of renal failure [23]. Renal insufficiency due 2022–05485–01).
to myeloma may affect the levels of FLC but not the κ/λ-ratio [23].
Also, none of the relevant 31 cases had end-stage kidney disease [19]. PATIENT CONSENT STATEMENT
We have no indications of specific assay batch problems, since the The authors have confirmed patient consent statement
discrepancy when present occurred repeatedly during the observa- is not needed for this submission
tion period in a fraction of the patients, and not across the whole
CLINICAL TRIAL REGISTRATION
study population. Furthermore, there was no sign of or reason to sus-
The authors have confirmed clinical trial registration is not needed for
pect machine-related issues since both assays were run on the same
this submission.
nephelometer. Laboratory diligence along with parallel testing with
both serum electrophoresis and FLC analysis, enabled the discovery
of discrepant FLC measurement in 2020, proving on the one hand the ORCID
importance of serum electrophoresis, and on the other hand, revealing Ljupco Veskovski https://orcid.org/0009-0003-0334-5282
the insufficiency of FLC methods. Although our findings demonstrate Ulf-Henrik Mellqvist https://orcid.org/0009-0004-0456-2657
an advantage for one of the FLC methods, only a validating study at
another center could potentially support our results. REFERENCES
To the best of our knowledge, this is the first study to describe 1. Greipp PR, San Miguel JF, Durie BGM, Crowley JJ, Barlogie B, Bladé
clinical consequences for patients due to the reported issues with J, et al. International staging system for multiple myeloma. J Clin
FLC analysis. Even if the study has a limited number of patients, Oncol. 2005;23(15):3412–20. https://doi.org/10.1200/JCO.2005.04
.242
we can show clinical, relevant discrepancies in a substantial num-
2. Palumbo A, Avet-Loiseau H, Oliva S, Lokhorst HM, Goldschmidt H,
ber of cases. The intention of the authors is to point toward this Rosinol L, et al. Revised international staging system for multiple
issue, and we recommend constant vigilance and close collaboration myeloma: a report from International Myeloma Working Group. J Clin
between laboratory and clinical physicians, particularly for cases of Oncol. 2015;33(26):2863–69. https://doi.org/10.1200/JCO.2015.61.
2267
malignant plasma cell disorder relying primarily on FLC analysis for
3. Rajkumar SV. Multiple myeloma: 2022 update on diagnosis, risk strat-
diagnosis and monitoring, regardless of the choice of method for FLC ification, and management. Am J Hematol. 2022;97(8):1086–107.
analysis. https://doi.org/10.1002/ajh.26590
4. Salomon-Perzyński A, Jamroziak K, Głodkowska-Mrówka E. Clonal
evolution of multiple myeloma—clinical and diagnostic implica-
AUTHOR CONTRIBUTIONS
tions. Diagnostics. 2021;11(9):1534. https://doi.org/10.3390/
Ljupco Veskovski, Ingvar Jakobsson, Ulf-Henrik Mellqvist, and Per- diagnostics11091534
Ola Andersson conceived and designed the study concept. All authors 5. Rajkumar SV, Dimopoulos MA, Palumbo A, Blade J, Merlini G, Mateos
acquired, analyzed, or interpreted the data. Ljupco Veskovski drafted MV, et al. International Myeloma Working Group updated criteria for
the diagnosis of multiple myeloma. Lancet Oncol. 2014;15:e538–48.
the manuscript and all authors critically revised and finally approved
https://doi.org/10.1016/S1470-2045(14)70442-5
the submitted version.
6. Durie BGM, Harousseau JL, Miguel JS, Bladé J, Barlogie B, Anderson
K, et al. International uniform response criteria for multiple
ACKNOWLEDGMENTS myeloma. Leukemia. 2006;20(9):1467–73. https://doi.org/10.1038/
Christina Suhonen and Annika Falk at the Laboratory for Clinical sj.leu.2404284
7. Bradwell AR, Carr-Smith HD, Mead GP, Tang LX, Showell PJ, Drayson
Chemistry SÄS Borås were essential for identifying the discrepant
MT, et al. Highly sensitive, automated immunoassay for immunoglobu-
index cases that formed the basis of this study. Karin Appelgren lin free light chains in serum and urine. Clin Chem. 2001;47(4):673–80.
was involved in the identification of significant discrepant cases at https://doi.org/10.1093/clinchem/47.4.673
 26886146, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jha2.886 by Bangladesh Hinari NPL, Wiley Online Library on [19/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
VESKOVSKI ET AL . 461

8. Mead GP, Carr-Smith HD, Drayson MT, Morgan GJ, Child JA, serum free light chain analysis. Clin Chem Lab Med. 2019;58:85–94.
Bradwell AR. Serum free light chains for monitoring multiple myeloma. https://doi.org/10.1515/cclm-2019-0533
Br J Haematol. 2004;126(3):348–54. https://doi.org/10.1111/j.1365- 18. Bosmann M, Kössler J, Stolz H, Walter U, Knop S, Steigerwald U.
2141.2004.05045.x Detection of serum free light chains: the problem with antigen excess.
9. Dimopoulos M, Kyle R, Fermand JP, Rajkumar SV, San Miguel J, Clin Chem Lab Med. 2010;48(10):1419–22. https://doi.org/10.1515/
Chanan-Khan A, et al. Consensus recommendations for standard CCLM.2010.283
investigative workup: report of the International Myeloma Workshop 19. Kennard A, Hawley C, Tate J, Klingberg S, Pretorius C, Hutchinson
Consensus Panel 3. Blood. 2011;117(18):4701–5. https://doi.org/10. C, et al. Comparison of Freelite™ and N Latex serum free light chain
1182/blood-2010-10-299529 assay in subjects with end stage kidney disease on haemodialysis. Clin
10. Velthuis HT, Knop I, Stam P, Van den Broek M, Bos HK, Hol S, Chem Lab Med. 2016;54(6):1045–52. https://doi.org/10.1515/cclm-
et al. N Latex FLC – new monoclonal high-performance assays for 2015-0799
the determination of free light chain kappa and lambda. Clin Chem 20. Caponi L, Romiti N, Koni E, Fiore AD, Paolicchi A, Franzini M. Inter-
Lab Med. 2011;49(8):1323–32. https://doi.org/10.1515/CCLM.2011 assay variability in automated serum free light chain assays and their
.624 use in the clinical laboratory. Crit Rev Clin Lab Sci. 2020;57(2):73–85.
11. Campbell JP, Heaney JLJ, Shemar M, Baldwin D, Griffin AE, Oldridge E, https://doi.org/10.1080/10408363.2019.1670133
et al. Development of a rapid and quantitative lateral flow assay for the 21. Fleming CKA, Swarttouw T, de Kat Angelino CM, Jacobs JFM,
simultaneous measurement of serum kappa and lambda immunoglob- Russcher H. Method comparison of four clinically available assays for
ulin free light chains (FLC): Inception of a new near-patient FLC serum free light chain analysis. Clin Chem Lab Med. 2019;58:85–94.
screening tool. Clin Chem Lab Med. 2017;55(3):424–34. https://doi. https://doi.org/10.1515/cclm-2019-0533
org/10.1515/cclm-2016-0194 22. Caponi L, Koni E, Romiti N, Paolicchi A, Franzini M. Different
12. Jacobs JFM, de Kat Angelino CM, Brouwers HMLM, Croockewit SA, immunoreactivity of monomers and dimers makes automated free
Joosten I, Van der Molen RG. Evaluation of a new free light chain light chains assays not equivalent. Clin Chem Lab Med. 2018;57:221–
ELISA assay: bringing coherence with electrophoretic methods. Clin 29. https://doi.org/10.1515/cclm-2018-0412
Chem Lab Med. 2018;56(2):312–22. https://doi.org/10.1515/cclm- 23. Moreau C, Autier B, Cavey T, Rouger E, Norwood J, Bendavid C,
2017-0339 Escoffre M, et al. Evaluation of the Impact of Renal Failure on Cor-
13. Muluhngwi P, Sharp CN, Pozzi N, Elin RJ, Jortani SA. Verification of relation and Concordance Between 2 Free Light Chain Assays. Clin
Newly FDA-Approved Kappa and Lambda Free Light Chain Assays on Lymphoma Myeloma Leuk. 2016;16(12):693–704. https://doi.org/10.
a Previously Untested Platform. J Appl Lab Med. 2019;4(3):323–30. 1016/j.clml.2016.08.012
https://doi.org/10.1373/jalm.2019.029215
14. Carr-Smith HD, Jenner EL, Evans JAR, Harding SJ. Analytical issues
of serum free light chain assays and the relative performance of SUPPORTING INFORMATION
polyclonal and monoclonal based reagents. Clin Chem Lab Med.
Additional supporting information can be found online in the Support-
2016;54(6):997–1003. https://doi.org/10.1515/cclm-2015-1068
15. Van Hoovels L, Vercammen M, Nevejan L, Cornette M, Briers PJ,
ing Information section at the end of this article.
Deeren D, et al. Serum free light chain analysis: persisting limitations
with new kids on the block. Clin Chem Lab Med. 2022;60(9):1440–48.
https://doi.org/10.1515/cclm-2022-0347
16. Messiaen A-S, De Sloovere MMW, Claus P-E, Vercammen M, Van How to cite this article: Veskovski L, Jakobsson I, Andersson
Hoovels L, Heylen O, et al. Performance Evaluation of Serum Free Light P-O, Gustafsson T, Sedigh A, Knut-Bojanowska D, et al. The
Chain Analysis: Nephelometry vs Turbidimetry, Monoclonal vs Poly-
choice of serum-free light chain analysis method could
clonal Reagents. Am J Clin Pathol. 2017;147(6):611–22. https://doi.
potentially have clinical consequences for myeloma patients.
org/10.1093/ajcp/aqx037
17. Fleming CKA, Swarttouw T, de Kat Angelino CM, Jacobs JFM, eJHaem. 2024;5:455–61. https://doi.org/10.1002/jha2.886
Russcher H. Method comparison of four clinically available assays for