Annals of http://acb.sagepub.

com/
Clinical Biochemistry

A multicentre study comparing two methods for serum free light chain analysis
RJ Lock, R Saleem, EG Roberts, MJ Wallage, TJ Pesce, A Rowbottom, SJ Cooper, ED McEvoy, JL Taylor and S Basu
Ann Clin Biochem 2013 50: 255
DOI: 10.1177/0004563212473447

The online version of this article can be found at:

http://acb.sagepub.com/content/50/3/255

Published by:

http://www.sagepublications.com

On behalf of:
Association for Clinical Biochemistry and Laboratory Medicine

Netherlands Society for Clinical Chemistry and Laboratory

Japan Society of Clinical Chemistry

Additional services and information for Annals of Clinical Biochemistry can be found at:

Email Alerts: http://acb.sagepub.com/cgi/alerts

Subscriptions: http://acb.sagepub.com/subscriptions

Reprints: http://www.sagepub.com/journalsReprints.nav

Permissions: http://www.sagepub.com/journalsPermissions.nav

>> Version of Record - May 1, 2013

What is This?

Downloaded from acb.sagepub.com at Faculte de Medecine de Monastir on August 25, 2014

Original Article

A multicentre study comparing two methods for serum free

light chain analysis

RJ Lock1, R Saleem2, EG Roberts1, MJ Wallage1, TJ Pesce3, A Rowbottom2, SJ Cooper4,

ED McEvoy4, JL Taylor4 and S Basu4
1
Immunology and Immunogenetics, Pathology Sciences, North Bristol NHS Trust, Southmead Hospital, Westbury-on-Trym, Bristol BS10
5NB; 2Lancashire & Lakeland Regional Immunology Service, Royal Preston Hospital, Sharoe Green Lane, Preston PR2 9HT; 3Blood
Science Specials Laboratory, Northampton General Hospital, Cliftonville, Northampton, Northamptonshire NN1 5BD; 4Immunology and
Haematology Departments, Royal Wolverhampton NHS Hospitals Trust, New Cross Hospital, Wednesfield Road, Wolverhampton WV10
0QP, UK
Corresponding author: Dr RJ Lock. Email: Robert.lock@nbt.nhs.uk

Abstract
Background: Serum free light chain analysis is now well established in the investigation of monoclonal gammopathies. In the
UK there has, until recently, been a single supplier of kits for such analysis. Recently, a second method using monoclonal
antisera was introduced. We have compared the performance of these two kits in four routine laboratories.
Method: Samples submitted for routine analysis (327 samples, 258 [79%] from patients with B-cell lymphoproliferative
disease) for serum free light chains were tested by both technologies (Freelite, Binding Site and N Latex FLC, Siemens),
according to the manufacturers’ instructions.
Results: Qualitative data were available by both methods on 313 samples for serum free kappa chains and 324 samples for
lambda free light chains. We found poor correspondence of 81% for kappa and 74% for lambda. Five percent of samples were
significantly discordant in these assays.
Conclusions: These assays perform very differently in clinical practice. They cannot be used interchangeably, especially if
monitoring patient responses to therapy.

Ann Clin Biochem 2013; 50: 255–261. DOI: 10.1177/0004563212473447

Introduction unlikely that all monoclonal proteins would behave identi-

The use of serum free light chain assays in the investigation
of patients suspected of monoclonal gammopathy is now
well established and use of the assay is advocated in inter-
national guidelines.1 Until recently only one assay was com-
mercially available in the UK (FreeliteTM , Binding Site,
Birmingham, UK), adapted for use on a variety of nephelo-
metric and turbidimetric platforms. A second commercial
assay has now come to market (N Latex FLC, Siemens,
Camberley, Surrey, UK). Limited data are available regard-
ing the performance of this assay.2,3 In view of the described
problems with the inter-laboratory reproducibility of the
FreeliteTM assay,4 it is important to establish whether the
new assay offers any advantages.
It was also important to establish whether there were sig-
nificant differences between the two assays in terms of their
ability to identify and quantify monoclonal immunoglobu-
lin light chains. Crucially, there is a fundamental difference
in the formulation of the two assays. The Binding Site assay
is based on the use of polyclonal antisera whereas the
Siemens assay uses monoclonal antisera. We considered it
cally in the two assays.
We present here evaluation data from four laboratories;
two using the Siemens BN II nephelometer platform and
two using the Siemens ProSpec nephelometer.
Methods
Unselected routine samples submitted for serum light
chain analysis were analysed in parallel for both the Binding
Site (FreeliteTM Human Kappa and Lambda Free Kits, The
Binding Site) and Siemens (N latex FLC Kappa and
Lambda, Siemens Healthcare Diagnostics Ltd) assays, accord-
ing to the manufacturers’ instructions (hereafter referred to as
Freelite and N latex FLC). Serum dilutions, both initial and
subsequent (where results were outside of the reportable
range) were as recommended by the manufacturers.
The majority of serum samples were stored at 48C for no
more than two weeks before analysis. For operational
reasons (i.e. batch analysis) aliquots of some serum samples
were stored at 2208C prior to analysis, where the two-week

Annals of Clinical Biochemistry 2013; 50: 255–261

Downloaded from acb.sagepub.com at Faculte de Medecine de Monastir on August 25, 2014

256 Annals of Clinical Biochemistry Volume 50 May 2013
................................................................................................................................................

Table 1 Samples available for analysis by site

Number available for
Number Number of samples from patients with
regression analysis
Site Platform of samples clonal B cell disorders
Kappa Lambda
North Bristol BNII 124 101 111 108 (88 myeloma, 9 amyloid, 3 MGUS, 8 other)
Northampton ProSpec 51 42 43 46 (34 myeloma, 3 amyloid, 7 MGUS, 2 other)
New Cross BNII 60 60 60 54 (31 myeloma, 17 MGUS, 6 other)
Preston ProSpec 111 110 110 50 (36 myeloma, 3 amyloid, 7 MGUS, 4 other)
Total 327 313 324 258

Note that some samples were not included in the regression or bias analysis. This was either through restrictions of sample volume available or where indeterminate
results arose (either less than detection limit or extremely high). MGUS, monoclonal gammopathy of undetermined significance

Table 2 Reproducibility data based on 10 replicates except N latex FLC between-batch where only three replicates were possible
Kappa serum free light chains Lambda serum free light chains
Coefficient of Coefficient of
Mean (SD) (mg/L) variation (%) Mean (SD) (mg/L) variation (%)
Within-batch
Freelite 11.17 (1.09) 9.76 9.06 (0.69) 7.64
N Latex FLC 13.29 (0.29) 2.17 13.39 (0.25) 1.84

Between-batch
Freelite 16.96 (1.21) 7.12 27.42 (1.92) 7.02
Freelite 30.37 (1.75) 5.76 54.36 (3.49) 6.4
N Latex FLC 13.93 (0.71) 5.09 12.20 (0.44) 3.57
N Latex FLC 36.57 (2.34) 6.39 35.50 (2.00) 5.63

period might be exceeded. Frozen samples were thawed once Results

and thoroughly mixed prior to analysis. Two centres used a
Siemens BN II nephelometer and two a Siemens ProSpec
nephelometer (Table 1). Samples with data for either kappa
or lambda light chains by both assays were included in the
analysis. A summary of the samples included is detailed in
Table 1.
Reproducibility assessment (within-batch and between-
batch coefficients of variation) was based on 10 replicates,
except for the N latex FLC between-batch where only
three replicates were possible. Serum samples were selected
to fall either in the reference range or to be moderately elev-
ated. This ensured both assays were assessed similarly and
not at the extremes of the analytical sensitivity for either.
Correlation was by Deming’s regression analysis.
The Clinical and Laboratory Standards Institute (CLSI)
(EP09-A2-IR) guidelines suggest that a correlation coefficient
r 2 . 0.95 indicates good correlation between two assays;5
therefore, we also performed standard linear regression
to determine this parameter. Only definitive data were
included in this part of the analysis (e.g. data recorded as
less than the detection limit were excluded). Bias was deter-
mined by the Altman – Bland method. Owing to the large
dynamic range of the data, log transformed data were
used for correlation and bias calculations. Qualitative
results (less than reference range, within reference range,
greater than reference range) were assessed for correspon-
dence. The kappa statistic was calculated to quantify corre-
spondence. The Analyse-IT for Excel statistics package was
used (version 2.21, Analyse-It Software Ltd, Leeds, UK).
Table 1 indicates the number of samples entered into the
study by each site. Note that the majority (79%) are from
patients with B-cell clonal disorders. This reflects the
requesting patterns as seen in each centre, e.g. in North
Bristol the majority are either new diagnoses or are monitor-
ing samples for myeloma trial patients.
Reproducibility of both assays was acceptable (Table 2).
Figures 1a, b, 2a and b indicate that while the assays are
correlated, the assays are not identical and there is a sig-
nificant number of outliers. Details of the individual data
for outliers are given in Table 3. Standard linear regression
(not shown) gave r 2 of 0.86 for kappa and 0.71 for
lambda, both well below the requirements of the CLSI.5
Qualitative data were available by both methods on
317 samples for serum free kappa chains and 335 samples
for lambda free light chains. Data are shown in Table 4.
Categories were assigned according to the manufacturers’
designated reference ranges. For the Binding Site we used
Katzmann’s data6 (kappa 3.3 –19.4 mg/L, lambda 5.7–
26.3 mg/L) and for Siemens the manufacturer’s stated
ranges (kappa 6.7– 22.4 mg/L, lambda 8.3 –27.0 mg/L).
Reference ranges were verified by probability plotting as
previously described (data not shown).7 Again the poor cor-
relation is reflected in relatively poor correspondence of 81%
for kappa and 74% for lambda. The kappa statistic for
lambda FLC was 0.58 (interpreted as ‘fair’) and for kappa
FLC 0.62 (interpreted as ‘moderate’).
Within-batch and between-batch variations are given in
Table 2 (North Bristol data).

Downloaded from acb.sagepub.com at Faculte de Medecine de Monastir on August 25, 2014

 Lock et al. Serum free light chain methods comparison 257
................................................................................................................................................

(a) Scatter plot with Deming fit

5

4
Log kappa (N Latex FLC) (log mg/L)

3 Identity

Deming fit
(0.33 + 0.81x)
2
95% CI bands

0
0 1 2 3 4 5
Log kappa (Freelite) (log mg/L)

Scatter plot with Deming fit

(b) 4
Log lambda (N Latex FLC) (log mg/L)

Identity

2 Deming fit
(0.42 + 0.78x)

95% CI bands

0
0 1 2 3 4
Log lambda (Freelite) log mg/L

Figure 1 Panels a and b show the Deming regression curves for kappa and lambda serum free light chains respectively. For kappa y ¼ 0.33 þ 0.81x and for
lambda y ¼ 0.42 þ 0.78x

Discussion into the International Working Group for Multiple

There are several studies published showing the usefulness Myeloma guidance.1 All these studies are based on the
of the analysis of serum free light chains in lymphoprolifera- use of a single analysis system (Freelite). With the introduc-
tive disease (e.g. refs.8,9). The analysis is now incorporated tion of a second system to the market (N Latex FLC), it is

Downloaded from acb.sagepub.com at Faculte de Medecine de Monastir on August 25, 2014

258 Annals of Clinical Biochemistry Volume 50 May 2013
................................................................................................................................................

(a) Difference plot

1.5
Identity

1
Difference (log kappa [Siemens] – log kappa [BS])

Bias (0.0189706763)

0.5
95% limits of agreement
(–0.6101632518 to 0.6481046045)

–0.5

–1

–1.5

–2

–2.5
–1 0 1 2 3 4
Mean of All

(b) Difference plot

1.5
Identity
Difference (log lambda [N Latex FLC] – log lambda

1
Bias (0.1086558587)

0.5
95% limits of agreement
(–0.6358375873 to 0.8531493048)

0
[Freelite])

–0.5

–1

–1.5

–2

–2.5
–0.5 0.5 1.5 2.5 3.5
Mean of All

Figure 2 Panels a and b show the Altman –Bland plots for kappa and lambda serum free light chains, respectively. Those samples which were most discrepant
(samples with high mean [.log 1.5; .31.6 mg/L] and outside of the 95% limits of agreement) are detailed in Table 3

Downloaded from acb.sagepub.com at Faculte de Medecine de Monastir on August 25, 2014

 Table 3 Samples with high serum free light chains identified as outliers by Altman – Bland plots
Kappa Lambda
Kappa (N Latex Lambda (N Latex N Latex Para- Serum
(Freelite) FLC) (Freelite) FLC) Freelite FLC Age protein Paraprotein creatinine eGFR
Source Patient (mg/L) (mg/L) (mg/L) (mg/L) ratio ratio (y) Gender (g/L) type Diagnosis (mmol/L) (mL/min)
New Cross 1 4.66 19.5 6070 59.7 ,0.01 0.33 74 M ,2 Lambda Myeloma 86 79
New Cross 2 2611 24.2 59.3 48.8 44.03 0.50 77 M 3 IgA-kappa MGUS 49 .90
Northampton 3 3570 746 11.8 23.5 302.54 31.74 77 F TS IgG-kappa Myeloma 150 35
Northampton 4 33.3 ins 8870 234 ,0.01 54 F beta region IgA-lambda Myeloma 486 9
21.7 13.4 3600 120 ,0.01 0.11 beta region Myeloma 490 9
28.1 ins 2940 167 0.01 beta region Myeloma 433 10
Northampton 5 24.3 ins 348.0 50.7 0.07 80 F TS Lambda FLC myeloma 153 41
Northampton 6 8.1 ins .14000 2300 ,0.01 61 F TS Lambda FLC myeloma 198 24
Northampton 7 1.2 ins 832 35.9 ,0.01 83 M 28 IgG-lambda Myeloma 60 88
North Bristol 8 0.3 6.99 1430 31.9 ,0.01 0.22 54 F 13 IgA-lambda Myeloma 73 72
North Bristol 9 ,6.3 5.27 932 118 ,0.01 0.04 76 M TS Lambda Treated 76 86
myeloma?
North Bristol 10 7.9 12.8 422 40.2 0.02 0.32 71 F TS Lambda FLC myeloma on 123 37
treatment
8.4 14.9 248 40.2 0.03 0.37 71 TS FLC myeloma on 133 34
treatment
North Bristol 11 657 85.4 7.2 4.7 91.25 18.29 69 F TS Kappa FLC myeloma 117 40
North Bristol 12 6.3 7.96 267 42.7 0.02 0.19 57 M 1 IgG-lambda Myeloma on 77 90
treatment

Downloaded from acb.sagepub.com at Faculte de Medecine de Monastir on August 25, 2014

Preston 13 6.83 6.89 436 41.1 0.02 0.17 80 F 1 (whole IgG-lambda þ free Treated myeloma 66 75
molecule) lambda
Preston 14 169 30.6 2.03 4.79 83.25 6.39 60 M 4 IgG-kappa Treated myeloma 41 .90

Ins, insufficient sample for analysis; eGFR, estimated glomerular filtration rate; beta region, unable to quantify owing to interference from other beta region proteins; TS, too small to quantify. Note three samples were
available for patient 4 and two samples for patient 10
Lock et al. Serum free light chain methods comparison
................................................................................................................................................
259
260 Annals of Clinical Biochemistry Volume 50 May 2013
................................................................................................................................................
Table 4 Correspondence data for kappa and lambda free light chain
analysis
N Latex FLC
Low Normal
(a) Lambda FLC
Freelite Low 37 15
Normal 19 85
High 0 2
Correspondence 74%
(b) Kappa FLC
Freelite Low 4 11
Normal 9 79
High 0 23
Correspondence 81%
The categories Low, Normal and High are defined by the manufacturers’
reference ranges. Kappa statistic for lambda FLC was 0.58 (interpreted as
‘fair’) and for kappa FLC 0.62 (interpreted as ‘moderate’)
essential to determine whether this system, based on the use
of monoclonal, rather than polyclonal, antisera is equally
able to detect and quantify monoclonal free light chains in
serum. Thus far, we know of only two papers comparing
the use of these two assays on routine clinical samples.2,3
Our major concern was that there was a theoretical possi-
bility that, with some monoclonal free light chains, the use
of monoclonal antisera with restricted epitope specificity
might not produce sufficiently large immune complexes to
allow detection by light scattering in the nephelometric
systems used for analysis. In contrast to Hoedemakers
et al.,2 our study included a large percentage of samples
from patients with monoclonal gammopathies. The percen-
tage of samples from monoclonal patients is unclear in the
study of Pretorius et al.,3 but we note that many potential
cases may have been excluded by their selection criteria
(samples ,50 mg/L on Freelite were excluded). The three
studies are thus not directly comparable. Nevertheless,
some themes emerge.
Correspondence between the two assays was quite low
(kappa 81%, lambda 74%), certainly lower than in the
selected data of Pretorius et al. 3 as indicated by the lower
kappa statistic, but in many samples this might be expected
to have little clinical impact (e.g. normal by one assay and
low by the other). Note however that this only applies
if the sensitivities of the assays for detecting disease state
serum samples are the same, for which there are as yet no
conclusive data. There were, however, some worrying
results, as shown in Table 3. In some the major concern
is that patients transferring between centres could not be
monitored effectively if different methods were used in
the different centres.
It should be noted that it is unclear as to which result is
the more accurate in many of these patients. There are no
international standards for the assays. There are well docu-
mented problems of non-linearity and antigen excess detec-
tion. Providing that the assay used is consistent and able
to detect all clonal free light chains then the numeric
differences are academic. However, many of the recommend-
ations in the literature are based on experience with the
Freelite assay and in the light of our data may not be
directly transferable to the Siemens assay (for example,
Downloaded from acb.sagepub.com at Faculte de Medecine de Monastir on August 25, 2014
the absolute values for FLC that are recommended as risk
stratification markers in all B-cell malignancies). This
would include the value of .500 mg/L used to identify
High patients who potentially have acute renal failure secondary
to multiple myeloma.10 In addition, international guidelines
0 recommend a concentration of .100 mg/L to accurately
52 monitor patients, this is particularly useful for AL
125 amyloid and oligosecretory/non-secretory multiple
myeloma patients. This could have considerable impact on
the clinical interpretation of results found to be disparate
0 between the two assays.
18 We found 17 samples (5.2% of all samples) from 14
173
patients where the clonal light chains were poorly detected
by the monoclonal antisera (Table 3), in one case with a
highly misleading normal ratio (Table 3, patient 10) or,
more worryingly, not detected at all (Table 3, patient 2).
More significant outliers were found for lambda than
kappa. It is interesting to note that Pretorius et al. 3 described
three patients with lymphoproliferative disease in whom
normal FLC ratios were seen in the N Latex FLC assay
(1 AL amyloid patient, 1 lambda light chain myeloma
patient and 1 chronic myelomoncytic leukaemia patient
with a positive kappa Bence Jones protein). All three were
detected as having an abnormal FLC ratio by Freelite.
Similarly, Hoedemakers et al. 2 noted one patient with
lambda light chain myeloma with a normal ratio by N
Latex FLC that was abnormal by Freelite.2 So, although
the numbers are small, and therefore not conclusive, both
these and our own study support our contention that
some clones might be missed by the more limited epitope
specificity expected of monoclonal antisera.
While in general terms the two assays perform similarly
in the detection of serum free light chains, we believe that
there are sufficient data here to suggest the assays are not
equivalent. We would stress that this last point is very
important. As there is poor correlation, the current inter-
national guidelines cannot be cross applied to the Siemens
assay. More data are required to establish equivalent FLC
concentrations, as quantified by the N Latex FLC assay,
for the stratification and management of patients with
B-cell lymphoproliferative disorders.
DECLARATIONS
Competing interests: None.
Funding: None.
Ethical approval: Not applicable.
Guarantor: RJL.
Contributorship: EGR, MJW, TJP, RS, EM and JT performed
the analyses. EGR, MJW, SC, RS, TJP, EM and JT helped
locate, source and compile data. RJL analysed the data.
All authors contributed to the planning, writing and
approval of final draft.
Acknowledgements: The authors would like to thank both
the Binding Site and Siemens for the support provided to
allow this study to take place. Our thanks go to Francis
Smith, Gloucester Royal Hospital for supplying some of
the data for Table 3 and to Jayne Parkes, Haematology,
New Cross for invaluable liaison and sourcing some of

................................................................................................................................................
the data. N Latex FLC kits were kindly donated free of
charge by Siemens.
REFERENCES
1 Dispenzieri A, Kyle R, Merlini G, et al. International Myeloma
Working Group guidelines for serum-free light chain analysis in multiple
myeloma and related disorders. Leukemia 2009;23:215 –24
2 Hoedemakers RM, Pruijt JF, Hol S, et al. Clinical comparison
of new monoclonal antibody-based nephelometric assays for free
light chain kappa and lambda to polyclonal antibody-based
assays and immunofixation electrophoresis. Clin Chem Lab Med
2011;50:489 –95
3 Pretorius CJ, Klingberg S, Tate J, Wilgen U, Ungerer JP. Evaluation of the
N Latex FLC free light chain assay on the Siemens BN analyser:
precision, agreement, linearity and variation between reagent lots. Ann
Clin Biochem 2012;49:450 – 5
4 UK NEQAS. Immunology, Immunochemistry and Allergy Annual
Report 2010. See http://www.kpmd.co.uk/immqas/downloads/
Annual_Report_2010_V1.1_FINAL.pdf (last checked 26 July 2012)
Downloaded from acb.sagepub.com at Faculte de Medecine de Monastir on August 25, 2014
Lock et al. Serum free light chain methods comparison 261
5 CLSI. Method comparison and bias estimation using patient samples;
Approved guideline – second edition (interim revision). CLSI document
EP09-A2-IR. Wayne, PA, USA: Clinical Laboratory Standards Institute,
2010
6 Katzmann JA, Clark RJ, Abraham RS, et al. Serum reference intervals and
diagnostic ranges for free k and free l immunoglobulin light chains:
relative sensitivity for detection of monoclonal light chains. Clin Chem
2002;48:1437 – 44
7 Lock RJ, Unsworth DJ. Immunoglobulins and Immunoglobulin
subclasses in the elderly. Ann Clin Biochem 2003;40:143 –8
8 Robson EJD, Taylor J, Beardsmore C, Basu S, Mead G, Lovatt T. Utility of
serum free light chain analysis when screening for lymphoproliferative
disorders. Lab Med 2009;40:325 –9
9 Palladini GDA, Gertz MA, Kumar S, et al. Validation of the criteria of
response to treatment in AL amyloidosis. Blood 2010;11:1364a
10 Leung N, Gertz MA, Zeldenrust SR, et al. Improvement of cast
nephropathy with plasma exchange depends on the diagnosis and on
reduction of serum free light chains. Kidney Int 2008;73:1282 –8
(Accepted 17 November 2012)