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Scandinavian Journal of Clinical and Laboratory
Investigation
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Automated alarm to detect antigen excess in
serum free immunoglobulin light chain kappa and
lambda assays
Petter Urdal, Erik K. Amundsen, Karin Toska & Olav Klingenberg
To cite this article: Petter Urdal, Erik K. Amundsen, Karin Toska & Olav Klingenberg (2014)
Automated alarm to detect antigen excess in serum free immunoglobulin light chain kappa and
lambda assays, Scandinavian Journal of Clinical and Laboratory Investigation, 74:7, 575-581, DOI:
10.3109/00365513.2014.915426
To link to this article: http://dx.doi.org/10.3109/00365513.2014.915426
Published online: 09 Jul 2014.
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Date: 28 August 2017, At: 03:24
Scandinavian Journal of Clinical & Laboratory Investigation, 2014; 74: 575–581
ORIGINAL ARTICLE
Automated alarm to detect antigen excess in serum free
immunoglobulin light chain kappa and lambda assays
PETTER URDAL1,3, ERIK K. AMUNDSEN1, KARIN TOSKA1 & OLAV KLINGENBERG2
of Medical Biochemistry, Oslo University Hospital, Ullevål and 2Rikshospitalet, Oslo, and 3Institute of
Clinical Medicine, University of Oslo, Oslo, Norway
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1Department
Abstract
Background. Antigen excess causing a falsely low concentration result may occur when measuring serum free immunoglobulin light chains (SFLC). Automated antigen excess detection methods are available only with some analyzers. We
have now developed and verified such a method. Methods. Residuals of sera with known SFLC-κ and -λ concentrations
were analyzed using Binding Site reagents and methods adapted to the Roche Cobas® c.501 analyzer. Results. We analyzed 117 sera for SFLC-κ and -λ and examined how the absorbance increased with time during the 7 minutes of reaction (absorbance reading points 12–70). From this an antigen excess alarm factor (ratio of absorbance increases between
reading points 68–60 and 20–12, multiplied by 100) was defined. Upon our request, Roche added to our two SFLC
assays a program which calculated this antigen excess alarm factor and triggered an alarm when the factor was below a
defined value. We verified this antigen excess alarm function by analyzing serum from 325 persons of whom 143 were
multiple myeloma patients. All samples with a known concentration above 30 mg/L triggered either an antigen excess
alarm, an ‘above test’ alarm or both. Also, all samples above 200 mg/L (SFLC-λ) and 300 mg/L (SFLC-κ) triggered the
antigen excess alarm and all but one triggered the above test alarm. Conclusions. The antigen excess alarm function
presented here detected all known antigen excess samples at no increased time of analysis, a reduced workload and
reduced reagent cost.
Key Words: Multiple myeloma, turbidimetry, nephelometry, antigen excess, free light chains
Introduction
In a serum sent to us for analysis we measured
immunoglobulin free light chains kappa (SFLC-κ)
and lambda (SFLC-λ) to be 14 and 9 mg/L. The
correct result for SFLC-κ was however later shown
to be 1320 mg/L.
Antigen excess occurs when antibody of a defined
limited capacity is mixed with too high amounts of
antigen. Antigen excess may occur with turbidimetric
or nephelometric immunological assays for SFLC-κ
and SFLC-λ [1–6] as well as with other proteins and
different assay types [7–9], causing a false and mostly
low concentration result. To detect antigen excess,
the two main manufacturers of SFLC reagents and
methods – Binding Site and Siemens Diagnostics –
have included antigen excess alarm functions in
their SFLC assays adapted to each of their analyzers
SPAplus and BN ProSpec instruments [10,11].
Binding Site has also included this function in their
assay adapted to Cobas Integra® 800 [10] but not to
other analyzers from Roche Diagnostics or from
other companies.
Laboratories using Binding Site reagents adapted
to other analyzers are instead advised to dilute and
reanalyze the serum when the SFLC result and/or
the SFLC-κ/ SFLC-λ ratio is outside its reference
range, in patients with previous antigen excess or if
other clinical or laboratory findings indicate that the
SFLC result might be falsely low [10]. These rules
are work- and reagent-consuming. In addition, the
rules would not have prevented us from reporting the
wrong SFLC-κ result described above. In the present
study we document an automated alarm function
which detects a high SFLC concentration also in the
Correspondence: Petter Urdal, Department of Medical Biochemistry, Oslo University Hospital, Ullevål, Postboks 4956 Nydalen, 0424 Oslo, Norway.
Fax: ! 47 22118189. E-mail: petterur@online.no
(Received 22 May 2013 ; accepted 25 March 2014 )
ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare
DOI: 10.3109/00365513.2014.915426
576
P. Urdal et al.
presence of antigen excess. This alarm function is
based on Binding Site reagents and a Roche Cobas®
c.501 analyzer, and uses absorbances already obtained
when measuring SFLC-κ or -λ. It is similar but not
identical to the antigen excess alarm functions of
Binding Site, while Siemens Diagnostics uses a prereaction protocol [11].
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Materials and methods
Sera for use in the present study were selected among
those sent to the department of medical biochemistry for analysis of SFLC-κ and SFLC-λ. During two
time periods (December 2011–March 2012 and
March–December 2012), two sets of sera (117 and
325 sera from 117 and 325 individuals) were collected. The first set was used for developing an antigen excess alarm function and the second set for
verifying this alarm function. In principle, all sera
above 200 mg/L in SFLC-κ or -λ were included, but
some sera high in SFLC-κ, SFLC-λ and creatinine
were not included in order to avoid a large dominance of sera from patients with renal disease. Sera
below 200 mg/L in both SFLC-κ and -λ were
included randomly. The sera were either stored at
! 4°C for at most 7 days or stored at " 70°C for at
most 6 months before being further analyzed as
described below.
The sera were collected at Oslo University
Hospital, Ullevål and Rikshospitalet, after measurement of SFLC-κ and SFLC-λ concentrations by use
of Binding Site (‘FreeLite’) reagents and methods,
either adapted to a Roche Cobas® c.501 analyzer
(Ullevål) or to a Siemens BN ProSpec® nephelometer (Rikshospitalet). These methods, used prior to
inclusion, were run strictly as recommended by
Binding Site. They included an initial within-analyzer
dilution of sample and automatic reanalysis at a fivefold lower sample fraction if the result of the first
analysis was above 30 mg/L. If there was an ‘above
test’ alarm reported together with the result of the
automatic reanalysis, manual dilution and analysis of
the diluted serum was performed. Samples in which
either SFLC-κ or -λ were below 5 mg/L were also
subjected to manual dilution and analysis. With one
exception, i.e. SFLC-κ of the patient described in the
Introduction, the SFLC concentrations measured
before inclusion are the ones used in the Figures and
Tables of the Results section. The patient mentioned
in the Introduction who was included as a normal
patient, was later found by the antigen excess alarm
function to have a high SFLC-κ concentration, dilution and reanalysis found this to be 1320 mg/L.
After inclusion we analyzed once more the
117 ! 325 samples for SFLC-κ and SFLC-λ concentrations using the same reagents and the same method
adapted to Roche Cobas® c.501. However, manual
dilution and reanalysis, described above, was not performed since this had already been done prior to
inclusion and was not necessary for development and
verification of the antigen excess alarm function. This
alarm function is described in more detail in the
Results section. The project was approved by the local
ethics committee.
Results
Figure 1 shows the increasing absorbance from reading point 10 of eight samples analyzed for SFLC-κ.
In sera with SFLC-κ concentration below 150 mg/L
the absorbance increased almost linearly with time
whereas in the sera well above this concentration the
Figure 1. Absorbance increase with time during analysis for SFLC-κ. The Roche Cobas® c.501 analyzer reads the absorbance 70 times
with intervals of 8 seconds. After adding reagent 1 and sample, the reaction starts when the antibody-containing reagent 2 is added between
reading points 10 and 11. The SFLC-κ concentrations (mg/L) of the eight sera examined are given on the right hand side. The 1320 mg/L
sample is the one described in the Introduction.
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Antigen excess alarm in SFLC assays
early absorbance increase, between reading points 12
and 20, was higher and the late increase, between
points 60 and 68, more moderate.
In 117 sera we examined more closely how the
early and late absorbance changes were related to
the SFLC-κ and SFLC-λ concentration. The early
absorbance change was continuously increasing
with increasing SFLC-κ concentration (Figure 2a).
The late absorbance change increased with increasing SFLC-κ concentrations up to approximately
100 mg/L but decreased for increasing SFLC-κ
above 200 mg/L (Figure 2b).
From all 117 SFLC-κ and -λ absorbance curves
we calculated an antigen excess alarm factor (late
absorbance change divided by early absorbance
change multiplied by 100). The SFLC-κ alarm factor
value and its relationship to the SFLC-κ concentration is shown in Figure 2c. A cut-off at 75 for
the factor would discriminate most samples below
100 mg/L from all samples above 200 mg/L. Similarly, with SFLC-λ an antigen excess alarm factor of
50 would discriminate most samples below 100 mg/L
from all samples above 250 mg/L (Figure 3).
Upon our request, Roche added to the SFLC-κ
and -λ assays a program which calculated the antigen
excess alarm factor defined above and triggered an
alarm when the factor was below 75 (SFLC-κ) or 50
(SFLC-λ). This alarm initiated an automatic rerun
at a five-fold lower sample fraction. The alarm factor
was printed as part of the result report.
577
The combined use of the above test alarm and
the antigen excess alarm function was examined by
analyzing the 325 samples of the second sample set,
of which 143 were from multiple myeloma patients,
on 27 separate days during 8 months. The sample
described in the Introduction, considered at inclusion
to have a SFLC-κ concentration of 14 mg/L, triggered the antigen excess alarm but not the above test
alarm. Of the remaining 324 samples, none below
and all above 30 mg/L in SFLC triggered the above
test alarm. As for the antigen excess alarm, of
the 324 samples none with concentrations in the
range 15–100 mg/L triggered this alarm, whereas a
small majority of the samples in the range 100–300
mg/L (SFLC-κ) and 100–200 mg/L (SFLC-λ) did
(Figures 4 and 5). Above 300 mg/L (SFLC-κ) and
200 mg/L (SFLC-λ) all but two samples, one analyzed for SFLC-κ and the other for SFLC-λ, triggered the antigen excess alarm (Figures 4 and 5). If
the antigen excess alarm function is modified as suggested below, these two samples would have also
triggered an antigen excess alarm.
A ‘false’ antigen excess alarm appeared with several samples below 11 mg/L, more frequently with
the lambda (n # 12, Figure 5) than with the kappa
assay (n # 2, Figure 4). The results of Figures 2a
and 3a suggests that these alarms could all have
been avoided if the antigen excess function had
included a threshold value for the early absorbance
change, the antigen excess factor being calculated
Figure 2. Early absorbance change (a), late absorbance change (b) and alarm factor value (c): Their relationship to SFLC-κ concentration.
For 117 sera analyzed for SFLC-κ the early and late absorbance changes were estimated as indicated in Figure 1. The alarm factor value
was defined as ratio between late and early absorbance change multiplied by 100.
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578
P. Urdal et al.
Figure 3. Early absorbance change (a), late absorbance change (b) and alarm factor value (c): Their relationship to SFLC-λ concentration.
For 117 sera analyzed for SFLC-λ the early and late absorbance changes and the alarm factor value were estimated as described in the
Figure 2 legend.
only for sera above this threshold. By modifying the
alarm function by (i) introducing early absorbance
change threshold values of 0.030 for κ (cfr Figure
2a) and 0.060 for λ (cfr Figure 3a), and (ii) increasing the antigen excess alarm cut-off values to 100
(κ) and to 60 (λ) there would be no false alarms
(Figures 4 and 5).
The assay ranges of SFLC-κ and SFLC-λ as
used with the Roche Cobas® c.501 analyzer were
approximately 5–60 mg/L and 5–75 mg/L, but Binding Site recommends rerun for samples above the
upper reference limit, we use above 30 mg/L, to
reduce the risk for undetected antigen excess. With
seven of the 325 samples the first result was reported
as a value below 80 mg/L (Table I, column 4) though
their real SFLC concentration was much higher
(Table I, column 3). These samples with antigen
excess were the ones closest not to be detected if only
Figure 4. Verification of the antigen alarm function: The antigen excess factor of 325 samples analyzed for SFLC-κ. Shown as open or
closed circles dependent upon whether the initial absorbance change was below or above 0.030, respectively.
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Antigen excess alarm in SFLC assays
579
Figure 5. Verification of the antigen alarm function: The antigen excess factor of 325 samples analyzed for SFLC-λ. Shown as open or
closed circles dependent upon whether the initial absorbance change was below or above 0.060, respectively.
an above test alarm was used. The antigen excess
factors calculated with these samples are summarized in Table I, column 5. The five sera with high
SFLC-κ concentrations (samples 1–5 in Table I) all
showed very low antigen excess alarm factor values.
The two high SFLC-λ sera showed moderately low
antigen excess alarm factor values, but still below the
alarm cut-off value of 60. Sample 3 in Table I is
identical to sample 1320 mg/L in Figure 1 and is the
sample described in the Introduction.
On each day of analysis of the verification period
a two-level quality control was run. During this
period three different reagent lots were used both
with the kappa and the lambda assays. This neither
clearly affected the measured concentrations of the
controls nor their antigen excess factor values. The
quality control results for the whole verification
period are summarized in Table II.
The antigen excess factor values of SFLC-λ
obtained in the development and the verification
Table I. Selected* samples with antigen excess.
SFLC
1
2
3
4
5
6
7
κ
κ
κ
κ
κ
λ
λ
Concentration
(mg/L)
Result (mg/L)
reported after the
first automatic
analysis
Antigen
excess factor
53400
3220
1320
14390
725
2200
6070
46.5
65.3
14.0
77.8
69.0
63.3
76.5
13
11
5
3
37
51
36
SFLC, Serum free immunoglobulin light chains.
*Selected among the 325 verification samples as samples above
750 mg/L in SFLC-κ or -λ and where the result of the first
analysis, before any rerun, was reported as below 80 mg/L.
time periods were largely similar. Thus, for samples
with concentrations of SFLC-λ of 20–60 mg/L collected during the development time period the
mean $ standard deviation of the antigen excess
factors (77.9 $ 5.7, n # 41) was close to that of the
verification period (80.2 $ 6.6, n # 75). For samples
with SFLC-κ concentrations of 20–60 mg/L the
antigen excess factor values of the development
time period (185.1 $ 23.1, n # 88) differed only
moderately from those of the verification period
(195.0 $ 23.7, n # 83).
Discussion
Our antigen excess alarm is, in principle, triggered
by any serum high in SFLC irrespective of whether
antigen excess is also present. It thus supplements
the ‘above test’ alarm which might not always give
an alarm when there is antigen excess. Both alarms
suggest reanalysis at a lower sample fraction, which
when performed will reveal a high SFLC concentration if present.
Table II. Quality control results of the verification period.
Concentration
(mg/L)
Antigen excess
factor
Control*
No
Mean
SD
Mean
SD
κ
κ
λ
λ
27
22
27
26
14.6
29.3
27.0
54.9
0.72
2.09
0.97
1.28
233
256
89.7
85.3
21
16
5.3
3.9
low
high
low
high
*The control sera used were those included in the Freelite human
kappa (or lambda) free kit for use with Roche Cobas® c systems.
SD, Standard deviation.
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580
P. Urdal et al.
The most reliable way to detect antigen excess is
by analyzing all sera at two dilutions. In the present
study all sera above 30 mg/L, and those below 5
mg/L, were analyzed at two or more dilutions prior
to inclusion whereas most sera in the SFLC concentration range 5–30 mg/L were analyzed at only one
dilution. Our antigen excess alarm functioned reliably. All sera above 30 mg/L triggered one or both
of the alarms when analyzed without manual predilution. The antigen excess alarm was not released by
any serum in the concentration range 15–100 mg/L
and by all sera above 300 mg/L. If in use as part of
the routine assay, this reliable absence of antigen
excess alarm when SFLC was above 30 but below
100 mg/L would have prevented many unnecessary
manual dilutions and reanalysis. And the results
shown in Figures 4 and 5 and in Table I do suggest
that the antigen excess alarm has a quality for detecting high SFLC in the presence of antigen excess well
above that of the ‘above test’ alarm.
For sera with SFLC concentration in the range
5–30 mg/L, as measured prior to inclusion, we cannot exclude antigen excess present in some sera but
missed by both types of alarm. As reported, we did
find one sample included with initially reported normal SFLC-κ and SFLC-λ concentrations. When
reanalyzed as part of the study, it triggered the antigen excess alarm and after dilution of sample the
correct SFLC-κ concentration was found to be 1320
mg/L. Despite this case, we believe, from scarcity of
reports and from our own previous experience, that
antigen excess leading to a high SFLC concentration
being reported as below 30 mg/L, is rare. Still, it is
vital that we detect such cases of antigen excess. If
we were to document that our antigen excess alarm
effectively detected these probably rare cases, we
would have to include a very large number of sera,
and to analyze all at two dilutions. We found this to
be outside the capacity of the present study, but still
something that ought to be done before the antigen
excess alarm may be taken into common use.
We obtained many antigen excess alarms in sera
below 15 mg/L in either SFLC-λ or -κ concentration. It would, however, be possible to avoid the
unwanted alarms simply by restricting the antigen
excess alarm function to samples where the early
absorbance change is above a defined value. Such a
restriction would also allow the use of a higher alarm
cut-off value for the antigen excess factor, which
might be of importance especially with the SFLC-λ
assay where some samples with high concentrations
have only moderately reduced antigen excess factor
values.
One might expect that a change in reagent lot
may affect the antigen excess factor values measured.
With the Binding Site antigen excess application the
discriminatory value of the factor is established with
each lot of reagents [12]. Our experience did not
suggest lot adjustment to be necessary; the antigen
excess factor values of normal samples were only
moderately affected by change in reagent lot.
A similar antigen excess control is used by Roche
to avoid antigen excess when measuring serum ferritin. However, the antigen excess control of SFLC
may, one might argue, function less well given that
the light chains of different myeloma patients each
are monoclonal. They might differ in how they are
recognized by the antibodies of the reagent. Figure
2a does not suggest a high degree of such variability
for the early absorbance change, whereas Figures
4 and 5 do indicate some variability in alarm factor
values. Still, excluding sera below approximately
15 mg/L by introducing an early absorbance change
threshold (cfr Figures 2a, 3a, 4 and 5), the remaining
sera below 100 mg/L in SFLC all showed high
alarm factor values, clearly separable from the lower
alarm factor values of sera above 200–300 mg/L
in SFLC.
Today, laboratories using Binding Site reagents
adapted to other analyzers are advised to reanalyze
at a lower sample fraction all samples in which the
initial SFLC value is above the upper reference limit,
usually above 20–30 mg/L [10,13]. Such samples are
quite common among those received for SFLC analysis. For laboratories, it will be a benefit to gain
access to SFLC methods that include an automated
antigen excess detection. The antigen excess detection described here detected all antigen excess samples that we knew of at no increased time of analysis.
If taken into use we expect it will give a safe way of
detecting antigen excess, a reduced workload, and a
reduced reagent cost.
Acknowledgements
We thank Mohammed Jasim and Tore Øie, Roche
Norway, for applying the automated antigen excess
detection methods to the Roche Cobas® c.501 analyzer. We thank the medical technologists at the
Department of Medical Biochemistry, Oslo University Hospital, Ullevål and Rikshospitalet, for their
kind help during the project.
Declaration of interest: The authors report no
conflict of interest. The authors alone are responsible
for the content and writing of the paper.
References
[1] Murata K, Clark RJ, Lockington KS, Tostrud LJ, Greipp PR,
Katzmann JA. Sharply increased serum free light-chain concentrations after treatment for multiple myeloma. Clin
Chem 2010;56:16–20.
[2] Daval S, Tridon A, Mazeron N, Ristori JM, Evrard B. Risk
of antigen excess in serum free light chain measurements.
Clin Chem 2007;53:1985–6.
[3] Vercammen M, Meirlaen P, Broodtaerts L, Broek IV,
Bossuyt X. Effect of sample dilution on serum free light
Antigen excess alarm in SFLC assays
[4]
[5]
[6]
Downloaded by [The University of Edinburgh] at 03:24 28 August 2017
[7]
chain concentration by immunonephelometric assay. Clin
Chim Acta 2011;412:1798–804.
McCudden CR, Voorhees PM, Hammet-Stabler CA.
A case of hook effect in the serum free light chain assay
using the Olympus AU400e. Clin Biochem 2009:42:
121–4.
Bosmann M, Kössler J, Stolz H, Walter U, Knop S,
Steigerwald U. Detection of serum free light chains:
the problem with antigen excess. Clin Chem Lab Med
2010;48:1419–22.
Murng SHK, Follows L, Whitfield P, Snowden JA,
Swallow K, Green K, Sargur R, Egner W. Defining the
impact of individual sample variability on routine immunoassay of serum free light chains (sFLC) in multiple myeloma.
Clin Exp Immunol 2013;171:201–9.
Papik K, Molnar B, Fedorcsak P, Schaefer R, Lang F,
Sreter L, Feher J, Tulassay Z. Automated prozone effect
detection in ferritin homogeneous immunoassays using
neural network classifiers. Clin Chem Lab Med 1999;
37:471–6.
581
[8] Al-Mahdili HA, Jones GR. High-dose hook effect in six
automated human chorionic gonadotrophin assays. Ann
Clin Biochem 2010;47:383–5.
[9] Akamatsu S, Tsukazaki H, Inoue K, Nishio Y. Advanced
prostate cancer with extremely low prostate-specific antigen
value at diagnosis: an example of high dose hook effect. Int
J Urol 2006;13:1025–7.
[10] Jenner E, Levoguer A, Evans J, Harding S. Serum free light
chain immunoassays: a guide to antigen excess detection.
Clin Chim Acta 2012;413:949.
[11] Te Velthuis H, Knop I, Stam P, van den Broek M, Bos HK,
Hol S, Teunissen E, Fischedick KS, Althaus H, Schmidt B,
Wagner C, Melsert R. N Latex FLC – new monoclonal highperformance assays for the determination of free light chain
kappa and lambda. Clin Chem Lab Med 2011;49:1323–32.
[12] Product data sheet for human kappa free kit (for use on the
SPAplus). Insert code: SIN 113DS, version 12 May 2011.
[13] Product data sheet for human kappa free kit (for use on the
Roche Cobas® systems). Insert code: SIN 129Dk, version 1
June 2011.