Clinical Chemistry 45: 62-67, 1999;
(Clinical Chemistry. 1999;45:62-67.)
© 1999 American Association for Clinical Chemistry, Inc.
Evaluation of Interferences in Rate and Fixed-Time Nephelometric Assays of Specific Serum Proteins
Xavier Bossuyta and
Norbert Blanckaert
Department of Clinical Pathology, University Hospital Leuven, Kapucijnenvoer 33, B-3000 Leuven, Belgium.
a Author for correspondence. Fax 32 16 332896; e-mail xavier.bossuyt{at}uz.kuleuven.ac.be.
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Abstract
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We performed interference studies for IgG, IgA, IgM, haptoglobin, and
1-antitrypsin assayed in serum, using either
fixed-time nephelometry on the BN 100 from Behring or rate
nephelometry on two analyzers from Beckman Instruments. For clear serum
samples, results for IgG, IgA, IgM, and haptoglobin obtained with the
three nephelometers showed good agreement. Values for
1-antitrypsin in clear sera were lower with the BN 100
than with the Array 360 or Immage. In lipemic samples, the BN 100 gave
higher values than the Array 360 or Immage for all analytes except IgG.
Addition of Intralipid to serum produced atypical reactions with the BN
100 (fixed-time nephelometry) but not with the Array 360 or Immage
(rate nephelometry). The interference of lipemia on the BN 100 was also
seen when the Beckman antibody was used, indicating that the effect was
reagent-independent. For hemolyzed samples, the BN 100 gave higher
values than the Array 360 or Immage for haptoglobin but not for the
other analytes. Addition of increasing amounts of a hemolysate to serum
revealed a negative interference in all assay systems. This effect was
more pronounced with the Beckman reagent than with the Behring reagent
in all three nephelometers and was independent of the type of
instrument (fixed-time vs rate nephelometry).
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Introduction
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Immunochemical determination of individual serum proteins serves
as an important tool in the diagnosis and follow up of various
pathological conditions (e.g., acute phase reaction and multiple
myeloma). Automated methods involving nephelometry or turbidimetry are
widely used in clinical laboratories for this purpose. These assays
have suffered from poor intermethod and interlaboratory agreement, in
spite of the availability over the last four decades of numerous
national and international protein reference materials [Ref.
(1), and the references therein]. A few years ago, IFCC
introduced the international reference preparation CRM 470 with
certified target values for 14 plasma proteins (2). The
universal use of CRM 470 as a master calibrator should increase
interlaboratory and intermethod agreement. However, in addition to
calibration problems, there are several other sources of differences in
assay results, such as epitope recognition, assay conditions, and
interferences.
To assess calibrator-independent, reagent-specific, and
technique-related differences, we compared the results of several CRM
470-calibrated nephelometric assays. We assayed serum samples by
fixed-time nephelometry on a BN 100 from Behring and by rate
nephelometry on an Array 360 or an Immage from Beckman Instruments.
Special attention was given to evaluation of interferences because
fixed-time methods are reportedly subject to atypical reactions
(3).
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Materials and Methods
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Specific serum proteins were quantified by BN 100 or BNA
nephelometers from Behringwerke AG and by Array 360 and/or Immage
nephelometers from Beckman Instruments. Except when specified
otherwise, reagents used on the BN 100 were from Behringwerke AG and
reagents used on the Array 360 and Immage were from Beckman
Instruments. Total protein and triglyceride concentrations were
determined by Boehringer Mannheim reagent kits on a Hitachi 747
analyzer (Boehringer Mannheim GmbH). The hemoglobin concentration in
the hemolysate was determined with a Cell-Dyn 3500 (Abbott
Laboratories); the hemoglobin concentration in the patient samples was
calculated using the H-index on a Hitachi 747. This calculation was
based on the linear relation (r = 0.98) between the
H-index and the hemoglobin concentration determined on the Cell-Dyn
3500. Intralipid 20% was obtained from Pharmacia Biotech.
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Results
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interference by lipid
IgG, IgA, IgM, 1-antitrypsin, and haptoglobin
were determined in clear sera and in hemolyzed or lipemic samples,
using BN 100, Array 360, and Immage nephelometers. These instruments
were calibrated according to the manufacturers' instructions, using
calibrators traceable to the CRM 470 standard. The method comparison
results for IgG, IgA, and IgM are shown in Fig. 1
; the results for haptoglobin and 1-antitrypsin
are shown in Fig. 2
. For samples with a clear aspect on macroscopic examination
(left panels), the results for IgG, IgA, and IgM (Fig. 1
), and
haptoglobin (Fig. 2
) obtained with the three methods showed
excellent agreement. Values for 1-antitrypsin were lower
with the BN 100 than with the Array 360 or Immage. The slope and
intercept for the linear regression between the BN 100
(y-value) and the Array 360 (x-value) for
1-antitrypsin were 1.09 and -0.38 g/L, respectively.
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Figure 1. Comparison of the Immage, Array 360, and BN 100 methods
for immunoglobulins.
We assayed serum samples that were clear on macroscopic examination
(left panels), hemolyzed samples (median hemoglobin
concentration, 1.6 g/L; range, 0.9–23.5 g/L; middle
panels), and lipemic samples (median triglyceride concentration,
3.7 mmol/L; range, 2.5–6.6 mmol/L; right panels). IgG
(top panels), IgA (middle panels), and IgM
(bottom panels) were determined on an Immage
(x-axis), an Array 360 (y-axis,  ), and a BN
100 (y-axis,  ).
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Figure 2. Comparison of the Immage, Array 360, and BN 100 methods
for haptoglobin and 1-antitrypsin.
We assayed samples that were clear on macroscopic examination
(left panels), hemolyzed samples (median hemoglobin
concentration, 1.6 g/L; range, 0.9–23.5 g/L; middle
panels), and lipemic samples (median triglyceride concentration,
3.7 mmol/L; range, 2.5–6.6 mmol/L; right panels).
Haptoglobin (top panels) and 1-antitrypsin
(bottom panels) were determined on Immage
(x-axis), Array 360 (), and BN 100 () analyzers.
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For lipemic samples (right panels), values for IgA and IgM (Fig. 1
),
and haptoglobin and 1-antitrypsin (Fig. 2
) obtained
using the BN 100 were consistently higher than the values obtained
using the Immage and the Array 360. In selected samples, a twofold
difference for IgM was observed between the BN 100 results and the
Array 360 or Immage results.
In the next step, we examined whether the differences in assay results
for IgA, IgM, haptoglobin, and 1-antitrypsin in
lipemic samples were reagent-dependent (most likely antibody-dependent)
or were related to the type of assay, i.e., peak rate vs fixed-time
measurement. We suspected that the fixed-time nephelometric measurement
was affected by lipemia. Increasing concentrations of Intralipid 20%
were added to a control serum, and the specific proteins were measured.
As shown in Fig. 3
, increasing Intralipid concentrations caused increasing
interference in the haptoglobin, 1-antitrypsin, IgM, and
IgA assays in the BN 100 fixed-time nephelometer but not in the
rate-nephelometric Array 360 and Immage assays. The effect was most
pronounced for IgM, followed by IgA, 1-antitrypsin, and
haptoglobin. In contrast, neither the fixed-time nor the rate method
showed interference by lipemia in the IgG assay.
To evaluate the independent effect of intralipid on the Behring
instrument (BNA), we performed a control experiment in which we added 6
g/L Intralipid but replaced the IgM antibody reagent with buffer alone.
The signal measured in the presence and in the absence of the antibody
reagent corresponded to 3.25 and 0.61 g/L, respectively. The IgM value
in the absence of Intralipid was 1.55 g/L.
To exclude the possibility that the interference by lipemia was
reagent-dependent, we also studied the effect of Intralipid on IgM and
IgA measurements on the BN 100, using the Beckman reagent instead of
the Behring reagent. As shown in Fig. 3
, addition of Intralipid caused
antibody-independent increases in the signal on the BN 100, confirming
that the effect of lipid was instrument- or methodology-related and not
reagent-dependent.
interference by hemolysis
IgG, IgA, IgM, 1-antitrypsin, and haptoglobin
were determined in clear serum samples, using three
nephelometers (Figs. 1
and 2
, left panels) and in hemolyzed
samples (Figs. 1
and 2
, middle panels; median hemoglobin concentration,
1.6 g/L; range, 0.9–23.5 g/L). For IgG, IgA, and IgM, the results were
transferable between the BN 100, the Array 360, and the Immage
analyzers for clear sera as well as for hemolyzed samples. The BN 100
gave lower values for 1-antitrypsin in both clear and
hemolyzed samples. For the haptoglobin assay, results between the three
instruments were superimposable for clear sera but not for hemolyzed
samples, in which the Array 360 and Immage gave lower values than the
BN 100. When the antibody reagent was replaced with buffer alone, no
signal was measured with the Behring instrument or the Beckman
instrument (data not shown).
To determine the reason for the differences in assay results between
fixed-time and rate nephelometry for haptoglobin in hemolyzed
specimens, we investigated the effect of in vitro hemolysis on
haptoglobin determinations by adding increasing amounts of hemolysate
to nonhemolyzed serum. The haptoglobin concentration in the samples
with added hemolysate was determined with (a) the Immage,
Array 360, and BN 100, using the manufacturers' reagents;
(b) the Array 360, using the Behring reagent; and
(c) the BN 100, using the Beckman reagent. The results shown
in Fig. 4
demonstrate a gradual decrease of the haptoglobin result when
increasing concentrations of hemoglobin were present in the sample.
That the degree of this negative interference was reagent-dependent and
not analyzer-dependent was shown as follows. When the Behring reagent
was used on the BN 100 and on the Array 360, the haptoglobin signal was
reduced by 20% (0.27 g/L) in the presence of 2 g/L added hemoglobin.
When the Beckman reagent was used on both nephelometers, the
haptoglobin signal was reduced by 50% (0.65 g/L) in the presence of 2
g/L added hemoglobin. Thus, this negative interference was more
pronounced with the Beckman reagent than with the Behring reagent,
independent of the type of nephelometry (fixed-time or rate) used. It
can be speculated that the hemoglobin-haptoglobin complex is less
well-recognized by the Beckman reagent than by the Behring reagent.
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Discussion
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In nephelometric assays, specific antibodies react with the
antigen and form insoluble complexes that scatter light. The scattered
light is measured at 17° for the BNA, 70° for the Array 360, and
90° for the Immage. The wavelengths used are 840 nm, 670 nm, and
between 400 and 620 nm for the BNA, the Immage, and the Array 360,
respectively. In fixed-time nephelometry (Behring), two readings of
scattered light are made. The first reading is made 7.5 s after
the distribution of sample and antibody in the reaction buffer, and the
second reading is made 6 min later. The scattering measured in the
first reading is subtracted from the scattering measured in the second
reading. In this way, nonspecific scattering produced by the antibody
and the sample is minimized. In peak-rate nephelometry, the first
derivative of the variation of scattered light vs time during the
immunoprecipitation of antigen-antibody complexes is used for
calculation of the antigen concentration. The maximum intensity of the
peak is proportional to the antigen concentration. In the Array 360,
readings are performed every 20 ms. In the Immage, at least 18 cycles
of readings are made every 5 s. The variation of signal vs time is
used to calculate results.
Interferences in nephelometric assays depend on several factors, such
as the quality of the antibody used, the final sample dilution, and the
kinetic approach used. The present study clearly demonstrates that the
negative interference by in vitro hemolysis on haptoglobin
determination was analyzer-independent but reagent-dependent. The
interference was more pronounced with the Beckman reagent than with the
Behring reagent.
The higher the sample dilution, the lower the risk of nonspecific
reactions. For example, interferences by lipemia in the Behring
instrument were observed with IgA and IgM, for which the sample
dilution was 1:20, but not with IgG, for which the sample dilution was
1:400. With the Beckman instruments, the sample dilution was 1:36 for
IgM and IgA, and 1:216 for IgG.
Nonspecific interference that varies with reaction time cannot be
discerned by fixed-time nephelometry. The only way to detect such
nonspecific reactions is by replacing the antibody with diluent. Such a
control, however, is not part of the assay procedure presented by the
manufacturer. In the Array 360 system, nonspecific reactions are not
detected because the antibody is not added to the flow cell until the
scattered light signal after addition of the sample to the buffer is
stable. With the Immage, nonspecific reactions are offset by the
automatic measurement of a sample blank in the absence of antibody in a
separate cuvette. These blanks are performed when small sample
dilutions are used (1:6) and are done at each measurement.
The most pronounced nonspecific interference observed in this study was
with the fixed-rate method for the IgM assay in lipemic samples. In a
group of lipemic samples (n = 13), the median IgM value was 0.8
g/L (range, 0.5–1.8 g/L) with the Beckman instrument and 1.4 g/L
(range, 1.0–3.2 g/L) with the Behring instrument. Thus, the Behring
values were 75% higher than the Beckman values. This difference, which
was attributable to interference by lipemia, was larger than the
intraindividual (within-subject) biological CV (9.3%) and the
interindividual (between-subject) biological CV (34.9%) (4)
and, therefore, is clinically significant. For IgA, the median value in
a group of lipemic samples (n = 14) was 1.48 g/L (range, 0.5–3.8
g/L) for the Beckman instrument and 1.85 g/L (range, 0.8–4.4 g/L) for
the Behring instrument. The Behring values were 27% higher than the
Beckman values; this difference was larger than the intraindividual
biological CV (8.6%) (4).
The reagent inserts from Behring mention that turbidity and particles
can interfere with the test and that very lipemic samples must be
clarified by centrifugation. However, neither the marked effect of
lipemia on the IgM and IgA assays nor the limited interference of
hemoglobin on the haptoglobin assay are mentioned. The Beckman inserts
state that bilirubin, lipid, and hemoglobin do not interfere with the
IgA, IgG, and IgM assays and that hemoglobin produces a negative
interference of 15–50% on the haptoglobin assay, which is in
agreement with our findings.
In conclusion, nonspecific interactions can be limited by use of
specific antibodies, large sample dilutions, and a good estimation of
the sample blank.
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Acknowledgments
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We thank M. Artoos, H. Raveschot, A. Berghmans, V. Boets, and S.
Despiegeleer for expert technical assistance. We thank Analis (Belgium)
for providing the Beckman reagents.
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Abstract
Introduction
), an Immage (
), and an
Array 360 (
), and using
Beckman reagent on a BN 100 (