Download reaction volume of 10 L with 0.5 M each of the

Clinical Chemistry 47, No. 5, 2001
reaction volume of 10 ␮L with 0.5 ␮M each of the primers
5⬘-AGCACCAAGCAATATCATTTATG-3⬘ and 5⬘-GCCTCCAAGTATCTGCACAG-3⬘, 50 ng of genomic DNA,
and 0.5 ␮M each of the anchor and detection probes (TIB
MOLBIOL). The anchor probe 5⬘-CCAAGCTGCTCTCTGGTAACTCATT-3⬘ was labeled at the 3⬘ end with fluorescein; the sensor probe 5⬘-AGCTAGTTCAACTTTCATTGCCAT-3⬘ was labeled with LightCycler Red 640 at its 5⬘
end and modified at its 3⬘ end by phosphorylation to
block extension. As reaction buffer in the PCR, the LightCycler DNA-Master hybridization Probes 10⫻ buffer
(Roche Diagnostics) with a final MgCl2 concentration of
3.5 mM was used. Cycling conditions were as follows:
95 °C for 1 min; and 40 cycles of 95 °C for 0 s, 59 °C for
20 s, and 72 °C for 20 s (ramping rate, 20 °C/s). Fluorescence was monitored at the end of each 20-s annealing
phase. After amplification, melting curves were generated
by denaturation at 95 °C for 0 s, holding the samples at
50 °C for 20 s, and then heating the sample to 75 °C at
0.2 °C/s, simultaneously monitoring the decline in fluorescence. Melting curves were converted to melting peaks
by calculating the negative derivative of the fluorescence
with respect to temperature (-dF/dT) against temperature
(T).
Typical results for genotyping using this method are
shown in Fig. 1. The melting peak of the wild-type sample
(curve 1) was at 66.9 °C, whereas the mutant homozygous
sample (curve 2) produced a melting peak at 64.5 °C. The
heterozygous sample produced two melting peaks at 66.9
and 64.5 °C (curve 3).
The whole process, including DNA extraction, was
completed within 70 min.
With this method we analyzed 18 individuals from four
different MCAD-deficient families and 25 healthy controls
(50 chromosomes). The results were consistent with those
obtained previously by restriction fragment length polymorphism analysis.
In conclusion, this new method combines simple sample processing and rapid analysis; it therefore affords both
high-throughput genotyping and rapid results.
Fig. 1. Melting peaks for MCAD genotyping.
Curves 1, 2, and 3 represent wild-type, homozygous mutant, and heterozygous
samples, respectively. Each analysis included a heterozygous DNA control and a
water control, which was negative (data not shown).
959
References
1. Iafolla AK, Thompson RJ, Roe CR. Medium-chain acyl-coenzyme A dehydrogenase deficiency: clinical course in 120 affected children. J Pediatr
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of the newborn populations in Belgium, Germany, Poland, Czech Republic,
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locus: clinical and evolutionary consideration. Pediatr Res 1997;41:201–9.
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1990;171:498 –505.
New Generation Cardiac Troponin I Assay for the
Access Immunoassay System, Per Venge,1* Bertil Lindahl,2
and Lars Wallentin2 (Department of Medical Sciences,
1
Clinical Chemistry and 2 Internal Medicine, University
of Uppsala, SE-751 85 Uppsala, Sweden; * address correspondence to this author at: Department of Clinical
Chemistry, University Hospital, SE-751 85 Uppsala, Sweden; fax 46-186113703, e-mail [email protected])
The measurement of troponins in blood has rapidly
become an alternative to conventional methods of detecting myocardial damage (1– 8 ), particularly in unstable
angina, and several studies have indicated the prognostic
importance of increased troponins in various clinical
settings (9 –14 ). These studies, however, have also pointed
out the need for more sensitive methods because patients
with even small increases of troponin seem to be at
increased risk of cardiac events. Currently, cardiac troponin I (cTnI) can be quantified by assays from several
manufacturers (15–19 ), whereas only one company currently commercializes a cardiac troponin T assay
(2, 20, 21 ). The aim of this work was to evaluate the
analytical performance of a new generation of the Access
cTnI assay. We also provide data on values in apparently
healthy subjects.
Venous blood was drawn from 70 patients admitted to
our Coronary Care Unit because of suspicion of an acute
coronary syndrome. Only patients found to have increased myocardial markers such as creatine kinase-MB
and troponin I were included. The study was approved by
the ethics committee of the Medical Faculty of Uppsala
University. Serum samples were also obtained from 122
apparently healthy subjects (70 women and 52 men;
median age, 41 years; range, 26 –73 years) as part of a
health-screening program.
The new ACCESS cTnI assay (Beckman Coulter, Inc.,
960
Technical Briefs
Chaska, MN) is a two-site immunoenzymatic (sandwich)
immunoassay. Paramagnetic particles coated with mouse
monoclonal anti-cTnI, mouse monoclonal anti-cTnI-alkaline phosphatase conjugate, and sample are added to a
reaction vessel to form a particle-cTnI-conjugate sandwich. The cTnI in the sample binds to the immobilized
anti-cTnI on the solid phase. The mouse anti-cTnI conjugate reacts with a different antigenic site on the cTnI
molecule. Separation in a magnetic field and washing
remove materials not bound to the solid phase. A chemiluminescent substrate (dioxetane Lumigen PPD) is added
to the reaction vessel, and light generated by the reaction
is measured with a luminometer. The photon production
is proportional to the quantity of cTnI in the sample. The
amount of analyte is determined by means of a stored
multipoint calibration curve.
We defined the detection limit as the concentration of
cTnI at a signal 2 SD above the mean signal of 10
replicates of the zero calibrator, as calculated from the
calibration curve. Three studies were performed on one
lot of reagents. The mean detection limit was 0.0036 ␮g/L
(range, 0.0024 – 0.005 ␮g/L). The lower limit of the reporting range was defined as the concentration at which the
variation in duplicate samples was ⱕ20% (CV) and was
calculated by means of the computer software Multicalc®
(Wallac Oy). It was estimated by assaying serial dilutions
of five different patient LiHeparin (cat. no. 367993; 3 mL
PET tube; 72 IU of lithium heparinate; final concentration
in filled tube, 48 IU/mL of blood; BD Vacutainer Systems)
plasma samples with increased cTnI (range, 0.21– 0.81
␮g/L). The lower limit was 0.0085 ␮g/L. In the same
experiment, the cTnI at a CV of 10% was 0.03 ␮g/L.
To determine assay imprecision, in one set of experiments trilevel controls provided by Beckman Coulter
(range, 0.582–29.2 ␮g/L) were analyzed in triplicate in a
total of 36 assays representing 6 tests per day during 6
different days. The model used to estimate imprecision
was a one-way ANOVA, assuming random effect. Estimates were calculated for intraassay, interassay, and total
imprecision for each control level and showed intraassay
variations (CVs) of 2.2–3.2%, interassay variations of
1.2– 4.4%, and total imprecision of 2.5–5.4%. In an additional study, three patient samples (LiHeparin) with mean
cTnI values (range) of 0.098 ␮g/L (0.09 – 0.11␮g/L), 0.069
␮g/L (0.06 – 0.07 ␮g/L), and 0.034 ␮g/L (0.03– 0.04 ␮g/L)
were assayed six times on each of 2 separate days, and the
total imprecision was calculated. The CVs were 6.4%,
4.3%, and 15%, respectively.
A linearity study was performed with LiHeparinplasma samples from six subjects with increased cTnI
(0.86 –75 ␮g/L). The samples were measured in quadruplicate at five different dilutions (0.8, 0.6, 0.4, 0.2, and 0.1).
The mean apparent recovery was 98.5% (95% confidence
interval, 95.9 –101.0%). To analyze the linearity at lower
concentrations, we used the results for the estimation of
the functional sensitivity as above. As shown in Fig. 1A,
dilutions of the five patient samples were linear down to
cTnI concentrations ⬍0.03 ␮g/L (r ⫽ 0.9991).
In vitro sample stability was tested using matched
serum and LiHeparin-plasma samples obtained from
eight patients with cTnI concentrations ranging from 0.81
to 52.0 ␮g/L. Accepting a ⫾ 10% deviation from the
values at 0 h, cTnI was stable in LiHeparin-plasma
samples left at room temperature for 48 h after blood
sampling, although the values at 48 h showed increased
variation (median, 91%; range, 85–111%). After ⱖ72 h at
room temperature, mean results were 80% of the initial
concentration. Values in LiHeparin-plasma samples
stored at 4 °C also changed ⬍10% at 48 h, and median
values were 89% of control (range, 85–98%) at 72 h or
more. The in vitro stability of cTnI in serum was similar to
that in LiHeparin plasma at both temperatures. In all
sample types, the changes after storage at either room
temperature or 4 oC were independent of the initial cTnI
concentration. Five freeze-thaw cycles with four samples
Fig. 1. Linearity after dilution of five patient samples (LiHeparin
plasma; A) and comparison of cTnI in matched serum and LiHeparinplasma samples (B).
(A), the compiled correlation coefficient (r) for all data was 0.9991. The x axis
shows the dilution, and the y axis shows the measured cTnI concentrations. (B),
difference plot [(LiHeparin plasma ⫺ serum)/serum ⫻ 100] prepared according
to the Bland-Altman method (25 ), as modified by Pollock et al. (26 ). The
correlation coefficient (r) for the comparison of results in serum and LiHeparin
plasma was 0.9975, with the equation: serum ⫽ ⫺0.5011 ⫹ 1.0050(LiHeparin
plasma). The solid line indicates the mean difference (%); the hatched area
indicates the 95% confidence interval.
Clinical Chemistry 47, No. 5, 2001
at different concentrations had no significant effect on
cTnI recovery (median, 100%; range, 92–107%).
A sample type comparison was performed in matched
serum and LiHeparin-plasma samples (n ⫽ 54) collected
at the same time from the same subject (Fig. 1B). Results
in LiHeparin plasma and serum were not significantly
different [2.4% lower in serum (95% confidence interval,
⫺1.3% to 6.1%)]. Plasma and serum showed a correlation
coefficient of 0.9975.
In 122 sera from apparently healthy subjects, the cTnI
concentrations of all except 2 subjects were ⬍0.01 ␮g/L.
Thus, the 95th and 99th percentiles were 0.01 and 0.02
␮g/L, respectively, in this group.
We conclude that the new generation of Access cTnI
assay has several desirable features. The lower limit of the
reported range is below the 95th percentile of our preliminary reference range for a healthy population. Both
serum and LiHeparin plasma may be used for measurement of cTnI, although there was a tendency to lower
values in LiHeparin plasma at lower cTnI concentrations.
[Part of this difference might be explained by the loss in
cTnI that has been shown to occur for both cTnI and
troponin T during the early phase of acute myocardial
infarction (22, 23 ). The data presented in this study,
however, do not allow conclusions on this point.] When
measured by this assay, cTnI is stable in vitro at both
room temperature and 4 °C and can be stored up to 48 h
under these conditions without any major effect on recovery. It seems, however, reasonable to recommend storage
for ⬍24 h. [The in vitro stability observed with the new
generation cTnI assay contrasts with our experience with
the Access first-generation cTnI assay. Indeed, storage of
samples at room temperature for 1–2 h produced decreases of 20 –30% of the measured cTnI concentration
(unpublished observations and Ref. (15 ).]
The improved in vitro stability may be explained by the
selection of different monoclonal antibodies to develop
the new generation cTnI assay. According to Beckman
Coulter, Inc., the monoclonal antibodies used in the new
generation cTnI assay were selected based on published
information that suggested that the antibodies recognize
epitopes located within a region encompassing amino
acids 30 –110 in the N-terminal half of the cTnI molecule.
In recent studies, the region encompassing amino acids
30 –110 was shown to be more resistant to proteolytic
cleavage (15, 24 ).
This study was supported by grants from Beckman
Coulter Inc. The technical expertise of Ing-Britt Persson is
gratefully appreciated.
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