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Journal of Clinical Microbiology, May 2001, p. 1975-1977, Vol. 39, No. 5
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.1975-1977.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Evaluation of an Automated Liquid-Handling
System (Tecan Genesis RSP 100) in the Abbott LCx Assay for
Chlamydia trachomatis
Kevan L.
Hanson1 and
Charles P.
Cartwright1,2,*
Department of Laboratory Medicine and Pathology, Hennepin
County Medical Center, Minneapolis, Minnesota
55415,1 and Department of Laboratory
Medicine and Pathology, University of Minnesota Medical School,
Minneapolis, Minnesota 554552
Received 27 November 2000/Returned for modification 14 January
2001/Accepted 18 February 2001
 |
ABSTRACT |
The present study investigated the feasibility of automating the
specimen-pipetting component of sample preparation in the LCx Chlamydia
assay (LCx-CT assay; Abbott Laboratories, Chicago, Ill.) by using a
commercially available liquid-handling system (Tecan Genesis RSP100;
Tecan Inc., Research Triangle Park, N.C.). The Tecan instrument proved
to be comparable in both precision and accuracy to a manual
multipipettor (Eppendorf model 4850; Eppendorf Scientific, Westbury,
N.Y.). The Tecan instrument was extensively checked for evidence of
specimen-to-specimen transfer, and no level of contamination sufficient
to generate a signal above the background in the LCx-CT assay was
detected. Finally, pipetting speed was significantly improved by using
the Tecan instrument. A mean time of 2.5 min was required to pipette a
complete LCx-CT assay carousel (20 samples and 4 controls) with the
Tecan instrument, whereas 8.4 min was required to pipette a comparable number of samples manually (P < 0.001).
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TEXT |
Detection of Chlamydia
trachomatis in urogenital specimens by nucleic acid amplification
(NAA) assays, including the LCx Chlamydia assay (LCx-CT assay; Abbott
Laboratories, Chicago, Ill.), has become firmly established as the
standard of care for the diagnosis of infection with this organism
(3, 6, 9). Although the LCx-CT assay has excellent
performance characteristics (4, 7, 8), a significant
drawback of this and many other first-generation NAA tests for C. trachomatis is their relative lack of automation, making them
significantly more labor intensive than alternative methodologies for
detection of this organism. The specimen preparation component of the
LCx-CT assay includes pipetting of fixed volumes of processed samples
into amplification vials containing prealiquoted amplification
reagents. This process is typically performed with a manually operated
single-channel pipettor, a laborious and ergonomically problematic
operation for moderate- to high-volume laboratories. Automated
liquid-handling devices, such as the Tecan Genesis RSP100 (Tecan Inc.,
Research Triangle Park, N.C.) automated pipettor, have been shown to be
highly effective at increasing throughput and decreasing labor
expenditure in a number of high-volume applications (1,
5). There have, however, been no published evaluations describing the use of this type of instrument in NAA applications, with
their more stringent requirements for pipetting accuracy and precision
and concern about cross-specimen transfer leading to false-positive
results (2).
Study institution.
The clinical microbiology laboratory at
Hennepin County Medical Center (HCMC) has been performing the LCx NAA
assays for C. trachomatis (LCx-CT) and Neisseria
gonorrhoeae (LCx-GC) since January 1997. The annual volume
of tests was 38,034 (22,485 for the LCx-CT assay and 15,549 for the
LCx-GC assay) in 1997 and has risen to a projected (on the basis of
data collected from January through September 2000) 53,877 tests
(30,897 for the LCx-CT assay and 22,980 for the LCx-GC assay) in 2000. The typical daily work flow is illustrated in Fig.
1 and is performed by a single technologist with four thermocyclers and four LCx analyzers. The mean
positivity rates for the populations served by HCMC since the
introduction of the LCx assays are 8.07 and 3.26% for C. trachomatis and N. gonorrhoeae, respectively.
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FIG. 1.
Typical daily LCx assay workflow at HCMC enables
analysis of a maximum of 120 samples tested for both C. trachomatis and N. gonorrhoeae in an 8-h shift by one
technologist using four thermocyclers and four LCx analyzers. a, each
run consists of 40 samples (80 tests) plus controls; b, completion of
run 3 from the previous day; c, results reporting and instrument
cleanup. The following assay procedures are represented by the
indicated symbols: sample preparation, solid gray bars; sample transfer
to amplification vials, open bars; thermocycler loading, solid black
bars; amplification, dotted bars; LCx analyzer loading, vertically
hatched bars; product detection, diagonally hatched bars.
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All clinical samples used for determination of the performance
characteristics of the Tecan automated pipettor in the Abbott
LCx-CT
assay were endocervical swab specimens submitted to the
HCMC clinical
microbiology laboratory for testing for
C. trachomatis.
After routine testing was completed, discarded materials were
retained
(frozen at
70°C) for use in the
study.
The precisions of the Tecan automated pipettor and the Eppendorf 4850 manual pipettor were determined gravimetrically. Multiple
(
n = 24) 100-µl aliquots of water were pipetted, using 1,000-µl
maximum-volume pipette tips, into preweighed LCx amplification
vials,
and the volume dispensed was determined with an analytical
balance
(AE-163; Mettler Instrument Corp., Highstown, N.J.). Coefficients
of
variation (CV) are shown in Table
1 and
were comparable for
both instruments.
The reproducibility of the Tecan automated pipettor in the LCx-CT assay
was assessed in three independent studies. Serial
dilutions of known
C. trachomatis-positive specimens were prepared
by using
pooled negative specimens as a diluent. A total of 10
such positive
pools were then multiply pipetted (10 replicates)
into LCx-CT assay
amplification vials by using the Tecan automated
pipettor, and the mean
signal and percent CV were calculated for
each pool (Table
1).
A total of 69 previously analyzed specimens (15 specimens positive for
C. trachomatis and 54 specimens negative for
C. trachomatis)
were repeatedly pipetted (three replicates) with the
Tecan automated
pipettor and then retested by the LCx-CT assay. The
mean signal
and CV for each group of replicates were determined (Table
1).
One hundred previously analyzed clinical samples (24 samples positive
for
C. trachomatis and 76 samples negative for
C. trachomatis)
were simultaneously pipetted into amplification vials
with either
the Tecan automated pipettor or the Eppendorf 4850 manual
pipettor
and then analyzed by the LCx-CT assay. Entirely concordant
results
were obtained, with no statistically significant differences in
qualitative results obtained with the two pipettors. The mean
± standard deviation (SD) signals generated from positive specimens
were
1,708.3 ± 184.4 cps for manually pipetted samples and
1,694.6
± 164.4 cps for samples pipetted with the Tecan automated
pipettor.
The corresponding values for negative specimens were 19.2 ± 4.7
and 16.6 ± 4.0 cps for the Eppendorf and Tecan pipettors,
respectively.
Two studies were conducted to evaluate the risk of cross-sample
contamination when the Tecan automated pipettor was used.
In the first
study, a total of 16 high-level-positive samples
(mean signal,
1871.8 ± 132.8 cps; mean signal-to-cutoff ratio,
3.24 ± 0.28) were evenly interspersed among 64 uninoculated sample
collection
vials. All 80 samples were then pipetted into LCx-CT
assay
amplification vials with the Tecan automated pipettor and
analyzed. The
mean ± SD signal generated from the negative samples
was
15.7 ± 1.9 cps (range, 12.3 to 22.7 cps). The second study
was
performed immediately after the completion of the first cross-sample
contamination study. A total of 80 uninoculated sample collection
vials
were placed on the Tecan instrument, and an aliquot (100
µl) of
sample diluent was transferred from each collection vial
into an LCx-CT
amplification vial. The activated vials were then
run through the
standard LCx amplification and product detection
protocol. These vials
yielded a mean signal of 14.6 ± 1.4 cps
(range, 12.0 to 17.8 cps). In addition, upon completion of the
evaluation of the Tecan
automated pipettor, multiple areas of
the instrument including the base
plate, permanent pipettors,
specimen vial racks, amplification vial
racks, pipette tip discard
tray, and wash station were sampled by using
LCx specimen collection
swabs. Swabs were then expressed into sample
collection vials
and analyzed for the presence of both
C. trachomatis and
N. gonorrhoeae DNA by standard LCx
protocols. All swab samples taken from the
Tecan instrument were
negative for
C. trachomatis and
N. gonorrhoeae when subjected to testing by the LCx
assays.
An initial comparison of the time required to pipette samples manually
and with the Tecan automated pipettor was performed
by pipetting the
contents of four complete LCx carousels (a total
of 80 samples) by both
methods and recording the time required
to complete the process. A mean
time of 2.5 min was required to
pipette the contents of a complete
LCx-CT carousel (20 samples
and 4 controls) with the Tecan automated
pipettor, whereas 8.4
min was required to pipette a comparable number
of samples manually
(
P < 0.001). Upon completion of a
probationary training and evaluation
period, a real-time analysis of
the workload requirement for routine
testing by the LCx assay in our
laboratory (a combination of the
LCx-CT and the LCx-GC assays) was
performed with the Tecan automated
pipettor. Technologists performing
LCx assays recorded the total
number of billable analyses performed
(excluding all required
controls and repeat tests) and the time taken
to complete all
daily tasks, including accessioning of specimens,
reporting of
results, and instrument maintenance, for 30 consecutive
workdays.
The data generated during that study were compared with those
obtained during a similar study conducted in October 1997 when
all
specimens were manually pipetted (Table
2).
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TABLE 2.
Results of independent real-time evaluations of the
workload requirement for the Abbott LCx assays when addition of
samples to amplification vials was performed with a manual pipettor
(Eppendorf 4850) or an automated pipettor (Tecan Genesis RSP100
automated pipettor)
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|
The results of the present study demonstrate that the use of an
automated pipetting instrument, in this instance, the Tecan
Genesis
RSP100 instrument, can improve the efficiency of the Abbott
LCx NAA
assays for
C. trachomatis and
N. gonorrhoeae
without compromising
the integrity of the results obtained. Use of the
Tecan automated
pipettor in our laboratory has enabled us to
accommodate increases
in test volumes for the LCx-CT and the LCx-GC
assays without concomitant
increases in technologist time. Indeed,
routine use of the Tecan
automated pipettor has resulted in a decrease
in the typical daily
workload from 8.22 h in 1997 (in which
pipetting was exclusively
manual) to 6.92 h in 2000 (Table
2) over
a time period in which
the annual volume of LCx tests has increased by
almost 20,000.
The performance of the Tecan automated pipettor in the
LCx-CT
assay was, as far as we were able to ascertain, entirely
comparable
to that achieved with a manual pipettor (the Eppendorf 4850 pipettor).
There was no evidence that the use of an automated pipettor
adversely
affected any of the performance characteristics of the assay.
Although the level of additional automation afforded by use of
the
Tecan automated pipettor is relatively small, the labor savings
realized by using the automated pipettor in this high-volume molecular
biology-based application were still considerable. In addition,
although difficult to measure quantitatively, the ergonomic benefits
of
using an automated pipettor for high-volume molecular biology-based
assays are significant and further increase the benefit-to-cost
ratio
of acquiring such a device. The present study illustrates
that NAA
assays are amenable to automation and that the increased
instrumentation cost can often be more than offset by a concomitant
decrease in labor
cost.
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ACKNOWLEDGMENTS |
This study was supported in part by a grant from Abbott Laboratories.
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FOOTNOTES |
*
Corresponding author. Mailing address: Clinical
Laboratories, MC #812, Hennepin County Medical Center, 701 Park Ave.,
Minneapolis, MN 55415. Phone: (612) 347-3026. Fax: (612) 904-4229. E-mail: charles.cartwright{at}co.hennepin.mn.us.
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REFERENCES |
| 1.
|
Ertingshausen, G.,
S. I. Shapiro,
G. Green, and G. Zborowski.
1975.
Adaptation of a T3-uptake test and of radioimmunoassays for serum digoxin, thyroxine, and triiodothyronine to an automated radioimmunoassay system "Centria".
Clin. Chem.
21:1305-1313[Abstract].
|
| 2.
|
Gronowski, A. M.,
S. Copper,
D. Baorto, and P. R. Murray.
2000.
Reproducibility problems with the Abbott Laboratories LCx assay for Chlamydia trachomatis and Neisseria gonorrhoeae.
J. Clin. Microbiol.
38:2416-2418[Abstract/Free Full Text].
|
| 3.
|
Guaschino, S., and F. De Seta.
2000.
Update on Chlamydia trachomatis.
Ann. N. Y. Acad. Sci.
900:293-300[CrossRef][Medline].
|
| 4.
|
Lee, H. H.,
M. A. Chernesky,
J. Schachter,
J. D. Burczak,
W. W. Andrews,
S. Muldoon,
G. Leckie, and W. E. Stamm.
1995.
Diagnosis of Chlamydia trachomatis genitourinary infection in women by ligase chain reaction assay of urine.
Lancet
345:213-216[CrossRef][Medline].
|
| 5.
|
Liebl, B.,
T. Anhaupl,
E. Haen,
B. Gunster, and M. Georgieff.
1993.
A partially automated radioligand binding assay system for use in clinical and pharmaceutical research.
J. Recept. Res.
13:369-378[Medline].
|
| 6.
|
Quinn, T. C.
1995.
DNA amplification assays: a new standard for diagnosis of Chlamydia trachomatis infections.
Ann. Acad. Med. Singapore
24:627-633[Medline].
|
| 7.
|
Schachter, J.,
W. E. Stamm,
T. C. Quinn,
W. W. Andrews,
J. D. Burczak, and H. H. Lee.
1994.
Ligase chain reaction to detect Chlamydia trachomatis infection of the cervix.
J. Clin. Microbiol.
32:2540-2543[Abstract/Free Full Text].
|
| 8.
|
Stary, A.,
E. Schuh,
M. Kerschbaumer,
B. Gotz, and H. Lee.
1998.
Performance of transcription-mediated amplification and ligase chain reaction assays for detection of chlamydial infection in urogenital samples obtained by invasive and noninvasive methods.
J. Clin. Microbiol.
36:2666-2670[Abstract/Free Full Text].
|
| 9.
|
Taylor-Robinson, D.
1997.
Evaluation and comparison of tests to diagnose Chlamydia trachomatis genital infections.
Hum. Reprod.
12(11 Suppl.):113-120[Abstract].
|
Journal of Clinical Microbiology, May 2001, p. 1975-1977, Vol. 39, No. 5
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.1975-1977.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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