AMH immunoassay:
A multi center evaluation of the precision of a novel fully automated
AMH assay under routine conditions
Johan Schiettecatte1; Murat Öktem2; Arne Thies3; Martine Cohen-Bacrie4; Ellen Anckaert1; Cyrill Müller5; Deniz
Topcu2; Arndt Gröning3, Sabine Engelmann6; Christoph Milczynski5
1. UZ Brussels, Laarbeeklaan 101, 1090 Brussels, Belgium, 2. Duzen Laboratories Group, Tunus Cad. No. 95 Kavaklidere, Ankara, Turkey, 3. MVZ
wagnerstibbe für Laboratoriumsmedizin und Pathologie GmbH, Georgstraße 50, 30159 Hannover, Germany, 4. Laboratoire Eylau Saint Didier, 55,
rue Saint Didier, 75116 Paris, France, 5. Limbach Laboratory, Medical Care Center, Im Breitspiel 15, 69126 Heidelberg, Germany, 6. Roche
Diagnostics GmbH, Sandhoferstr. 116, 68305 Mannheim
Background
21-day precision according to CLSI-EP5: AMH immunoassay
The CLSI EP5 precision performance experiment (21 days) was performed on cobas e 601,
cobas e 411 and MODULAR® ANALYTICS E170. The experiment included PC AMH 1, PC
AMH 2 as control material and 5 serum pools with different levels.
Anti-Müllerian-Hormone (AMH) is an established marker for assessing the ovarian reserve
and optimization of in-vitro fertilization treatment. Current existing manual AMH assays exhibit
limitations with respect to throughput and reliability of results.
Recently a fully automated AMH assay has been developed on the Elecsys®/cobas e
electrochemiluminescence immunoassay platform (product launch in preparation).
The objective of the technical performance study was to evaluate the precision of this highthroughput assay under routine conditions.
Repeatability of AMH immunoassay according to CLSI-EP5
e 411
e 601
E170
2.0
1.8
Study design and methods applied
1.6
1.4
1.2
CV (%)
The precision performance of AMH immunoassay was evaluated in five different routine
laboratories across Europe. The evaluation of the immunoassay was performed on a total of
two cobas e 601 instruments, three cobas e 411 instruments and one MODULAR®
ANALYTICS E170 and compared to the respective routinely used manual method. Each
laboratory evaluated the performance of the immunoassay independently.
The study design encompassed a within-run-precision experiment and the evaluation of the
repeatability and intermediate precision, following the procedure recommended according to
CLSI-EP5.
For conducting the experiments, each laboratory was provided with an identical set of spiked
sample and control material covering major parts of the measuring range (sample
concentration between 0.24 ng/mL up to 18.5 ng/mL).
1.0
0.8
0.6
0.4
0.2
0.0
Figure
Figure 2:
2: Repeatability
Repeatability precision
precisionperformance
performance (CV
(CV(%))
[%])ofofAMH
AMHimmunoassay
immunoassayon
onRoche
Roche
®
® ANALYTICS
ANALYTICS
E170,cobas
cobas
e 411
cobas
e 601.
analyzers such
such as
as MODULAR
MODULAR
E170,
e 411
andand
cobas
e 601.
Within-run-precision: AMH immunoassay
The within-run-precision experiment was performed by measuring 21 replicates in one run
using 5 different serum level pools. On cobas e 601 and MODULAR® ANALYTICS E170
randomly assigned measuring cells were used to determine the within-run-precision
coefficient of variation (CV) [%].
Intermediate precision of AMH immunoassay according to CLSI-EP5
e 601
Within-run-precision
AMH
immunoassay
Within-run-precision AMH
immunoassay
E170
4.5
4.0
3.0
3.5
CV (%)
CV (%)
e 411
5.0
3.5
2.5
2.0
3.0
2.5
2.0
1.5
1.5
1.0
1.0
Instrument
0.5
19.17 Laboratory 3
Laboratory 3
12.75 Laboratory 3
Laboratory 3
e 601
2.52
Laboratory 3
0.73
Laboratory 2
0.24
Laboratory 1
Laboratory 2
19.17
Laboratory 1
Laboratory 2
12.75
Laboratory 1
Laboratory 2
e 411
2.52
Laboratory 1
Laboratory 2
0.73
Laboratory 1
Laboratory 5
0.24
Laboratory 3
Laboratory 5
19.17 Laboratory 4
Laboratory 3
12.75 Laboratory 4
Laboratory 5
Laboratory 4
Laboratory 3
2.52
Laboratory 5
Laboratory 4
Laboratory 3
Laboratory 5
0.73
0.24
Mean (ng/mL)
Laboratory 4
Laboratory 3
0.5
Laboratory
Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab.
1
2
2
1
2
2
1
2
2
1
2
2
1
2
2
1
2
2
1
2
2
0.24 ng/mL
0.72 ng/mL
1.1 ng/mL
2.4 ng/mL
5.5 ng/mL
12.5 ng/mL
18.8 ng/mL
0.0
Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab.
1
2
2
1
2
2
1
2
2
1
2
2
1
2
2
1
2
2
1
2
2
0.24 ng/mL
0.72 ng/mL
1.1 ng/mL
2.4 ng/mL
5.5 ng/mL
12.5 ng/mL
18.8 ng/mL
Figure 3:
3: Intermediate
Intermediate precision
precision performance
performance (CV
(CV (%))
[%]) ofofAMH
AMHimmunoassay
immunoassayon
onRoche
Roche
Figure
®
411
and
cobas
e 601.
analyzers such
such as
analyzers
as MODULAR
MODULAR®ANALYTICS
ANALYTICSE170,
E170,cobas
cobase e
411
and
cobas
e 601.
E170
Figure
Within-run-precision performance
on Roche
Rocheanalyzers
analyzerssuch
such
Figure 1: Within-run-precision
performance of
of AMH
AMH immunoassay on
®®
as
ANALYTICS
ANALYTICSE170,
E170,cobas
cobasee411
411and
andcobas
cobase e601.
601.
as cobas
cobas ee601
601and
andMODULAR
MODULAR
The AMH immunoassay’s precision performance following the CLSI-EP5 protocol resulted in a
maximum day-to-day variability CV of 3.7% and part-to-part variability CV of 2.1%. The
repeatability was below CV 1.8%. At all testing sites the intermediate-precision exhibited CVs
below 4.4%.
The within-run-precision CV observed on Roche routine analyzers using AMH immunoassay
was below 3.4 % in all five laboratories.
5-day precision according to CLSI-EP5 scheme: comparison between AMH immunoassay and ELISA methods under routine conditions
6
4
2
0
-2
Gobal mean
(ng/mL) of sample
0.29
0.92
0.93
3.00
4.51
13.96
Laboratory 5
Laboratory 3
Laboratory 1
Laboratory 2
manual method 1
Laboratory 2
Laboratory 1
Laboratory 5
E170 Laboratory 3
e 601
Laboratory 3
e 411 Laboratory 4
Laboratory 5
Laboratory 3
manual method 2 Laboratory 4
Laboratory 1
Laboratory 2
manual method 1
Laboratory 2
E170 Laboratory 3
Laboratory 5
Laboratory 1
e 601
Laboratory 3
e 411 Laboratory 4
Laboratory 5
manual method 2 Laboratory 4
Laboratory 3
Laboratory 1
Laboratory 2
manual method 1
Laboratory 2
Laboratory 1
Laboratory 5
E170 Laboratory 3
e 601
Laboratory 3
e 411 Laboratory 4
Laboratory 5
manual method 2 Laboratory 4
Laboratory 3
Laboratory 2
manual method 1
Laboratory 1
Laboratory 2
Laboratory 1
Laboratory 5
E170 Laboratory 3
e 601
Laboratory 3
e 411 Laboratory 4
manual method 2 Laboratory 4
Laboratory 5
Laboratory 3
Laboratory 2
manual method 1
Laboratory 1
E170 Laboratory 3
Laboratory 2
Laboratory 1
e 601
Laboratory 5
e 411 Laboratory 4
Laboratory 3
manual method 2 Laboratory 4
Laboratory 5
Laboratory 3
Laboratory 2
manual method 1
Laboratory 1
E170 Laboratory 3
Laboratory 2
e 601
Laboratory 1
Laboratory 5
Laboratory 3
e 411 Laboratory 4
manual method 2 Laboratory 4
Laboratory 5
Laboratory 3
manual method 1
Laboratory 2
Laboratory 1
E170 Laboratory 3
Laboratory 2
e 601
Instrument/
Method
Laboratory 1
Laboratory
Laboratory 5
-6
Laboratory 3
-4
e 411 Laboratory 4
Difference to global
mean (ng/mL)
Precision data were collected over a time period of 5 days at all sites, using PreciControl AMH level 1 and 2 and five serum level pools as sample material. Two different ELISA assays were
compared to the AMH assay. For the ELISA each day two independent measurements with each two aliquots of control and serum level pool material were performed to imitate the CLSI guidance.
17.96
Figure
5 days
measuring
7 sample
material
concentrations
with with
ELISA
methods
(black)
and AMH
immunoassay
(blue) (blue).
Figure4:4:Summary
Summaryofofresults
resultsobtained
obtainedduring
during
5 days
measuring
7 sample
material
concentrations
ELISA
methods
(black)
and AMH
immunoassay
Under routine conditions the AMH immunoassay’s repeatability CV was below 5.1%, the intermediate precision CV did not exceed 6.9%. For the both ELISA methods the repeatability was up to
53.1% (method 1) and 150.1% (method 2), the intermediate precision CV was up to 63.8% (method 1) and 185.3% (method 2).
Conclusion
The fully automated AMH immunoassay demonstrated a within-run-precision CV of below 3.4% for native and spiked sample material across all five laboratories. The assay’s precision performance
following the 21-day-CLSI-EP5 protocol resulted in an intermediate precision CV below 4.4%. The repeatability precision did not exceed 1.8%. At all testing sites the maximum day-to-day variability
CV was 3.7%, the part-to-part variability CV below 2.1%. In a comparison with commercially available ELISA tests, the automated AMH immunoassay exhibited a manyfold better precision
performance than the routinely used manual methods.
The availability of the fully automated AMH immunoassay run on routine Elecsys®/cobas e instruments will represent a fast and precise alternative to manual AMH assay testing.