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Product Information

Testosterone IITestosterone

R E F 05200067 190

Elecsys 2010 system, MODULAR ANALYTICS E170cobas e analyzers

Product InformationElecsys and cobas e analyzers

Elecsys Testosterone II Immunoassay

Please note: As a result of differing printing dates, it is possible that differences may occur between the information given here and that appearing inthe instruction for use. In such cases the data given in the insert enclosed with the kit applies. Please also note that the product information documentcontains more extensive information than the instruction for use. Significant additions or changes are indicated by a change bar in the margin.This document may contain claims which are not cleared by the FDA and is therefore not intended for use in the USA.

2



Table of contents

1 Intended use 4

2 Summary 4

2.1 Biochemistry, clinical indications, methodology1,2,3,4,5,6,7 42.2 Elecsys test principle 6

3 Elecsys Testosterone II assay testing procedure 9

3.1 Materials provided, materials required (but not provided) 93.2 Assay procedure 93.3 Calibration 10

3.3.1 Standardization — traceability 103.3.2 Calibration frequency 11

3.4 Quality control 113.5 Calculation 123.6 Limitations – interference 123.7 Measuring range 133.8 Dilution 133.9 Expected values 13

3.9.1 Tanner stages 16

4 Specific performance data of the test 17

4.1 Precision 174.2 Analytical sensitivity 194.3 Analytical specificity 194.4 Method comparison16 20

4.4.1 Comparison Elecsys Testosterone and Elecsys Testosterone II to LC-MS/MS 204.4.2 Elecsys Testosterone vs Elecsys Testosterone II 21

4.5 Competitor comparison 224.5.1 Elecsys Testosterone II vs Abbott AxSym 224.5.2 Elecsys Testosterone II vs Beckman Access 234.5.3 Elecsys Testosterone II vs Siemens Centaur 244.5.4 Elecsys Testosterone II vs Siemens DPC RIA 254.5.5 Elecsys Testosterone II vs Siemens Immulite 26

4.6 Analyzer and lot-to-lot method comparisons 274.6.1 Elecsys 2010 vs MODULAR ANALYTICS E170 274.6.2 Lot-to-lot comparison 27

5 References 28

6 Notes 29

Short guide to Elecsys Testosterone II assay 32

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1 Intended use

Immunoassay for the in vitro quantitative determination of testosterone in human serum and plasma.The electrochemiluminescence immunoassay “ECLIA” is intended for use on Elecsys and cobas eimmunoassay analyzers.

2 Summary

2.1 Biochemistry, clinical indications, methodology1,2,3,4,5,6,7

The androgen testosterone (17β-hydroxyandrostenone) has a molecular weight of 288 daltons.

Testosterone in men is synthesized almost exclusively by the Leydig cells of the testes, small amounts bythe zona reticularis of the adrenal cortex. Androgens are involved in the differentiation of the somaticgender in the prenatal phase. If androgens are not present in this developmental phase, male genitaliaare not formed. In subsequent development of the male, testosterone promotes the development of thesecondary sex characteristics in men and serves to maintain the function of the prostate and seminalvesicles.

In women, small quantities of testosterone are formed by the thecal cells of the ovaries, by the placenta,as well as by the zona reticularis of the adrenal cortex. Most of the testosterone, however, is formedvia conversion from other androgens (dehydroepiandrosterone and androstenedione). Testosteroneis converted in the ovaries to estradiol. Female androgen deficiency is found eg. in women withsurgical menopause, with hypopituitarism, with adrenal insufficiency and in women with completeandrogen insensitivity syndrome (CAIS). Increased endogenous concentrations of androgens in womencan cause hirsutism and virilization.

In addition to gender-specific effects, testosterone also has anabolic effects.

Production rate of testosterone in men is around 5-7 mg per day, in women around 0.2-0.3 mg per day.Therefore, testosterone in men is the androgen with the highest concentration.

Testosterone in blood is present as free testosterone (FT) (1-4 %) or bound testosterone. The boundtestosterone is loosely bound to albumine, a serum protein (30-40 %) or to sex hormone bindingglobulin, SHBG (60-70 %). The affinity between testosterone and albumin is not very high and iseasily reversible. The term bioavailable testosterone (BAT) refers to the sum of free testosterone plusalbumin-bound testosterone. Testosterone bound to SHBG is biologically inactive because of thestrong affinity between SHBG and testosterone.

The secretion of testosterone is regulated by luteinizing hormone (LH), and is subject to negativefeedback via the pituitary and hypothalamus.

The determination of testosterone in women is helpful in the diagnosis of androgenic syndrome(AGS), polycystic ovaries (Stein-Leventhal syndrome) and when an ovarian tumor, adrenal tumor,adrenalhyperplasia or ovarian insufficiency is suspected.

Testosterone is determined in men when reduced testosterone production is suspected, e.g. inhypogonadism, estrogen therapy, chromosome aberrations (as in the Klinefelter’s syndrome) andliver cirrhosis.

4



Fig. 1: Human steroidogenesis, adapted from Walter F. Boron, Emile L. Boulpaep (2003). MedicalPhysiology: A Cellular And Molecular Approach, page 1300, Elsevier/Saunders

Hypothalamus

TestosteroneEstradiol

Testosterone-ABP

Spermatogenesis

GnRH

Pituitarygland

induces LH

receptors

Leydigcells

Sertolicells

inhibited inhibited

Inhibin

LH FSH

ABP

ABP = Androgen Binding Prote in

Fig. 2: Regulation of spermatogenesis

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The Elecsys Testosterone II assay is based on a competitive test principle using a high affinitymonoclonal antibody (sheep) specifically directed against testosterone. Endogenous testosteronereleased from the sample by 2-bromoestradiol competes with the added testosterone derivative labeledwith a ruthenium complexa for the binding sites on the biotinylated antibody. Compared to the ElecsysTestosterone assay, the Elecsys Testosterone II assay shows improved agreement to LC-MS/MS in thefemale concentration range.

2.2 Elecsys test principle

Competition principle. Total duration of assay: 18 minutes at 37 °C.In the first incubation step of the Elecsys Testosterone II assay 20 μL of sample are incubated with abiotinylated monoclonal testosterone-specific antibody. The binding sites of the labeled antibodybecome occupied by the sample analyte (depending on its concentration). After addition ofstreptavidin-coated microparticles and a testosterone derivative labeled with a ruthenium complex inthe second step, the complex becomes bound to the solid phase via interaction of biotin and streptavidin.The reaction mixture is aspirated into the measuring cell where the microparticles are magneticallycaptured onto the surface of the electrode. Unbound substances are then removed with ProCell.Application of a voltage to the electrode then induces chemiluminescent emission which is measuredby a photomultiplier. Results are determined via a calibration curve which is instrument-specificallygenerated by 2-point calibration and a master curve provided via the reagent barcode.

Anti-Testosterone-SMA~ Bi

Testosterone-Peptid~ Ru conjugate

Antigen in sample

9 min

Bi

9 minDetection

SA-microparticle

Fig. 3: Test principle Elecsys Testosterone II assay

Elecsys measuring cell

The electrochemiluminescence measuring cell is the most important element of the entire system. It isconstructed as flow chamber and has three main tasks:

• Separation of unbound and bound substances. The streptavidin-coated microparticles laden withimmune complexes are drawn to the surface of the electrode with the help of a magnet and held theretemporarily. Unbound reagent components and excess sample material are then removed fromthe measuring cell with ProCell system buffer.

• Generation of electrochemiluminescence. Application of a defined voltage induces theelectrochemiluminescent reaction and the resulting light emission is measured directly by thephotomultiplier.

• At the end of the electrochemiluminescent reaction the microparticles are removed with measuringcell cleaning solution (CleanCell). The measuring cell is then ready for the next measurement(figure 4).

a) Tris(2,2’-bipyridyl)ruthenium(II)-complex (Ru(bpy)2+3

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Photomultiplier

Working electrode

Magnet

UnboundRu-label

Direction offlow

Counter elec-trodes

Microparticles withbound immuno-complexes

Fig. 4: Elecsys measuring cell

Electrochemiluminescence detection: reaction scheme8,9,10,11,12,13

The electrochemiluminescent label is a ruthenium complex (Ru(bpy)2+3 ). In the electrochemical

reaction (Ru(bpy)2+3 ) is first oxidized to Ru(bpy)3+

3 at the surface of the electrode. At the same timetripropylamine (TPA, component of ProCell) which is present in excess, is oxidized to form the cationradical TPA+* which spontaneously loses a proton. The powerful oxidant Ru(bpy)3+

3 reacts with thefree radical TPA*, a strong reductant, to form the excited state of the ruthenium complex, Ru(bpy)2+

3 ).This returns to the ground state by emitting a photon at 620 nm and is then again available for a newlight-generating reaction cycle (figure 5).

e

e

TPA+*

TPA

- H+

TPA*

e

Ru(bpy)2+

Groundstate

Photon(620nm)

Ru(bpy)3+

-

-

-

3

3

Ru(bpy)2+

Excited state3

Fig. 5: Reaction scheme

7



Measurement

A photomultiplier measures the intensity of the signal in RLU (Relative Light Units). The voltageapplication phase lasts 1.4 seconds. For measurement purposes the signal produced over a period of 0.4seconds (0.02 – 0.42 s) is integrated. Part of the voltage application phase showing this measurementwindow is presented in figure 6.

Time [s]

Voltage [V]RLU intensity

Fig. 6: RLU intensity

Evaluation

The results are determined on the basis of a calibration curve.

Concentration

RLU

Sandwich assay

RLU

Concentration

Competitive assay

Fig. 7: Signal concentration curve

8



3 Elecsys Testosterone II assay testing procedure

3.1 Materials provided, materials required (but not provided)

Please find detailed information in the current instruction for use.

3.2 Assay procedure

For optimum performance of the assay follow the directions given in this document for the analyzerconcerned. Refer to the appropriate operator’s manual for analyzer-specific assay instructions.Resuspension of the microparticles takes place automatically before use. Read in the test-specificparameters via the reagent barcode. If in exceptional cases the barcode cannot be read, enter the 15-digitsequence of numbers. The information contained in the barcodes is shown in figure 8.

Master curve (dependent on test principle)Test parameter Test data Application data Calibration data Quality control data Stability data - reagent - calibration

Reagent identification

2-D bar code Bottle bar code

Fig. 8: Calibration concept

MODULAR ANALYTICS E170 and cobas e 601 analyzers: PreClean M solution is necessary.

MODULAR ANALYTICS E170, Elecsys 2010 and cobas e analyzers:Bring the cooled reagents to approx. 20 °C and place on the reagent disk (20 °C) of the analyzer. Avoidthe formation of foam. The system automatically regulates the temperature of the reagents and theopening/closing of the bottles.

Calibrators:Elecsys Testosterone II CalSet II consists of lyophilized human serum with added testosterone in twoconcentration ranges. The CalSet can be used with all reagent lots.

The exact lot-specific calibrator values are encoded in the barcode as well as printed on the enclosed (orelectronically available) calibrator barcode sheet.

Dissolve carefully the contents of one bottle by adding exactly 1.0 mL of distilled or deionized water andallow to stand closed for 15 minutes to reconstitute. Mix carefully, avoiding the formation of foam.Transfer aliquots of the reconstituted calibrator into empty labeled snap-cap bottles (CalSet Vials).Attach the supplied labels to the additional bottles. Store the aliquots immediately at -20 °C.Perform only one calibration procedure per aliquot.

Store the calibrators at 2-8 °C. The lyophilized calibrators are stable up to the stated expiration date.Stability of the reconstituted calibrators:

• at -20 °C 3 months (freeze only once)

• on the analyzers at 20-25 °C use only once

For use on the analyzers, place the reconstituted calibrators (in the system-compatible bottles withbarcoded labels) in the sample zone. Read in all the information necessary for calibrating the assay;these data are encoded in the supplied barcodes, the reagent barcode, and the barcode on the calibratorbottle label. Ensure the calibrators are at ambient temperature (20-25 °C) before measurement.

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3.3 Calibration

3.3.1 Standardization — traceability

This method has been standardized via ID-GC/MS (“Isotope Dilution - Gas Chromatography/MassSpectrometry”)14,15 with the following results for samples from male:16Passing/Bablok17,18,19: y = 1.016x - 0.027, τ = 0.928Linear regression: y = 1.012x - 0.003, r = 0.992

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

ID-GC/ MS (ng/ mL)

Elec

sys

Test

ost

ero

ne

II

(n

g/m

L)

Fig. 9: Elecsys Testosterone vs ID-GC/MS, primary calibration

Detail of figure 9 for testosterone samples < 2 ng/mL16

Passing/Bablok: y = 0.959x + 0.005, τ = 0.780Linear regression: y = 0.969x + 0.007, r = 0.992

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

ID-GC/ MS (ng/ mL)

Elec

sys

Test

ost

ero

ne

II

(ng

/mL)

Fig. 10: Elecsys Testosterone II vs ID-GC/MS, primary calibration (values < 2 ng/mL)

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Every Elecsys Testosterone II reagent set has a barcoded label containing the specific information forcalibration of the particular reagent lot. The predefined master curve is adapted to the analyzer by theuse of Elecsys Testosterone II CalSet II.

3.3.2 Calibration frequency

Calibration must be performed once per reagent lot using fresh reagent (i.e. not more than 24 hourssince the reagent kit was registered on the analyzer). Renewed calibration is recommended as follows:

• after 1 month (28 days) when using the same reagent lot

• after 7 days (when using the same reagent kit on the analyzer)

• as required: e.g. quality control findings outside the specified limits

3.4 Quality control

For quality control, use Elecsys PreciControl Universal 1 and 2, which contains lyophilized controlserum based on human serum in two concentration ranges. The controls are used for monitoring theaccuracy and precision of Elecsys immunoassays.The exact lot-specific target values and ranges are encoded in the barcodes as well as printed on theenclosed (or electronically available) value sheet.The target values and ranges were determined and evaluated by Roche. They were obtained using theElecsys assay reagents and analyzers available at the time. If the target values and control ranges areupdated at a later time, this information is conveyed via the reagent barcodes and on an additional valuesheet included in the reagent kit. This value sheet lists all control lots to which the new values apply. Ifsome of the values remain unchanged, the original values conveyed via the CBC (Control Barcode) andon the value sheet included in the control kit (or provided electronically), continue to be valid. Resultsmust be within the corresponding specified ranges. All test steps must be checked when increasing ordecreasing trends or suddenly occurring deviations beyond the range limits are seen.Other suitable control material can be used in addition. Controls for the various concentration rangesshould be run as single determinations at least once every 24 hours when the test is in use, once perreagent kit, and after every calibration. The control intervals and limits should be adapted to eachlaboratory’s individual requirements. Values obtained should fall within the defined limits. Eachlaboratory should establish corrective measures to be taken if values fall outside the limits. Follow theapplicable government regulations and local guidelines for quality control.

HandlingDissolve carefully the contents of one bottle by adding exactly 3.0 mL of distilled water and allow to standclosed for 30 minutes to reconstitute. Mix carefully, avoiding the formation of foam. In accordance withthe individual laboratory requirements, transfer the reconstituted control immediately into the empty,labeled snap-cap bottles supplied (ControlSet Vials) and freeze aliquots in additional ControlSet Vials.Attach the supplied labels to these additional bottles. Perform only one control procedure per aliquot.

Storage and stabilityStore at 2-8 °C.The lyophilized control serum is stable up to the stated expiration date.

Storage and stability in the reconstituted control serum:

at -20 °C 1 month (freeze only once)

or at 2-8 °C 3 days

on the analyzers at 20-25 °C up to 5 hours

11



3.5 Calculation

The analyzer automatically calculates the analyte concentration of each sample (either in ng/mL, ng/dLor nmol/L).

Conversion factors:

• ng/mL x 3.47 = nmol/L

• ng/mL x 100 = ng/dL

• nmol/L x 0.288 = ng/mL

3.6 Limitations – interference

The assay is unaffected by the following:16

Substance Tested up to No effects up to

Bilirubin 66 mg/dL 30 mg/dL

Hemoglobin 1000 mg/dL 600 mg/dL

Intralipid 2000 mg/dL < 1000 mg/dL

Biotin 70 ng/mL 30 ng/mL

Rheumatic factor 1500 IU/mL 1000 IU/mL

Criterion: Recovery within ± 10 % of initial value (concentration range > 1-15 ng/mL), recoverywithin ± 15 % of initial value (concentration range > 0.5-1 ng/mL) and recovery of ± 0.075 ng/mL(concentration range of 0.150-0.500 ng/mL).In patients receiving therapy with high biotin doses (i.e. > 5 mg/day), no sample should be taken until atleast 8 hours after the last biotin administration.

In vitro tests were performed on 18 commonly used pharmaceuticals. Three special drugs wereadditionally tested. A strong interaction with Nandrolone (INN international nonproprietary name,WHO) was found. Do not use samples from patients under Nandrolone treatment. For the other 20pharmaceuticals, no interference with the assay was found.In isolated cases, elevated testosterone levels can be seen in samples from female patients with endstage renal disease (ESRD).

Active agentConc.tested(mg/L)

Active agentConc.tested(mg/L)

1 Acetylcystein 150 11 Metronidazole 200

2 Ampicillin-Na 1000 12 Phenylbutazone 400

3 Ascorbic acid 300 13 Doxycyclin 50

4 Ca-Dobesilate 200 14 Acetylsalicylic Acid 1000

5 Cyclosporine 5 15 Rifampicin 60

6 Cefoxitin 2500 16 Acetaminophen 200

7 Heparin 5000 U 17 Ibuprofen 500

8 Intralipid 10000 18 Theophyllin 100

9 Levodopa 20 Special 1 Heparin Clexane 5000 U

10 Methyldopa 20 Special 2 Dexamethason 20

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In rare cases, interference due to extremely high titers of antibodies to analyte-specific antibodies,streptavidin or ruthenium can occur. These effects are minimized by suitable test design.

For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medicalhistory, clinical examination and other findings. Implausible elevated testosterone values in womenshould be verified by an extraction method or a validated LC-MS/MS tandem method.5

3.7 Measuring range

0.025-15.0 ng/mL or 0.087-52.0 nmol/L (defined by the limit of detection and the maximum of themaster curve). Values below the limit of detection are reported as < 0.025 ng/mL or < 0.087 nmol/L.Values above the measuring range are reported as > 15.0 ng/mL or > 52.0 nmol/L.

3.8 Dilution

Generally not necessary due to the broad measuring range.

3.9 Expected values

The following table shows the results obtained with the Elecsys Testosterone II assay in an apparentlyhealthy group of 214 males and 160 females without intake of contraceptiva and prescription drugs(study number CIM 000669). Blood samples were taken between 6.30 am and 1.00 p.m. This clinicalstudy with focus on the Elecsys Testosterone II assay included measurements in parallel with the ElecsysSHBG assay. The results were evaluated for the Elecsys Testosterone II and Elecsys SHBG assays andcommonly used parameters derived from different calculation procedures, including albumin as animportant parameter involved.20

• Free testosterone index (% FTI) or free androgen index (% FAI) as calculated on a molar/molar basis:FTI (%) = (testosterone in nmol/L divided by SHBG in nmol/L) x 100

• Free testosterone calculated (FTc) in nmol/L and %

• Bioavailable testosterone calculated (BATc) in nmol/L and %

FTc and BATc were calculated by means of individual concentrations for total testosterone, SHBG, andalbumin and via the association constant of albumin to testosterone. A detailed description of thecalculation procedure is available on request. Refer also to the homepage of www.issam.ch/freetesto.htm.

Each laboratory should investigate the transferability of the expected values to its own patientpopulation and if necessary determine its own reference ranges.

The following results were obtained:16

Percentiles

Median 5-95th Median 5-95thTest subjects N

ng/mL nmol/L

Males20-49 years 136 5.36 2.49-8.36 18.6 8.64-29.0

Males≥ 50 years 78 4.76 1.93-7.40 16.5 6.68-25.7

Females20-49 years 89 0.271 0.084-0.481 0.941 0.290-1.67

Females≥ 50 years 71 0.162 0.029-0.408 0.563 0.101-1.42

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Figure 11 shows the distribution of testosterone values in the apparently healthy male group based onage (n = 214). Solid line: 50 % percentile, upper line: 95 % percentile, lower line: 5 % percentile.

Ele

csys

Te

sto

ste

ron

e I

I -

ma

le(n

g/m

L)

Age (yea rs )

0

2

4

6

8

10

12

20 25 30 35 40 45 50 55 60 65 70

Fig. 11: Distribution of testosterone values in the apparently healthy male group

Figure 12 shows the distribution of testosterone values in the apparently healthy female group based onage (n = 160). Solid line: 50 % percentile, upper line: 95 % percentile, lower line: 5 % percentile.

Ele

csys

Te

sto

ste

ron

e I

I -

fem

ale

(ng

/mL)

Age (yea rs )

0.0

0.1

0.2

0.3

0.4

0.5

0.6

20 25 30 35 40 45 50 55 60 65 70

Fig. 12: Distribution of testosterone values in the apparently healthy female group

14



SHBG

Percentiles

Median 5-95thTest subjects N

nmol/L

Males 20-49 years 136 33.5 16.5-55.9

Males ≥ 50 years 78 40.8 19.3-76.4

Females 20-49 years 89 64.3 24.6-122

Females ≥ 50 years 71 57.4 17.3-125

Free testosterone index or free androgen index

Percentiles

Median 5-95thTest subjects N

FTI or FAI (%)

Males 20-49 years 136 57.2 35.0-92.6

Males ≥ 50 years 78 38.2 24.3-72.1

Females 20-49 years 89 1.53 0.297-5.62

Females ≥ 50 years 71 1.15 0.187-3.63

Free testosterone, calculated

Percentiles


FTc (nmol/L) FTc (%)

Males20-49 years 136 0.379 0.198-0.619 2.10 1.53-2.88

Males≥ 50 years 78 0.304 0.163-0.473 1.91 1.23-2.59

Females20-49 years 89 0.011 0.003-0.033 1.19 0.701-2.19

Females≥ 50 years 71 0.008 0.001-0.020 1.26 0.685-2.64

15



Bioavailable testosterone, calculated

Percentiles


BATc (nmol/L) BATc (%)

Males20-49 years 136 9.10 4.36-14.3 49.8 35.0-66.3

Males≥ 50 years 78 6.63 3.59-11.0 42.1 27.5-60.7

Females20-49 years 89 0.246 0.059-0.756 25.7 15.3-47.7

Females≥ 50 years 71 0.168 0.030-0.430 28.0 15.1-55.2

3.9.1 Tanner stages

The following table shows the Tanner stages for males for the Elecsys Testosterone II assay (CIM StudyNo: RD000669; Leipzig).16

Percentiles

Median 5-95thTanner stage N

ng/mL

1 26 < 0.025 < 0.025

2 18 0.597 < 0.025 - 4.32

3 15 2.45 0.648 - 7.78

4 16 3.44 1.80 - 7.63

5 20 4.46 1.88 - 8.82

The table below shows the Tanner stages for females for the Elecsys Testosterone II assay (CIM StudyNo: RD000669; Leipzig).16

Percentiles

Median 5-95thTanner stage N

ng/mL

1 37 < 0.025 < 0.025 - 0.061

2 12 < 0.025 < 0.025 - 0.104

3 12 0.079 < 0.025 - 0.237

4 12 0.122 < 0.025 - 0.268

5 27 0.197 0.046 - 0.383

16



4 Specific performance data of the test

Representative performance data on the analyzers are given below. Results obtained in individuallaboratories may differ.

4.1 Precision

Precision was determined using Elecsys reagents, human sera and controls in a protocol (EP5-A2) ofthe CLSI (Clinical and Laboratory Standards Institute): 2 runs per day in duplication each for 21 days(n = 84). The following results were obtained:16

Elecsys 2010 and cobas e 411 analyzers

Repeatabilityb Intermediate precision

Mean SD CV SD CVSample

ng/mL nmol/L ng/mL nmol/L % ng/mL nmol/L %

HSc 1 0.095 0.330 0.004 0.014 4.7 0.008 0.028 8.4

HS 2 0.691 2.40 0.014 0.048 2.1 0.022 0.076 3.2

HS 3 2.16 7.50 0.042 0.146 1.9 0.060 0.208 2.8

HS 4 8.67 30.1 0.229 0.795 2.6 0.243 0.843 2.8

HS 5 13.0 45.1 0.158 0.548 1.2 0.440 1.53 3.4

PC Ud1 6.30 21.9 0.088 0.305 1.4 0.182 0.632 2.9

PC U2 2.65 9.20 0.047 0.163 1.8 0.097 0.337 3.7

MODULAR ANALYTICS E170 and cobas e 601 analyzers

Repeatability Intermediate precision

Mean SD CV SD CVSample

ng/mL nmol/L ng/mL nmol/L % ng/mL nmol/L %

HS 1 0.091 0.316 0.014 0.049 14.8 0.017 0.059 18.1

HS 2 0.696 2.42 0.029 0.097 4.1 0.030 0.104 4.4

HS 3 2.13 7.39 0.059 0.017 2.8 0.067 0.232 3.2

HS 4 8.79 2.53 0.236 0.833 2.7 0.292 1.01 3.3

HS 5 13.1 45.8 0.281 0.081 2.1 0.331 1.15 2.5

PC U1 6.08 21.1 0.179 0.625 2.9 0.190 0.659 3.1

PC U2 2.56 8.88 0.067 0.229 2.6 0.093 0.323 3.6

b) Repeatability = within-run precisionc) HS = human serumd) PC U = PreciControl Universal

17



Figure 13 shows 6 human sera pools with different concentration ranges measured in 6 labs on1 cobas e 411, 2 Elecsys 2010 and 3 MODULAR ANALYTICS E170 analyzers; 21 times during MCEstudy.

0

2

4

6

8

10

12

0 2 4 6 8 10 12 14 16

Mean Va lues Tes tos te rone [ng/ mL]

CV

[%

]

Elecsys Testosterone on cobas e 411 & Elecsys 2010

Elecsys Testosterone II on cobas e 411 & Elecsys 2010

Elecsys Testosterone on MODULAR ANALYTICS E170

Elecsys Testosterone II on MODULAR ANALYTICS E170

Repeatability data (n = 21)

Fig. 13: Repeatability - MCE-Study

Figure 14 shows 6 human sera pools with different concentration ranges measured in 6 labs on1 cobas e 411, 2 Elecsys 2010 and 3 MODULAR ANALYTICS E170 analyzers; 2 replicates in 2 seriesduring MCE study.

0

4

8

12

16

20

0 2 4 6 8 10 12 14 16

Mean Va lues Tes tos te rone [ng/ mL]

CV

[%

]

Within - laboratory (�to tal�) precision da ta

Elecsys Testosterone on cobas e 411 & Elecsys 2010

Elecsys Testosterone II on cobas e 411 & Elecsys 2010

Elecsys Testosterone on MODULAR ANALYTICS E170

Elecsys Testosterone II on MODULAR ANALYTICS E170

Fig. 14: Intermediate precision - MCE-Study

18



4.2 Analytical sensitivity

Limit of Blank (LoB), Limit of Detection (LoD) and Limit of Quantitation (LoQ)

• Limit of Blank: = 0.012 ng/mL or 0.042 nmol/L

• Limit of Detection: = 0.025 ng/mL or 0.087 nmol/L

• Limit of Quantitation: = 0.120 ng/mL or 0.416 nmol/L

The limit of blank and limit of detection were determined in accordance with the CLSI (Clinical andLaboratory Standards Institute) EP17-A requirements. The limit of quantitation was determined usingthe result of functional sensitivity testing.The limit of blank is the 95th percentile value from n ≥ 60 measurements of analyte-free samplesover several independent series. The limit of blank corresponds to the concentration below whichanalyte-free samples are found with a probability of 95 %.The limit of detection is determined based on the limit of blank and the standard deviation of lowconcentration samples.The limit of detection corresponds to the lowest analyte concentration which can be detected (valueabove the limit of blank with a probability of 95 %).The limit of quantitation (functional sensitivity) is the lowest analyte concentration that can bereproducibly measured with a intermediate precision coefficient of variation of ≤ 20 %. It has beendetermined using low concentration testosterone samples.

4.3 Analytical specificity

For the antibody derivative used, the following cross-reactivities were found (in %).16

Substance Conc.(ng/mL)

Cross-reactivity(%)

Substance Conc.(ng/mL)

Cross-reactivity(%)

Androstendione 100 ≤ 2.5 Ethisterone 1000 ≤ 2.40

Cortisol 1000 ≤ 0.01 Norgestrel 1000 ≤ 0.91

Cortisone 2000 n.d.e Testosteronepropionate 100 ≤ 2.46

Danazol 1000 ≤ 0.5 5-α-Androstane-3β,17β-diol 1000 ≤ 2.11

Dexamethasone 2000 n.d. 5-α-Dihydro-testosterone 500 ≤ 0.86

DHEA 1000 ≤ 0.016 11-β-Hydroxy-testosterone 100 ≤ 18.0

DHEA-S 50000 ≤ 0.003 11-Keto-testosterone 1000 ≤ 3.22

D-5-Androstene-3β,17β-diol 1000 ≤ 0.29 Prednisone 1000 n.d.

Estradiol 1000 ≤ 0.16 Prednisolone 1000 ≤ 0.002

Estrone 1000 ≤ 0.004 Progesterone 1000 n.d.

e) n.d. = not detectable

19



4.4 Method comparison16

4.4.1 Comparison Elecsys Testosterone and Elecsys Testosterone II to LC-MS/MS

Figure 15 and 16 presents the comparison of the Elecsys Testosterone and the Elecsys Testosterone II toLC-MS/MS (Roche internal method: liquid chromatography tandem mass spectroscopy). Ratio plotsconfirm the improved results in accuracy of samples from females with the Elecsys Testosterone II(figure 16) if compared to LC-MS/MS.The ratio plots show a comparison of the Elecsys Testosterone (figure 15) and Elecsys Testosterone II(figure 16) with LC-MS/MS using UK-NEQAS female and male ring trial samples (UK NEQAS Distr.334-343, 345-348; 33 females and 40 samples from males; no spiked serum samples). The x-axis (inng/mL) represents the testosterone concentration measured by LC-MS/MS; the y-axis represents theimmunoassay testosterone concentration divided by the LC-MS/MS testosterone concentration.Ratio of 1 means equal results of both methods.

0.0

0.5

1.0

1.5

2.0

2.5

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

LC-MS/ MS

Ele

csys

Te

sto

ste

ron

e /

LC

-MS

/MS

Fig. 15: Elecsys Testosterone vs LC-MS/MS

Ele

csys

Te

sto

ste

ron

e I

I /

LC-M

S/M

S

0.0

0.5

1.0

1.5

2.0

2.5

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

LC-MS/ MS

Fig. 16: Elecsys Testosterone II vs LC-MS/MS

20



4.4.2 Elecsys Testosterone vs Elecsys Testosterone II

The comparison between Elecsys Testosterone (x) and Elecsys Testosterone II (y) on cobas e 411 andElecsys 2010 analyzers show a good comparability in 239 samples from males with the following results:Number of samples measured (N): 239Passing/Bablok: y = 0.977x + 0.032; τ = 0.870

0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14

Elecsys Tes tos te rone (ng/ mL)on cobas e 411 & Elecsys 2010

Passing/Babloky = 0.977x + 0.032md(95) = 0.655N = 239; r = 0.985τ = 0.870

Ele

csys

Te

sto

ste

ron

e I

I (n

g/m

L)on

co

ba

s e

411

& E

lecs

ys 2

010

Fig. 17: Elecsys Testosterone vs Elecsys Testosterone II, male (MCE)

Figure 18 shows the comparison between Elecsys Testosterone (x) and Elecsys Testosterone II (y) onElecsys 2010 with 149 samples from females for the range up to 2 ng/mL.

0.0

0.5

1.0

1.5

2.0

0.0 0.5 1.0 1.5 2.0

Elecsys Tes tos te rone (ng/ mL)on Elecsys 2010

Ele

csys

Te

sto

ste

ron

e I

I (n

g/m

L)on

Ele

csys

201

0


Fig. 18: Elecsys Testosterone vs Elecsys Testosterone II, female (MCE)

Average value of samples from females (slope in Passing/Bablok regression) in the Elecsys Testosterone IIassay shows approximately 30 % lower results when compared to the “old” Elecsys Testosteroneimmunoassay.

21



4.5 Competitor comparison

4.5.1 Elecsys Testosterone II vs Abbott AxSym

Figure 19 shows the comparison between Elecsys Testosterone II (x) and Abbott Testosterone onAxSym (y) with 152 samples from males.

Elecsys Tes tos te rone II (ng/ mL)

Ab

bo

tt A

xSym

Test

ost

ero

ne

(n

g/m

L)


0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14

Fig. 19: Elecsys Testosterone II vs Abbott AxSym, male (MCE Study)

Figure 20 shows the comparison between Elecsys Testosterone II (x) and Abbott Testosterone onAxSym (y) with 53 samples from females for the range up to 2 ng/mL.

Elecsys Tes tos te rone II (ng/ mL)

Ab

bo

tt A

xSym

Test

ost

ero

ne

(n

g/m

L)

Passing/Babloky = 1.026x - 0.007md(95) = 0.299N = 53; r = 0.873τ = 0.830

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

Fig. 20: Elecsys Testosterone II vs Abbott AxSym, female (MCE Study)

22



4.5.2 Elecsys Testosterone II vs Beckman Access

Figure 21 shows the comparison between Elecsys Testosterone II (x) and Beckman Access (y) with152 samples from males.

Elecsys Tes tos te rone II(ng/ mL)

Be

ckm

an

Acc

ess

Test

ost

ero

ne

(n

g/m

L)


0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14

Fig. 21: Elecsys Testosterone II vs Beckman Access, male (MCE Study)

Figure 22 shows the comparison between Elecsys Testosterone II (x) and Beckman Access (y) with53 samples from females for the range up to 2 ng/mL.


Be

ckm

an

Acc

ess

Test

ost

ero

ne

(n

g/m

L)


0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

Fig. 22: Elecsys Testosterone II vs Beckman Access, female (MCE Study)

23



4.5.3 Elecsys Testosterone II vs Siemens Centaur

Figure 23 shows the comparison between Elecsys Testosterone II (x) and Siemens Centaur Testosterone(y) with 152 samples from males.


Sie

me

ns

Ce

nta

ur

Test

ost

ero

ne

(n

g/m

L)


0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14

Fig. 23: Elecsys Testosterone II vs Siemens Centaur, male (MCE Study)

Figure 24 shows the comparison between Elecsys Testosterone II (x) and Siemens Centaur Testosterone(y) with 53 samples from females for the range up to 2 ng/mL.


Sie

me

ns

Ce

nta

ur

Test

ost

ero

ne

(n

g/m

L)


0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

Fig. 24: Elecsys Testosterone II vs Siemens Centaur, female (MCE Study)

24



4.5.4 Elecsys Testosterone II vs Siemens DPC RIA

Figure 25 shows the comparison between Elecsys Testosterone II (x) and Siemens DPC RIA (y) with152 samples from males.


Sie

me

ns

DP

C R

IATe

sto

ste

ron

e (

ng

/mL)


0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14

Fig. 25: Elecsys Testosterone II vs Siemens DPC RIA, male (MCE Study)

Figure 26 shows the comparison between Elecsys Testosterone II (x) and Siemens DPC RIA (y) with53 samples from females for the range up to 2 ng/mL.


Sie

me

ns

DP

C R

IATe

sto

ste

ron

e (

ng

/mL)


0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

Fig. 26: Elecsys Testosterone II vs Siemens DPC RIA, female (MCE Study)

25



4.5.5 Elecsys Testosterone II vs Siemens Immulite

Figure 27 shows the comparison between Elecsys Testosterone II (x) and Siemens Immulite (y) with135 samples from males.


Sie

me

ns

Imm

ulit

e 2

00

0Te

sto

ste

ron

e (

ng

/mL)


0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14

Fig. 27: Elecsys Testosterone II vs Siemens Immulite, male (MCE Study)

Figure 28 shows the comparison between Elecsys Testosterone II (x) and Siemens Immulite (y) with36 samples from females for the range up to 2 ng/mL.


Sie

me

ns

Imm

ulit

e 2

00

0Te

sto

ste

ron

e (

ng

/mL)


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

Fig. 28: Elecsys Testosterone II vs Siemens Immulite, female (MCE Study)

26



4.6 Analyzer and lot-to-lot method comparisons

4.6.1 Elecsys 2010 vs MODULAR ANALYTICS E170

Figure 29 shows the comparison between Elecsys Testosterone II on the MODULARANALYTICS E170 (x) and the Elecsys 2010 (y).16

Elecsys Tes tos te rone II (ng/ mL)on MODULAR ANALYTICS E170

Ele

csys

Te

sto

ste

ron

e I

I (n

g/m

L)o

n E

lecs

ys 2

01

0


0

2

4

6

8

10

12

14

16

18

0 2 4 6 8 10 12 14 16 18

Fig. 29: Elecsys Testosterone II on the MODULAR ANALYTICS E170 vs the Elecsys 2010

4.6.2 Lot-to-lot comparison

Figure 30 shows the comparison between the Elecsys Testosterone II P02-lot (x) and the ElecsysTestosterone II P03-lot (y).16

Elecsys Tes tos te rone II (ng/ mL)P02-lot

Ele

csys

Te

sto

ste

ron

e I

I (n

g/m

L)P

03

-lo

t


0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6 7 8

Fig. 30: Elecsys Testosterone II P02-lot vs P03-lot

27



5 References

1. Nieschlag E, Behre HM. Testosteron Action, Deficiency, Substitution. Cambridge University Press,2004. ISBN 0 521 83390 9.

2. Runnebaum B, Rabe T. Gynäkologische Endokrinologie und Fortpflanzungsmedizin SpringerVerlag 1994; Band 1:36-38,70,116 Band 1:39-40, 520-521, 593-594, 422-423. ISBN 3-540-57345-3,ISBN 3-540-57347-x.

3. Wheeler MJ. The determination of bio-available testosterone. Ann Clin Biochem 1995;32:345-357.4. Kane J, Middle J, Cawood M. Measurement of serum testosterone in women; what should we

do? Ann Clin Biochem 2007;44:5-15.5. Rosner W, Auchus RJ, Azzis R, Sluss PM, Raff H. Position Statement: Utility, Limitations, and

Pitfalls in Measuring Testosterone: An Endocrine Society Positions Statement. J Clin EndocrinolMetab 2007;92(2):404-413.

6. Arlt W. Androgen Therapy in Women. Eur J Endocrinol 2006;154(1):1-11.7. Wu AHB. Tietz Clinical Guide To Laboratory Tests. 4th Edition, WB Saunders Co, 2006:1010 pp.8. Blackburn GF, Shah HP, Kenten JH, et al. Electrochemiluminescence Detection for Development of

Immuno-assays and DNA Probe Assays for Clinical Diagnostics. Clin Chem 1991;37(9):1534-39.9. Kenten JH, Casadei J, Link J, et al. Rapid Electrochemiluminescence Assays of Polymerase Chain

Reaction Products. Clin Chem 1991;37(9):1626–1632.10. Kenten JH, Gudibande S, Link J, et al. Improved Electrochemiluminescent label for DNA Probe

Assays: Rapid Quantitative Assays of HIV-1 Polymerase Chain Reaction Products. Clin Chem1992;38(6):873–879.

11. Leland JK, Powell MJ. Electrogenerated Chemiluminescence: An Oxidative-Reduction Type ECLReaction/Sequence Using Tripropyl Amine. J Electrochem Soc 1990;137(10):3127–31.

12. Obeng YS, Bard AJ. Electrogenerated Chemiluminescence. 53 Electrochemistry and Emissionfrom Adsorbed Monolayers of a Tris (bipyridyl) ruthenium (II)-Based Surfactant on Gold and TinOxide Electrodes. Langmuir 1991;7:195-201.

13. Xu X-H, Bard AJ. Electrogenerated Chemiluminescence. 55 Emission from Adsorbed Ru(bpy)2+3

on Graphite, Platinum, and Gold. Langmuir 1994;10:2409-2414.14. Thienpont LM, De Brabandere VI, Stöckl D, De Leenheer AP. Use of cyclodextrins for

prepurification of progesterone and testosterone from human serum prior to determination withisotope dilution-gas chromatography/mass spectrometry. Anal Chem 1994;66:4116-4119.

15. Thienpont LM, Franzini C, Kratochvila J, Middle J, Ricos C, Sicekmann L. Analytical qualityspecifications for reference methods and operating specifications for networks of referencelaboratories. Recommendations of the European EQA-Organizers Working Group B. Eur J ClinChem and Clin Biochem 1995;33:949-957.

16. Data on file at Roche.17. Passing H, Bablok W, Bender R, Schneider B. A General Regression Procedure for Method

Transformation. J Clin Chem Clin Biochem 1988;26:783-790.18. Passing H, Bablok W. A New Biometrical Procedure for Testing the Equality of Measurements from

Two Different Analytical Methods. J Clin Chem Clin Biochem 1983;21:709-20.19. Passing H, Bablok W. Comparison of Several Regression Procedures for Method Comparison

Studies and Determination of Sample Sizes. J Clin Chem Clin Biochem 1984;22:431-445.20. Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation

of free testosterone in serum. J Clin Endocrinol Metab 1999;84:3666-3672.

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6 Notes

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Insert current IFU here

31



Short guide to Elecsys Testosterone II assay

Name of test Elecsys Testosterone II

Intended use Immunoassay for the in vitro quantitative determination oftestosterone in human serum and plasma.The electrochemiluminescence immunoassay „ECLIA“ isintended for use on Elecsys and cobas e immunoassayanalyzers.

Method ECLIA, competition principle

Duration of test; temperature 18 minutes, 37 °C

Evaluation quantitative

Reagents ready for use

Stability reagent 2-8 °C unopend: up to the stated expiration dateafter opening at 2-8 °C: 12 weekson the analyzers: 8 weeks

Calibrators Elecsys Testosterone II CalSet II (Cal 1 + 2), lyophilized;reconstitution necessary

Calibration frequency once per reagent lot using fresh reagent (i.e. not morethan 24 hours since the reagent kit was registered on theanalyzer)after 1 month (28 days) when using the same reagent lotafter 7 days (when using the same reagent kit on theanalyzer)as required: e.g. quality control findings outside thespecified limits

Stability calibrator lyophilized calibrators: up to the stated expiration datereconstituted calibrators: at -20 °C: 3 months (freeze onlyonce)on the analyzers at 20-25 °C: use only once

Controls Elecsys PreciControl PreciControl Universal 1 + 2,lyophilized; reconstitution necessary

Stability controls lyophilized controls: up to the stated expiration datereconstituted calibrators: at -20 °C 1 month (freeze onlyonce) oror at 2-8 °C: 3 dayson the analyzers at 20-25 °C: up to 5 hours

Sample material Serum and Li-heparin, K2- and K3-EDTA plasma

Sample volume 20 µL

Measuring range 0.025-15.0 ng/mL or 0.087-52.0 nmol/L

LoB = 0.012 ng/mL or 0.042 nmol/L

LoD = 0.025 ng/mL or 0.087 nmol/L

LoQ = 0.120 ng/mL or 0.416 nmol/L

32



Laboratory-specific info

Laboratory-specific reference ranges

Critical values

Measures to be taken if critical values occur

Back-up method

Confirmatory tests

Checked by

Name Name

Function Function

Date Date

Name Name

Function Function

Date Date

33



Edition: 2010–03

ELECSYS, MODULAR, COBAS, COBAS E and LIFE NEEDS ANSWERS are trademarks of Roche.Other brand or product names are trademarks of their respective holders.

© 2010, Roche Diagnostics

Roche Diagnostics GmbHSandhofer Strasse 116D-68305 MannheimGermanywww.roche.com

05900131 001 (1.0)

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