Product Information. Testosterone II. Testosterone. Elecsys 2010 system, MODULAR ANALYTICS E170 cobas e analyzers

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1 Testosterone II Testosterone Elecsys 2010 system, MODULAR ANALYTICS E170 cobas e analyzers

2 Please note: As a result of differing printing dates, it is possible that differences may occur between the information given here and that appearing in the instruction for use. In such cases the data given in the insert enclosed with the kit applies. Please also note that the product information document contains 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

3 Table of contents 1 Intended use 4 2 Summary Biochemistry, clinical indications, methodology 1,2,3,4,5,6, Elecsys test principle 6 3 Elecsys Testosterone II assay testing procedure Materials provided, materials required (but not provided) Assay procedure Calibration Standardization traceability Calibration frequency Quality control Calculation Limitations interference Measuring range Dilution Expected values Tanner stages 16 4 Specific performance data of the test Precision Analytical sensitivity Analytical specificity Method comparison Comparison Elecsys Testosterone and Elecsys Testosterone II to LC-MS/MS Elecsys Testosterone vs Elecsys Testosterone II Competitor comparison Elecsys Testosterone II vs Abbott AxSym Elecsys Testosterone II vs Beckman Access Elecsys Testosterone II vs Siemens Centaur Elecsys Testosterone II vs Siemens DPC RIA Elecsys Testosterone II vs Siemens Immulite Analyzer and lot-to-lot method comparisons Elecsys 2010 vs MODULAR ANALYTICS E Lot-to-lot comparison 27 5 References 28 6 Notes 29 Short guide to Elecsys Testosterone II assay 32 3

4 1 Intended use 2 Summary 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 e immunoassay analyzers. 2.1 Biochemistry, clinical indications, methodology 1,2,3,4,5,6,7 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 by the zona reticularis of the adrenal cortex. Androgens are involved in the differentiation of the somatic gender in the prenatal phase. If androgens are not present in this developmental phase, male genitalia are not formed. In subsequent development of the male, testosterone promotes the development of the secondary sex characteristics in men and serves to maintain the function of the prostate and seminal vesicles. 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 formed via conversion from other androgens (dehydroepiandrosterone and androstenedione). Testosterone is converted in the ovaries to estradiol. Female androgen deficiency is found eg. in women with surgical menopause, with hypopituitarism, with adrenal insufficiency and in women with complete androgen insensitivity syndrome (CAIS). Increased endogenous concentrations of androgens in women can 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 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 bound testosterone is loosely bound to albumine, a serum protein (30-40 %) or to sex hormone binding globulin, SHBG (60-70 %). The affinity between testosterone and albumin is not very high and is easily reversible. The term bioavailable testosterone (BAT) refers to the sum of free testosterone plus albumin-bound testosterone. Testosterone bound to SHBG is biologically inactive because of the strong affinity between SHBG and testosterone. The secretion of testosterone is regulated by luteinizing hormone (LH), and is subject to negative feedback 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. in hypogonadism, estrogen therapy, chromosome aberrations (as in the Klinefelter s syndrome) and liver cirrhosis. 4

5 Product Information Fig. 1: Human steroidogenesis, adapted from Walter F. Boron, Emile L. Boulpaep (2003). Medical Physiology: A Cellular And Molecular Approach, page 1300, Elsevier/Saunders Hypothalamus GnRH Pituitary gland inhibited FSH LH inhibited induces LH receptors Leydig cells Testosterone Estradiol Sertoli cells ABP Inhibin Testosterone-ABP Spermatogenesis ABP = Androge n Binding Prote in Fig. 2: Regulation of spermatogenesis 5

6 The Elecsys Testosterone II assay is based on a competitive test principle using a high affinity monoclonal antibody (sheep) specifically directed against testosterone. Endogenous testosterone released from the sample by 2-bromoestradiol competes with the added testosterone derivative labeled with a ruthenium complex a for the binding sites on the biotinylated antibody. Compared to the Elecsys Testosterone assay, the Elecsys Testosterone II assay shows improved agreement to LC-MS/MS in the female 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 a biotinylated monoclonal testosterone-specific antibody. The binding sites of the labeled antibody become occupied by the sample analyte (depending on its concentration). After addition of streptavidin-coated microparticles and a testosterone derivative labeled with a ruthenium complex in the 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 magnetically captured 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 measured by a photomultiplier. Results are determined via a calibration curve which is instrument-specifically generated by 2-point calibration and a master curve provided via the reagent barcode. 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 is constructed as flow chamber and has three main tasks: Separation of unbound and bound substances. The streptavidin-coated microparticles laden with immune complexes are drawn to the surface of the electrode with the help of a magnet and held there temporarily. Unbound reagent components and excess sample material are then removed from the measuring cell with ProCell system buffer. Generation of electrochemiluminescence. Application of a defined voltage induces the electrochemiluminescent reaction and the resulting light emission is measured directly by the photomultiplier. At the end of the electrochemiluminescent reaction the microparticles are removed with measuring cell 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) 6

7 Fig. 4: Elecsys measuring cell Electrochemiluminescence detection: reaction scheme 8,9,10,11,12,13 The electrochemiluminescent label is a ruthenium complex (Ru(bpy) ). In the electrochemical reaction (Ru(bpy) ) is first oxidized to Ru(bpy) at the surface of the electrode. At the same time tripropylamine (TPA, component of ProCell) which is present in excess, is oxidized to form the cation radical TPA + * which spontaneously loses a proton. The powerful oxidant Ru(bpy) reacts with the free radical TPA*, a strong reductant, to form the excited state of the ruthenium complex, Ru(bpy) ). This returns to the ground state by emitting a photon at 620 nm and is then again available for a new light-generating reaction cycle (figure 5). e- TPA + * - H + TPA e- Ru(bpy) 3+ 3 TPA* - Ru(bpy) 2+ 3 Excited state Ru(bpy) 2+ 3 Ground state Photon (620nm) Fig. 5: Reaction scheme 7

8 Measurement A photomultiplier measures the intensity of the signal in RLU (Relative Light Units). The voltage application phase lasts 1.4 seconds. For measurement purposes the signal produced over a period of 0.4 seconds ( s) is integrated. Part of the voltage application phase showing this measurement window is presented in figure 6. Fig. 6: RLU intensity Evaluation The results are determined on the basis of a calibration curve. Fig. 7: Signal concentration curve 8

9 3 Elecsys Testosterone II assay testing procedure 3.1 Materials provided, materials required (but not provided) 3.2 Assay procedure Please find detailed information in the current instruction for use. For optimum performance of the assay follow the directions given in this document for the analyzer concerned. 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-specific parameters via the reagent barcode. If in exceptional cases the barcode cannot be read, enter the 15-digit sequence of numbers. The information contained in the barcodes is shown in figure 8. 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. Avoid the formation of foam. The system automatically regulates the temperature of the reagents and the opening/closing of the bottles. Calibrators: Elecsys Testosterone II CalSet II consists of lyophilized human serum with added testosterone in two concentration 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 (or electronically available) calibrator barcode sheet. Dissolve carefully the contents of one bottle by adding exactly 1.0 ml of distilled or deionized water and allow 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 C use only once For use on the analyzers, place the reconstituted calibrators (in the system-compatible bottles with barcoded 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 calibrator bottle label. Ensure the calibrators are at ambient temperature (20-25 C) before measurement. 9

10 3.3 Calibration Standardization traceability This method has been standardized via ID-GC/MS ( Isotope Dilution - Gas Chromatography/Mass Spectrometry ) 14,15 with the following results for samples from male: 16 Passing/Bablok 17,18,19 : y = 1.016x , τ = Linear regression: y = 1.012x , r = Fig. 9: Elecsys Testosterone vs ID-GC/MS, primary calibration Detail of figure 9 for testosterone samples < 2 ng/ml 16 Passing/Bablok: y = 0.959x , τ = Linear regression: y = 0.969x , r = Fig. 10: Elecsys Testosterone II vs ID-GC/MS, primary calibration (values < 2 ng/ml) 10

11 Every Elecsys Testosterone II reagent set has a barcoded label containing the specific information for calibration of the particular reagent lot. The predefined master curve is adapted to the analyzer by the use of Elecsys Testosterone II CalSet II Calibration frequency 3.4 Quality control Calibration must be performed once per reagent lot using fresh reagent (i.e. not more than 24 hours since 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 For quality control, use Elecsys PreciControl Universal 1 and 2, which contains lyophilized control serum based on human serum in two concentration ranges. The controls are used for monitoring the accuracy and precision of Elecsys immunoassays. The exact lot-specific target values and ranges are encoded in the barcodes as well as printed on the enclosed (or electronically available) value sheet. The target values and ranges were determined and evaluated by Roche. They were obtained using the Elecsys assay reagents and analyzers available at the time. If the target values and control ranges are updated at a later time, this information is conveyed via the reagent barcodes and on an additional value sheet included in the reagent kit. This value sheet lists all control lots to which the new values apply. If some of the values remain unchanged, the original values conveyed via the CBC (Control Barcode) and on the value sheet included in the control kit (or provided electronically), continue to be valid. Results must be within the corresponding specified ranges. All test steps must be checked when increasing or decreasing 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 ranges should be run as single determinations at least once every 24 hours when the test is in use, once per reagent kit, and after every calibration. The control intervals and limits should be adapted to each laboratory s individual requirements. Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the limits. Follow the applicable government regulations and local guidelines for quality control. Handling Dissolve carefully the contents of one bottle by adding exactly 3.0 ml of distilled water and allow to stand closed for 30 minutes to reconstitute. Mix carefully, avoiding the formation of foam. In accordance with the 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 stability Store 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 or at 2-8 C on the analyzers at C 1 month (freeze only once) 3 days up to 5 hours 11

12 3.5 Calculation The analyzer automatically calculates the analyte concentration of each sample (either in ng/ml, ng/dl or nmol/l). Conversion factors: ng/ml x 3.47 = nmol/l ng/ml x 100 = ng/dl nmol/l x = 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), recovery within ± 15 % of initial value (concentration range > ng/ml) and recovery of ± ng/ml (concentration range of ng/ml). In patients receiving therapy with high biotin doses (i.e. > 5 mg/day), no sample should be taken until at least 8 hours after the last biotin administration. In vitro tests were performed on 18 commonly used pharmaceuticals. Three special drugs were additionally 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 20 pharmaceuticals, no interference with the assay was found. In isolated cases, elevated testosterone levels can be seen in samples from female patients with end stage renal disease (ESRD). Active agent Conc. tested (mg/l) Active agent 1 Acetylcystein Metronidazole Ampicillin-Na Phenylbutazone Ascorbic acid Doxycyclin 50 Conc. tested (mg/l) 4 Ca-Dobesilate Acetylsalicylic Acid Cyclosporine 5 15 Rifampicin 60 6 Cefoxitin Acetaminophen Heparin 5000 U 17 Ibuprofen Intralipid Theophyllin Levodopa 20 Special 1 Heparin Clexane 5000 U 10 Methyldopa 20 Special 2 Dexamethason 20 12

13 3.7 Measuring range 3.8 Dilution 3.9 Expected values 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 medical history, clinical examination and other findings. Implausible elevated testosterone values in women should be verified by an extraction method or a validated LC-MS/MS tandem method ng/ml or nmol/l (defined by the limit of detection and the maximum of the master curve). Values below the limit of detection are reported as < ng/ml or < nmol/l. Values above the measuring range are reported as > 15.0 ng/ml or > 52.0 nmol/l. Generally not necessary due to the broad measuring range. The following table shows the results obtained with the Elecsys Testosterone II assay in an apparently healthy group of 214 males and 160 females without intake of contraceptiva and prescription drugs (study number CIM ). Blood samples were taken between 6.30 am and 1.00 p.m. This clinical study with focus on the Elecsys Testosterone II assay included measurements in parallel with the Elecsys SHBG assay. The results were evaluated for the Elecsys Testosterone II and Elecsys SHBG assays and commonly used parameters derived from different calculation procedures, including albumin as an important 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, and albumin and via the association constant of albumin to testosterone. A detailed description of the calculation procedure is available on request. Refer also to the homepage of Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges. The following results were obtained: 16 Test subjects Males years Males 50 years Females years Females 50 years Percentiles N Median 5-95 th Median 5-95 th ng/ml nmol/l

14 Figure 11 shows the distribution of testosterone values in the apparently healthy male group based on age (n = 214). Solid line: 50 % percentile, upper line: 95 % percentile, lower line: 5 % percentile. 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 on age (n = 160). Solid line: 50 % percentile, upper line: 95 % percentile, lower line: 5 % percentile. 0.6 Elecsys Testosterone II - female (ng/ ml) Age (years) Fig. 12: Distribution of testosterone values in the apparently healthy female group 14

15 SHBG Percentiles Test subjects N Median 5-95 th nmol/l Males years Males 50 years Females years Females 50 years Free testosterone index or free androgen index Percentiles Test subjects N Median 5-95 th FTI or FAI (%) Males years Males 50 years Females years Females 50 years Free testosterone, calculated Test subjects Males years Males 50 years Females years Females 50 years Percentiles N Median 5-95 th Median 5-95 th FTc (nmol/l) FTc (%)

16 Bioavailable testosterone, calculated Test subjects Males years Males 50 years Females years Females 50 years Percentiles N Median 5-95 th Median 5-95 th BATc (nmol/l) BATc (%) Tanner stages The following table shows the Tanner stages for males for the Elecsys Testosterone II assay (CIM Study No: RD000669; Leipzig). 16 Tanner stage N Median Percentiles ng/ml 5-95 th 1 26 < < < The table below shows the Tanner stages for females for the Elecsys Testosterone II assay (CIM Study No: RD000669; Leipzig). 16 Tanner stage N Median Percentiles ng/ml 5-95 th 1 37 < < < < < <

17 4 Specific performance data of the test 4.1 Precision Representative performance data on the analyzers are given below. Results obtained in individual laboratories may differ. Precision was determined using Elecsys reagents, human sera and controls in a protocol (EP5-A2) of the CLSI (Clinical and Laboratory Standards Institute): 2 runs per day in duplication each for 21 days (n = 84). The following results were obtained: 16 Sample Elecsys 2010 and cobas e 411 analyzers Repeatability b Intermediate precision Mean SD CV SD CV ng/ml nmol/l ng/ml nmol/l % ng/ml nmol/l % HS c HS HS HS HS PC U d PC U MODULAR ANALYTICS E170 and cobas e 601 analyzers Repeatability Intermediate precision Sample Mean SD CV SD CV ng/ml nmol/l ng/ml nmol/l % ng/ml nmol/l % HS HS HS HS HS PC U PC U b) Repeatability = within-run precision c) HS = human serum d) PC U = PreciControl Universal 17

18 Figure 13 shows 6 human sera pools with different concentration ranges measured in 6 labs on 1 cobas e 411, 2 Elecsys 2010 and 3 MODULAR ANALYTICS E170 analyzers; 21 times during MCE study. Fig. 13: Repeatability - MCE-Study Figure 14 shows 6 human sera pools with different concentration ranges measured in 6 labs on 1 cobas e 411, 2 Elecsys 2010 and 3 MODULAR ANALYTICS E170 analyzers; 2 replicates in 2 series during MCE study. Within - laboratory ( total ) precision da ta CV [%] Mean Values Testosterone [ng/ ml] 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

19 4.2 Analytical sensitivity Limit of Blank (LoB), Limit of Detection (LoD) and Limit of Quantitation (LoQ) Limit of Blank: = ng/ml or nmol/l Limit of Detection: = ng/ml or nmol/l Limit of Quantitation: = ng/ml or nmol/l The limit of blank and limit of detection were determined in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP17-A requirements. The limit of quantitation was determined using the result of functional sensitivity testing. The limit of blank is the 95th percentile value from n 60 measurements of analyte-free samples over several independent series. The limit of blank corresponds to the concentration below which analyte-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 low concentration samples. The limit of detection corresponds to the lowest analyte concentration which can be detected (value above the limit of blank with a probability of 95 %). The limit of quantitation (functional sensitivity) is the lowest analyte concentration that can be reproducibly measured with a intermediate precision coefficient of variation of 20 %. It has been determined 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) Crossreactivity (%) Substance Conc. (ng/ml) Crossreactivity (%) Androstendione Ethisterone Cortisol Norgestrel Cortisone 2000 n.d. e Testosterone propionate Danazol Dexamethasone 2000 n.d. DHEA α-Androstane-3β, 17β-diol DHEA-S Keto-testosterone α-Dihydrotestosterone 11-β-Hydroxytestosterone D-5-Androstene- 3β,17β-diol Prednisone 1000 n.d. Estradiol Prednisolone Estrone Progesterone 1000 n.d. e) n.d. = not detectable 19

20 4.4 Method comparison 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 to LC-MS/MS (Roche internal method: liquid chromatography tandem mass spectroscopy). Ratio plots confirm 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 , ; 33 females and 40 samples from males; no spiked serum samples). The x-axis (in ng/ml) represents the testosterone concentration measured by LC-MS/MS; the y-axis represents the immunoassay testosterone concentration divided by the LC-MS/MS testosterone concentration. Ratio of 1 means equal results of both methods. Fig. 15: Elecsys Testosterone vs LC-MS/MS Fig. 16: Elecsys Testosterone II vs LC-MS/MS 20

21 4.4.2 Elecsys Testosterone vs Elecsys Testosterone II The comparison between Elecsys Testosterone (x) and Elecsys Testosterone II (y) on cobas e 411 and Elecsys 2010 analyzers show a good comparability in 239 samples from males with the following results: Number of samples measured (N): 239 Passing/Bablok: y = 0.977x ; τ = τ Fig. 17: Elecsys Testosterone vs Elecsys Testosterone II, male (MCE) Figure 18 shows the comparison between Elecsys Testosterone (x) and Elecsys Testosterone II (y) on Elecsys 2010 with 149 samples from females for the range up to 2 ng/ml. 2.0 Elecsys Testosterone II (ng/ ml) on Elecsys Elecsys Testosterone (ng/ ml) on Elecsys 2010 Passing/Bablok y = 0.715x md(95) = N = 149; r = τ = 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 II assay shows approximately 30 % lower results when compared to the old Elecsys Testosterone immunoassay. 21

22 4.5 Competitor comparison Elecsys Testosterone II vs Abbott AxSym Figure 19 shows the comparison between Elecsys Testosterone II (x) and Abbott Testosterone on AxSym (y) with 152 samples from males. τ Fig. 19: Elecsys Testosterone II vs Abbott AxSym, male (MCE Study) Figure 20 shows the comparison between Elecsys Testosterone II (x) and Abbott Testosterone on AxSym (y) with 53 samples from females for the range up to 2 ng/ml τ Fig. 20: Elecsys Testosterone II vs Abbott AxSym, female (MCE Study) 22

23 4.5.2 Elecsys Testosterone II vs Beckman Access Figure 21 shows the comparison between Elecsys Testosterone II (x) and Beckman Access (y) with 152 samples from males. τ 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) with 53 samples from females for the range up to 2 ng/ml τ Fig. 22: Elecsys Testosterone II vs Beckman Access, female (MCE Study) 23

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

25 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) with 152 samples from males. τ 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) with 53 samples from females for the range up to 2 ng/ml. τ Fig. 26: Elecsys Testosterone II vs Siemens DPC RIA, female (MCE Study) 25

26 4.5.5 Elecsys Testosterone II vs Siemens Immulite Figure 27 shows the comparison between Elecsys Testosterone II (x) and Siemens Immulite (y) with 135 samples from males. τ 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) with 36 samples from females for the range up to 2 ng/ml τ Fig. 28: Elecsys Testosterone II vs Siemens Immulite, female (MCE Study) 26

27 4.6 Analyzer and lot-to-lot method comparisons Elecsys 2010 vs MODULAR ANALYTICS E170 Figure 29 shows the comparison between Elecsys Testosterone II on the MODULAR ANALYTICS E170 (x) and the Elecsys 2010 (y). 16 τ Fig. 29: Elecsys Testosterone II on the MODULAR ANALYTICS E170 vs the Elecsys Lot-to-lot comparison Figure 30 shows the comparison between the Elecsys Testosterone II P02-lot (x) and the Elecsys Testosterone II P03-lot (y) τ Fig. 30: Elecsys Testosterone II P02-lot vs P03-lot 27

28 5 References 1. Nieschlag E, Behre HM. Testosteron Action, Deficiency, Substitution. Cambridge University Press, ISBN Runnebaum B, Rabe T. Gynäkologische Endokrinologie und Fortpflanzungsmedizin Springer Verlag 1994; Band 1:36-38,70,116 Band 1:39-40, , , ISBN , ISBN x. 3. Wheeler MJ. The determination of bio-available testosterone. Ann Clin Biochem 1995;32: Kane J, Middle J, Cawood M. Measurement of serum testosterone in women; what should we do? Ann Clin Biochem 2007;44: 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 Endocrinol Metab 2007;92(2): Arlt W. Androgen Therapy in Women. Eur J Endocrinol 2006;154(1): 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): Kenten JH, Casadei J, Link J, et al. Rapid Electrochemiluminescence Assays of Polymerase Chain Reaction Products. Clin Chem 1991;37(9): 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 Chem 1992;38(6): Leland JK, Powell MJ. Electrogenerated Chemiluminescence: An Oxidative-Reduction Type ECL Reaction/Sequence Using Tripropyl Amine. J Electrochem Soc 1990;137(10): Obeng YS, Bard AJ. Electrogenerated Chemiluminescence. 53 Electrochemistry and Emission from Adsorbed Monolayers of a Tris (bipyridyl) ruthenium (II)-Based Surfactant on Gold and Tin Oxide Electrodes. Langmuir 1991;7: Xu X-H, Bard AJ. Electrogenerated Chemiluminescence. 55 Emission from Adsorbed Ru(bpy) on Graphite, Platinum, and Gold. Langmuir 1994;10: 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 with isotope dilution-gas chromatography/mass spectrometry. Anal Chem 1994;66: Thienpont LM, Franzini C, Kratochvila J, Middle J, Ricos C, Sicekmann L. Analytical quality specifications for reference methods and operating specifications for networks of reference laboratories. Recommendations of the European EQA-Organizers Working Group B. Eur J Clin Chem and Clin Biochem 1995;33: 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: 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: 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: 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:

29 6 Notes 29

30

31 Insert current IFU here 31

32 Short guide to Elecsys Testosterone II assay Name of test Intended use Method Elecsys Testosterone II 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 e immunoassay analyzers. ECLIA, competition principle Duration of test; temperature 18 minutes, 37 C Evaluation Reagents Stability reagent Calibrators Calibration frequency Stability calibrator quantitative ready for use 2-8 C unopend: up to the stated expiration date after opening at 2-8 C: 12 weeks on the analyzers: 8 weeks Elecsys Testosterone II CalSet II (Cal 1 + 2), lyophilized; reconstitution necessary once per reagent lot using fresh reagent (i.e. not more than 24 hours since the reagent kit was registered on the analyzer) 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 lyophilized calibrators: up to the stated expiration date reconstituted calibrators: at -20 C: 3 months (freeze only once) on the analyzers at C: use only once Controls Elecsys PreciControl PreciControl Universal 1 + 2, lyophilized; reconstitution necessary Stability controls Sample material Sample volume 20 µl Measuring range LoB LoD LoQ lyophilized controls: up to the stated expiration date reconstituted calibrators: at -20 C 1 month (freeze only once) or or at 2-8 C: 3 days on the analyzers at C: up to 5 hours Serum and Li-heparin, K2- and K3-EDTA plasma ng/ml or nmol/l = ng/ml or nmol/l = ng/ml or nmol/l = ng/ml or nmol/l 32

33 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 Function Date Name Function Date Name Function Date Name Function Date 33

34 Edition: 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 GmbH Sandhofer Strasse 116 D Mannheim Germany (1.0) 34