Evaluation of direct and indirect markers to assess the androgen status in healthy males during aging.

Evaluation of the male androgen status requires a marker that reflects the biologically active fraction of plasma testosterone. The serum sex hormone-binding globulin (SHBG) concentration is not suitable here because of its wide inter-individual scatter. As potential biological markers of the active testosterone fraction we compared indirect methods calculated on the basis of SHBG and total testosterone measured by fully automated IMMULITE 2000 assays (DPC, Los Angeles, CA, USA), and total testosterone alone with direct free testosterone measured by RIA (DPC). Indirect methods were the free androgen index FAI, calculated free testosterone cFT, and calculated bio-available testosterone cBT. Further androgens measured were DHEAS and androstenedione. Blood samples were collected from a cohort of 446 healthy men aged between 20-99 years. All parameters except SHBG decreased significantly during aging. The direct free testosterone assay was significantly correlated with the indirect androgen parameters. This is in accordance with earlier results using LC-MS as the gold standard method. The strongest correlation was seen with cBT/measured albumin (r=0.750), though the direct testosterone RIA does not measure the entire unbound fraction of testosterone, and total testosterone can rapidly be measured with an automated assay system. It was found that a fixed albumin concentration of 43 g/L is a reasonable calculation basis for cBT in subjects of <70 years. In the elderly >70 years or persons with known pathologies of the androgen axis, it is commendable to measure the albumin concentration individually. In conclusion, calculated bio-available testosterone (cBT) is the best marker to reflect the bioactive testosterone fraction, i.e. the androgen status in males.

Reference intervals for testosterone, androstenedione and SHBG levels in healthy females and males from birth until old age.

The measurement of androgen levels is important in the follow-up of sexual development and in the diagnosis of disturbances of the gonadal function in children and adults. The aim of this study was to evaluate the age dependence of the serum concentrations of testosterone, androstenedione, and SHBG from birth until old age using the IMMULITE 2000 automated assay system (DPC, Los Angeles). Testosterone and androstenedione median levels were very high during the first weeks of life due to residual maternal hCG and decreased to low basal levels around the detection limit of the assay. With the onset of puberty around the age of 10 years both parameters increased strongly, reaching a maximum at about 17 years (testosterone: > 20-fold in boys, 2-fold in girls; androstenedione: 10-fold in boys, 5-fold in girls). In both girls and boys, we measured a decline in the SHBG medians during sexual maturation. This decline was more pronounced in boys (median 78.3 to 26.2 nmol/l from Tanner stage 1 to 5) since the higher androgen levels are thought to down-regulate SHBG. In male adults a continuous decrease was seen for testosterone from a median of 16.1 nmol/l in age group 21-30 years to 9.7 nmol/l in the age group > 70 years. In women the testosterone levels which were only about 5% of that of men from the same age group decreased only slightly, starting from a median of 0.9 to 0.6 nmol/l. In both sexes androstenedione levels decreased continuously during aging. In contrast to the androgen levels, the median SHBG levels increased steadily in men from 20.8 to 44.5 nmol/l, while the median SHBG levels in women decreased from 78.3 to 44.5 nmol/l in the age group of 61-70 years. Interestingly, the SHBG levels rose again in women of the group > 70 years. The reference intervals elaborated here may help in the assessment of the status of sexual development, and to diagnose pathologies of the gonadal axis or hypogonadism during aging.

Stability of insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3 measured by the IMMULITE automated chemiluminescence assay system in different blood specimens.

Insulin-like growth factor I (IGF-I) and IGF binding protein 3 (IGFBP-3) are measured to diagnose disorders of the somatotropic axis in children and adults. In clinical studies samples for IGF-I and IGFBP-3 measurement must often be stored for months and sent to specialized laboratories. Therefore, we tested the stability of IGF-I and IGFBP-3 in whole blood, serum and plasma from 12 volunteers at 4 degrees, 22 degrees, and 37 degrees C for several hours and at -25 degrees C for several months. The effect of only one protease inhibitor (Aprotinin = Trasylol, Bayer, Germany ) on IGF-I and IGFBP-3 measured in the automated IMMULITE assay system (DPC, Los Angeles) was tested. IGF-I and IGFBP-3 were stable in heparinized whole blood, plasma and serum at 22 degrees C up to 24 hours. IGF-I was stabilized by aprotinin for up to 72 hours at 37 degrees C. Factor concentrations were not altered after storage at -25 degrees C for at least 12 months. Recognition of IGFBP-3 fragments by the antibody used in the automated IGFBP-3 IMMULITE was excluded by measurement in 26 sera from pregnant women which usually contain IGFBP-3 fragments. In conclusion, samples for measurement of IGF-I and IGFBP-3 should be kept on ice and cooled if shipment takes more than 48 hours or alternatively 5000 IU/ml aprotinin should be added. IMMULITE assays are also valid to measure IGF-I and IGFBP-3 after at least 12 months storage at -25 degrees C.

Reference ranges for two automated chemiluminescent assays for serum insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3).

Assays for insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3) have become essential tools in the diagnostic work-up of disorders of the somatotropic axis in children and adults. The aim of this study was to evaluate the automated IMMULITE IGF-I and IGFBP-3 assays and to establish reference limits--central 95% intervals, median, 0.1 and other centiles as clinically relevant--as a function of age from 797 females and 787 males, from the first week of life through the ninth decade. Pubertal children were classified by sex and by sexual maturation (Tanner stage). IGF-I and IGFBP-3 levels were also assayed in 20 pediatric patients each with growth hormone deficiency (GHD) and Turner syndrome (UTS), before and during 12 months of recombinant growth hormone (rhGH) therapy, as well as in 11 adult patients with GHD and seven with acromegaly before therapy. Both the IGF-I and IGFBP-3 assays were accurate, specific and sufficiently sensitive to measure IGF-I and IGFBP-3 in serum with good linearity and recovery. In the IGF-I assay, potential interference from IGFBPs was eliminated by blocking with excess IGF-II. Circulating IGF-I and IGFBP-3 concentrations, and their ratio IGF-I/IGFBP-3, were age-dependent, showing low levels immediately after birth, a typical pubertal peak for girls and boys, and a pronounced decline after puberty, reaching a plateau in early adulthood. In adults IGF-I and IGFBP-3 levels decreased smoothly but steadily with age. Children with GHD and UTS had low circulating IGF-I and IGFBP-3 levels which increased to normal reference limits under therapy with rhGH. Adult GHD patients showed IGF-I levels below the age-related median; untreated acromegalic patients mostly had IGF-I and IGFBP-3 levels above the age-related 97.5th centile. In conclusion, the automated IMMULITE IGF-I and IGFBP-3 assays are reliable tools in the diagnosis of pathologies of the GH/IGF axis and in the follow-up of their therapies.

Evaluation of the chemiluminescence immunoassays for the measurement of troponin I, myoglobin and CK-MB using the IMMULITE system in comparison to other measuring systems.

We evaluated the chemiluminescence immunoassays for the detection of the cardiac markers troponin I, myoglobin and CK-MB on the IMMULITE System (Diagnostic Products Corporation) in comparison to the same analytes of other companies. The IMMULITE assays are two-site solid phase immunometric assays using a murine monoclonal capture antibody on the solid phase and a polyclonal antibody conjugated with alkaline phosphatase (except CK-MB monoclonal, murine) for detection. Precision was investigated using serum pools with a low, a cutoff and a high concentration of the respective analyte. The results were satisfactory with an intra-assay precision coefficient of variation, CV of 1.7% - 3.2% for troponin I, 2.6% - 5.1% for myoglobin, 2.7% - 5.3% for CK-MB and an interassay precision of 5.1% - 6.9% for troponin I, 5.7% - 7.3% for myoglobin and 3.8% - 8.4% for CK-MB. In linearity studies with various dilution steps, a mean value of 105% was found for troponin I, 103% for myoglobin and 117% for CK-MB. The average recovery was 85% for troponin I, 100% for myoglobin and 95% for CK-MB. The clinical validity of the assays in the diagnosis and therapy of myocardial infarction was investigated in 120 patients who were sent to the hospital with suspected myocardial infarction. Four hours after admission all patients with clinically verified myocardial infarction showed troponin I and troponin T values above the cutoff value. A maximum rate of 32% of the patients (IMMULITE Troponin I) with an instable angina pectoris showed troponin values above the cutoff for myocardial infarction (1.0 microg/L), 4 hours after admission. A cutoff-reduction to 0.2 pg/L for troponin I increased the number of patients to 45%. The negative predictive value was constantly 67%. The results obtained by IMMULITE assays were compared to the Elecsys cardiac assays (Roche Diagnostics) and the AxSYM-cardiac assays (Abbott Diagnostics). The highest correlation (r = 0.99) was found for IMMULITE Troponin I (DPC) and Troponin I (Abbott). The Abbott-Troponin I showed the highest diagnostic sensitivity within 4 hours after admission. All compared methods showed a similar diagnostic sensitivity (close to 100%) > 4 hours after admission. For all investigated methods the percentage of discrepant results decreased to a minimum 4 hours after admission.

Reference ranges for serum concentrations of lutropin (LH), follitropin (FSH), estradiol (E2), prolactin, progesterone, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), cortisol and ferritin in neonates, children and young adults.

The aim of this study was to establish reference ranges for children (neonates to young adults), for serum lutropin (LH), follitropin (FSH), estradiol (E2), progesterone, prolactin, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), cortisol and ferritin, using the nonisotopic, automated chemiluminescence immunoassay system, Immulite (DPC). Serum samples from 762 children (369 female; age 1 day to 19 years) were examined. Of these, 381 were classified as pubertal. Due to non-normal distribution, the 2.5th, 50th and 97.5th percentiles (central 95% interval) were calculated for each group. Statistical differences between the reference ranges were analyzed with respect to age, sex and the stage of sexual maturation. The median concentrations of E2, prolactin, progesterone, DHEAS, cortisol and ferritin were higher during the first 2 weeks post-partum than thereafter. The largest difference was seen with prolactin, which showed up to 27-fold higher values during this period. In contrast, before the onset of puberty, hardly any sex difference was observed and all analyte concentrations remained relatively constant, apart from SHBG which increased steadily after the neonatal period. The increase of gonadal activity in females with the onset of sexual maturation included an increase in LH and FSH, which was accompanied by a strong increase in E2, progesterone and prolactin. Cortisol increased to a lesser extent during puberty. In males, the increase in the median concentrations of the hormones was smaller, except for DHEAS. The concentration of ferritin was high in the neonatal period but did not change during sexual maturation. Our findings agree with earlier studies. The calculated reference intervals can be used to assess the development of children, particularly for measurements performed by the Immulite and Immulite 2000 chemiluminescence assay systems.