Transference of CALIPER pediatric reference intervals to biochemical assays on the Roche cobas 6000 and the Roche Modular P.

OBJECTIVES: The Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) has recently established pediatric age- and sex-specific reference intervals for over 85 biochemical markers on the Abbott Architect system. Previously, CALIPER reference intervals for several biochemical markers were successfully transferred from Abbott assays to Roche, Beckman, Ortho, and Siemens assays. This study further broadens the CALIPER database by performing transference and verification for 52 biochemical assays on the Roche cobas 6000 and the Roche Modular P. DESIGN AND METHODS: Using CLSI C28-A3 and EP9-A2 guidelines, transference of the CALIPER reference intervals was attempted for 16 assays on the Roche cobas 6000 and 36 on the Modular P. Calculated reference intervals were further verified using 100 healthy CALIPER samples. RESULTS: Most assays showed strong correlation between assay systems and were transferable from Abbott to the Roche cobas 6000 (81%) and the Modular P (86%). Bicarbonate and magnesium were not transferable on either system and calcium and prealbumin were not transferable to the Modular P. Of the transferable analytes, 62% and 61% were verified on the cobas 6000 and the Modular P, respectively. CONCLUSIONS: This study extends the utility of the CALIPER database to two additional analytical systems, which facilitates the broad application of CALIPER reference intervals at pediatric centers utilizing Roche biochemical assays. Transference studies across different analytical platforms can later be collectively analyzed in an attempt to develop common reference intervals across all clinical chemistry instruments to harmonize laboratory test interpretation in diagnosis and monitoring of pediatric disease.

Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry.

BACKGROUND: Currently there is no reliable method suitable for routine measurement of serum free testosterone (FT). AIM: To develop such a method involving liquid chromatography tandem mass spectrometry (LC-IDMS/MS) that directly detects and quantifies the FT present in serum. METHODS: Ultrafiltrate testosterone obtained from 0.5 mL of serum was partially purified by liquid/liquid extraction and quantified using an Agilent 1200 Series HPLC system coupled to an API 5000 mass spectrometer equipped with an atmospheric pressure chemical ionization ion source. Using split samples serum free testosterone was compared between direct ultrafiltration (UF) coupled LC-MS/MS, analogue FT immunoassay, free testosterone calculated from mass action equations (cFT) and with equilibrium dialysis (ED) coupled LC-MS/MS. RESULTS: Total imprecision determined over twenty runs was <6% at 67 pmol/L and 158 pmol/L FT. The dynamic response was linear up to at least 2500 pmol/L while physical LLOQ (18 % CV) equaled 16 pmol/L. The UF method agreed poorly with analogue immunoassay (correlation coefficient 0.667; bias -81%), somewhat better against cFT when total testosterone was determined by immunoassay (correlation coefficient 0.816, bias 21% ) and still better yet against cFT when total testosterone was determined by LC-MS/MS (correlation coefficient 0.8996, bias 10%). Agreement was closest with ED method (correlation coefficient 0.9779, bias 2.4%). CONCLUSION: We present a relatively simple UF coupled LC-MS/MS definitive method that measures serum free testosterone. The method is relatively fast, reliable and is suitable for the routine clinical laboratory practice.

Rapid determination of serum testosterone by liquid chromatography-isotope dilution tandem mass spectrometry and a split sample comparison with three automated immunoassays.

OBJECTIVES: To develop a rapid convenient-to-implement high performance liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) method for determination of serum testosterone concentration in routine clinical laboratories. METHODS: Following extraction by organic solvents, an Agilent 1200 Series HPLC system coupled to an API 5000 mass spectrometer equipped with an atmospheric pressure chemical ionization ion source was used to separate, detect and quantify serum testosterone. Ion-transitions of m/z 289.2-->109.1 and 294.2-->113.2 were used to monitor testosterone and testosterone-2,2,4,6,6-d(5), respectively. RESULTS: Functional sensitivity was 0.056 nmol/L (CV 20%). Within-run and total imprecision were 4.6% and 5.2% at 1.3 nmol/L, 2.4% and 4.3% at 11.0 nmol/L, and 1.9% and 1.9% at 23.4 nmol/L respectively. The LC-MS/MS method agreed closely with three automated immunoassays when the concentration of testosterone exceeded 3 nmol/L. However, the immunoassays showed a positive bias at concentrations below 3 nmol/L. CONCLUSION: This method provides a rapid, simple, highly selective and sensitive procedure that can be easily used for determination of serum testosterone in routine clinical laboratories. It measures serum testosterone precisely and accurately at concentrations found in children and adults of both genders.

National Academy of Clinical Biochemistry laboratory medicine practice guidelines for use of tumor markers in testicular, prostate, colorectal, breast, and ovarian cancers.

BACKGROUND: Updated National Academy of Clinical Biochemistry (NACB) Laboratory Medicine Practice Guidelines for the use of tumor markers in the clinic have been developed. METHODS: Published reports relevant to use of tumor markers for 5 cancer sites--testicular, prostate, colorectal, breast, and ovarian--were critically reviewed. RESULTS: For testicular cancer, alpha-fetoprotein, human chorionic gonadotropin, and lactate dehydrogenase are recommended for diagnosis/case finding, staging, prognosis determination, recurrence detection, and therapy monitoring. alpha-Fetoprotein is also recommended for differential diagnosis of nonseminomatous and seminomatous germ cell tumors. Prostate-specific antigen (PSA) is not recommended for prostate cancer screening, but may be used for detecting disease recurrence and monitoring therapy. Free PSA measurement data are useful for distinguishing malignant from benign prostatic disease when total PSA is <10 microg/L. In colorectal cancer, carcinoembryonic antigen is recommended (with some caveats) for prognosis determination, postoperative surveillance, and therapy monitoring in advanced disease. Fecal occult blood testing may be used for screening asymptomatic adults 50 years or older. For breast cancer, estrogen and progesterone receptors are mandatory for predicting response to hormone therapy, human epidermal growth factor receptor-2 measurement is mandatory for predicting response to trastuzumab, and urokinase plasminogen activator/plasminogen activator inhibitor 1 may be used for determining prognosis in lymph node-negative patients. CA15-3/BR27-29 or carcinoembryonic antigen may be used for therapy monitoring in advanced disease. CA125 is recommended (with transvaginal ultrasound) for early detection of ovarian cancer in women at high risk for this disease. CA125 is also recommended for differential diagnosis of suspicious pelvic masses in postmenopausal women, as well as for detection of recurrence, monitoring of therapy, and determination of prognosis in women with ovarian cancer. CONCLUSIONS: Implementation of these recommendations should encourage optimal use of tumor markers.

National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines for use of tumor markers in clinical practice: quality requirements.

BACKGROUND: This report presents updated National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines summarizing quality requirements for the use of tumor markers. METHODS: One subcommittee developed guidelines for analytical quality relevant to serum and tissue-based tumor markers in current clinical practice. Two other subcommittees formulated recommendations particularly relevant to the developing technologies of microarrays and mass spectrometry. RESULTS: Prerequisites for optimal use of tumor markers in routine practice include formulation of the correct clinical questions to ensure selection of the appropriate test, adherence to good clinical and laboratory practices (e.g., minimization of the risk of incorrect patient and/or specimen identification, tube type, or timing), use of internationally standardized and well-characterized methods, careful adherence to manufacturer instructions, and proactive and timely reactions to information derived from both internal QC and proficiency-testing specimens. Highly desirable procedures include those designed to minimize the risk of the reporting of erroneous results attributable to interferences such as heterophilic antibodies or hook effects, to facilitate the provision of informative clinical reports (e.g., cumulative and/or graphical reports, appropriately derived reference intervals, and interpretative comments), and when possible to integrate these reports with other patient information through electronic health records. Also mandatory is extensive validation encompassing all stages of analysis before introduction of new technologies such as microarrays and mass spectrometry. Provision of high-quality tumor marker services is facilitated by dialogue involving researchers, diagnostic companies, clinical and laboratory users, and regulatory agencies. CONCLUSIONS: Implementation of these recommendations, adapted to local practice, should encourage optimization of the clinical use of tumor markers.

Free thyroxine reference interval in each trimester of pregnancy determined with the Roche Modular E-170 electrochemiluminescent immunoassay.

OBJECTIVE: To determine using a simplified study design trimester-specific FT4 reference intervals in pregnancy with the Roche Modular immunoassay in routine use. DESIGN AND METHODS: Surplus blood from 300 women in each trimester, drawn at documented times in the gestation, and from 40 age-matched nonpregnant women were assayed for FT4, thyroid stimulating hormone (TSH) and antithyroid peroxidase autoantibody (anti-TPO). RESULTS: After excluding women positive for anti-TPO and with abnormal TSH, reference intervals were calculated as 12.5-19.1 pmol/L (nonpregnant group), 11-19 pmol/L (first trimester), 9.7-17.5 pmol/L (second trimester) and 8.1-15.3 pmol/L (third trimester). 3rd trimester FT4 was significantly lower than that of the second trimester (p<0.001) which, in turn, was lower than that of the first trimester (p<0.001). FT4 reference intervals in pregnancy were significantly lower than in the nonpregnant women (p<0.002). CONCLUSIONS: The observed trimester-specific FT4 reference intervals progressively decline with advancing gestation and differ significantly from one another.

Light and exercise and melatonin production in women.

Melatonin may protect against breast cancer. Light and other factors influence melatonin, but the evidence is limited. The authors conducted a study to determine factors related to melatonin. Women volunteers recruited in Toronto, Canada, from 2002 to 2004 collected urine for three nights (winter and summer), took periodic light measurements, and recorded exposures in a diary. The relation of each variable to log-transformed creatinine-corrected 6-sulfatoxymelatonin in overnight urine was determined by use of generalized estimating equation linear regression. The final model was based on 1,054 measurement days from 213 participating women. None of the light variables was related to the log of 6-sulfatoxymelatonin. A significant interaction between season and day length was included in the final model. The most significant factor was duration of exercise (beta = 0.072; p = 0.004, two-tailed), which increased the amount of melatonin produced. Exercise duration later in the day was more significant (beta = 0.108; p = 0.0009, two-tailed). There was no difference between moderate or strenuous exercise. The failure to find a relation between light brightness and melatonin may be due to the difficulty of measuring this, as well as the importance of the light spectrum, which could not be measured. It is possible that the protective effect of exercise with respect to breast cancer may operate in part through an effect on melatonin.