Novel threshold value of midnight serum cortisol for diagnosis of hypercortisolism using the Roche Cortisol II assay.

BACKGROUND: The diagnosis of hypercortisolism requires multiple biochemical investigations, due to variations in cortisol production during the 24-hour circadian cycle. Midnight serum cortisol is difficult to interpret since the threshold value is dependent on the analytical method used and is often not provided by the manufacturer. Second-generation assays are more specific than first-generation assays and may have lower threshold values. OBJECTIVES: The aim of this study was to determine a novel threshold value of midnight serum cortisol for the biochemical diagnosis of hypercortisolism, using the Roche Cobas Cortisol® second-generation assay. METHODS: This study was performed in adult patients hospitalized in the endocrinology unit of a university hospital. Patients had a complete assessment of their 24-hour cortisol cycle, i.e., a serum cortisol test every four hours and at least two first-line tests: late night salivary cortisol, dexamethasone suppression test and/or 24-hour urinary free cortisol. First-line tests were used to identify patients with hypercortisolism. Serum samples were analyzed by second-generation electrochemiluminescence immunoassays (ECLIA) from Roche Cobas Cortisol®. RESULTS: Midnight serum cortisol samples were obtained from 175 hospitalized patients. The novel threshold value obtained was 157 nmol/L with a sensitivity of 82.9% (68.6 to 94.3%) and a specificity of 90.0% (85.0 to 95.0%). CONCLUSION: Our study confirms that the threshold value of midnight serum cortisol is not comparable between first- and second-generation Roche Cobas Cortisol® assays and that the threshold value is lower with the second-generation assay.

Development and validation of a candidate reference method for serum cortisol by isotope dilution liquid chromatography-tandem mass spectrometry combined with dextran sulfate-Mg2+ precipitation.

Accurate and precise cortisol measurements are requisite for ensuring appropriate diagnosis and management of diseases related with adrenal or pituitary gland disorders. Prompted by the needs in characterization of certified reference materials and quality assurance for serum cortisol measurements, we developed and evaluated a highly reliable measurement procedure based on isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) combined with dextran sulfate-Mg2+ precipitation as the sample pretreatment. An appropriate amount of serum was accurately weighed and spiked with the isotope labelled internal standard. After precipitation, massive lipids and lipoproteins were removed from serum matrix. The clear supernatant was transferred and extracted with ethyl acetate-hexane solution. The cortisol was analyzed with LC-MS/MS in positive electrospray ionization mode. The within-run and total coefficient of variations (CVs) ranged from 0.3 to 0.6% and 0.7 to 1.2%, respectively, for a concentration range of 76.30 to 768.04 nmol/L. A regression comparison of the results obtained by the present method and the certified values of ERM-DA451 showed agreement with no statistical difference (Y = 1.0092 X-0.7455; 95% CI for the slope, 0.9940 to 1.0212; 95% CI for the intercept, - 3.6575 to 2.6390, r2 = 0.999). All structural analogs of cortisol tested were well resolved from cortisol in 12 min on a phenyl ligand column under an isocratic elution. The limit of quantification was estimated to 5 pg cortisol in absolute amount. This method is accurate and simple and can be served as a candidate reference measurement procedure in establishment of serum cortisol reference system.

Cortisol cut-points for the glucagon stimulation test in the evaluation of hypothalamic pituitary adrenal axis.

Diagnosis of adrenal insufficiency requires evaluation by dynamic stimulation tests. The insulin tolerance test (ITT) is accepted as the gold-standard test for the evaluation of hypothalamo-pituitary-adrenal (HPA) axis but the test is unpleasant and dangerous. Although it takes more time, glucagon stimulation test (GST) is a good alternative to ITT. The primary aim of this study was to compare the ITT and GSTs in the evaluation of HPA axe in patients with pituitary disorders. We conducted a prospective study in which ITT and GST were performed within 7 days in 81 patients. Serum cortisol was measured. We divided our population in Group 1 (G1): Adrenal Insufficiency (Peak cortisol under ITT <200 ng/mL) and Group 2 (G2): normal response (Peak cortisol under ITT >200 ng/mL). Receiver-operating characteristic (ROC) analysis was performed to identify the thresholds for GST. The mean peak of cortisol under GST was not significantly different from that obtained after ITT in the whole cohort (182.67 ± 89.07 ng/mL vs. 179.75 ± 79.01 ng/mL), and it was significantly reduced in patients of G1 (p < 10-3). ROC curve analysis showed that the best diagnostic accuracy was obtained with a peak cortisol cut-off to GST of 167 ng/mL (sensitivity, 89%; specificity, 79%). Using this cut-off, 86.4% of the patients were correctly classified. In our prospective series, GST is a potential accurate and safe alternative test for the assessment HPA. Test-specific cut-offs should be applied to avoid misinterpretation.

Stability of an Alcohol-free, Dye-free Hydrocortisone (2 mg/mL) Compounded Oral Suspension.

The stability of hydrocortisone in a commercially available dye-free oral vehicle was monitored to establish a beyond-use date for hydrocortisone oral suspension 2 mg/mL. Hydrocortisone oral suspension (2 mg/mL) was prepared from 10-mg tablets in a dye-free oral vehicle (Oral Mix, Medisca) and stored at 4°C and 25°C for 90 days in amber, plastic prescription bottles and oral syringes. The suspendability and dose repeatability of the oral suspension were evaluated. The solubility of hydrocortisone in the dye-free vehicle was determined. Over 90 days, pH and concentration of hydrocortisone in the oral suspension were measured. The stability-indicating nature of a high-pressure liquid chromatographic assay was evaluated in detail. The solubility of hydrocortisone in the dye-free vehicle was 230 mcg/mL at 25°C. This means that about 90% of the drug remains in the solid state where it is less susceptible to degradation. The preparation suspended well to support dose repeatability. The chromatographic assay resolved hydrocortisone from cortisone, excipients in the vehicle, and all degradation products. The assay passed United States Pharmacopeia system suitability tests. Hydrocortisone oral suspension (2 mg/mL) compounded using a dye-free, alcohol-free oral vehicle, Oral Mix, was stable in amber plastic bottles and syringes stored at 4°C and 25°C for 90 days within a 95% confidence interval.

Reference intervals for six salivary cortisol measures based on the Croatian Late Adolescence Stress Study (CLASS).

INTRODUCTION: The aim of this nested study is to provide the reference intervals for already published measurements of salivary cortisol from the Croatian Adolescence Stress Study (CLASS). MATERIAL AND METHODS: A total of 969 individuals (372 males and 597 females) were included in the reference sample (age range: 18-21 years). Salivary cortisol concentrations were determined by the enzyme immunoassay (LUCIO-Medical ELISA Salivary Cortisol Kit, Nal von Minden, Germany) in the Department of Medical Laboratory Diagnostics, University Hospital Split. Nonparametric statistics were used for calculating the reference intervals (RIs) and 90% confidence intervals (90% CIs). RESULTS: The lower limits of RIs determined by the direct method were higher in females (> 10%) than in males for the cortisol concentrations at awakening (SCC0), 30 to 45 after awakening (SCC30-45) and at bedtime (SCCbedtime). The upper limits of RIs for the SCCbedtime were higher (> 10%) in males than in females. Females also had higher upper limits of RIs for the cortisol awakening response (CAR) and the diurnal cortisol slope (DCS) and higher lower limits of RIs for the CAR and the area under the curve with respect to ground (AUCG). The lower limits of RIs for the DCS were higher in males than in females. CONCLUSIONS: Obtained reference values open the arena for introducing salivary bioscience in Croatian clinical laboratory practice and provide important data for better understanding of gender differences in adaptation to stress during late adolescence.

Chinese Pediatric Reference Intervals for Serum Cortisol on IMMULITE 2000.

Clinical interpretation of the test results for cortisol based on continuous reference intervals with appropriate partitions improves pediatric diagnosis; however, these values are available only for Caucasians. To develop the pediatric reference intervals for Chinese population, we examined the serum cortisol levels in 1,143 healthy Chinese children aged 4-18 years (566 boys and 577 girls), using an IMMULITE 2000 Immunoassay System (Siemens Healthcare GmbH). Phlebotomy was performed at 7-9 a.m. for 284 boys and 287 girls and at 1-3 p.m. for the others. They were divided into four age groups according to the Clinical and Laboratory Standards Institute guideline EP28-A3c, with the last group further stratified according to sampling time. Separate reference intervals of 49.6-323.7, 70.9-395.3, and 90.1-448.7 nmol/L were established for children aged 4-8, 9-12, and 13-15 years, respectively. Further, reference intervals of 118.2-464.7 and 71.4-446.7 nmol/L were established for morning and afternoon cortisol levels, respectively, in children aged 16-18 years. Further studies are necessary to transfer and validate these reference intervals in other analytical systems and pediatric populations, and to allow for broader applications.

A time-adjusted cortisol cut-off can reduce referral rate for Synacthen stimulation test whilst maintaining diagnostic performance.

OBJECTIVE: Cortisol cut-offs can predict requirement for Synacthen stimulation tests (SST). We assessed the performance of a standard cortisol cut-off (375 nmol/L) across the morning and compared this with a time-adjusted cut-off. DESIGN: Retrospective audit PATIENTS: Community reference set (n=12 550) and SST patients (n=757). MEASUREMENTS: In the reference population, time-specific cortisol medians were calculated and used to convert cortisol to time-adjusted Multiples of the Median (MoM). In 757 SST patients, the predictive performance of a standard cortisol cut-off (375 nmol/L) and its time-adjusted MoM equivalent were compared. RESULTS: Median cortisol decreased by ~30 nmol/L per hour between 0700 and 1200h. In the reference population, proportions below the 375 nmol/L cut-off increased throughout the morning (range 35%-64%), whereas using the time-adjusted MoM cut-off proportions were consistent (range 46%-50%), with a 17% maximal difference in referral rates between the two cut-offs after 1100h. A similar pattern was noted in the SST cohort. When a cortisol MoM cut-off was used to predict SST success, the excess proportion of patients tested and misclassification rates were lower and more consistent than when the standard cut-off was used. A median cortisol of 375 nmol/L equated to 444 and 313 nmol/L before 0800 and after 1100 h, respectively. CONCLUSION: The use of a standard cortisol cut-off results in 17% more patients being referred for SST later in the morning. A time-adjusted cortisol cut-off provides consistent and lower referral rates, whilst maintaining similar or better performance than a standard single cut-off in predicting outcome of SST.

Quality of compounded hydrocortisone capsules used in the treatment of children.

OBJECTIVES: Due to the lack of paediatric-licensed formulations, children are often treated with individualized pharmacy-compounded adult medication. An international web-based survey about the types of medication in children with adrenal insufficiency (AI) revealed that the majority of paediatric physicians are using pharmacy-compounded medication to treat children with AI. Observations of loss of therapy control in children with congenital adrenal hyperplasia with compounded hydrocortisone capsules and regained control after prescribing a new hydrocortisone batch led to this 'real world' evaluation of pharmacy-compounded paediatric hydrocortisone capsules. METHODS: Capsule samples were collected randomly from volunteering parents of treated children suffering from congenital adrenal hyperplasia from all over Germany. Analysis of net mass and hydrocortisone content by high-performance liquid chromatography with ultraviolet (HPLC-UV) detection method was performed based on the European Pharmacopeia. RESULTS: In a total of 61 batches that were sent, 5 batches could not be analysed because of missing dose information, insufficient number of capsules or were not possible to be evaluated. Fifty-six batches containing 1125 capsules were evaluated. 21.4% of the batches revealed insufficiency in uniformity of net mass or drug content and additional 3.6% failed because they did not contain the labelled drug. CONCLUSIONS: Compounded medication is a possible cause of variation of steroid doses in children with adrenal insufficiency or congenital adrenal hyperplasia, putting these vulnerable patients at risk of poor disease control and adrenal crisis. These data may apply to other individualized compounded oral medication as well, emphasizing the need for development of licensed paediatric formulations approved by regulatory authorities.

Cortisol and cortisone ratio in urine: LC-MS/MS method validation and preliminary clinical application.

BACKGROUND: The determination of urinary cortisol/cortisone ratio is of clinical utility in cases of Cushing's syndrome, apparent mineralocorticoid excess, and also provides information on 11ß-hydroxysteroid dehydrogenase (11ß-HSD) type 2 activity. It is therefore of utmost importance to ensure accurate cortisol and cortisone measurement and establish appropriate reference ranges. METHODS: After the isotopic dilution of urine, sample cleanups were obtained with on-line solid-phase extraction and cortisol and cortisone, separated using a Zorbax Eclipse XDB-C18 HPLC analytical column, were analyzed by tandem mass spectrometry with an electrospray ionization source in positive ion mode operation. RESULTS: The method was linear, with concentrations of up to 625 and 1125 nmol/L and lower limit of quantitation (LLOQ) of 5 and 6 nmol/L, for cortisol and cortisone, respectively. Within-run and between-run coefficients of variation were <5% and 6% for cortisol and 6% and 8% for cortisone, respectively. No ion suppression was observed. The non-parametric reference range for the cortisol/cortisone ratio was 0.14-1.09. CONCLUSIONS: A simple and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the measurement of cortisol and cortisone in urine. Our findings indicate that the proposed analytical method is suitable for routine purposes and useful in many pathological conditions.

Target analyte quantification by isotope dilution LC-MS/MS directly referring to internal standard concentrations--validation for serum cortisol measurement.

BACKGROUND: Isotope dilution LC-MS/MS methods used in the clinical laboratory typically involve multi-point external calibration in each analytical series. Our aim was to test the hypothesis that determination of target analyte concentrations directly derived from the relation of the target analyte peak area to the peak area of a corresponding stable isotope labelled internal standard compound [direct isotope dilution analysis (DIDA)] may be not inferior to conventional external calibration with respect to accuracy and reproducibility. METHODS: Quality control samples and human serum pools were analysed in a comparative validation protocol for cortisol as an exemplary analyte by LC-MS/MS. Accuracy and reproducibility were compared between quantification either involving a six-point external calibration function, or a result calculation merely based on peak area ratios of unlabelled and labelled analyte. RESULTS: Both quantification approaches resulted in similar accuracy and reproducibility. CONCLUSIONS: For specified analytes, reliable analyte quantification directly derived from the ratio of peak areas of labelled and unlabelled analyte without the need for a time consuming multi-point calibration series is possible. This DIDA approach is of considerable practical importance for the application of LC-MS/MS in the clinical laboratory where short turnaround times often have high priority.

Salivary cortisol monitoring: determination of reference values in healthy children and application in asthmatic children.

Venipuncture testing of adrenocortical function in asthmatic infants and young children receiving inhaled corticosteroids can raise cortisol levels and mask physiological responses. This study aimed to establish reference ranges for salivary cortisol levels and evaluate the safety and effects of jet-nebulized budesonide inhalation suspension (BIS) on salivary cortisol levels and patient outcomes in infants and young children with mild or persistent asthma. Reference salivary cortisol levels were determined in healthy children aged 6 months to 4 years old. A 12-week multicenter, randomized, parallel-group, open-label study was performed involving 53 age-matched asthmatic children who received either 0.5 mg/day of BIS or 40-60 mg/day of cromolyn sodium inhalation suspension (CIS) via compressor nebulizer. The effective measuring range of salivary cortisol concentration in asthmatic children was 0.12-3.00 micrograms/dL. The upper and lower limits of the reference range were 0.827 and 0.076 micrograms/dL, respectively. No significant difference was seen from baseline through week 12 in the CIS and BIS groups. BIS was safe in these patients, with no inhibitory effects on adrenocortical function. Salivary cortisol measurement offers a useful and accurate tool for testing adrenocortical function in infants and young children. Longer-term studies that incorporate testing of the hypothalamic-pituitary-adrenal axis are warranted to confirm our findings.

[Report of the NEDO project "Research and development to promote the creation and utilization of an intellectual infrastructure: development of reference materials for laboratory medicine" "Development of pure substance-type certified reference materials"].

There is an increasing demand to establish a metrological traceability system for in vitro diagnostics and medical devices. Pure substance-type reference materials are playing key roles in metrological traceability, because they form the basis for many traceability chains in chemistry. The National Metrology Institute of Japan (NMIJ), in the National Institute of Advanced Industrial Science and Technology (AIST), has been developing purity-certified reference materials (CRMs) in this field, such as cholesterol, creatinine, and urea. In the New Energy and Industrial Technology Development Organization (NEDO) project, entitled: "Research and Development to Promote the Creation and Utilization of an Intellectual Infrastructure: Development of Reference Materials for Laboratory Medicine", several pure substance-type CRMs were developed. For a pure protein solution CRM, amino acid analysis and nitrogen determination were chosen as the certification methods. The development and certification processes for the C-reactive protein (CRP) solution CRM were completed, with the recombinant human CRP solution as a candidate material. This CRP solution CRM is now available as NMIJ CRM. For cortisol CRM, a purified candidate material and highly pure primary reference material were prepared. Each impure compound in the materials was identified and quantified. The pure cortisol CRM will be available in 2009. These two CRMs provide a traceability link between routine clinical methods and the SI unit.

[Basic Review Committee for Clinical Laboratory Standardization. Column 1. Activity report of 2007. Group items of authentic sample, Working Group J2].

(1) Concerning measurement of serum levels of insulin, C-peptide, and cortisol, a joint experiment in 2006 suggested that a reference material derived from authentic samples was effective for the convergence of data. (2) To reconfirm the convergence of data using a candidate reference material derived from authentic sample, we generated pooled serum samples of patients as candidate materials, and the levels of insulin and C-peptide in the sera of healthy subjects and the level of cortisol in panel serum IRMM ERM-DA45I-1-DA45I-3 were measured. (3) The serum levels measured with commercially available reagents were reconfirmed to lead to data convergence. (4) We employed reagents which were traceable in reference materials of authentic samples, calibrated each reagent, and confirmed the convergence of data, as verified in the previous examination. (5) The prototype reference material of authentic sample was proven to be effective as a standard reference material.

Production of gender-specific morning salivary cortisol reference intervals using internationally accepted procedures.

BACKGROUND: Salivary cortisol concentrations correlate well with biologically active unbound free plasma cortisol concentrations. Despite its practical and analytical advantages, salivary cortisol measurement has been used mainly as a research tool rather than for the routine evaluation of adrenal function. This may be partly explained by the lack of robust reference data in the literature. METHODS: Using the recommended procedures for the production of reference intervals published by the International Federation of Clinical Chemistry, we aimed to produce morning salivary cortisol reference intervals for males and females. Salivary cortisol was measured in 496 specimens collected from 248 reference individuals (128 males, median age 41 years, range 16-86; and 120 females, median age 44 years, range 16-98) attending an otorhinolaryngology clinic. Reference individuals mailed saliva specimens sampled on two consecutive mornings to our laboratory, where cortisol concentrations were measured. RESULTS: Statistical analysis showed no significant correlation with age or body mass index. The following 95% gender-partitioned reference intervals were produced: males 10.9-40.3 nmol/l; and females 9.3-40.3 nmol/l. CONCLUSION: Knowledge of these salivary cortisol reference intervals helps us monitor the adrenal function of outpatients using topical intranasal glucocorticoids for rhinosinusitis.

Preparation of horseradish peroxidase hydrazide and its use in immunoassay.

Preparation of horseradish peroxidase (HRP) hydrazide that is HRP linked to adipic acid dihydrazide (HRP-ADH) and its use in enzyme immunoassay (EIA) is described. In this new strategy, horseradish peroxidase was conjugated to adipic acid dihydrazide using a carbodiimide coupling method. The resulting HRP-ADH was then coupled to cortisol-21-hemisuccinate (Cortisol-21-HS) to prepare enzyme conjugate. The prepared cortisol-21-HS coupled ADH-HRP (Cortisol-21-HS-ADH-HRP) enzyme conjugate was used for the development of an enzyme linked immunosorbent assay (ELISA) for direct estimation of cortisol. To the cortisol antibody coated microtiter wells, standard or serum samples (50 microL), along with cortisol-21-HS-ADH-HRP enzyme conjugate (100 microL) were incubated for 1 h at 37 degrees C. Bound enzyme activity was measured by using tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. The sensitivity of the assay was 0.05 microg/dL and the analytical recovery ranged from 92.9 to 101.7%.

[Hydrocortisone Sodium Phosphate Reference Standard (Control 001) of National Institute of Health Sciences].

The raw material of hydrocortisone sodium phosphate was examined for the preparation of the "Hydrocortisone Sodium Phosphate Reference Standard (Control 001)". The analytical data obtained were: pH, 8.3: optical rotation, [alpha]D20 = +126.2 degrees; UV spectrum, lambda max of 248 nm and specific absorbance in water at 248 nm = 338.6; IR spectrum, same as that of the Hydrocortisone Sodium Phosphate Reference Standard (Control 891); free phosphoric acid, 0.2%; free hydrocortisone, 0.01%; thin-layer chromatography, no impurity was detected until 200 micrograms; high-performance liquid chromatography, total amount of impurities estimated to be less than 0.2%; residual solvent, 0.0% (acetone) and 0.02% (ethanol); loss on drying, 1.5%. Based on the above results, the raw material was authorized as the Hydrocortisone Sodium Phosphate Reference Standard (Control 001) of the National Institute of Health Sciences.

A reference system for cortisol.

OBJECTIVES: The International Federation of Clinical Chemistry (IFCC) initiated a pilot study, with cortisol as an example, that aims to implement the concept of standardization of hapten immunoprocedures on the basis of metrological traceability. In fact, this standardization concept comes down to correct calibration of measurement procedures, so that measurement results for patient samples can be traced back to the metrologically highest reference of a result, i.e., an SI unit as embodied in the primary reference material. In consequence, demonstration of standardization of a test system on the basis of traceability requires evaluation of measurement results pertaining to patient samples for accuracy. Such an evaluation shall be done by a correlation study of the routine test system with an accuracy-based reference measurement procedure. DESIGN AND METHODS: The IFCC project will select a panel of single donation human serum samples, and assign them with values for cortisol by measurement with at least two isotope dilution-gas chromatography/mass spectrometry reference methods. Limited amounts of the panel will be distributed to manufacturers, with the object to measure the samples with their calibrated immunoprocedures. The patient correlation studies between the routine and the reference methods will then be interpreted in terms of specificity and accuracy. It will also be investigated whether the performed comparison can be used as a basis for re-calibration. From the experience gained through this pilot study for cortisol, IFCC will formulate recommendations and guidance to manufacturers on how to perform and reliably interpret patient correlation studies. In this way, it is to be expected that the project might form a basis for general implementation of the concept of standardization of hapten immunoassays on the basis of metrological traceability.

[Hydrocortisone Acetate Reference Standard (Control 961) of National Institute of Health Sciences].

The raw material of hydrocortisone acetate was tested for the preparation of the "Hydrocortisone Acetate Reference Standard (Control 961)". Analytical data obtained were as follows: IR spectrum, specific absorption wave numbers at 3428, 1748, 1723, 1631, and 1375 cm-1; specific absorbance, E1cm1% (242 nm) = 406; thin-layer chromatography, no impurities were detected until 0.05 microgram; high-performance liquid chromatography (HPLC), 2-3 impurities were detected and the amount of the total impurities was estimated to be about 0.2%; assay by HPLC, 100.7%. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Reference Standard (Control 961).

Development, validation, and certification by isotope dilution gas chromatography-mass spectrometry of lyophilized human serum reference materials for cortisol (CRM 192 and 193) and progesterone (CRM 347 and 348).

The Community Bureau of Reference of the European Communities has produced four batches of lyophilized serum Certified Reference Materials, two for cortisol (CRM 192 and 193) and two for progesterone (CRM 347 and 348). For cortisol, one of the pools consisted of serum from healthy blood donors, whereas the second batch was supplemented with pure cortisol. The progesterone Reference Materials contained only endogenous hormone concentrations. Assessment of vial-to-vial variability in the cortisol and progesterone concentrations showed no between-sample inhomogeneity, and the materials were stable. The quality of the materials was therefore considered sufficient for certification of the values for the cortisol and progesterone concentrations by a collaborative study involving several laboratories from the European Communities, using isotope dilution gas chromatography-mass spectrometry. Inaccuracy in reconstitution of the lyophilized materials was less than 0.3%; imprecision of sampling was less than 0.2%. For determinations of cortisol and progesterone concentrations, the mean within-laboratory coefficients of variation (CVs) were 1.76% (CRM 192), 1.19% (CRM 193), 1.64% (CRM 347), and 1.75% (CRM 348). The between-laboratory CVs were greater: CRM 192, 1.79%; CRM 193, 1.48%; CRM 347, 2.08%; and CRM 348, 2.16%. The concentrations in the reconstituted Reference Materials were certified to be 273 nmol/L in CRM 192 and 763 nmol/L in CRM 193 for cortisol and 10.13 nmol/L in CRM 347 and 40.3 nmol/L in CRM 348 for progesterone. Uncertainties at the 0.95 confidence level--6 (CRM 192), 14 (CRM 193), 0.21 (CRM 347), and 1.0 nmol/L (CRM 348)--were considered compatible with the intended use of the materials.

Serum cortisol in adrenal hirsutism as estimated by five different methods.

Serum cortisol had been estimated in 152 hirsute women complaining of fertility problems, of whom 36 were subsequently diagnosed as having adrenal hirsutism and 10 as having congenital adrenal hyperplasia (steroid 21-hydroxylase deficiency), using five methods: an in-house tritium radioimmunoassay after extraction with ethanol; the Diagnostic Products Corp. "Coat-a-count" iodinated direct radioimmunoassay; the Pharmacia-LKB "DELFIA" lanthanum-enhanced fluoroimmunoassay; the Amersham "Amerlite" luminescence immunoassay; and the Walker "Synelisa" enzyme-linked immunoassay. Although stripped pool serum samples containing weighed amounts of cortisol produced acceptable values in all assays, the patient samples showed a number of high results, much greater than the accepted normal upper limit of 250 ng/ml (25 micrograms/dl, 690 nmol/l). This was especially so in 21-hydroxylase deficiency, when cortisol values should be very low. Only the luminescence and iodinated assays produced very low values after dexamethasone suppression. After the outliers had been excluded, only the iodinated assay showed a good statistical agreement with the more elaborate tritium assay. The most specific assay was the luminescence method, which produced generally lower results in most cases. This was selected as the new routine method. The unreliable cortisol results in adrenal hirsutism are attributed to high cross-reaction of the antiserum in each of the assays with 17-hydroxyprogesterone, progesterone and 21-deoxyderivatives of cortisol and deoxycorticosterone. In general, all standard and commercially available cortisol assays appears to be unsuitable for cortisol estimation in 21-hydroxylase deficiency, and probably also for neonates.