Adrenal venous sampling criteria for chemiluminescent enzyme immunoassay as a preferable alternative to radioimmunoassay in primary aldosteronism.

Plasma aldosterone concentration (PAC) was routinely measured using radioimmunoassay (RIA); however, the RIA kit was discontinued in March 2021 in Japan. This study examined PAC conversion in adrenal venous sampling (AVS) and AVS criteria when measured using chemiluminescent enzyme immunoassay (CLEIA). PAC of 415 adrenal venous blood samples from AVS (including segmental AVS) of 63 patients with primary aldosteronism was measured using RIA (Spac-S aldosterone kit; Fujirebio Inc.) and CLEIA (Lumipulse Presto Aldosterone; Fujirebio Inc.). PAC of 70 AVS samples was also measured using liquid chromatography-mass spectrometry (LC-MS/MS, ASKA Pharma Medical Co., Ltd.). PAC conversion formulas were determined for each AVS sample assay. PAC measured using CLEIA was significantly correlated with that measured using RIA (correlation coefficient = 0.971). The PAC conversion formula was PAC (CLEIA) = PAC (RIA) × 0.772 - 1,199 pg/mL. The PAC of 14,000 pg/mL in RIA was equivalent to 9,613 pg/mL in CLEIA. PAC measured using CLEIA was also correlated with that measured using LC-MS/MS, and the PAC conversion formula was PAC (CLEIA, pg/mL) = 0.97 × PAC (LC-MS/MS, pg/mL) + 211. The inter-assay coefficient of variability (CV) was 1.1-1.3% and intra-assay CV was 1.0-1.7%, measured using CLEIA. The PAC conversion formula for AVS samples was obtained using CLEIA and RIA, and the conversion formula was different from that for peripheral blood. PAC values measured by CLEIA showed preferable accuracy and high concordance with those measured by LC-MS/MS, even in AVS samples. The study outcomes are useful for interpreting AVS results using non-RIA measurement methods.

Effect of sample delivery conditions on Renin-Angiotensin-Aldosterone System (RAAS) assay.

Renin-Angiotensin-Aldosterone System (RAAS) measurements are influenced by several factors. We investigated the effect of sample delivery conditions on RAAS measurements including sample storage temperature and time. Blood samples were collected from thirty participants using enzyme inhibitor tubes and serum separation gel evacuated tubes. Plasma and serum from fresh blood samples without further storage (as baseline), and from blood samples that were stored at either 0 °C, 4 °C, or 25 °C for 3 h, 6 h and 24 h, respectively, were extracted and stored at -30 °C for batch measurements using radioimmunoassay. Concentrations of Aldosterone (Ald) decreased following delivery temperature and time, and were significantly different when samples were set aside at 0 °C for 24 h (p < .01), 4 °C for 6 h (p < .01), and 25 °C for 3 h (p < .05). However, levels of Angiotensin (Ang I) increased following delivery temperature and time, and were significantly different when samples were set aside at 0 °C and 4 °C for 6 h (p < .05) and at 25 °C for 3 h (p < .001). However, no changes were observed for the concentrations of plasma renin activity (PRA) and Ang II, except for Ang II which increased significantly when samples were set aside at 25 °C for 24 h (p < .001). Our results indicate that samples used for RAAS measurement should be placed at a low temperature and analyzed as soon as possible after collection.

Quality planning and control strategy for AQT90 flex Radiometer® in point of care testing.

In the context of point of care testing (PoCT) and ISO 22870, internal quality control (IQC) is a crucial part of PoCT accreditation processes. Quality Control materials shall be periodically examined with a frequency that is based on the robustness of the analytical procedure and the risk of harm to the patient from an erroneous result. We propose to apply the statistical quality control (SQC) procedure to develop an individualized QC plan for AQT90 flex instrument used in PoCT. The robustness is determined by the sigma-metric and analytical goal represented by an allowable total error (TEa) is evaluated using a Varela graphic tool. A Sigma-metric SQC run size nomogram for estimating the number of patient samples between IQC events. According to the calculated robustness we can distinguish 3 groups of parameters: HCG and CRP with large sample size per event, D-Dimer and Procalcitonin with an average sample size per event and Myoglobin. NT-proBNP. and Troponin T with a limited sample size per event. In PoCT, the SQC strategy can promote more effective, and not necessarily more frequent, IQC.

Calcitonin levels by ECLIA correlate well with RIA values in higher range but are affected by sex, TgAb, and renal function in lower range.

Calcitonin (CT) is a marker for both initial diagnosis and monitoring of patients with residual or recurrent medullary thyroid carcinoma (MTC). In Japan, serum CT had been measured by radioimmunoassay (RIA) until recently. Electrochemiluminescence immunoassay (ECLIA) became commercially available in 2014, and this technique is now the only method used to examine CT concentration. The purposes of this study were to investigate the correlations between the CT concentration measured with ECLIA (ECLIA-CT) and RIA (RIA-CT) and to explore the clinical characteristics of patients with elevated ECLIA-CT. CT concentrations of 348 sera samples from 334 patients with various thyroid disorders including nine MTC were measured using both assays. The correlation analysis revealed an excellent correlation between ECLIA-CT and RIA-CT among the cases with CT level >150 pg/mL by both assays (rs = 0.991, p < 0.001). However, 63% of all samples exhibited undetectable ECLIA-CT, while their RIA-CTs were measured between 15 and 152 pg/mL. The ECLIA-CTs in all patients who underwent total thyroidectomy for non-MTC showed low concentrations. High ECLIA-CT was observed in patients with MTC or pancreas neuroendocrine tumor. ECLIA-CT was also increased in 14 other male patients with non-MTC, including four with renal failure. Multivariate logistic regression analysis showed that male sex, negative TgAb, and lower estimated glomerular filtration rate were independent factors to predict detectable ECLIA-CT (≥0.500 pg/mL). These results indicate that ECLIA-CT correlates well with RIA-CT in higher range and is affected by sex, TgAb, and renal function.

Comparison of the New, Rapid, and Fully Automated Kryptor TSH receptor Antibodies Assay (B.R.A.H.M.S.) with the Radioimmunological Assay (B.R.A.H.M.S.).

BACKGROUND: Radioimmunoassays, which are often not automated and time-consuming, are gradually being re-placed in medical laboratories by non-radioactive methods that need to be evaluated. The purpose was to compare the measurement of thyroid-stimulating hormone receptor antibodies (TRAb) by the new Brahms' kit using Kryptor TRACE technology and the Brahms' radioimmunoassay. METHODS: We prospectively collected all samples from patients who received thyroid-stimulating hormone receptor antibodies testing in July 2018 at the University Hospital of Brest. The radioimmunoassay used was the Dynotest TRAK human by BRAHMS Diagnostica (Berlin, Germany). The Kryptor method used the BRAHMS TRAK human Kryptor kit performed with the Kryptor Compact Plus system. RESULTS: The inter-assay coefficient variations for the radioimmunological and Kryptor methods were 11.07% and 8.36%, respectively, with the low level quality control and 8.36% and 4.38%, respectively, with the high level quality control. Forty-four patients were included in the study including thirty-two Graves' disease patients in follow-up. The sensitivity of the radioimmunological method for the detection of Graves' disease was 0.94 and the specificity was 0.73. The sensitivity of the Kryptor method was 0.91 and the specificity was 0.91. A non-proportional systematic bias in favor of higher values of TRAb concentrations with the radioimmunological method was observed: slope of 0.93 (0.74 - 1.07, 95% confidence interval) and an intercept of -0.69 IU/L (-1.58 to -0.30, 95% confidence interval). Compared to the Kryptor method, the radioimmunological method tends to overestimate TRAb concentrations by up to 120%. CONCLUSIONS: The fully automated Brahms Kryptor kit using TRACE technology to measure TRAb reduces sampling time and intra- as well as inter-assay variations. The Kryptor kit underestimates the results of TRAb leading to a lower sensitivity and higher specificity compared to the radioimmunoassay. Thus, the new Brahms Kryptor kit has good laboratory performances but the interpretation of the results must still be performed with caution.

Harmonisation of serum dihydrotestosterone analysis: establishment of an external quality assurance program.

BACKGROUND: Serum dihydrotestosterone (DHT) is an important analyte for the clinical assessment of disorders of sex development. It is also reportedly a difficult analyte to measure. Currently, there are significant gaps in the standardisation of this analyte, including no external quality assurance (EQA) program available worldwide to allow for peer review performance of DHT. We therefore proposed to establish a pilot EQA program for serum DHT. METHODS: DHT was assessed in the 2015 Royal College of Pathologists of Australasia Quality Assurance Programs' Endocrine program material. The material's target (i.e. "true") values were established using a measurement procedure based on isotope dilution gas chromatography (GC) tandem mass spectrometry (MS/MS). DHT calibrator values were based on weighed values of pure DHT material (>97.5% purity) from Sigma. The allowable limits of performance (ALP) were established as ±0.1 up to 0.5 nmol/L and ±15% for targets >0.5 nmol/L. RESULTS: Target values for the six levels of RCPAQAP material for DHT ranged from 0.02 to 0.43 nmol/L (0.01-0.12 ng/mL). The material demonstrated linearity across the six levels. There were seven participating laboratories for this pilot study. Results of the liquid chromatography (LC) MS/MS methods were within the ALP; whereas the results from the immunoassay methods were consistently higher than the target values and outside the ALP. CONCLUSIONS: This report provides the first peer comparison of serum DHT measured by mass spectrometry (MS) and immunoassay laboratories. Establishment of this program provides one of the pillars to achieve method harmonisation. This supports accurate clinical decisions where DHT measurement is required.

Assay-dependent variability of serum insulin concentrations: a comparison of eight assays.

Although insulin measurement is essential for both clinical and research purposes, there is currently no reference method for insulin assays. The aim of this study was to compare results of serum insulin determined by a number of commercially available assays. We compared eight insulin assays by analyzing 165 serum samples. Assays included two chemiluminescence (Roche and DiaSorin), four ELISA (Tosoh, Mercodia, Monobind, and Diametra), and two IRMA (Izotop and BioSource) methods. Each assay was compared with the mean of all assay methods and Bland-Altman difference plots were used to measure agreement between each assay and overall mean. Least squared perpendicular distance regression analysis (Deming's method) was used to calculate slope and intercept for bias and also for each assay vs. mean of eight assays. Findings showed that the lowest and highest median insulin concentrations varied by a factor of 1.8. Maximum and minimum correlations with mean of assays were observed for Roche (0.992) and BioSource (0.844), respectively. Significant bias was observed in six assays. In pairwise comparisons of different assays, the highest and least mean differences were 7.78 µU/mL and -0.14 µU/mL, respectively. In conclusion, serum insulin measurement with different assays showed a maximum of 1.8-fold difference, a point that should be taken into consideration in the interpretation of circulating insulin levels in both clinical and research fields.

Hypocretin measurement: shelf age of radioimmunoassay kit, but not freezer time, influences assay variability.

The hypothalamic peptide hypocretin 1 (orexin A) may be assayed in cerebrospinal fluid to diagnose narcolepsy type 1. This testing is not commercially available, and factors contributing to assay variability have not previously been comprehensively explored. In the present study, cerebrospinal fluid hypocretin concentrations were determined in duplicate in 155 patient samples, across a range of sleep disorders. Intra-assay variability of these measures was analyzed. Inter-assay correlation between samples tested at Emory and at Stanford was high (r = 0.79, p < 0.0001). Intra-assay correlation between samples tested in duplicate in our center was also high (r = 0.88, p < 0.0001); intra-assay variability, expressed as the difference between values as a percentage of the higher value, was low at 9.4% (SD = 7.9%). Although both time the sample spent in the freezer (r = 0.16, p = 0.04) and age of the kit used for assay (t = 3.64, p = 0.0004) were significant predictors of intra-kit variability in univariate analyses, only age of kit was significant in multivariate linear regression (F = 4.93, p = 0.03). Age of radioimmunoassay kit affects intra-kit variability of measured hypocretin values, such that kits closer to expiration exhibit significantly more variability.

Harmonization Study Between LC-MS/MS and Diasorin RIA for Measurement of 25-Hydroxyvitamin D Concentrations in a Large Population Survey.

BACKGROUND: Population-based research on vitamin D has increased dramatically in recent years. Such studies are typically reliant on assay procedures to measure reliable and comparable levels of 25-hydroxyvitamin D [25(OH)D] concentrations. METHODS: Concentrations of 25(OH)D3 and 25(OH)D2 were measured using LC-MS/MS in 5,915 participants (aged 31 years) of Northern Finland Birth Cohort 1966. Blood samples were assayed in batches over a course of 18 months. As anomalies were present in the measurements, 200 samples were reassayed using Diasorin RIA. Agreement between measurements was assessed by Passing-Bablok regression and limits of agreement (LoA). To harmonize LC-MS/MS with Diasorin RIA measurements, formulae were derived from the LoA. RESULTS: Concentrations measured by LC-MS/MS were much higher than those measured by Diasorin RIA, with a mean difference of 12.9 ng/ml. Constant variation was evident between batch measurements after log transformation. Statistical formula was applied separately for each batch of LC-MS/MS measurements, enabling us to remove both the constant and proportional bias that was evident prior to the transformation. CONCLUSION: Despite the introduction of schemes/programs to improve accuracy of assays to measure 25(OH)D, significant differences can still happen. In these instances, methods to harmonize measurements based on a relatively small number of replicates can be successfully applied to establish confidence and to enable between-study comparisons.

In vitro evaluation of concentration, labeling effectiveness and stability for 131 I-labeled radioimmunoassay ligand using real-time detection technology.

Radioimmunoassay belongs to the analytical method enabling highly specific and sensitive quantification of molecules. The verification of the real-time radioimmunoassay technology usefulness for ligand-quality characteristics evaluation such as concentration, influence of radiolabeling on binding affinity and stability was estimated. The anti-epidermal growth factor receptor antibody 131 I-cetuximab was employed as the ligand antibody. The concentration of 131 I-cetuximab was derived from the shape of binding curves coming from the ligand-receptor interaction. The binding curves also allowed the estimation of 131 I-cetuximab binding affinity for different radiolabeling procedures (incubation times 1, 5, and 10 minutes) in stability testing up to 96 hours at 4°C. The stability testing also included comparative analysis by size exclusion high-performance liquid chromatography. The assessment of cetuximab concentrations using real-time method showed acceptable accordance between real and calculated values. The real-time method revealed that 1-minute radiolabeling proved to be the optimal incubation time for direct radioiodination of cetuximab. Stability testing showed the significant change in radioligand affinity by one order at the longest incubation times (72 and 96 hours). Characterization of stability and binding behavior of radiolabeled monoclonal antibodies by the verified real-time method before use in other assays may be employed to eliminate variability and suboptimal antibody performance.

The estimation of calibration equations for variables with heteroscedastic measurement errors.

In clinical chemistry and medical research, there is often a need to calibrate the values obtained from an old or discontinued laboratory procedure to the values obtained from a new or currently used laboratory method. The objective of the calibration study is to identify a transformation that can be used to convert the test values of one laboratory measurement procedure into the values that would be obtained using another measurement procedure. However, in the presence of heteroscedastic measurement error, there is no good statistical method available for estimating the transformation. In this paper, we propose a set of statistical methods for a calibration study when the magnitude of the measurement error is proportional to the underlying true level. The corresponding sample size estimation method for conducting a calibration study is discussed as well. The proposed new method is theoretically justified and evaluated for its finite sample properties via an extensive numerical study. Two examples based on real data are used to illustrate the procedure.

25-hydroxy-Vitamin D status: limitations in comparison and clinical interpretation of serum-levels across different assay methods.

Background: Over the last decade, clinical interest to evaluate human 25-hydroxy-vitamin D (25[OH]D) serum levels has increased exponentially. In the present study, four chemiluminescence immunoassays (CLIA), one radioimmunoassy (RIA), and one high performance liquid chromatography (HPLC) method were compared and also with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in view of 25(OH)D serum level determination. Methods: For the method comparison, blood samples from 133 consecutive patients were prospectively collected. All participants gave written informed consent for their blood samples to be used in this study. They came to the Department of Nuclear Medicine of the Central Hospital Steyr (Austria) for osteodensidometric measurement as part of their preventive medical check-up. Pearson's correlation coefficients, Bland-Altman plots, and paired t-tests were calculated. Assay-specific reference ranges were considered using blood samples from persons with normal parathormone, calcium, and total-protein values (n = 97). Results: The highest correlation was between the HPLC and the LC-MS/MS method (r = 0.96). The lowest correlation was between the cobas Vitamin D3 assay (Roche) and any of the evaluated assays (r = 0.46 - 0.63). Bland-Altman plots revealed a big negative mean bias in three assays (cobas Vitamin D3 assay [Roche]: -22.8; DiaSorin LIAISON [25[OH]D total CLIA [Diasorin]: -18.4; Diasorin 25[OH]D125 I RIA [Diasorin]: -23.8 [nmol/L]) and a much smaller positive mean bias in the other assays (ClinRep complete 25[OH]D2/D3 HPLC kit [Recipe]: 2.7; ADVIA Centaur Vitamin D total assay [Siemens]: 8.2; IDS total vitamin D assay [Immunodiagnostic Systems]: 12.1 [nmol/L]) compared to the LC-MS/MS method. Meanwhile, the manufacturer has withdrawn the cobas Vitamin D3 assay from the market. Conclusions: Poor antibody specificity with cross-reactivity to other vitamin D metabolites, incomplete extraction of the 25(OH)D analyte from the vitamin D-binding protein (DBP), and confounding matrix substances such as lipids could be potential reasons for the unacceptable performance of the cobas Vitamin D3 assay (Roche) and also the significant differences in the 25(OH)D determination between various assays. Standardization and harmonization of 25(OH)D measurements are therefore urgently needed. The widespread introduction of well standardized assays in clinical laboratories is the challenge in the next years. (Clin. Lab. 2014;60:1541-1550. DOI: 10.7754/Clin.Lab.2014.131114)

Free testosterone by direct and calculated measurement versus equilibrium dialysis in a clinical population.

INTRODUCTION: The value of clinically available free testosterone (FT) assays remains controversial. Here, we evaluate the agreement between the radioimmunoassay (RIA) and calculated FT (cFT) versus equilibrium dialysis (EqD), considered the gold standard. METHODS: Fifty-six consecutive men (aged 26-77) had blood samples assessed for FT, including men with treated and untreated testosterone deficiency (TD) and men without TD. Samples were split and tested by the two methodologies at a Quest Diagnostics national reference laboratory. cFT was calculated by the Vermeulen method. RESULTS: A robust correlation was noted for RIA and EqD (r = 0.966) and for cFT and EqD (r = 0.986). Strong correlations were observed for men receiving testosterone therapy and for men in the lowest and highest quartiles for total and FT. The correlation of total testosterone with FT was similar for cFT (r = 0.843), RIA (r = 0.806), and EqD (r = 0.809). Sex-hormone binding globulin (SHBG) was not correlated with any measure of FT. Bland-Altman analysis demonstrated similar bias for both cFT and RIA, although cFT consistently overestimated FT. Numerical values for RIA were approximately one seventh of EqD values. CONCLUSIONS: These results support the clinical use of both RIA and cFT as measures of FT. Due to numerical differences, each test requires its own set of reference values.

CA 19-9: handle with care.

Since its inception in the mid-1980s of the 20th century testing for carbohydrate antigen 19-9 (CA 19-9) has raised expectation for an earlier diagnosis and accurate monitoring of several malignant diseases. After almost 30 years, the available evidences have confirmed the appropriateness and usefulness of determining CA 19-9 levels as a prognostic indicator and as a reliable tool for monitoring pancreatic and gastrointestinal cancer, but concerns have been raised about its applications in screening, which is actually not recommended, and in the diagnosis of malignancies, due to several interferences that limit the specificity and to the insufficient sensitivity of this marker. In this paper we aimed to review the basic concepts of CA 19-9 testing and its current applications, with a major focus on the most recent evidences dealing with assay interference, methods comparison and monitoring of malignant diseases. The prognostic value and monitoring recommendations for pancreatic, gastric and colorectal cancers are described in depth.

Proposed diagnostic criteria for subclinical Cushing's syndrome associated with adrenal incidentaloma.

Subclinical Cushing's syndrome (SCS) associated with adrenal incidentaloma is usually characterized by autonomous cortisol secretion without overt symptoms of Cushing's syndrome (CS). Although the diagnostic criteria for SCS differ among countries, the 1 mg dexamethasone suppression test (DST) is essential to confirm the presence and the extent of cortisol overproduction. Since 1995, SCS has been diagnosed in Japan based on serum cortisol levels ≥3 µg/dL (measured by radioimmunoassay [RIA]) after a 1 mg DST. However, the increasing use of enzyme immunoassays (EIA) instead of RIA has hindered the diagnosis of SCS because of the differing sensitivities of commercially available assays, particularly for serum cortisol levels of around 3 µg/dL. One way to overcome this problem is to lower the cortisol threshold level after a 1 mg DST. In the present study, we examined the clinical applicability of lowering the cortisol threshold to 1.8 µg/dL, similar to the American Endocrine Society's guidelines for CS, by reanalyzing 119 patients with adrenal incidentaloma. Our findings indicate that serum cortisol levels ≥1.8 µg/dL after 1 mg DST are useful to confirm the diagnosis of SCS if both of the following criteria are met: (1) basal ACTH level <10 pg/mL (or poor plasma ACTH response to corticotrophin-releasing hormone) and (2) serum cortisol ≥5 µg/dL at 21:00 to 23:00 h. If only one of (1) and (2) are met, we recommend that other clinical features are considered in the diagnosis of SCS, including serum dehydroepiandrosterone sulfate levels, urine free cortisol levels, adrenal scintigraphy, and clinical manifestation.

Validation of a fecal glucocorticoid metabolite assay to assess stress in the budgerigar (Melopsittacus undulatus).

The budgerigar (Melopsittacus undulatus) is a small parrot native to Australia that is commonly held in zoos, laboratories, and private homes. Assessment of budgerigar stress levels would aid welfare monitoring and improve our understanding of their biology. Analyzing fecal glucocorticoid metabolites provides a noninvasive method to measure stress levels in birds. For this method to be reliable, the antibody to be used in an immunoassay must be carefully selected for each species, and validation must be performed. A common limitation in many existing assays is the inability to accurately detect variable fecal glucocorticoid metabolites in minute quantities of feces, requiring small samples to be combined. We have developed a double antibody radioimmunoassay protocol based on a commercially available (125) I-corticosterone radioimmunoassay kit for use in detecting fecal glucocorticoid metabolites in small quantities (<20 mg) of budgerigar droppings. The assay was validated pharmacologically with an adrenocorticotropic hormone challenge and with oral administration of corticosterone. Our validation has demonstrated our assay is both sensitive and a reliable approach to noninvasive monitoring of stress in budgerigars.

Noninvasive monitoring of adrenocortical function in captive jaguars (Panthera onca).

Jaguars are threatened with extinction throughout their range. A sustainable captive population can serve as a hedge against extinction, but only if they are healthy and reproduce. Understanding how jaguars respond to stressors may help improve the captive environment and enhance their wellbeing. Thus, our objectives were to: (1) conduct an adrenocorticotrophic hormone (ACTH) challenge to validate a cortisol radioimmunoassay (RIA) for noninvasive monitoring of adrenocortical function in jaguars; (2) investigate the relationship between fecal corticoid (FCM) and androgen metabolite (FAM) concentrations in males during the ACTH challenge; and (3) establish a range of physiological concentrations of FCMs for the proposed protocol. Seven jaguars (3 M, 4 F) received 500 IU/animal of ACTH. Pre- and post-ACTH fecal samples were assayed for corticoid (M and F) and androgen metabolites (M) by RIA. Concentrations of FCMs increased (P80.01) after ACTH injection (pre-ACTH: 0.90 ± 0.12 µg/g dry feces; post-ACTH: 2.55 ± 0.25 µg/g). Considering pre- and post-ACTH samples, FCM concentrations were higher (P80.01) in males (2.15 ± 0.20 µg/g) than in females (1.30 ± 0.20 µg/g), but the magnitude of the response to ACTH was comparable (P>0.05) between genders. After ACTH injection, FAMs increased in two (of 3) males; in one male, FCMs and FAMs were positively correlated (0.60; P80.01). Excretion of FCMs was assessed in 16 jaguars (7 M, 9 F) and found to be highly variable (range, 80.11-1.56 µg/g). In conclusion, this study presents a cortisol RIA for monitoring adrenocortical function in jaguars noninvasively.

[Report of immunoassay control surveys conducted in the past 30 years by the Subcommittee for Radioisotope in vitro Test, the Medical Science and Pharmaceutical Committee, and the Japan Radioisotope Association].

Immunoassay control surveys, were conducted by the Subcommittee for Radioisotope in vitro Test, the Medical Science and Pharmaceutical Committee, and the Japan Radioisotope Association, between 1978 to 2008. A total of 40 analytes for 26 hormones, 14 tumor markers and pharmaceutical drugs were investigated in participating facilities. In the first immunoassay control survey in 1978, samples were measured using only RI kits, however, non-RI kits increased gradually during the next 30 years. In the 30th immunoassay control survey, more than 90% samples were measured using non-RI kits. Coefficient variation (CV) of intra-kits has been decreasing yearly in all analytes for hormones as well as tumor markers. However, improvement of CV in inter-kits has not been seen in the past 30 years by a lack of international standards, although there has been continuous effort over the years for the standardization of immunoassay. Growth hormone (GH) deficiency has been diagnosed using various loading tests. However, the clinical diagnosis varies according to the GH kit used. Standardization for GH measurement has been possible by using recombinant GH as the standard among commercial GH kits. The diagnosis of subclinical Cushing's syndrome also varies according to the cortisol kits being used. Candidate reference measurement procedure and low level cortisol standards have been developed by the Biomedical Standard Section, of the National Metrology Institute of Japan. Standardization of measurement is necessary for improvement of immunoassay.