Comparison and commutability study between standardized liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and chemiluminescent enzyme immunoassay for aldosterone measurement in blood.

A commutability confirmation test for the blood aldosterone measurement was performed on liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) as a designated comparison method (DCM) and four chemiluminescent enzyme immunoassay (CLEIA) measurement procedures based on metrological traceability. A conventional radioimmunoassay (RIA) and two measurement procedures of CLEIA which obtains RIA equivalent values were also compared. The relationship between the DCM value and the CLEIA value with respect to 120 pg/mL of the RIA value, which is the screening criterion of primary aldosteronism (PA) was clarified. For the correlation test, 75 samples of patient serum and plasma were used. Regression analysis revealed that the standardized LC-MS/MS and four CLEIA measurement procedures were in good agreement. This is the effect of measurement specificity and calibration using by certified reference material (CRM). The median of the LC-MS/MS corresponding to 120 pg/mL of RIA was 48.5 pg/mL. In the mean of standardized four CLEIA values corresponding to the 48.5 pg/mL of LC-MS/MS value was 47.51 pg/mL and the standard deviation (SD) was 2.93 pg/mL. However, the correlation between the RIA value and the RIA equivalent of the two measurement procedures by CLEIA differed depending on the measurement procedure. This is due to the influence of RIA measurement performance. Standardized CLEIA measurements are suitable for routine measurement procedure. When converting the LC-MS/MS equivalent value by the standardized CLEIA to the conventional RIA value, it is necessary to use the conversion formula.

Calibration and evaluation of routine methods by serum certified reference material for aldosterone measurement in blood.

We attempted to study the standardization of aldosterone measurement in blood. The serum certified reference material (serum CRM) was established by spiking healthy human serum with pure aldosterone. ID-LC/MS/MS as a reference measurement procedure was performed by using the serum CRM. LC-MS/MS as a comparison method (CM) was routinely used for clinical samples, and the values with and without calibration by the serum CRM were compared. The serum CRM demonstrated similar reactivity with peripheral blood plasma as clinical samples in routine methods (RM) of RIA, ELISA, and CLEIA. In comparison between RM and CM, the results in regression analysis indicated that the range of the correlation coefficient (r) was 0.913 - 0.991, the range of y intercept was 0.9 - 67.3 pg/mL and the range of slope was 0.869 - 1.174. The values by RM in 100 - 150 pg/mL for the diagnostic level, had a significant calibration effect, and the relative difference between calibrated value in RM and result by CM was within ±20%. Furthermore, the calibrated value using the serum CRM was 10,187 pg/mL, which corresponds to measured value of 14,000 pg/mL using RIA for the adrenal venous sampling. Measured values between plasma and serum as a sample for the aldosterone measurement from clinical samples showed no significant differences. In conclusion, we succeeded to prepare the certified reference material of aldosterone for RM. Then, we can accurately calculate corrected values by using our equation for four RMs of determination of aldosterone.

[Internal Quality Control and External Quality Assessment on POCT].

The quality management (QM) of POCT summarizes its internal quality control (IQC) and external quality assessment (EQA). For QM requirements in POCT, ISO 22870-Point-of-care testing (POCT) -Requirements for quality and competence and ISO 15189-Medical laboratories-Requirements for quality and competence, it is performed under the guidance of the QM committee. The role of the POC coordinator and/or medical technologist of the clinical laboratory is important. On measurement performance of POCT devices, it is necessary to confirm data on measurement performance from the manufacturer other than those in the inserted document. In the IQC program, the checking and control of measurement performance are the targets. On measurements of QC samples by the manufacturer, it is essential to check the function of devices. In addition, regarding the EQA program, in 2 neighboring facilities, there is an effect to confirm the current status of measurement and commutability assessment in these laboratories using whole blood along with residual blood samples from daily examinations in the clinical laboratory.

[From the Point of View of the Production of Reference Material].

In the international standardization of lipid measurement for TC, HDL-C, LDL-C, and TG in serum, the establishment of a measurement system and certified serum reference materials (CRM) are the most important fundamental factor. They have been fully established. Routine measurement procedures using manufacturers' reagent kits must be calibrated with the CRM and are standardized to maintain the commutability of measured values. In TC and HDL-C measurements, the commutability of measured values from reagent kits has been maintained by calibration with the CRM. However, it is likely that different values will be obtained due to the reactivity with abnormal specimens based on measurement principles and performance of the reagent kits. This different reactivity is a typical phenomenon in abnormal specimens on conducting lipid measurements. This phenomenon cannot be avoided using the CRM. On selection of the reagent kit, it is necessary to perform evaluation studies using abnormal specimens based on the comparison method. Direct methods standardized for LDL-C measurement are selected based on evaluation studies. In TG measurements for international standardization, it is necessary to change the procedure to measure the total glycerides instead of the elimination of free glycerol by the JSCC method. This is done to avoid the influence of LPL activity on heparin treatment for cardiovascular disease patients.

[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.

[Feasibility study for development of automatic calibration system for clinical laboratory analyzers].

This report summarizes the results of a "Feasibility Study for Development of Automatic Calibration System for Clinical Laboratory Analyzers", that was entrusted to the JCCLS (Japanese Committee for Clinical Laboratory Standards) as a project for 2007FY by the Mechanical Social Systems Foundation. The purpose of this study was to establish a calibration system for clinical laboratory analyzers that are commonly used in Japanese laboratories, in order to promote the standardization of clinical tests. Standardization of an automatic calibration system for clinical laboratory analyzers will facilitate accuracy control without having to depend on the skill of laboratory operators, which will achieve the further standardization of clinical tests.

[Establishment of working reference materials in laboratory medicine].

It is recognized that the establishment of reference materials is the most important factor for standardization in laboratory medicine. In particular, working reference materials for routine measurement procedures are effective tools to maintain the commutability of measured values. Working reference materials are dealt with in a traceability chain system issued by ISO 17511 (in vitro diagnostic medical devices--measurement of quantities in biological samples--metrology traceability of values assigned to calibrators and control materials). The JCCLS is strongly promoting standardization in laboratory medicine by using working reference materials. Working groups for standardization are organized by the JCCLS in cooperation with the NMIJ (National Metrology Institute of Japan) with a grant from the NEDO (New Energy and Industrial Technology Development Organization). The objective is the establishment of working reference materials for routine use. Standardization criteria using the reference materials are being established. Research and development and investigation studies of matrix reference materials have been started. In investigation studies, the production protocol of reference materials are being prepared. After being established, reference materials will be presented to the JCTLM (Joint Committee on Traceability in Laboratory Medicine). These activities will enhance standardization in laboratory medicine. As a results, they will contribute to maintaining the reliability of measured values in routine laboratory procedures.

[Noninvasive blood glucose monitoring: new technology using metabolic heat conformation method].

Self-monitoring of blood glucose has become an essential aspect of management of patients with diabetes mellitus. Although several approaches for noninvasive blood glucose monitoring(NIGM) have been proposed including near infrared spectrophotometry. Body heat generated by glucose oxidation is based on the subtle balance of capillary glucose and oxygen supply to the cells. Hence, the blood glucose can be estimated by measuring the body heat and the oxygen supply. Development of the metabolic heat conformation (MHC) method consists of a sensor pickup and a calibration model. The calibration model incorporates mathematical procedures to process signals from the sensor pickup to final glucose value. The patients group was classified into clusters (calibration functions). Each subject patient was assigned to one of calibration functions. The assigned calibration function for the patient was later used for calculating the glucose values. Regression analysis involving 127 data points at random timing (109 data points from diabetic patients, 18 data points from non-diabetic patients) ranging 54mg/dl to 405mg/dl by the non-invasive method against the hexokinase photometric method for plasma as a reference method was performed. The correlation coefficient (r) was 0.91. Repeatability of the non-invasive method was measured for healthy fasting persons. The standard deviations were ranged from 5 to 6mg/dl around the concentration of 100mg/dl. These data provide preliminary evidence that the MHC method can be used to estimate blood glucose concentrations non-invasively.

Committee on Diabetes Mellitus Indices of the Japan Society of Clinical Chemistry-recommended reference measurement procedure and reference materials for glycated albumin determination.

BACKGROUND: Glycated albumin is an intermediate glycaemic control marker for which there are several measurement procedures with entirely different reference intervals. We have developed a reference measurement procedure for the purpose of standardizing glycated albumin measurements. METHODS: The isotope dilution liquid chromatography/tandem mass spectrometry method was developed as a reference measurement procedure for glycated albumin. The stable isotopes of lysine and fructosyl-lysine, which serve as an internal standard, were added to albumin isolated from serum, followed by hydrogenation. After hydrolysis of albumin with hot hydrochloric acid, the liberated lysine and fructosyl-lysine were measured by liquid chromatography/tandem mass spectrometry, and their concentrations were determined from each isotope ratio. The reference materials (JCCRM611) for determining of glycated albumin were prepared from pooled patient blood samples. RESULTS: The isotope dilution-tandem mass spectrometry calibration curve of fructosyl-lysine and lysine showed good linearity (r = 0.999). The inter-assay and intra-assay coefficient of variation values of glycated albumin measurement were 1.2 and 1.4%, respectively. The glycated albumin values of serum in patients with diabetes assessed through the use of this method showed a good relationship with routine measurement procedures (r = 0.997). The relationship of glycated albumin values of the reference material (JCCRM611) between these two methods was the same as the relationship with the patient serum samples. CONCLUSION: The Committee on Diabetes Mellitus Indices of the Japan Society of Clinical Chemistry recommends the isotope dilution liquid chromatography/tandem mass spectrometry method as a reference measurement procedure, and JCCRM611 as a certified reference material for glycated albumin measurement. In addition, we recommend the traceability system for glycated albumin measurement.

Japanese standard reference material for JDS Lot 2 haemoglobin A1c. I: Comparison of Japan Diabetes Society-assigned values to those obtained by the Japanese and USA domestic standardization programmes and by the International Federation of Clinical Chemistry reference laboratories.

BACKGROUND: The Committee on Standardization of Laboratory Testing Related to Diabetes Mellitus of the Japan Diabetes Society (JDS) previously recommended use of the primary calibrator (JDS Lot 1) prepared by the former Committee for Standardization of Glycohemoglobin for standardizing the measurement of haemoglobin A1c (HbA1c). Owing to the depletion of vials of Lot 1 in March 2001, the present committee certified a new reference material, Lot 2, now distributed by the Health Care Technology Foundation (HECTEF). The standardization programme for HbA1c measurement in Japan is currently based on Lot 2, which has values assigned from within Lot 1; the Lot 1 values were consensus values based on assays by laboratories in the Japanese national quality control programme. In this study, for the purpose of international comparison and standardization, Lot 2 was assayed by the JDS reference laboratories, the National Glycoprotein Standardization Program (NGSP) in the USA, and by reference laboratories approved by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). METHOD: The HbA1c values of JDS Lot 2 were transferred from those assigned to Lot 1 using KO500, a high-resolution HPLC method, at three laboratories approved by the JDS committee. Subsequently, vials of JDS Lot 2 were shipped to and assayed by the NGSP in the USA and 10 IFCC reference laboratories. RESULT: The JDS-assigned HbA1c values (from Lot 1) are 4.04 for Level 1, 5.38 for Level 2, 7.32 for Level 3, 9.88 for Level 4, and 12.63 for Level 5, all expressed as a percentage of total haemoglobin. The values obtained by NGSP and the IFCC laboratories gave the following formulas: NGSP value(%)=JDS value(%)+0.3%; IFCC value(%)=1.068xJDS value(%)-1.741%. CONCLUSION: Although the values obtained by the IFCC laboratories are significantly lower than the values assigned to Lot 2 by the JDS, the relationship is linear. In addition, standardization of HbA1c based on JDS Lot 2 is currently at a satisfactory level in Japan. As a result, the reassignment of values for Lot 2 to agree with the IFCC values should be relatively easy and will be done after all relevant parties agree to the change.

Japanese standard reference material JDS Lot 2 for haemoglobin A1c. II: Present state of standardization of haemoglobin A1c in Japan using the new reference material in routine clinical assays.

BACKGROUND: In 2001, the Committee on Standardization of Laboratory Testing Related to Diabetes Mellitus of the Japan Diabetes Society (JDS) prepared and certified a new reference material for haemoglobin A1c (HbA1c), Lot 2. The standardization programme for HbA1c measurement in Japan is currently based on Lot 2, although some laboratories still use the previous material (Lot 1). The values assigned to Lot 2 were based on the consensus values for Lot 1 and should give the same results. Therefore, there should be no difference in the measured values no matter which calibrators are used. The Committee conducted a domestic survey in order to confirm this relationship. METHOD: In November 2002, four samples for HbA1c assay were sent to 795 laboratories as part of a national survey in Japan. Assays were performed using the laboratories' routine clinical methods. The coefficients of variation (CVs) of the reported values from all laboratories for the samples were calculated in order to determine the current level of standardization in Japan. RESULTS: The overall CVs in the measured values for the four samples ranged from 2.7% to 4.0%. Values from laboratories using calibrators based on Lots 1 and 2 were similar. CONCLUSION: The present state of standardization for the routine measurement of HbA1c in Japan, as indicated by the 2002 survey, is excellent. This should aid in the eventual conversion of Lot 2 to IFCC-based values from the results of the 2002 national HbA1c survey.

[Procedure for accurate evaluation of laboratory data and its international trends].

From the work at ISO/TC212 (Clinical laboratory testing and in vitro diagnostic test systems), the international standards on medical laboratories--particular requirements for quality and competence and reference materials were issued. The accreditation of clinical laboratory was started using ISO 15189. Furthermore, on setting and usage of reference materials were constituted with compatibility global harmonization by newly organizing JCTLM (Joint Committee on Traceability in Laboratory Medicine). As the results, the work of the standardization based on reliability such as validation of reagent's kits and the measured values and uncertainty evaluation would be internationally advanced.

Report on HbA1c Proficiency Testing in Asia in 2012.

In 2010, the Japan Diabetes Society decided to introduce the National Glycohemoglobin Standardization Program (NGSP) values into clinical practice. Accordingly, NGSP Certification of Japanese manufacturers of HbA1c-related diagnostic reagents and instruments was initiated in February, 2012, through an NGSP network laboratory, the Asian Secondary Reference Laboratory (ASRL) #1. Traceability to the NGSP reference system can be endorsed by manufacturer certification, as well as by the College of American Pathologists (CAP) survey. Nevertheless, only a few manufacturers participate in the CAP survey in Japan. Thus, proficiency testing (PT) was proposed and executed by ASRL #1. Single-donor whole-blood samples were used for the PT. The participated measurement systems were NGSP certified. Twenty-two laboratories obtained certification through ASRL #1; 2 through the Secondary Reference Laboratory (SRL) #8; and 9 through the SRL #9. The combination plots of the bias data in this PT and in the NGSP certification performed in March and May in 2012 were consistent with each other: mean NGSP values at each level agreed well with the target value. In conclusion, PT using whole blood is useful in endorsing NGSP certification.

[Laboratory accreditation and proficiency testing].

ISO/TC 212 covering clinical laboratory testing and in vitro diagnostic test systems will issue the international standard for medical laboratory quality and competence requirements, ISO 15189. This standard is based on the ISO/IEC 17025, general requirements for competence of testing and calibration laboratories and ISO 9001, quality management systems-requirements. Clinical laboratory services are essential to patient care and therefore should be available to meet the needs of all patients and clinical personnel responsible for human health care. If a laboratory seeks accreditation, it should select an accreditation body that operates according to this international standard and in a manner which takes into account the particular requirements of clinical laboratories. Proficiency testing should be available to evaluate the calibration laboratories and reference measurement laboratories in clinical medicine. Reference measurement procedures should be of precise and the analytical principle of measurement applied should ensure reliability. We should be prepared to establish a quality management system and proficiency testing in clinical laboratories.

IFCC guideline for sampling, measuring and reporting ionized magnesium in plasma.

Analyzers with ion-selective electrodes (ISEs) for ionized magnesium (iMg) should yield comparable and unbiased results for iMg. This IFCC guideline on sampling, measuring and reporting iMg in plasma provides a prerequisite to achieve this goal [in this document, "plasma" refers to circulating plasma and the forms in which it is sampled, namely the plasma phase of anticoagulated whole blood (or "blood"), plasma separated from blood cells, or serum]. The guideline recommends measuring and reporting ionized magnesium as a substance concentration relative to the substance concentration of magnesium in primary aqueous calibrants with magnesium, sodium, and calcium chloride of physiological ionic strength. The recommended name is "the concentration of ionized magnesium in plasma". Based on this guideline, results will be approximately 3% higher than the true substance concentration and 4% lower than the true molality in plasma. Calcium ions interfere with all current magnesium ion-selective electrodes (Mg-ISEs), and thus it is necessary to determine both ions simultaneously in each sample and correct the result for Ca2+ interference. Binding of Mg in plasma is pH-dependent. Therefore, pH should be measured simultaneously with iMg to allow adjustment of the result to pH 7.4. The concentration of iMg in plasma may be physiologically and clinically more relevant than the concentration of total magnesium. Furthermore, blood-gas analyzers or instruments for point-of-care testing are able to measure plasma iMg using whole blood (with intact blood cells) as the sample, minimizing turn-around time compared to serum and plasma, which require removal of blood cells.

Approved IFCC recommendation on reporting results for blood glucose: International Federation of Clinical Chemistry and Laboratory Medicine Scientific Division, Working Group on Selective Electrodes and Point-of-Care Testing (IFCC-SD-WG-SEPOCT).

In current clinical practice, plasma and blood glucose are used interchangeably with a consequent risk of clinical misinterpretation. In human blood, glucose is distributed, like water, between erythrocytes and plasma. The molality of glucose (amount of glucose per unit water mass) is the same throughout the sample, but the concentration is higher in plasma, because the concentration of water and therefore glucose is higher in plasma than in erythrocytes. Different devices for the measurement of glucose may detect and report fundamentally different quantities. Different water concentrations in the calibrator, plasma, and erythrocyte fluid can explain some of the differences. Results for glucose measurements depend on the sample type and on whether the method requires sample dilution or uses biosensors in undiluted samples. If the results are mixed up or used indiscriminately, the differences may exceed the maximum allowable error for glucose determinations for diagnosing and monitoring diabetes mellitus, thus complicating patient treatment. The goal of the International Federation of Clinical Chemistry and Laboratory Medicine, Scientific Division, Working Group on Selective Electrodes and Point of Care Testing (IFCC-SD-WG-SEPOCT) is to reach a global consensus on reporting results. The document recommends reporting the concentration of glucose in plasma (in the unit mmol/L), irrespective of sample type or measurement technique. A constant factor of 1.11 is used to convert concentration in whole blood to the equivalent concentration in plasma. The conversion will provide harmonized results, facilitating the classification and care of patients and leading to fewer therapeutic misjudgments.

Recommendation for measuring and reporting chloride by ISEs in undiluted serum, plasma or blood.

The proposed recommendation for measuring and reporting chloride in undiluted plasma or blood by ion-selective electrodes (ISEs) will provide results that are identical to chloride concentrations measured by coulometry for standardized normal plasma or blood samples. It is applicable to all current ISEs dedicated to chloride measurement in undiluted samples that meet the requirements. However, in samples with reduced water concentration, results by coulometry are lower than by ion-selective electrode due to volume displacement. The quantity measured by this standardized ISE procedure is called the ionized chloride concentration. It may be clinically more relevant than the chloride concentration as determined by coulometry, photometry or by ISE after dilution of the sample.