Reference Material Institute for Clinical Chemistry Standards (ReCCS)

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1 Certified Reference Material for Measurement of Glucose, Creatinine, Uric Acid and Urea-N in Human Serum JCCRM Certificate of Analysis Intended use This Certified Reference Material CRM is intended primarily for use in calibrating and evaluating the accuracy of routine methods and validating working reference materials. In order to ensure the reliability of evaluation of routine methods, this CRM has three concentration levels: medium, high and abnormally high. Since neither preservatives nor additives are used, and for preparing this CRM fresh serum is used, this CRM shows s good commutability to enzymatic methods and chemical quantification methods such as Jaffe method. Major intended uses are as follows: (1) To validate working reference materials or calibrators. (2) To evaluate the accuracy of various routine methods. (3) To evaluate the internal and external quality of measurements. Instructions for use (1) Take out a serum vial tube of this CRM and thaw the frozen serum by allowing the tube to stand with the capside up at room temperature for approximately one hour. (2) While holding the serum tube vertically with the cap-side up, hold the cap with fingers, and confirm that the cap is tightly screwed on. If the cap is loose, tighten it securely. Then mix the serum by gently rotating the tube approximately 20 times. Next, turn the tube upside-down slowly at least 40 times to secure homogeneity. Note: Insufficient mixing leads to incorrect measurement results. (3) Conduct sampling of the mixed serum for measurements. s. Unless s used immediately, tighten the vial cap and refrigerate it for use in the same day. Note: Once thawed, the serum should not be frozen again for future use. Precautions for use in vitro use only This CRM is prepared from human serum and shown to be negative to the HBs antigens, HCV antibodies and HIV antibodies. However, since other infectious agents are not completely ruled out, handle this CRM as a biohazardous material capable of transmitting infectious diseases, and take necessary precautions just like working with any biological samples at Biosafety in microbiological and biomedical laboratories. Storage and Expiration ion This CRM is distributed or shipped on dry ice. After receiving, store it immediately in a freezer (below 50 ). Do not use the material if no dry ice remains upon arrival. The shelf life of this material is 6 months from the arrival date when stored at below 50 at an end user. Specifications One of this CRM consists of 6 plastic vial tubes; 2 tubes for each of three different concentration levels (: medium, : high, : abnormally high). Each tube contains 1 ml of human serum. : medium, : high, : abnormally high, H ( producer ) The Laboratory of ReCCS is an ISO Guide 34 (reference materials producer), ISO/IEC (calibration laboratory), and ISO (reference laboratory in Laboratory Medicine) certified Institute 1 / 5

2 Certificate of Analysis Certified Concentration Values in JCCRM The certified values (25 ) and expanded uncertainties of concentrations of Creatinine, Uric acid, Urea-N and Glucose are shown below; Certified concentration values 1) and uncertainties 2) for Creatinine Since these values are certified according to ISO/IEC and ISO 15195, those are accepted internationally via ILAC/APLAC MRA. Table 1a Item Concentration mg/dl H Creatinine 0.86 ± ± ± 0.13 mg/dl Certified concentration values 1) and uncertainties 2) for Uric acid, Urea-N and Glucose Table 1b Concentration mg/dl H Uric acid 5.62 ± ± ± 0.17 mg/dl Urea-N 3) 11.4 ± ± ± 0.7 mg/dl Glucose ± ± ± 2.5 mg/dl Table 1c Concentration n mol/l Item H Creatinine 76± ± ± 11 mol/l Uric acid 334 ± ± ± 10 mol/l Table 1d Concentration mmol/l H Urea-N 3) 4.1 ± ± ± 0.2 mmol/l Glucose 5.60± ± ± 0.14 mmol/l Note 1) Metrological traceability: A standard solution for the measurements of the certified values was prepared using respective NIST SRM (Creatinine: SRM 914a, purity 99.7±0.3 %, Glucose: SRM 917c, purity 99.7±0.2 % and Urea: SRM912a, purity 99.9± %). A balance calibrated by Japan metrology reference system (JCSS: Japan Calibration Service System) was used for the preparation of serum samples and the standard solutions needed in the measurement of the certified values. 2) Uncertainty is expressed as expanded uncertainty, U, calculated as U=ku, where u is the combined standard uncertainty calculated according to the ISO Guide 1 where homogeneity, stability and others, etc. are all combined, and k is a coverage factor. The coverage factors (95 % level of confidence) are 2.2 for 13M, 2.6 for 13H, and 2.6 for 13HH, respectively. The coverage factors (95 % level of confidence) are all k=2. For uric acid and glucose uncertainties, long term reproducibility of each ID/MS was taken into estimation. 3) The certified value of urea nitrogen does not include endogenous ammonia nitrogen. 2 / 5

3 Measurement methods for certified values The certified values were determined by the following reference methods by the ReCCS. Creatinine: Isotope Dilution Mass Spectrometry according to the NIST measurement procedure for creatinine. 2 Uric acid: HPLC method after perchloric-acid deproteinization. 3 (The HPLC method was calibrated with our serum primary reference material for uric acid (JCCRM811) of which values were assayed by the Isotope Dilution Mass Spectrometry at the Laboratory of ReCCS). Urea-N: The urease-gldh enzymatic method in combination with Somogyi deproteinization method. 4 Glucose: The hexokinase-g-6-pd enzymatic method in combination with Somogyi deproteinization method proved to be traceable to ID/MS method (see Fig.1) for glucose. 5 Validation of Hexokinase-G-6-PDH reference method by ID/MS The JSCC reference method for quantification of plasma glucose, hexokinase-g-6-pdh enzymatic method in combination with Somogyi deproteinization method, was evaluated by the deviation from ID/MS. Glucose concentrations of two specimens, and mg/dl by hexokinase-g-6-pdh were and mg/dl by ID/MS, respectively; thus the deviations from ID/MS are calculated to be less than relatively 0.5%, showing that the hexokinase-g-6-pdh method is accurate. For this CRM, our Hexokinese-G-6-PDH e-g reference method procedure was checked by using samples with glucose concentrations quantified by ID/MS, of which biases were less than 1%. Preparation and Serum Property This CRM was prepared using fresh pooled human serum (ammonium nitrogen n is only 0.1 mmol/l as in fresh human serum). It has three concentration levels and the higher levels are prepared by adding high purity creatinine, uric acid, urea nitrogen and glucose into pooled serum. The materials are finally filtrated te using 0.2 μm filter for sterilization and securing homogeneity. JCCRM properties are as follows: Item JCCRM Method Ammonia nitrogen 0.1 mmol/l Colorimetric method Total protein 6.9 g/dl Biuret method Albumin 4.4 g/dl BCG colorimetric method Total cholesterol 166 mg/dl Enzymatic method Triglycerides 81 mg/dl Enzymatic method Density (25 ) g/cm 3 3 / 5

4 Comparison with certified values & measurement values in routine method Glucose Reagent A Glucose Reagent B Urea-N Reagent A Urea-N Reagent B Creatinine Reagent A Creatinine reagent B Uric acid Reagent A Uric acid Reagent B / 5

5 ID/MS Comparison Creatinine (1)Measurement values of SRM Our ID/MS technique and procedure were evaluated using NIST SRM 967a. A figure right shows each bias from the certified value by the NIST is within the each expanded uncertainty. (2)Comparisons with reference laboratories Each relative bias of three laboratories for creatinine is all 2 % or less from the all mean. References 1 Guide to the Expression of Uncertainty in Measurement, ISBN ,1st Ed. ISO., Geneva, Switzerland, Dodder NG, Tai SS, Sniegoski LT, Zhang NF, Welch MJ : Certification of creatinine in a human serum reference material by GC-MS and LC-MS, Clin Chem, 53(9) : , Reagent Committee, Japan Society of Clinical Chemistry: Recommended Methods for the measurement of Serum Uric Acid using HPLC Rinsho Kagaku,22 : , EJ Sampson, MA Baird, CA Burtis, EM Smith, DL Witte, DD Bayse : A Coupled-Enzyme Equilibrium Method for Measuring Urea in Serum: Optimization and Evaluation of the AACC Study Group on the Urea Candidate Reference Method, Clin Chem, 26 : , Reagent Committee, Japan Society of Clinical Chemistry: Recommended methods for measuring serum glucose, Rinsho Kagaku,, 20 : , 1991 Date of certification February 6, 2017 Provider of JCCRM Hirohito Umemoto, Ph.D. (President) Address: Azaminominami, Aoba-ku, Yokohama Japan Telephone: Facsimile: ando@reccs.net URL: 5 / 5