Reference Material Institute for Clinical Chemistry Standards (ReCCS)

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1 Reference Material Institute for Clinical Chemistry Standards (ReCCS) Certified Reference Material for Measurement of Total Cholesterol and Glycerides in Human Serum Certificate of Analysis Intended use This Certified Reference Material (CRM) is intended for use in evaluating the accuracy of Total Cholesterol, Total Glycerides and Triglycerides measurements in Laboratory Medicine, and validating secondary reference material. A vial is taken out from its case and is thawed at room temperature while placing it cap-side up. Next, the vial is left standing for about 30 minutes to bring the temperature of the serum to room temperature. Once this procedure is completed, hold the cap of the vial; gently rotate the vial 20 times in complete circles; and then mix the contents of the vial by turning the vial upside down at least 20 times (the reference material in this manner must be used within three hours. Once thawed, the serum cannot be frozen for reuse. Pipette Hold the cap and rotate the vial to homogenize e its contents. Thawing While placing vials cap-side up, leave them to stand until the temperature of the serum reaches near room m temperature. t After tightening the cap, mix the contents by inverting the vial at least 20 times Sample cup Precautions for use : In Vitro Use Only This CRM has been shown to be non-reactive n-re for HBs antigens, HCV and HIV antibodies. However, no known test method can give complete assurance of absence of HIV, HCV, HBs and antigens, and any other infectious agents. Thus assume that this CRM would be infectious, and exercise the same caution as for handling any other clinical specimens with the risk of infectious diseases. Preparation This CRM was prepared according to the protocol established by the Clinical and Laboratory Standards Institute (CLSI) [1] to ensure that, while avoiding lipoprotein degradation, its properties would be the same as those of fresh serum. Storage and expiration This CRM must be stored in a deep freezer (below -70) upon receipt. Under this condition, its shelf life is 6 months from the shipping date (see the label of the outer case). Product specifications A single set of consists of 4 vials indicated below, each vial contains 0.5ml of human serum. (TC/M) (TC/H) (TG/M,FG) (TG/H,FG) 1/5

2 Certified Concentration Values of Total Cholesterol The Total Cholesterol concentrations and uncertainties at 25below were measured by isotope dilution-mass spectrometry, ID/MS[3,4]. Since the analytical measurements were performed at the Laboratory of ReCCS as well as observance of ISO and ISO15195 quality assurance, these values are accepted internationally via ILAC/APLAC MRA. Levels Total Cholesterol ± mmol/l mol/ (TC/M) (184.2 ± 1.5 mg/dl) ± mmol/l (TC/H) (240.1 ± 2.0 mg/dl) The expanded uncertainty U (95% confidence interval) shown in the above table is obtained by U=ku c. u c is the combined standard uncertainty calculated according to the ISO GUM [2] (uncertainty of SRM and uncertainty of gravimetric method are also included) and k is a coverage factor (k = 2). Traceability to SI units was assured via calibration against NMIJ primary standards (purity ± 0.05%) and consistency was confirmed by comparing with other pertinent standard Materials. Standard solutions and sample solutions used in the measurements were prepared by a gravimetric method using a calibrated balance. Certified Concentration values of Total Glycerides and Triglycerides The Total Glycerides and Free Glycerol concentrations and uncertainties at 25below were measured by ID/MS [5,6]. Triglycerides (TG) are defined as TG = Total Glycerides - Free Glycerol. (TG/M, FG) (TG/H, FG) Total Glycerides mmol/l ( mg/dl) mmol/L ( mg/dL) Free Glycerol mmol/l ( mg/dl) mmol/l ( mg/dl) The expanded uncertainty U (95% confidence interval) shown in the above table is obtained by U=ku c. u c is the combined standard uncertainty calculated according to the ISO GUM [2] (uncertainty of SRM and uncertainty of gravimetric method are also included) and k is a coverage factor (Total Glycerides 2.0, TG 2.2). Traceability to SI units was assured via calibration against NIST Standard Reference Material 1595 (purity 99.5 ± 0.2%) and consistency was confirmed by comparing with other pertinent standard Materials. Standard solutions and sample solutions used in the measurements were prepared by a gravimetric method using a calibrated balance. Glycerides and Triglycerides concentrations are expressed as the triolein concentrations. 2/5

3 Supplemental Data Total Cholesterol concentrations by AK Total cholesterol is internationally standardized by the CDC reference method which is a modified Abell-Kendall method (AK) [7,8] implemented by CRMLN. Reference values by the AK are shown belowand herein compared with those by ID/MS. The total cholesterol concentrations measured by the CDC reference method was higher for both levels than those by the ID/MS. The reason for this is that the color fixing agent (Liebermann-Burchard reagent) used in the CDC reference method reacts with those similar compounds of cholesterol. Table 1. Total cholesterol concentrations by the CDC reference method (AK) CDC Reference Method* Bias of the CDC Reference Method (mg/dl) from the ID/MS Method (TC/M) % (TC/H) % Triglycerides concentrations by the ID/MS Table 2. Triglycerides concentrations and uncertainties by the ID/MS Triglycerides (mg/dl) (TG/M,FG) (TG/H, FG) Uncertainty is expressed as expanded uncertainty, U, calculated as U=ku, where u is the combined standard uncertainty calculated according to the ISO Guide2where homogeneity, stability ity and others, etc. are all combined, and k is a coverage factor. The coverage factors (95 % level of confidence) are all k=2. The triglycerides concentration equals the total concentration of triglycerides, diglycerides and monoglycerides, where free glycerol is not included in triglycerides. IFCC RELA Proficiency Test As shown below, ReCCS demonstrates good results in 2012 (Total Cholesterol) and 2013 (Total glycerides) RELA Proficiency test. [9] 3/5

4 Characteristics of This CRM Table 2. The characteristics of (AK) TC/M TC/H TG/M, FG TG/H, FG Density (g/cm 3 ) Pycnometer method (25 C) Total protein (g/dl) Biuret method Albumin (g/dl) BCG method Triglycerides (mg/dl) Enzymatic method Free fatty acids (meq/l) Enzymatic method Cholesterol ester ratio (%) Enzymatic method Uric Acid (mg/dl) Uricase-POD method Total bilirubin (mg/dl) Vanadic acid oxidation method Lp(a) (mg/dl) Latex agglutination Turbidity Abs Optical density method (710nm, 10mm) (TC/M) (TC/H) α preβ β Chylomicrons α preβ β Chylomicrons Fig 1. Agarose gel electrophoresis pattern References [1]NCCLS Publication C37-A: Preparation and validation of commutable frozen serum pools as secondary reference materials for cholesterol measurement procedures,clsi,wayne,pa,1999. [2]Guide to the Expression of Uncertainty in Measurement, ISBN , 1st Ed, ISO, Geneva, Switzerland (corrected and reprinted, 1995) [3]Cohen A, et al. : Total serum cholesterol by isotope dilution/mass spectrometry; A candidate definitive method, Clin. Chem, 26 : , [4]Ellerbe P, Meiselman S, Sniegoski LT, Welch MJ, White VE : Determination of serum cholesterol by a modification of the isotope dilution mass spectrometric definitive method, Anal. Chem, 61, , [5]Ellerbe P, Sniegoski LT, Welch MJ: Isotope dilution mass spectrometry as a candidate definitive method for determining total glycerides and triglycerides in serum, Clin Chem , [6]Bernert JT Jr, Bell CJ, McGuffey JE, Waymack PP : Determination of free glycerol in human serum reference materials by isotope-dilution [7]Abell LL, Levy BB, Brodie RB, Kendall FE : Simplified method for the estimation of total cholesterol in serum and demonstration of its specificity, J. Biol. Chem, 195, , [8]Cooper GR, Smith SJ, Duncan IW, et al. : Interlaboratory testing of the transferability of a candidate reference method for total cholesterol in serum, Clin. Chem, 32, , [9]RELA - IFCC External Quality assessment scheme for Reference Laboratories in Laboratory Medicine Home Page: 4/5

5 Date of certification October 16, 2017 Provider of Hirohito Umemoto, Ph.D.President Reference Material Institute for Clinical Chemistry Standards (ReCCS) Azaminominami, Aoba-Ku, Yokohama Japan Telephone: Facsimile : ando@reccs.net URL: 5/5