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Point Status of lipid and lipoprotein standardization

¹ Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington St., Boston, MA 02111.

² Pacific Biometrics Research Foundation, Seattle, WA.

³ H.S. Raffaele, Milan, Italy.

⁴ Rotterdam University Hospital, Rotterdam, The Netherlands.

⁵ Washington University School of Medicine, St. Louis, MO.

⁶ State Laboratory of Hygiene, Madison, WI.

⁷ Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan.

⁸ Institute of Biochemistry, Glasgow Royal Infirmary, Glasgow, Scotland, UK.

⁹ Canadian Reference Laboratory (1996) Ltd., Vancouver, BC, Canada.

¹⁰ Centers for Disease Control and Prevention, Atlanta, GA.
^a Author for correspondence. Fax 617-556-3103;

	Abstract

Top Abstract Introduction cdc-nhlbi lsp cholesterol reference method... ncep cutpoints: derivation and... Appendix 1 References

 
Cholesterol and triglyceride standardization procedures have been used extensively and continuously since the 1950s. Definitive and Reference Methods, as well as primary and secondary standards, have been developed and maintained as the basis for evaluating the accuracy of results by various methods in many laboratories. But, although standardization efforts for apolipoprotein A-I and B measurements have been reported in detail in the scientific literature, much less has been reported in the area of total and lipoprotein cholesterol and triglyceride standardization efforts. Standardized cholesterol and triglyceride concentrations, determined in multiple large epidemiological and clinical studies, have been instrumental to the National Cholesterol Education Program panels that have assessed the lipoprotein values associated with risk of coronary disease, and have determined the cutpoints that are now used extensively by physicians to guide diagnosis and treatment of individual patients.

	Introduction

Top Abstract Introduction cdc-nhlbi lsp cholesterol reference method... ncep cutpoints: derivation and... Appendix 1 References

 
The editorial in Clinical Chemistry by Sniderman and Cianflone (1) highlighted some of the potential advantages of measurement of apolipoproteins in clinical practice, most notably in its referral to the articles by Contois et al. on apolipoprotein (apo)¹ A-I and apo B reference intervals (2)(3). The improvements in apo A-I and apo B measurements in the past few years have been immense, thanks in great part to work by the IFCC Committee on Apolipoproteins, chaired by Santica Marcovina. This Committee developed World Health Organization (WHO)-IFCC International Reference Materials for apo A-I and apo B, which are now used internationally by manufacturers to set assay calibration (4)(5)(6). The editorial by Sniderman and Cianflone, however, also indicated, mistakenly, that similar standardization of lipid and lipoprotein lipids does not exist.

Although much has been written during the past few years regarding the progress in apolipoprotein measurement and standardization, it was clear from the editorial that public knowledge and understanding is lacking regarding international standardization of the lipid constituents of lipoproteins. In fact, the Lipid Standardization Program (LSP) of the Centers for Disease Control and Prevention–National Heart, Lung and Blood Institute (CDC-NHLBI) has provided standardization for lipid and lipoprotein cholesterol and triglyceride since 1957 in the US and in countries throughout the world (7). So that all may have a complete understanding of the importance of the standardization programs that exist for the cholesterol and triglyceride constituents of lipoproteins, we describe here the programs that are available.

	cdc-nhlbi lsp

Top Abstract Introduction cdc-nhlbi lsp cholesterol reference method... ncep cutpoints: derivation and... Appendix 1 References

 
Definitive and Reference Methods, purified primary standards, and serum reference materials have been developed and coordinated at the CDC as standardization resources for national and international standardization programs (7), in cooperation with NIST, NHLBI, AACC, NCCLS, and WHO. Reference materials have also been developed and evaluated in cooperation with NIST and the College of American Pathologists, and in consultation with manufacturers of diagnostic reagents.

The CDC-established Reference Methods for measuring total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglyceride (8) are used to set reference values for the serum pools used for LSP standardization. These frozen reference pools, stable in storage at -50 °C to -80 °C, are then used to transfer values assigned by the Reference Methods to research and epidemiological laboratories, as points of reference in the evaluation of coronary heart disease (CHD) risk. They have provided the basis for standardization of cholesterol and triglyceride analyses in >30 clinical trials conducted by the NHLBI (7), and in multiple US and international clinical trials, such as the West of Scotland Prevention Study (9) and the CARE Study (10). International standardization of cholesterol and triglyceride measurements has also been provided to laboratories supporting WHO projects and to international epidemiological central laboratories under WHO sponsorship, through the WHO Collaborating Center for Reference and Research in Blood Lipids established at the CDC.

This long-term standardization activity has documented the reliability of these Reference Methods, which are tied to the NIST Definitive Methods for cholesterol (11) and triglyceride (12). Thus, national and international standardization of serum lipid and lipoprotein measurements has been accomplished through CDC, NHLBI, and WHO collaboration, and comparable results are obtainable throughout the world because of these standardization efforts.

	cholesterol reference method laboratory network

Top Abstract Introduction cdc-nhlbi lsp cholesterol reference method... ncep cutpoints: derivation and... Appendix 1 References

 
With the advent of enzymic assays and alternative assay configurations such as dry chemistries, the effects of matrix interactions have required the use of fresh serum comparisons to ensure transfer of accuracy within National Cholesterol Education Program (NCEP)-specified guidelines (13)(14)(15)(16). For this purpose, in 1988, the CDC initiated the Cholesterol Reference Method Laboratory Network (CRMLN) to increase standardization support for manufacturers and clinical laboratories. The CRMLN comprises laboratories in the US, Canada, Europe, and Asia that are all tightly standardized to the CDC (see Appendix). The standardization protocol developed by the CRMLN for total cholesterol is now recognized as a model for standardization of other clinical chemistry analytes. The use of fresh specimens in analytical systems affected by matrix effects ensures accurate results on patients' specimens and comparability of results nationally and internationally. Manufacturers that participate in the program follow the NCCLS EP9 protocol (17), which involves comparing test methods with the Reference Method for 40 fresh sera. Comparisons that meet precision and accuracy requirements set by the CRMLN receive a certificate of traceability. A list of current certificate holders is maintained within the AACC homepage on the World Wide Web (http://www.aacc.org/standards). When establishing the fresh serum certification program, the CRMLN decided that the top priority was certification of manufacturers' systems, because the accuracy of each manufacturer's analytical system would impact on measurements in thousands of clinical laboratories. If clinical laboratories used the analytical systems as directed by their respective manufacturers, traceability to the Reference Methods could thus be transferred.

In addition to the program offered to manufacturers, however, an alternative program is available directly to clinical laboratories, which provides for a six-sample comparison and a certificate when requirements are met. The comparison program for clinical laboratories is less intensive than the manufacturers' protocol, for two reasons. First, because in most cases the manufacturer would have already completed the certification program on the system, the clinical laboratory comparison would simply confirm that the system was working as intended. In cases of heterogeneous systems, confirmation that the various entities work to produce accurate patients' results is obtained. The second reason was clearly one of economics: Clinical laboratories would generally not have the resources to afford the costs of a 40-sample comparison. To maximize the statistical power of evaluating clinical laboratories' six-sample comparisons, however, these laboratories are required to analyze each sample in duplicate on each of 3 days, whereas manufacturers must analyze each sample only once, in duplicate.

The CRMLN maintains direct traceability to the CDC through a rigorous standardization program. CRMLN laboratories perform the Abell–Kendall Reference Method for cholesterol (18), enzymic methods for triglyceride that are standardized to the chromotropic acid Reference Method at the CDC (19), and a designated comparison method for HDL-cholesterol that is tied to the HDL-cholesterol Reference Method (20)(21). The CRMLN laboratories are surveyed monthly to ensure that their analytical performance meets tightly defined specifications. Efforts by the CRMLN to help improve analytical performance of cholesterol and triglyceride measurements in clinical laboratories through the manufacturers is succeeding, as detailed in reports of improvement in results from proficiency testing programs (22).

Historically, LDL-cholesterol values, derived in most laboratories by mathematical calculation, have generally been assumed to be standardized whenever total cholesterol, HDL-cholesterol, and triglycerides are standardized. More recently, however, in conjunction with NCEP performance guideline development (13)(14)(15)(16), it was determined that within-limit performance of the latter three analyses did not ensure within-limit performance of the first. For that reason, the NCEP Working Group on Lipoprotein Measurement recommended that methods be developed that would allow actual LDL-cholesterol measurements to be made in clinical laboratories, and that those methods replace mathematical calculations of LDL-cholesterol. Methods suitable for clinical laboratory measurement of LDL-cholesterol have begun to be developed and to be compared with the Reference Method (23)(24)(25) and must now be added to lipid standardization programs. An unofficial standardization program instituted by Pacific Biometrics Research Foundation (Seattle, WA) in 1993 to standardize LDL-cholesterol determinations among laboratories performing beta-quantification (ultracentrifugation) has been expanded to include those results derived by other methods of measurement. The CDC has recently initiated the first steps of a similar program that would transfer traceability of the CDC Reference Method among CRMLN and LSP laboratories for eventual use in LDL-cholesterol certification protocols.

	ncep cutpoints: derivation and utilization

Top Abstract Introduction cdc-nhlbi lsp cholesterol reference method... ncep cutpoints: derivation and... Appendix 1 References

 
The Definitive and Reference Methods for lipoprotein cholesterol and triglyceride measurements that have been accepted by the NCEP Working Group on Lipoprotein Measurement (14)(15)(16) were developed at NIST and CDC, and have withstood the challenge of time as basic points of reference. The isolation procedures required in the LDL- and HDL-cholesterol Reference Methods, which have been developed and maintained at the CDC, have been clearly documented for validity with respect to the assessment of risk for CHD (26)(27)(28)(29). Standardized cholesterol and triglyceride measurements are the foundations for assessment of CHD risk. The relationships between total, LDL-, and HDL-cholesterol and CHD risk, prevention, and treatment have been documented extensively. Moreover, physicians and patients have become educated as to what analyte concentrations are desirable and what concentrations pose a risk of disease. These standardized lipid measurements can also be used in evaluating the relative importance of other potential markers. For example, in the studies by Contois et al. (2)(3), the recommended cutpoints for apo A-I and apo B, for determining CHD risk, were derived in part from the corresponding NCEP cutpoints already established for lipid and lipoprotein cholesterol concentrations. In addition, because standardization of lipids and lipoproteins is accomplished through Reference Methods that are traceable to NIST Definitive Methods, with use of primary standards and reference calibrators, the critical problem of accurate protein concentration determination of a surrogate standard, such as narrow-cut LDL for apo B (27), is obviated and there is agreement on acceptable reference methods—unlike the situation for apo B (28).

In conclusion, standardized cholesterol and triglyceride measurements provide long-standing basic characteristics for estimating CHD risk. The standardization programs allow direct comparison among the results of many studies, separated by time, country of origin, age, gender, and ethnicity. Because many of the differences in concentration are small, it is important to have, and to document, accuracy and precision limits so that significant associations can be evaluated. Lipid standardization programs provide this ability. Therefore, lipoprotein cholesterol and triglyceride values compiled from national and international trials in CDC-NHLBI-standardized laboratories are the basis by which the NCEP Adult Treatment Panel and other advisory committees throughout the world have classified desirable, borderline, and high-risk lipoprotein values. In the future, use of standardized lipid and apolipoprotein measurements will, we hope, provide even more detailed information for assessment of CHD risk and evaluation of individual dyslipidemic patients.

	Appendix 1

Top Abstract Introduction cdc-nhlbi lsp cholesterol reference method... ncep cutpoints: derivation and... Appendix 1 References

 
List of CRMLN Laboratories
in the us
Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington St., Boston, MA 02111, Contact: Judith R. McNamara, MT, (617) 556-3104 phone, (617) 556-3103 fax, mcnamara_li@hnrc.tufts.edu

Wadsworth Center for Laboratories and Research, Empire State Plaza, Albany, NY 12201, Contact: Robert Rej, PhD, (518) 473-0117 phone, (518) 473-2900 fax, bobrej@wadsworth.org

University of Washington Northwest Lipid Research Laboratories , 2121 N. 35th St., Seattle, WA 98103, Contact: Santica Marcovina, PhD, (206) 685-3331 phone, (206) 685-3279 fax, smm@u.washington.edu

Washington University School of Medicine Lipid Research Center , 660 S. Euclid Ave., St. Louis, MO 63110, Contact: Thomas G. Cole, PhD, (314) 362-3516 phone, (314) 362-7657 fax, thomcole@imgate.wustl.edu

Pacific Biometrics Research Foundation, 1100 Eastlake Ave., Seattle, WA 98109, Contact: Elizabeth Teng Leary, PhD, (206) 233-9151 phone, (206) 233-0198 fax, 102722.1040@compuserve.com

Wisconsin State Laboratory of Hygiene, E. 465 Henry Mall, Madison, WI 53706, Contact: David Hassemer, MS, (608) 833-1770, ext. 102 phone, (608) 833-2803 fax, hassemer@clia.slh.wisc.edu

outside the us
Canadian Reference Laboratory (1996) Ltd., 307–2083 Alma St., Vancouver, BC V6R 4N6 Canada, Contact: David W. Seccombe, MD, PhD, (604) 222-1879 phone, (604) 222-0134 fax, 73361.1047@compuserve.com

Osaka Medical Center for Cancer & Cardiovascular Diseases, 3 Nakamichi 1-chome Higashinari-ku, Osaka 537, Japan, Contact: Masakazu Nakamura, PhD, 81-6-972-1181, ext. 2211 phone, 81-6-972-7749 fax, xnakamur@iph.pref.osaka.jp

H.S. Raffaele Laboratorio Analisi Cliniche, Via Olgettina 60, 20132 Milan, Italy, Contact: Ferruccio Ceriotti, MD, 39-2-2643-2315 or -2313 phone, 39-2-2643-2640 fax, ceriotf@ rsisi.hsr.it

Rotterdam University Hospital Dijkzigt, Lipid Reference Laboratory, 3015 GD Rotterdam, The Netherlands, Contact: Christa M. Boersma-Cobbaert, PhD, 31-10-4633493 phone, 31-10-4367894 fax, boersma@ckcl.azr.nl

Institute of Biochemistry, Glasgow Royal Infirmary, 4th Floor, Alexandria Parade, Glasgow, G31 2ER Scotland, UK, Contact: Christopher J. Packard, PhD, 44-141-552-3535 phone, 44-141-553-2558 fax

currently undergoing standardization
Instituto Nacional de Saude Dr. Ricardo Jorge Av. Padre Cruz 1699 Lisbon, Portugal Contact: Maria do Carma Martins, PhD 351-1-757-7070 phone 351-1-759-0441 fax.

	Acknowledgments

 
We acknowledge consulting agreements by two of the authors with Genzyme Corp. (J.R.M.) and Sigma Diagnostics (J.R.M. and T.G.C.), developer and distributor, respectively, of a method for direct measurement of LDL-cholesterol.

	Footnotes

 
¹ Nonstandard abbreviations: apo, apolipoprotein; LSP, Lipid Standardization Panel; CDC-NHLBI, Centers for Disease Control and Prevention–National Heart, Lung and Blood Institute; CHD, coronary heart disease; NCEP, National Cholesterol Education Program; and CRMLN, Cholesterol Reference Method Laboratory Network.

	References

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This Article Abstract Full Text (PDF) Submit an electronic Letter to the Editor about this paper Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (15) Citing Articles via Google Scholar Google Scholar Articles by McNamara, J. R. Articles by Cooper, G. R. Search for Related Content PubMed PubMed Citation Articles by McNamara, J. R. Articles by Cooper, G. R. Related Collections Laboratory Management Lipids, Lipoproteins, and Cardiovascular Risk Factors