- Email: [email protected]
Lipid testing for the year 2000 and beyond
ATHEROSCLEROSIS Atherosclerosis 108 (Suppl.) (1994) S181 -Sl89
Lipid testing for the year 2000 and beyond Donald A. Wiebe Deparment
of Pathology anil Laboratory Medicine, ihhersity q/’ Wi.~coiain-Madison, Ma&.yorl, WI, USA
Abstract Lipid testing has progressed from early measurements of total lipid by extraction and weighing to assess the fat content of the specimen. This nonspecific approach to lipid testing has been replaced in clinical laboratories by automated and quantitative procedures that avoid the extraction process. Instead, selective enzymes are utilized in reaction schemes to quantitate the individual lipid classes present in patient specimens. For example. cholesterol esterase and oxidase are used on a routine basis to measure total cholesterol in plasma and serum specimens. Similar use of other enzyme systems has permitted triglycerides and phospholipids to be measured by clinical laboratories. Lipid and lipoprotein measurements have advanced considerably from the early nonspecific extraction and gravimetric analysis schemes to the specific automated procedures that are commonly used today. However, as lipids and lipoproteins increased in their clinical usefulness as cardiovascular risk assessment tools, the search intensified for newer approaches to measure these entities more easily and more accurately. The influence of National Cholesterol Education Program has played a key role in highlighting the importance of lipids and lipoprotein analysis. Today, lipid testing is available outside the traditional laboratory environments - drugstores sell units that individuals can use at home to assess cholesterol levels. Lipid testing has come a long way, and we have only begun to experience some of the remarkable changes for the future. Keywords:
Cholesterol; Triglycerides; HDL-C; Lipoproteins
1. Introduction Demand for accurate and precise lipid and lipoprotein tests is at an all-time high and will continue into the foreseeable future. Supported by standardized assessment and treatment schemes aided in part by the development of new hypolipidemic drugs, physicians have become more confident and aggressive in treating hyperlipidemia. All forms of mass media have inundated the public with educational and promotional material to be cognizant of cholesterol and its strong association with increased risk for cardiovascular disease. 0021-9150/94/$07.00
These activities have promoted a sense of hightened awareness surrounding lipid and lipoprotein testing. Laboratories that measure lipids and lipoproteins have been the beneficiary of this enthusiasm about cholesterol. Increased requests for laboratory services have been met with demands for more accurate cholesterol methods and development of new procedures for screening individuals. These challenges were met with overwhelming success for improved accuracy requirements and development of reliable screening methods which are used today. The questions remain: will this interest in lipids and lipoproteins remain high,
J.* 1994 Elsevier Science Ireland Ltd. All rights reserved
SSDI 0021-9150(94)05295-T
D.A. Wiebe / Atherosclerosb 108 (Suppl.) (1994) SISI -Si89
~-.~~~~
~~/IlI/l
m
‘“.,
Fig. 1. Four key development stages of laboratory tests from the early period of manual methods to the future of robotics systems.
and what can we expect for lipid testing for the year 2000 and beyond? The future for lipid and lipoprotein testing will obviously hinge directly on the reliability of these constituents to predict cardiovascular heart disease (CHD) risk for individuals and the population. The clinical usefulness of any given analyte or procedure ultimately will dictate the degree to which clinicians request the service. The assumption is made that the clinical importance of lipid and lipoprotein testing will remain high, and this presentation will focus on what the future has to offer in terms of laboratory developments with respect to lipids and lipoproteins. First, a look at our past and‘current approaches to lipid testing provides an appreciation of how much change can occur in only IO-30 years. In addition, a key component for judging what might happen in the future is to understand some of the factors that influence laboratories and the selection of new approaches. Finally, the crystal ball will be used to predict the future. Of course, the only certainty for the future is that change will occur. 2. Return to the past 2.1. Instrumentation A glimpse backward may provide some insight as to what might be in the future. Laboratories have undergone a tremendous evolution over the past 40 years, especially with respect to instrumentation used to perform various tests. Fig. 1
illussrates four transition periods that influenced development of instrumentation in laboratory medicine. Initially, laboratory tests were performed in test tubes using glass pipettes to transfer sample and reagents followed by manuai spectrophotometric readings. Imagine the slow throughput and tedious approach for medical technologists performing various analyses. During the early 1960s automated laboratory procedures were making their way into laboratories and change was just beginning. The Technicon Autoanlyzer is an excellent example of the new systems that laboratories could work with to improve their workflow and efficiency. These firstgeneration laboratory systems still required large volumes of patient specimens for the assays, but the throughput was considerably faster than achieved by manual approaches. A key aspect of these early instruments was the flexibility of using different sources of reagents to perform the desired testing. Instruments were open systems that permitted laboratorians to make decisions concerning reagents or procedural changes that would yield results to meet their needs. The manufacturer’s profit was realized primarily from selling instruments. Reagents and service for the instruments were secondary factors. The second generation of clinical instruments were closed systems in which only the manufacturer’s reagents could be used and the customer had to rely on their supplier for the overall quality of materials. Advantages were gained in the enhanced accuracy
D.A. Wiebe I Atherosclerosis 108 (Suppl.) (1994) S181-S189
and precision achieved by the end user, especially when used in small laboratory environments with minimal technical support staff. Manufacturers placed additional enhancements on these new analyzers that have been effective at lowering the manual operations required by the laboratory staff to yield a reliable and precise value. These features include: (1) random access analysis, (2) precise reagent and sample transfer, (3) microcomputer control, (4) electronic data transfer via computer interface, and (5) on-line diagnostic testing. In general, manufacturers provided laboratorians with complex systems that are computer-driven and capable of high throughput with superior precision and accuracy. What can the future have to offer that is currently unavailable? Robotics is the way of the future. Robotics will offer walk-away systems that will interact with multiple instruments, capable of primary tube sampling, and intelligent processing to perform the majority of processing techniques currently carried out by laboratory personnel. 2.2. Lipid analysis In years past, the only requirement in locating the lipid laboratory was a keen sense of smell: the acetic acid/acetic anhydride odour was a familiar aroma that emanated from these specialty laboratories. Every operation was a manual procedure requiring large quantities of organic solvents used to isolate lipid fractions from serum proteins and other serum constituents. Separatory funnels, large beakers, glass burettes, thin-layer chromatography tanks, digestive setups, extraction apparatus, an array of grade A glass pipettes, and a single- or dual-beam spectrophotometer with quartz cuvettes were just a few standard fixtures in these facilities. In general, caustic reagents were the basis for analytical methods employed to measure cholesterol, triglycerides, phospholipids, and fatty acids. Throughput and turnaround times were of less concern to the laboratories because these relatively new methods of laboratory productivity were expressed in days or weeks. Table 1 illustrates typical information reported from these early lipid laboratories. Note the use of the term mg% to express the concentration of the analytes - it brings back memories.
S183
Table 1 Lipid testsand normal ranges for assays typically performed in laboratories during the late 1950s Lipid assay
Normal range
Total cholesterol Free cholesterol Cholesterol esters Phospholipids Sphingomyelin Cephalin Lecithin Total fatty acids Neutral fat
150-280 mg% 30-60 mg% 70-75% of total 1SO-250 mg% 15-30 mg% O-20 mg% 1SO-230 mg% 200-700 mg% O-400 mg%
Even during the 1950s standardization of cholesterol data between laboratories was a con cem, as evidenced by this quote taken from a leading text on lipid disorders [ I] : “It may be emphasized that a comparison of figures of cholesterol and cholesterol esters is possible only if the same method is applied by all authors.’ Cholesterol standardization is one area in which significant progress and success has been achieved during recent years. The credit for the success achieved with cholesterol standardization must be given to the combined efforts of several organizations working toward this common goal. However, as we move from the past into today and on into the future, standardization is one activity that must be carried forth with inany other analytes besides cholesterol. All other lipid and lipoprotein assays have significant challenges before comparable success can be claimed. 3. Current status of lipid testiug Today’s laboratory has the benefit. of increased technology to perform lipid analyses. Instead of Table 2
Lipid tests typically performed in routine clinical laboratories during the 1990s Tests Cholesterol Triglycerides HDL-cholesterol Estimated LDL-C
Apolipoproteins Apo A-I Apo B Lp(a)
D.A.
S184
300
1
Wiebe / Atherosclero.~is 108 (Suppl.)
(1994) S181-Sl89
1990 - Lipid Workload
ELOL
l Chol q TG 0 HDL
In-patient
Out-patient
Fig. 2. Comparison of inpatient and outpatient requests for lipid tests. C&H, cholesterol and HDL-cholesterol; Chol, cholesterol; C&T. cholesterol and triglycerides: CT&H, cholesterol, triglycerides and HDL-cholesterol; ELDL, estimated LDL-C; HDL, HDL-cholesterol: SLPP, serum lipoprotein profile; TG, triglycerides; T&H, triglycerides and HDL-cholesterol.
the caustic reagents and tedious tasks, laboratories use enzymic assays that deliver high specificity along with increased sensitivity. These tests are readily available on computer-driven automated systems with high throughput which require a nominal amount of specimen to perform the service. Table 2 lists some of the usual lipid ant’ lipoprotein tests performed at the University of Wisconsin Hospital and Clinics (UWHC) and are representative of the typical laboratory. Single tests for cholesterol, triglycerides, and HDLcholesterol are only occasionally asked for by clinicians at UWHC. Instead, combinations or profiles of these various tests are ordered to facilitate the treatment or management of hyperlipidemia. The most frequently ordered profile is the package with cholesterol, triglyceride, and HDLcholesterol that uses the Friedewald e,quation to estimate the LDL-cholesterol. A less requested battery includes the same tests along with lipoprotein electrophoresis to assign a classical lipoprotein phenotype. UWHC is a medical school and an established tertiary care center: a monitor of ordering sources for lipid and lipoprotein tests revealed interesting information. Lipid and lipoprotein requests were monitored for a month (mid-October to mid-November) during 1990 to
determine what percentage of lipid orders were received from inpatients or outpatients (Fig. 2). Greater than 85% of these specialty tests were demonstrates received from outpatients: this proper utilization of lipid and lipoprotein requests by the UWHC clinical staff. In general, cardiovascular risk assessment lipid and lipoprotein tests should be performed on outpatients. An exception is triglyceride requests that may be useful to monitor patients receiving intralipid therapy; triglycerides represent over 50% of the orders for inpatients. Manufacturers of laboratory instrumentation and reagents have provided several options for cholesterol analysis with the necessary accuracy and precision to meet the NCEP guidelines. Many instruments are calibrated by the manufacturer to provide accurate cholesterol results for patient specimens, which simplifies the task for the iaboratory using the system. Triglycerides and HDLcholesterol assays are not well controlled among laboratories or from one method to another. These assays require a similar standardization eiTort to that which successfully brought cholesterol under control. Precision for triglycerides and HDL-cholesterol assays is acceptable, with 2% 3% coefficients of variation (CV) achievable with
D.A.
Webe
I Atltermdero.~is
most commercial methods. In general, much of the credit for success that laboratories have experienced with improved performance of lipid tests belongs to manufacturers and the highly automated systems they developed.
108 (SuppI.)
Table
(1994) Si81 -SJ89
3
Factors
that must be considered
laboratory Laboratory
issues
(new or existing) Reagent stability
Laboratory medicine and certainly the practice of clinical chemistry have only begun to feel the impact of the Clinical Laboratory Improvement Act (CLIA) passed by the US Congress in 1988. CLIA-88 deals primarily with performance issues of all testing executed by clinical laboratories, with licensure dependent on the success in meeting specified guidelines. Along with the performance issues of CLIA-88, laboratories are being scrutinized as cost centers since reimbursement for services is rapidly changing. In the past, issues that concerned the lipid laboratory included fasting versus nonfasting, serum versus piasma, precision accuracy, quality control materials and practices, and reference values. Today and for the foreseeable future, other issues must be factored into the decision process, including: screening, home testing, turnaround time, costs versus reimbursement, and biological variability. At the center of all these issues, the laboratory must deal with the test’s clinical utility. Obviously, clinical utility of a test should dictate the type and quality of service the laboratory delivers for the test. To meet this final issue requires close communication with clinicians ordering the service. The NCEP’s Adult Treatment Panel ( ATP) [2] established treatment guidelines to aid physicians who deal with hypercholesterolemic individuals and provide many of the clinical utility requirements. A few recent events will impact on fu?ure lipid and lipoprotein testing. One is the ATP, which revised its recommendations (ATP II) [3] and clearly establishes the importance of LDL-cholesterol analysis as the primary approach to monitoring effectiveness with either diet or drug intervention. Also, ATP II added HDL-cholesterol to cholesterol as the lipid parameters that should be screened to assess an individual’s risk for CHD. Thus, these new guidelines have increased the significance of both HDL and LDL
in order to establish a new
procedure
Instrumentation
4. Factors influencing laboratory practice
SI8.5
Cast/sample
Fastinglnonfasting Precision (short and long term) Accuracy
Sample size
Proficiency testing
Sample processing
Control
Spwimen
Data transfer
type
interferences Turnaround
materials
Reference values time
Reimbursement
testing within the practice of laboratory medicine. Certainly the new CLIA-88 regulations will further inlluence the utilization of lipid tests, especially the laboratory’s responsibility to document performance standards for all the lipid and lipoprotein tests. New and projected national health care policies may place greater uncertainty on the medical usefulness of lipid and lipoprotein measurements. However, treatment guidelines and health care policy issues aside, several laboratory initiatives will certainly have an impact on the future lipid laboratory. Traditionally, laboratories were primariiy concerned with analytical performance, such as accuracy and precision, of new assays. However, in today’s laboratory environment decisions to establish a new method in the laboratory will involve a series of issues as listed in Table 3. Certainly cost issues are high on the list of factors that influence which tests are offered in the laboratory - especially cost per test, which may dictate the specific instrument or reagent system to be used. Operational costs have to be determined, including direct labor and maintenance requirements that add to the cost of each test. A frequently overlooked issue is the amount of time required for calibration and troubleshooting of a particular method. A method requiring frequent attention or adjustments may cause more problems than an assay that is stable for a longer period of time. All these issues have become dominant factors for laboratories that wish to be competitive in laboratory medicine.
Sl86
D.A. Wiebe 1 Atherosclerosis 188 (Suppl.) (1994) Sl8lLSl89
5. Recent developments influencing lipid and lipoprotein tests 5.1. Direct LDL-cholesterol Until recently, laboratories had few options available for quantitation of LDL-cholesterol. The combination of ultracentrifugation and selective precipitation with heparinlmanganese chloride techniques serves as the basis of the which was the Lipid Re‘beta-quantification’ search Clinic’s approach for LDL-cholesterol [4]. However, few laboratories have access to an ultracentrifuge and, even if they have one, the method requires significant time and sample volume such that the process fails to be cost-effective. Thus, quantitating LDL-cholesterol via ultracentrifugation is usually reserved for research facilities. Instead, clinical laboratories have relied on the Friedewald equation for estimating a patient’s LDL-cholesterol from cholesterol, triglyceride, and HDL-cholesterol values [5]. The equation provides a reasonable estimation of LDL-cholesterol for samples that have typical lipoprotein phenotypes and mildly elevated triglycerides. However, the occasional hypertriglyceridemic (TG > 400 mg/dl) or atypical phenotype specimen results in a falsely low LDL-cholesterol value by Friedewald estimation. Until recently, the ultracentrifuge was the only alternative approach for quantitating LDL-cholesterol for these unusual specimens. An LDL-cholesterol assay is now commercially available that utilizes antibodies directed against VLDL and HDL to isolate LDL [6]. Thus, LDL-cholesterol can be quantitated directly and conveniently with standard automated analyzers with minimal sample pretreatment. This direct assay and others that are likely to follow offer significant advantages over the use of an equation. One advantage of the direct LDL assay is that either fasting or nonfasting samples can be crllected from patients and rhe results are valid. Immunoseparation schemes, such as the direct LDL-cholesterol assay, may offer specific and accurate approaches to several lipoprotein measurements. Look for additional development and exploitation of the selectivity of antibodies directed against specific lipoproteins for the future of lipoprotein profiling. Identifying
lipoproteins by their specific apolipoproteins may serve as the basis for future lipoprotein nomenclature. Such a nomenclature scheme would be dependent on immunoassays for both quantitation and speciiic separation lipoprotein classes. 5.2. Nuclear magnetic resonance (NMR)
Proton NMR was a key approach to structure identification for organic chemists during the past 20 years. Magnetic resonance imagery has become a significant tool to provide detailed views of tissues, organs, or body sections for diagnostic purposes without the use of X-rays or other invasive approaches. Now, NMR can be similarly applied in the laboratory to establish the lipoprotein profile of a given plasma specimen without having to perform a complex separation of individual components [ 71. Recent reports have documented acceptability and performance standards achievable by NMR. However, NMR requires elaborate instrumentation and is therefore likely to be used only in specialized laboratories. Keep in mind that the more complex procedures have ways of becoming more simplified in future generations of the technology, and more widely applied. Look for a specific NMR system developed with only the lipoprotein profiling aspect as the outcome and the instr:lmentation may be less demanding. 5.3. Magnetic par title separation An adaptation to the HDL separation scheme is the use of magnetized particles with dextran sulfate and magnesium chloride to achieve isolation without centrifugation. Magnetic HDL precipitation reagent is commercially available from Reference Diagnostics (Arlington, MA 02174). Several reagent systems are available to isolate HDL by precipitation of VLDL and LDL fractions. All these approaches have been developed with a centrifugation step to isolate the HDL supernatant. In fact, the variability of HDL procedures may be attributed to the myriad factors with incubation conditions such as time and temperature to achieve maximum reagent/lipoprotein interaction and centrifugation parameters such as temperature, time, and force of spin. Some HDL separation schemes are sensitive to slight varia-
D.A.
Wiehe
/ Atherosclerosis
tions in any of these conditions. The use of magnetic particles offers some improvements over the need for centrifugation. The most obvious advantage is in eliminating a need f’or the centrifuge to isolate HDL. However, the ability of the magnetic particles to separate both insoluble and soluble lipoprotein complexes with dextran particles may be more important. A key factor for separation of LDL and VLDL particles by centrifugation is the formation of insoluble precipitate with the reagents. Failure to form the insoluble complexes is a problem with some reagent systems, especially with hypertriglyceridemic specimens (TG > 400 mg/dl or higher). Thus, magnetic particles and other similar innovative techniques for separation or isolation of lipoproteins have a role in the future lipid laboratories. 5.4. Eiectrophoresis Lipoprotein electrophoresis has taken a back seat to newer techniques, especially selective precipitation techniques for separating and isolating lipoproteins. Electrophoresis was the primary technique used to assign the Fredrickson-Levy phenotype for a patient’s lipoprotein pattern. However, since the assignment of the patient’s phenotype offers little useful diagnostic information for the treatment of the hyperlipidemia, electrophoresis has been replaced by other techniques or discontinued altogether. Attempts to utilize electrophoresis as a method to quantitate lipoprotein fractions using specific enzgmic reagents have fallen short due to lack of sensitivity and poor reproducibility. So what is the future for lipoprotein electrophoresis? Lipoprotein electrophoresis, especially using agarose gel or cellulose acetate supports, will continue in a small way to serve as a qualitative tool to investigate lipoprotein abnormalities. But the real future of electrophoresis will depend on its ability to resolve complex lipoprotein families into fractions that provide greater diagnostic insight th,n current isolation schemes. Quantimetrix Corporation (Hawthorne, CA 90250) offers a high resolution polyacrylamide system capable of fractionating LDL into multiple subspecies. Thus, individuals with increased numbers of small. dense LDL particles that are more atherogenic
108 (Suppl.)
(1994) SIl?--S/89
5187
than the larger LDL particles can be easily identified and provided with more aggressive therapy. Other advances in electrophoresis techniques will have a positive impact on tahoratory medicine. Capillary electrophoresfi has received considerable attention in the biological field for separating proteins and metabolites with exceptional resolution. Such is the case with the recent report of electrophoresis performed on a microchip [g]. A mixture of six fluorescent-labeled amino acids was resolved by capillary electrophoresis in 15 s. The authors note that miniaturized electrophoretic systems offer considerable advantages over existing technology in terms of cost, reduced solvent and sample requirements, and analysis time. The potential exists for such an electrophoretic system to be fabricated to provide a complete analytical system on a microchip. Thus, electrophoresis will continue to be an integral part of the future laboratory; however, its specific role in lipid and lipoprotein testing remains to be determined.
Preparative ultracentrifugation remains the standard reference method for the isolation of sample relipoprotein classes. Unfortunately, quirements, spin times, expensive equipment, and overall technical complexity remain key obstacles that prevent ultracentrifugation from becoming a routine method in clinical laboratories. The bench ultracentrifuges which require 1.0 ml or less of sample provide an effective means to remove chylomicrons with short spin times (2-6 min), depending on the extent of turbidity, to eliminate the excessive lipid as an interferent for other tests. Other inherent problems and technical complexities prevent ultracentrifugation from achieving true status as a reference method or usefulness as a routine procedure. However, the ultracentrifuge will remain the standard for lipoprotein isolations in specialized research laboratories.
The use of antibodies directed against apo:ipoproteins associated with lipoproteins was mentioned previously with the development of a specific LDL assay. However, antibodies directed
S188
D.A.
Wiebc / .4tlterosclerosis 108 (Suppl.)
against these proteins is only one approach being explored in laboratories today. In addition, researchers use molecular probe techniques to generate diagnostic information from a subject’s DNA to discover possible genetic or functional changes in the protein synthesis that could explain a metabolic defect. Molecular diagnostic procedures are going to play a significant role in laboratory medicine, and certainly our understanding of lipid and lipoprotein abnormalities will be enhanced through these techniques. Similarly, researchers use biological receptor assays to gain additional information of abnormalities occurring at the cellular level. The role of LDL and its receptor to internalize the lipoprotein into the cell and ultimately down-regulate cholesterol synthesis is a key to understanding the abnormalities associated with familial hypercholesterolemia. The use of receptor assays in laboratory medicine has focused primarily on lymphocytes and hematologic issues to date, but the future may certainly have lipoprotein-related aspects.
6. The future Cardiovascular disease remains the number one cause of mortality, and with increased efforts directed at preventive medicine the need to measure lipid and lipoprotein fractions will expand. Our laboratories will continue to use more and more sophisticated techniques to quantitate many of the same analytes that we currently measure (cholesterol, triglycerides, HDL, and LDL). Issues concerning fasting or nonfasting will continue to be a factor for specific tests, but possible alternative sampling techniques will improve the reliability and interpretation of results. Possibly, indwelling sensors or a noninvasive technique might supplement our standard assays, but that these approaches will ever replace the need for a serum total cholesterol is difficult to envision. The certainties of the future will include smaller sample requirements, greater use of computers to operate our laboratories, and advanced information systems for archiving, retrieval, and interpretation of laboratory data (a universal nomenclature system remains in doubt).
(1994) SISI-S189
The centralized laboratory is currently being challenged by home testing and patient bedside services that can deliver results in a more timely manner. There is limited clinical need to provide more timely lipid measurements, therefore lipid test should be a minor player in this ‘home testing’ arena. However, these activities will continue to increase in the future and the search for simpler and less invasive techniques will be intensified and driven by incentives to keep medical costs under control. Obviously, reimbursement for laboratory services will be a critical issue. Specialized lipid and lipoprotein testing used to assess an individual’s risk for cardiovascular disease or to monitor treatment intervention for hyperlipoproteinemia can easily be performed outside a hospital setting and should be a clinic activity. Lipid test results should be provided on a STAT basis to physicians to have the most up-to-date information during the patient’s examination. Treatment of lipid and lipoprotein abnormalities requires behavior modification, and current tests results will reinforce a positive treatment program for the patient and increase the efficiency and utilization of physician time. Thus, in our future, lipid testing will need to be fast, accurate, and readily accessible in a clinic setting to meet the demands for patient care. What does the crystal ball hold for the future of lipid testing? As noted previously, demand for accurate and precise lipid and lipoprotein testing will continue to be strong. Cholesterol and triglycerides will continue to be the key lipid tests provided with the reliable enzymic assays that have been developed during the past decade. Expect to have vastly improved, stable, matrix-free quality control and serum calibrators available to deliver maximum performance of these assays. Lipoprotein testing will be the area in which greater change is likely to occur. First, a need may arise to develop reliable methods to determine the heterogeneity of the lipoprotein classes, especially if the atherogenicity of the lipoprotein changes as the size of the particle changes. Electrophoresis, especially capillary electrophoresis, may provide the basis for resolving different sizes of lipoproteins (such as LDL) to add to the physician’s menu of tests for assessment of a patient’s risk for cardiovascular disease
D.A. Wide
I Atherosclerosis 108 (Suppl.) (1994) Sl81-Sl89
The breakthrough will come with improved tests for specific lipoproteins. We have already observed increased activity in this area with the introduction of the direct-LDL assay using immunoseparation schemes to isolate select lipoprotein classes prior to cholesterol analysis. A homogeneous assay may be the next generation and the way of the future. Imagine an LDLcholesterol assay in which no separation or isolation step is required: only mixing the serum with reagent and cholesterol analysis is directed against the desired lipoprotein. The future is bright for lipid and lipoprotein testing. References 111 Thannhauser. S., Lipidoses: Diseases of the Intracellular PI
Lipid Metabolism, 3rd Edn., Gruune and Stratton, New York, 1958. The Expert Panel, Report of the National Cholesterol Education Program Expert Panel on Detection. Evaluation, and Treatment of High Blood Cholesterol in Adults, Arch. intern. Med.. 148 ( 1988) 36.
Sl89
[31 The Expert Panel. Sumrlmy of the Second Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation. and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II), J. Am. Med. Assoc., 269 (1993) 3015. 141 Hainline, A., Karen, J. and Lippel, K. (Eds.), Lipids Research Clinics Program, Lipid and Lipoprotein Analysis, Manual of Laboratory Operations, 2nd Edn., US Dept. HHS. Bethesda. MD, 1982. [51 Friedewald, W.T.. Levy, R.I. and Fredrickson, D.S., Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge, Clin. Chem., 18 (1972) 499. I61 Leary. E.T.. Tjersland. G., and Warnick, G.R.. Evaluation of the Genzyme immunoseparation reagent for direct quantitation of LDL cholesterol, Clin. Chem.. 39 ( 1993) 1124. 171 Otvos. J.D., Jeyarajah. E.J.. Bennett. D.W.. and Krauss, R.M., Development of a proton nuclear magnetic resonance spectroscopic method for determining plasma lipoprotein concentrations and subspecies distributions from a single. rapid measurement, Clin. Chem., 38 (1992) 1632. PI Harrison. D.J.. Fluri. K., Seiler, K. et al.. Micromaching a miniaturized capillary electrophoresis-based chemical analysis system on a chip. Science. 261 ( 1993) 895.
Lipid testing for the year 2000 and beyond Donald A. Wiebe Deparment
of Pathology anil Laboratory Medicine, ihhersity q/’ Wi.~coiain-Madison, Ma&.yorl, WI, USA
Abstract Lipid testing has progressed from early measurements of total lipid by extraction and weighing to assess the fat content of the specimen. This nonspecific approach to lipid testing has been replaced in clinical laboratories by automated and quantitative procedures that avoid the extraction process. Instead, selective enzymes are utilized in reaction schemes to quantitate the individual lipid classes present in patient specimens. For example. cholesterol esterase and oxidase are used on a routine basis to measure total cholesterol in plasma and serum specimens. Similar use of other enzyme systems has permitted triglycerides and phospholipids to be measured by clinical laboratories. Lipid and lipoprotein measurements have advanced considerably from the early nonspecific extraction and gravimetric analysis schemes to the specific automated procedures that are commonly used today. However, as lipids and lipoproteins increased in their clinical usefulness as cardiovascular risk assessment tools, the search intensified for newer approaches to measure these entities more easily and more accurately. The influence of National Cholesterol Education Program has played a key role in highlighting the importance of lipids and lipoprotein analysis. Today, lipid testing is available outside the traditional laboratory environments - drugstores sell units that individuals can use at home to assess cholesterol levels. Lipid testing has come a long way, and we have only begun to experience some of the remarkable changes for the future. Keywords:
Cholesterol; Triglycerides; HDL-C; Lipoproteins
1. Introduction Demand for accurate and precise lipid and lipoprotein tests is at an all-time high and will continue into the foreseeable future. Supported by standardized assessment and treatment schemes aided in part by the development of new hypolipidemic drugs, physicians have become more confident and aggressive in treating hyperlipidemia. All forms of mass media have inundated the public with educational and promotional material to be cognizant of cholesterol and its strong association with increased risk for cardiovascular disease. 0021-9150/94/$07.00
These activities have promoted a sense of hightened awareness surrounding lipid and lipoprotein testing. Laboratories that measure lipids and lipoproteins have been the beneficiary of this enthusiasm about cholesterol. Increased requests for laboratory services have been met with demands for more accurate cholesterol methods and development of new procedures for screening individuals. These challenges were met with overwhelming success for improved accuracy requirements and development of reliable screening methods which are used today. The questions remain: will this interest in lipids and lipoproteins remain high,
J.* 1994 Elsevier Science Ireland Ltd. All rights reserved
SSDI 0021-9150(94)05295-T
D.A. Wiebe / Atherosclerosb 108 (Suppl.) (1994) SISI -Si89
~-.~~~~
~~/IlI/l
m
‘“.,
Fig. 1. Four key development stages of laboratory tests from the early period of manual methods to the future of robotics systems.
and what can we expect for lipid testing for the year 2000 and beyond? The future for lipid and lipoprotein testing will obviously hinge directly on the reliability of these constituents to predict cardiovascular heart disease (CHD) risk for individuals and the population. The clinical usefulness of any given analyte or procedure ultimately will dictate the degree to which clinicians request the service. The assumption is made that the clinical importance of lipid and lipoprotein testing will remain high, and this presentation will focus on what the future has to offer in terms of laboratory developments with respect to lipids and lipoproteins. First, a look at our past and‘current approaches to lipid testing provides an appreciation of how much change can occur in only IO-30 years. In addition, a key component for judging what might happen in the future is to understand some of the factors that influence laboratories and the selection of new approaches. Finally, the crystal ball will be used to predict the future. Of course, the only certainty for the future is that change will occur. 2. Return to the past 2.1. Instrumentation A glimpse backward may provide some insight as to what might be in the future. Laboratories have undergone a tremendous evolution over the past 40 years, especially with respect to instrumentation used to perform various tests. Fig. 1
illussrates four transition periods that influenced development of instrumentation in laboratory medicine. Initially, laboratory tests were performed in test tubes using glass pipettes to transfer sample and reagents followed by manuai spectrophotometric readings. Imagine the slow throughput and tedious approach for medical technologists performing various analyses. During the early 1960s automated laboratory procedures were making their way into laboratories and change was just beginning. The Technicon Autoanlyzer is an excellent example of the new systems that laboratories could work with to improve their workflow and efficiency. These firstgeneration laboratory systems still required large volumes of patient specimens for the assays, but the throughput was considerably faster than achieved by manual approaches. A key aspect of these early instruments was the flexibility of using different sources of reagents to perform the desired testing. Instruments were open systems that permitted laboratorians to make decisions concerning reagents or procedural changes that would yield results to meet their needs. The manufacturer’s profit was realized primarily from selling instruments. Reagents and service for the instruments were secondary factors. The second generation of clinical instruments were closed systems in which only the manufacturer’s reagents could be used and the customer had to rely on their supplier for the overall quality of materials. Advantages were gained in the enhanced accuracy
D.A. Wiebe I Atherosclerosis 108 (Suppl.) (1994) S181-S189
and precision achieved by the end user, especially when used in small laboratory environments with minimal technical support staff. Manufacturers placed additional enhancements on these new analyzers that have been effective at lowering the manual operations required by the laboratory staff to yield a reliable and precise value. These features include: (1) random access analysis, (2) precise reagent and sample transfer, (3) microcomputer control, (4) electronic data transfer via computer interface, and (5) on-line diagnostic testing. In general, manufacturers provided laboratorians with complex systems that are computer-driven and capable of high throughput with superior precision and accuracy. What can the future have to offer that is currently unavailable? Robotics is the way of the future. Robotics will offer walk-away systems that will interact with multiple instruments, capable of primary tube sampling, and intelligent processing to perform the majority of processing techniques currently carried out by laboratory personnel. 2.2. Lipid analysis In years past, the only requirement in locating the lipid laboratory was a keen sense of smell: the acetic acid/acetic anhydride odour was a familiar aroma that emanated from these specialty laboratories. Every operation was a manual procedure requiring large quantities of organic solvents used to isolate lipid fractions from serum proteins and other serum constituents. Separatory funnels, large beakers, glass burettes, thin-layer chromatography tanks, digestive setups, extraction apparatus, an array of grade A glass pipettes, and a single- or dual-beam spectrophotometer with quartz cuvettes were just a few standard fixtures in these facilities. In general, caustic reagents were the basis for analytical methods employed to measure cholesterol, triglycerides, phospholipids, and fatty acids. Throughput and turnaround times were of less concern to the laboratories because these relatively new methods of laboratory productivity were expressed in days or weeks. Table 1 illustrates typical information reported from these early lipid laboratories. Note the use of the term mg% to express the concentration of the analytes - it brings back memories.
S183
Table 1 Lipid testsand normal ranges for assays typically performed in laboratories during the late 1950s Lipid assay
Normal range
Total cholesterol Free cholesterol Cholesterol esters Phospholipids Sphingomyelin Cephalin Lecithin Total fatty acids Neutral fat
150-280 mg% 30-60 mg% 70-75% of total 1SO-250 mg% 15-30 mg% O-20 mg% 1SO-230 mg% 200-700 mg% O-400 mg%
Even during the 1950s standardization of cholesterol data between laboratories was a con cem, as evidenced by this quote taken from a leading text on lipid disorders [ I] : “It may be emphasized that a comparison of figures of cholesterol and cholesterol esters is possible only if the same method is applied by all authors.’ Cholesterol standardization is one area in which significant progress and success has been achieved during recent years. The credit for the success achieved with cholesterol standardization must be given to the combined efforts of several organizations working toward this common goal. However, as we move from the past into today and on into the future, standardization is one activity that must be carried forth with inany other analytes besides cholesterol. All other lipid and lipoprotein assays have significant challenges before comparable success can be claimed. 3. Current status of lipid testiug Today’s laboratory has the benefit. of increased technology to perform lipid analyses. Instead of Table 2
Lipid tests typically performed in routine clinical laboratories during the 1990s Tests Cholesterol Triglycerides HDL-cholesterol Estimated LDL-C
Apolipoproteins Apo A-I Apo B Lp(a)
D.A.
S184
300
1
Wiebe / Atherosclero.~is 108 (Suppl.)
(1994) S181-Sl89
1990 - Lipid Workload
ELOL
l Chol q TG 0 HDL
In-patient
Out-patient
Fig. 2. Comparison of inpatient and outpatient requests for lipid tests. C&H, cholesterol and HDL-cholesterol; Chol, cholesterol; C&T. cholesterol and triglycerides: CT&H, cholesterol, triglycerides and HDL-cholesterol; ELDL, estimated LDL-C; HDL, HDL-cholesterol: SLPP, serum lipoprotein profile; TG, triglycerides; T&H, triglycerides and HDL-cholesterol.
the caustic reagents and tedious tasks, laboratories use enzymic assays that deliver high specificity along with increased sensitivity. These tests are readily available on computer-driven automated systems with high throughput which require a nominal amount of specimen to perform the service. Table 2 lists some of the usual lipid ant’ lipoprotein tests performed at the University of Wisconsin Hospital and Clinics (UWHC) and are representative of the typical laboratory. Single tests for cholesterol, triglycerides, and HDLcholesterol are only occasionally asked for by clinicians at UWHC. Instead, combinations or profiles of these various tests are ordered to facilitate the treatment or management of hyperlipidemia. The most frequently ordered profile is the package with cholesterol, triglyceride, and HDLcholesterol that uses the Friedewald e,quation to estimate the LDL-cholesterol. A less requested battery includes the same tests along with lipoprotein electrophoresis to assign a classical lipoprotein phenotype. UWHC is a medical school and an established tertiary care center: a monitor of ordering sources for lipid and lipoprotein tests revealed interesting information. Lipid and lipoprotein requests were monitored for a month (mid-October to mid-November) during 1990 to
determine what percentage of lipid orders were received from inpatients or outpatients (Fig. 2). Greater than 85% of these specialty tests were demonstrates received from outpatients: this proper utilization of lipid and lipoprotein requests by the UWHC clinical staff. In general, cardiovascular risk assessment lipid and lipoprotein tests should be performed on outpatients. An exception is triglyceride requests that may be useful to monitor patients receiving intralipid therapy; triglycerides represent over 50% of the orders for inpatients. Manufacturers of laboratory instrumentation and reagents have provided several options for cholesterol analysis with the necessary accuracy and precision to meet the NCEP guidelines. Many instruments are calibrated by the manufacturer to provide accurate cholesterol results for patient specimens, which simplifies the task for the iaboratory using the system. Triglycerides and HDLcholesterol assays are not well controlled among laboratories or from one method to another. These assays require a similar standardization eiTort to that which successfully brought cholesterol under control. Precision for triglycerides and HDL-cholesterol assays is acceptable, with 2% 3% coefficients of variation (CV) achievable with
D.A.
Webe
I Atltermdero.~is
most commercial methods. In general, much of the credit for success that laboratories have experienced with improved performance of lipid tests belongs to manufacturers and the highly automated systems they developed.
108 (SuppI.)
Table
(1994) Si81 -SJ89
3
Factors
that must be considered
laboratory Laboratory
issues
(new or existing) Reagent stability
Laboratory medicine and certainly the practice of clinical chemistry have only begun to feel the impact of the Clinical Laboratory Improvement Act (CLIA) passed by the US Congress in 1988. CLIA-88 deals primarily with performance issues of all testing executed by clinical laboratories, with licensure dependent on the success in meeting specified guidelines. Along with the performance issues of CLIA-88, laboratories are being scrutinized as cost centers since reimbursement for services is rapidly changing. In the past, issues that concerned the lipid laboratory included fasting versus nonfasting, serum versus piasma, precision accuracy, quality control materials and practices, and reference values. Today and for the foreseeable future, other issues must be factored into the decision process, including: screening, home testing, turnaround time, costs versus reimbursement, and biological variability. At the center of all these issues, the laboratory must deal with the test’s clinical utility. Obviously, clinical utility of a test should dictate the type and quality of service the laboratory delivers for the test. To meet this final issue requires close communication with clinicians ordering the service. The NCEP’s Adult Treatment Panel ( ATP) [2] established treatment guidelines to aid physicians who deal with hypercholesterolemic individuals and provide many of the clinical utility requirements. A few recent events will impact on fu?ure lipid and lipoprotein testing. One is the ATP, which revised its recommendations (ATP II) [3] and clearly establishes the importance of LDL-cholesterol analysis as the primary approach to monitoring effectiveness with either diet or drug intervention. Also, ATP II added HDL-cholesterol to cholesterol as the lipid parameters that should be screened to assess an individual’s risk for CHD. Thus, these new guidelines have increased the significance of both HDL and LDL
in order to establish a new
procedure
Instrumentation
4. Factors influencing laboratory practice
SI8.5
Cast/sample
Fastinglnonfasting Precision (short and long term) Accuracy
Sample size
Proficiency testing
Sample processing
Control
Spwimen
Data transfer
type
interferences Turnaround
materials
Reference values time
Reimbursement
testing within the practice of laboratory medicine. Certainly the new CLIA-88 regulations will further inlluence the utilization of lipid tests, especially the laboratory’s responsibility to document performance standards for all the lipid and lipoprotein tests. New and projected national health care policies may place greater uncertainty on the medical usefulness of lipid and lipoprotein measurements. However, treatment guidelines and health care policy issues aside, several laboratory initiatives will certainly have an impact on the future lipid laboratory. Traditionally, laboratories were primariiy concerned with analytical performance, such as accuracy and precision, of new assays. However, in today’s laboratory environment decisions to establish a new method in the laboratory will involve a series of issues as listed in Table 3. Certainly cost issues are high on the list of factors that influence which tests are offered in the laboratory - especially cost per test, which may dictate the specific instrument or reagent system to be used. Operational costs have to be determined, including direct labor and maintenance requirements that add to the cost of each test. A frequently overlooked issue is the amount of time required for calibration and troubleshooting of a particular method. A method requiring frequent attention or adjustments may cause more problems than an assay that is stable for a longer period of time. All these issues have become dominant factors for laboratories that wish to be competitive in laboratory medicine.
Sl86
D.A. Wiebe 1 Atherosclerosis 188 (Suppl.) (1994) Sl8lLSl89
5. Recent developments influencing lipid and lipoprotein tests 5.1. Direct LDL-cholesterol Until recently, laboratories had few options available for quantitation of LDL-cholesterol. The combination of ultracentrifugation and selective precipitation with heparinlmanganese chloride techniques serves as the basis of the which was the Lipid Re‘beta-quantification’ search Clinic’s approach for LDL-cholesterol [4]. However, few laboratories have access to an ultracentrifuge and, even if they have one, the method requires significant time and sample volume such that the process fails to be cost-effective. Thus, quantitating LDL-cholesterol via ultracentrifugation is usually reserved for research facilities. Instead, clinical laboratories have relied on the Friedewald equation for estimating a patient’s LDL-cholesterol from cholesterol, triglyceride, and HDL-cholesterol values [5]. The equation provides a reasonable estimation of LDL-cholesterol for samples that have typical lipoprotein phenotypes and mildly elevated triglycerides. However, the occasional hypertriglyceridemic (TG > 400 mg/dl) or atypical phenotype specimen results in a falsely low LDL-cholesterol value by Friedewald estimation. Until recently, the ultracentrifuge was the only alternative approach for quantitating LDL-cholesterol for these unusual specimens. An LDL-cholesterol assay is now commercially available that utilizes antibodies directed against VLDL and HDL to isolate LDL [6]. Thus, LDL-cholesterol can be quantitated directly and conveniently with standard automated analyzers with minimal sample pretreatment. This direct assay and others that are likely to follow offer significant advantages over the use of an equation. One advantage of the direct LDL assay is that either fasting or nonfasting samples can be crllected from patients and rhe results are valid. Immunoseparation schemes, such as the direct LDL-cholesterol assay, may offer specific and accurate approaches to several lipoprotein measurements. Look for additional development and exploitation of the selectivity of antibodies directed against specific lipoproteins for the future of lipoprotein profiling. Identifying
lipoproteins by their specific apolipoproteins may serve as the basis for future lipoprotein nomenclature. Such a nomenclature scheme would be dependent on immunoassays for both quantitation and speciiic separation lipoprotein classes. 5.2. Nuclear magnetic resonance (NMR)
Proton NMR was a key approach to structure identification for organic chemists during the past 20 years. Magnetic resonance imagery has become a significant tool to provide detailed views of tissues, organs, or body sections for diagnostic purposes without the use of X-rays or other invasive approaches. Now, NMR can be similarly applied in the laboratory to establish the lipoprotein profile of a given plasma specimen without having to perform a complex separation of individual components [ 71. Recent reports have documented acceptability and performance standards achievable by NMR. However, NMR requires elaborate instrumentation and is therefore likely to be used only in specialized laboratories. Keep in mind that the more complex procedures have ways of becoming more simplified in future generations of the technology, and more widely applied. Look for a specific NMR system developed with only the lipoprotein profiling aspect as the outcome and the instr:lmentation may be less demanding. 5.3. Magnetic par title separation An adaptation to the HDL separation scheme is the use of magnetized particles with dextran sulfate and magnesium chloride to achieve isolation without centrifugation. Magnetic HDL precipitation reagent is commercially available from Reference Diagnostics (Arlington, MA 02174). Several reagent systems are available to isolate HDL by precipitation of VLDL and LDL fractions. All these approaches have been developed with a centrifugation step to isolate the HDL supernatant. In fact, the variability of HDL procedures may be attributed to the myriad factors with incubation conditions such as time and temperature to achieve maximum reagent/lipoprotein interaction and centrifugation parameters such as temperature, time, and force of spin. Some HDL separation schemes are sensitive to slight varia-
D.A.
Wiehe
/ Atherosclerosis
tions in any of these conditions. The use of magnetic particles offers some improvements over the need for centrifugation. The most obvious advantage is in eliminating a need f’or the centrifuge to isolate HDL. However, the ability of the magnetic particles to separate both insoluble and soluble lipoprotein complexes with dextran particles may be more important. A key factor for separation of LDL and VLDL particles by centrifugation is the formation of insoluble precipitate with the reagents. Failure to form the insoluble complexes is a problem with some reagent systems, especially with hypertriglyceridemic specimens (TG > 400 mg/dl or higher). Thus, magnetic particles and other similar innovative techniques for separation or isolation of lipoproteins have a role in the future lipid laboratories. 5.4. Eiectrophoresis Lipoprotein electrophoresis has taken a back seat to newer techniques, especially selective precipitation techniques for separating and isolating lipoproteins. Electrophoresis was the primary technique used to assign the Fredrickson-Levy phenotype for a patient’s lipoprotein pattern. However, since the assignment of the patient’s phenotype offers little useful diagnostic information for the treatment of the hyperlipidemia, electrophoresis has been replaced by other techniques or discontinued altogether. Attempts to utilize electrophoresis as a method to quantitate lipoprotein fractions using specific enzgmic reagents have fallen short due to lack of sensitivity and poor reproducibility. So what is the future for lipoprotein electrophoresis? Lipoprotein electrophoresis, especially using agarose gel or cellulose acetate supports, will continue in a small way to serve as a qualitative tool to investigate lipoprotein abnormalities. But the real future of electrophoresis will depend on its ability to resolve complex lipoprotein families into fractions that provide greater diagnostic insight th,n current isolation schemes. Quantimetrix Corporation (Hawthorne, CA 90250) offers a high resolution polyacrylamide system capable of fractionating LDL into multiple subspecies. Thus, individuals with increased numbers of small. dense LDL particles that are more atherogenic
108 (Suppl.)
(1994) SIl?--S/89
5187
than the larger LDL particles can be easily identified and provided with more aggressive therapy. Other advances in electrophoresis techniques will have a positive impact on tahoratory medicine. Capillary electrophoresfi has received considerable attention in the biological field for separating proteins and metabolites with exceptional resolution. Such is the case with the recent report of electrophoresis performed on a microchip [g]. A mixture of six fluorescent-labeled amino acids was resolved by capillary electrophoresis in 15 s. The authors note that miniaturized electrophoretic systems offer considerable advantages over existing technology in terms of cost, reduced solvent and sample requirements, and analysis time. The potential exists for such an electrophoretic system to be fabricated to provide a complete analytical system on a microchip. Thus, electrophoresis will continue to be an integral part of the future laboratory; however, its specific role in lipid and lipoprotein testing remains to be determined.
Preparative ultracentrifugation remains the standard reference method for the isolation of sample relipoprotein classes. Unfortunately, quirements, spin times, expensive equipment, and overall technical complexity remain key obstacles that prevent ultracentrifugation from becoming a routine method in clinical laboratories. The bench ultracentrifuges which require 1.0 ml or less of sample provide an effective means to remove chylomicrons with short spin times (2-6 min), depending on the extent of turbidity, to eliminate the excessive lipid as an interferent for other tests. Other inherent problems and technical complexities prevent ultracentrifugation from achieving true status as a reference method or usefulness as a routine procedure. However, the ultracentrifuge will remain the standard for lipoprotein isolations in specialized research laboratories.
The use of antibodies directed against apo:ipoproteins associated with lipoproteins was mentioned previously with the development of a specific LDL assay. However, antibodies directed
S188
D.A.
Wiebc / .4tlterosclerosis 108 (Suppl.)
against these proteins is only one approach being explored in laboratories today. In addition, researchers use molecular probe techniques to generate diagnostic information from a subject’s DNA to discover possible genetic or functional changes in the protein synthesis that could explain a metabolic defect. Molecular diagnostic procedures are going to play a significant role in laboratory medicine, and certainly our understanding of lipid and lipoprotein abnormalities will be enhanced through these techniques. Similarly, researchers use biological receptor assays to gain additional information of abnormalities occurring at the cellular level. The role of LDL and its receptor to internalize the lipoprotein into the cell and ultimately down-regulate cholesterol synthesis is a key to understanding the abnormalities associated with familial hypercholesterolemia. The use of receptor assays in laboratory medicine has focused primarily on lymphocytes and hematologic issues to date, but the future may certainly have lipoprotein-related aspects.
6. The future Cardiovascular disease remains the number one cause of mortality, and with increased efforts directed at preventive medicine the need to measure lipid and lipoprotein fractions will expand. Our laboratories will continue to use more and more sophisticated techniques to quantitate many of the same analytes that we currently measure (cholesterol, triglycerides, HDL, and LDL). Issues concerning fasting or nonfasting will continue to be a factor for specific tests, but possible alternative sampling techniques will improve the reliability and interpretation of results. Possibly, indwelling sensors or a noninvasive technique might supplement our standard assays, but that these approaches will ever replace the need for a serum total cholesterol is difficult to envision. The certainties of the future will include smaller sample requirements, greater use of computers to operate our laboratories, and advanced information systems for archiving, retrieval, and interpretation of laboratory data (a universal nomenclature system remains in doubt).
(1994) SISI-S189
The centralized laboratory is currently being challenged by home testing and patient bedside services that can deliver results in a more timely manner. There is limited clinical need to provide more timely lipid measurements, therefore lipid test should be a minor player in this ‘home testing’ arena. However, these activities will continue to increase in the future and the search for simpler and less invasive techniques will be intensified and driven by incentives to keep medical costs under control. Obviously, reimbursement for laboratory services will be a critical issue. Specialized lipid and lipoprotein testing used to assess an individual’s risk for cardiovascular disease or to monitor treatment intervention for hyperlipoproteinemia can easily be performed outside a hospital setting and should be a clinic activity. Lipid test results should be provided on a STAT basis to physicians to have the most up-to-date information during the patient’s examination. Treatment of lipid and lipoprotein abnormalities requires behavior modification, and current tests results will reinforce a positive treatment program for the patient and increase the efficiency and utilization of physician time. Thus, in our future, lipid testing will need to be fast, accurate, and readily accessible in a clinic setting to meet the demands for patient care. What does the crystal ball hold for the future of lipid testing? As noted previously, demand for accurate and precise lipid and lipoprotein testing will continue to be strong. Cholesterol and triglycerides will continue to be the key lipid tests provided with the reliable enzymic assays that have been developed during the past decade. Expect to have vastly improved, stable, matrix-free quality control and serum calibrators available to deliver maximum performance of these assays. Lipoprotein testing will be the area in which greater change is likely to occur. First, a need may arise to develop reliable methods to determine the heterogeneity of the lipoprotein classes, especially if the atherogenicity of the lipoprotein changes as the size of the particle changes. Electrophoresis, especially capillary electrophoresis, may provide the basis for resolving different sizes of lipoproteins (such as LDL) to add to the physician’s menu of tests for assessment of a patient’s risk for cardiovascular disease
D.A. Wide
I Atherosclerosis 108 (Suppl.) (1994) Sl81-Sl89
The breakthrough will come with improved tests for specific lipoproteins. We have already observed increased activity in this area with the introduction of the direct-LDL assay using immunoseparation schemes to isolate select lipoprotein classes prior to cholesterol analysis. A homogeneous assay may be the next generation and the way of the future. Imagine an LDLcholesterol assay in which no separation or isolation step is required: only mixing the serum with reagent and cholesterol analysis is directed against the desired lipoprotein. The future is bright for lipid and lipoprotein testing. References 111 Thannhauser. S., Lipidoses: Diseases of the Intracellular PI
Lipid Metabolism, 3rd Edn., Gruune and Stratton, New York, 1958. The Expert Panel, Report of the National Cholesterol Education Program Expert Panel on Detection. Evaluation, and Treatment of High Blood Cholesterol in Adults, Arch. intern. Med.. 148 ( 1988) 36.
Sl89
[31 The Expert Panel. Sumrlmy of the Second Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation. and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II), J. Am. Med. Assoc., 269 (1993) 3015. 141 Hainline, A., Karen, J. and Lippel, K. (Eds.), Lipids Research Clinics Program, Lipid and Lipoprotein Analysis, Manual of Laboratory Operations, 2nd Edn., US Dept. HHS. Bethesda. MD, 1982. [51 Friedewald, W.T.. Levy, R.I. and Fredrickson, D.S., Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge, Clin. Chem., 18 (1972) 499. I61 Leary. E.T.. Tjersland. G., and Warnick, G.R.. Evaluation of the Genzyme immunoseparation reagent for direct quantitation of LDL cholesterol, Clin. Chem.. 39 ( 1993) 1124. 171 Otvos. J.D., Jeyarajah. E.J.. Bennett. D.W.. and Krauss, R.M., Development of a proton nuclear magnetic resonance spectroscopic method for determining plasma lipoprotein concentrations and subspecies distributions from a single. rapid measurement, Clin. Chem., 38 (1992) 1632. PI Harrison. D.J.. Fluri. K., Seiler, K. et al.. Micromaching a miniaturized capillary electrophoresis-based chemical analysis system on a chip. Science. 261 ( 1993) 895.