336 PART 2 ■ CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

CASE STUDY 14-1 the ATP-binding cassette transporter family52 that

A 52-year-old man went to his physician for a
physical examination. The patient had been a district
manager for an automobile insurance company for
the past 10 years and was 24 pounds overweight. He
had missed his last two appointments with the
physician because of business. The urinalysis
dipstick finding was not remarkable. His blood
pressure was elevated. The blood chemistry results
are listed in Case Study Table 14-1.1.
Questions Lipid profile
1. Given the abnormal tests, what additional
information would you like to have?
2. If this patient had triglycerides of 100 mg/dL
(1.1 mmol/L) and an HDL cholesterol of
23 mg/dL (0.6 mmol/L), what would be his
calculated LDL cholesterol value? 167, use the one with 5 via a mechanism that appears to be different than the
3. If, however, his triglycerides were 476 mg/dL
(5.4 mmol/L), with an HDL cholesterol of
23 mg/dL (0.6 mmol/L), what would be his
calculated LDL cholesterol value? triglycride above 400, DISTRIBUTIONS
pumps various ligands across the plasma membrane.
Defects in the gene for the ABCA1 transporter lead to
Tangier disease, a disorder associated with low HDL
and a predisposition to premature coronary heart dis-
ease.53 The exact mechanism of the ABCA1 transporter
is not known, but it is believed that the transporter
modifies the plasma membrane by transporting a lipid,
which then enables apo A-I that has dissociated from
HDL to bind to the cell membrane. In a detergent-like
extraction mechanism, apo A-I then removes excess
cholesterol and phospholipid from the plasma mem-
brane of cells to form a discoidal-shaped HDL parti-
cle.54 The newly formed HDL is then competent to ac-
cept additional cholesterol by the aqueous diffusion
pathway and is eventually converted into spherical HDL
by the action of LCAT (Fig. 14-3). Recently, ABCG1,
another ABC transporter, has been described to facili-
tate the efflux of cholesterol to lipid-rich spherical HDL
ABCA1 transporter.55
LIPID AND LIPOPROTEIN POPULATION

CASE STUDY TABLE 14-1.1 Serum lipoprotein concentrations differ between adult
LABORATORY RESULTS men and women, primarily as a result of differences in
sex hormone levels, with women having, on average,
ANALYTE PATIENT VALUE REFERENCE RANGE higher HDL cholesterol levels and lower total cholesterol
Na ⫹
151 135–143 mEq/L and triglyceride levels than men.56 The difference in total
K⫹
4.5 3.0–5.0 mEq/L cholesterol, however, disappears after menopause as es-
trogen decreases.57 Men and women both show a ten-
Cl⫺ 106 98–103 mEq/L
dency toward increased total cholesterol, LDL choles-
CO2 content 13 22–27 mmol/L terol, and triglyceride concentrations with age. HDL
Total protein 5.7 6.5–8.0 g/dL cholesterol concentrations generally remain stable after
Albumin 1.6 3.5–5.0 g/dL the onset of puberty and do not drop in women with the
onset of menopause. General adult reference ranges are
Ca2⫹ 7.9 9.0–10.5 mg/dL
shown in Table 14-3.
Cholesterol 210 140–200 mg/dL Circulating levels of total cholesterol, LDL choles-
Uric acid 6.2 3.5–7.9 mg/dL terol, and triglycerides in young children are generally
Creatinine 2.5 0.5–1.2 mg/dL much lower than those seen in adults.58 In addition, con-
centrations do not significantly differ between boys and
BUN 95 7–25 mg/dL
girls. HDL cholesterol levels for both boys and girls are
Glucose 88 75–105 mg/dL
Total bilirubin 1.2 0.2–1.0 mg/dL
Alkaline 27 7–59 IU/L TABLE 14-3 ADULT REFERENCE RANGES
phosphatase FOR LIPIDS
Lactate 202 90–190 IU/L ANALYTE REFERENCE RANGE
dehydrogenase
Total cholesterol 140–200 mg/dL
Aspartate 39 8–40 IU/L
HDL cholesterol 40–75 mg/dL
transaminase
LDL cholesterol 50–130 mg/dL
Amylase 152 76–375 IU/L
Triglyceride 60–150 mg/dL
340 PART 2 ■ CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

opening, or lumen, causing the blood to circulate in a

CASE STUDY 14-2
nonlaminar manner under greater and greater pressure,
A 30-year-old man with chest pain was brought to which further aggravates plaque formation. The final
the emergency department after a softball game. He event leading to complete occlusion of blood flow occurs
was placed in the coronary care unit when his ECG when there is a hemorrhage into the plaque, which re-
showed erratic waves in the ST region. A family sults in the formation of a thrombus that blocks blood
history revealed that his father died of a heart at- flow and precipitates a myocardial infarction.
tack at the age of 45 years. The patient had always Because lipid deposits in the vessel walls are fre-
been athletic in high school and college, so he had quently associated with increased serum concentrations
not concerned himself with a routine physical. The of LDL cholesterol or decreased HDL cholesterol, lower-
laboratory tests listed in Case Study Table 14-2.1 ing LDL is an important step in preventing and treating
73–75
were run. CHD. It is estimated that for every 1% decrease in
LDL cholesterol concentration, there is a 2% decrease in
LD1 &2 CKMB Creatinin, Lipid profile, Fasting required
Questions the risk of developing arteriosclerosis.76 For patients
with established heart disease, studies have shown that
1. Given the symptoms and the family history, what aggressive treatment to reduce LDL cholesterol levels
additional tests should be recommended? below 100 mg/dL (2.6 mmol/L) or even lower is effective
2. If his follow-up total cholesterol remains in the in the stabilization and sometimes regression of
same range after he is released from the hospital, plaques.77 Stabilization of plaque is thought to be at
and his triglycerides and HDL cholesterol are least as important as plaque regression in terms of rup-
within the normal range, what course of treat- ture potential.78
ment should be recommended? In some individuals, high levels of blood cholesterol
or triglycerides are caused by genetic abnormalities in
Statin. vitamin B3 which either too much is synthesized or too little is re-
CASE STUDY TABLE 14-2.1 moved.79–82 High levels of cholesterol and/or triglyc-
LABORATORY RESULTS abnormal : , albumin, cholestrol,
erides in most people, however, are a result of increased
consumption of foods rich in fat and cholesterol, smok-
PATIENT ANALYTE VALUES RANGE
ing, and lack of exercise or a result of other disorders or
Na⫹ 139 135–143 mEq/L disease states that affect lipid metabolism, such as dia-
K⫹ 4.1 3.0–5.0 mEq/L betes, hypertension, hypothyroidism, obesity, liver and
Cl⫺
101 98–103 mEq/L kidney diseases, and alcoholism. Low levels of HDL cho-
lesterol are also associated with increased risk of heart
CO2 content 29 22–27 mmol/L
disease, but there are currently limited ways to pharma-
Total protein 6.9 6.5–8.0 g/dL cologically raise HDL cholesterol levels. Existing drugs
Albumin 3.2 3.5–5.0 g/dL for increasing HDL-C are primarily fibric acid derivatives
Ca 2⫹
9.3 9.0–10.5 mg/dL (fibrates) and niacin-containing compounds.83,84 Newer
drugs that raise HDL-C by inhibiting CETP may play a
Cholesterol 278 140–200 mg/dL
role in the future; however, a recent clinical trial with the
Uric acid 5.9 3.5–7.9 mg/dL first CETP inhibitor drug unexpectedly showed in-
Creatinine 1.1 0.5–1.2 mg/dL creased CHD events.85 Other drugs using reconstituted
BUN 20 7–25 mg/dL HDL particles or administration of apo AI mimetic pep-
tides are also being actively investigated.86
Glucose 97 75–105 mg/dL
Laboratory analyses are an important adjunct to man-
Total 0.8 0.2–1.0 mg/dL aging patients with dyslipidemia, because accurate
bilirubin measurement of total, HDL, and LDL cholesterol and
Alkaline 20 7–59 IU/L triglyceride levels is needed to determine the most ap-
phosphatase propriate diet or diet and drug therapy. As shown in
Lactate 175 90–190 IU/L Table 14-5, individuals on a low-fat diet, who continue to
dehydrogenase have LDL cholesterol levels of 190 mg/dL (4.9 mmol/L)
Aspartate 35 8–40 IU/L or higher on repeated measurement will likely benefit
transaminase from drug intervention. If they have two or more CAD
risk factors and continue to have LDL cholesterol levels
Amylase 98 76–375 IU/L
of 160 mg/dL (4.1 mmol/L) or higher, they also would
benefit from drug therapy. And, if they have already been
 CHAPTER 14 ■ LIPIDS AND LIPOPROTEINS 341

previously diagnosed with heart disease, drug therapy Hypercholesterolemia

should be considered when the LDL cholesterol level is
130 mg/dL (3.4 mmol/L) or higher. The average of at Hypercholesterolemia is the lipid abnormality most
least two measurements, taken 1 to 8 weeks apart, should closely linked to heart disease.59 One form of the disease,
be used to determine the best treatment approach.66 which is associated with genetic abnormalities that pre-
Classic bile-acid sequestrant drug treatments, such dispose affected individuals to elevated cholesterol lev-
as cholestyramine, work by sequestering cholesterol els, is called familial hypercholesterolemia (FH).
in the gut so that it is not absorbed and, until recently, Homozygotes for FH are fortunately rare (1:1 million in
were considered to be the only safe drugs for use in chil- the population) and can have total cholesterol concen-
dren.76 Bile-acid sequestrants have uncomfortable ad- trations as high as 800 to 1,000 mg/dL (20–26 mmol/L).
verse effects such as bloating and constipation and are These patients frequently have their first heart attack
poorly tolerated. The most effective class of drugs for when still in their teenage years.90 Heterozygotes for the
managing patients with dyslipidemia are the HMG-CoA disease are seen much more frequently (1:500 in the
reductase drug inhibitors, such as lovastatin, simvastatin,
pravastatin, fluvastatin, atorvastatin, and rosuvastatin.
These drugs, commonly known as statins, block intracel-
lular cholesterol synthesis by inhibiting HMG-CoA re-
CASE STUDY 14-3
ductase, a rate-limiting enzyme in cholesterol biosynthe-
sis. The reduced level of cholesterol in hepatocytes
A 43-year-old white man was diagnosed with hyper-
increases the expression of the LDL receptor, which re-
lipidemia at age 13 years, when his father died of a
moves LDL from the circulation, thus reducing the depo-
myocardial infarction at age 34 years. The man’s
sition of LDL into vessels and the formation of plaques.
grandfather had died at age 43 years, also of a my-
The major safety issues with statins are myositis and he-
ocardial infarction. Currently, the man is active and
patotoxic effects; however, patient monitoring in clinical
asymptomatic with regard to CHD. He is taking
trials has shown that fewer than 2% of patients have sus-
40 mg of lovastatin (Mevacor), 2 times/day (maxi-
tained increases in liver enzymes. These drugs typically
mum dose). He had previously taken niacin but
reduce LDL cholesterol by as much as 20% to 40% and
could not tolerate it because of flushing and gas-
are generally well tolerated.73,87 Niacin is also a potent
trointestinal distress, nor could he tolerate
drug for reducing LDL cholesterol and is the only effec-
cholestyramine resin (Questran). His physical exami-
tive drug at this time for significantly raising HDL choles-
nation is remarkable for bilateral Achilles tendon
terol levels; however, it causes flushing and diarrhea in
thickening/xanthomas and a right carotid bruit.
many patients and can be hepatotoxic.88 Newer slow-
release formulations of niacin and combination drugs to
Questions Famlial hypercholestremia , heterozygous
reduce the flushing have been developed to improve com-
pliance.83 Fibric acid derivatives, such as clofibrate, gem- 1. What is his diagnosis?
fibrozil, fenofibrate, and etiofibrate, are most often used
2. Does he need further workup? Lp a , Excercise stress test, carotid ultra sound
to reduce triglyceride and VLDL cholesterol levels and in-
crease HDL cholesterol levels.84,88 Ezetimibe is a new 3. What other laboratory tests should be done? LP a , Homocystiene
drug that inhibits the absorption of cholesterol by in-
4. Does he need further drug treatment? If so, what?
hibiting the NPC1-L1 transporter in the intestine, and it Replace his drug, vitamin B3, Aspirin and vitamin E
is used often in conjunction with a statin.89
CASE STUDY TABLE 14-3.1
LABORATORY RESULTS
Hyperlipoproteinemia Triglycerides 91 mg/dL
Disease states associated with abnormal serum lipids are Total cholesterol 269 mg/dL
generally caused by malfunctions in the synthesis, trans-
HDL cholesterol 47 mg/dL
port, or catabolism of lipoproteins.80,88 Dyslipidemias can
be subdivided into two major categories: hyperlipopro- LDL cholesterol 204 mg/dL
teinemias, which are diseases associated with elevated Aspartate aminotransferase (AST) 34 U/L
lipoprotein levels, and hypolipoproteinemias, which are Alanine aminotransferase (ALT) 36 U/L
associated with decreased lipoprotein levels. The hyper-
Alkaline phosphatase (ACP) 53 U/L
lipoproteinemias can be subdivided into hypercholes-
terolemia, hypertriglyceridemia, and combined hyperlipi- Electrolytes and fasting glucose Normal
demia with elevations of both cholesterol and triglycerides.
 CHAPTER 14 ■ LIPIDS AND LIPOPROTEINS 343

Combined Hyperlipoproteinemia CASE STUDY 14-4

Combined hyperlipoproteinemia is generally defined
as the presence of elevated levels of serum total cholesterol
and triglycerides. Individuals presenting with this syn-
drome are considered at increased risk for CHD. In one ge-
netic form of this condition, called familial combined hy-
perlipoproteinemia (FCH), individuals from an affected
kindred may only have elevated cholesterol, whereas oth-
ers only have elevated triglycerides, and yet others, eleva-
tions of both. Another rare genetic form of combined hy-
perlipoproteinemia is called familial dysbetalipoproteine-
mia, or type III hyperlipoproteinemia. The name type III
A 60-year-old woman came to her physician because
she was having problems with urination. Her previ-
ous history included hypertension and episodes of
edema. The physician ordered various laboratory
tests on blood drawn in his office. The results are
shown in Case Study Table 14-4.1.
Questions
1. What are the abnormal results in this case?
She has kidney disease

hyperlipoproteinemia is a holdover from a lipoprotein typ- 2. Why do you think the triglycerides are abnormal?
If she takes hormonal therapy like estrogen she will has high triglyceride,
ing system developed by Fredrickson et al.101 that is oth- 3. What is the primary disease exhibited by this pa-
erwise generally no longer used. The disease results from tient’s laboratory data?
an accumulation of cholesterol-rich VLDL and chylomi- She has low albumin, Increased lipids, Low albumin, then it is nephrotic syndrome.
tri high, Hyper lipidemia, Hypo albuminemia.
cron remnants as a result of defective catabolism of those
CASE STUDY TABLE 14-4.1
particles. The disease is associated with the presence of a
LABORATORY RESULTS
relatively rare form of apo E, called apo E2/2. Individuals
with type III will frequently have total cholesterol values PATIENT REFERENCE
of 200–300 mg/dL (5–8 mmol/L) and triglycerides of ANALYTE VALUES RANGE
300–600 mg/dL (3–7 mmol/L). To distinguish them from Na⫹⫹ 149 135–143 mEq/L
other forms of combined hyperlipoproteinemias, it is first K⫹⫹ 4.5 3.0–5.0 mEq/L
necessary to isolate the VLDL fraction with ultracentrifu- ⫺
Cl 120 98–103 mEq/L
gation. A ratio derived from the cholesterol concentration
in VLDL to total serum triglycerides will be greater than CO2 content 12 22–27 mmol/L
⬎0.30 in the presence of type III hyperlipoproteinemia. If Total protein 5.7 6.5–8.0 g/dL
the VLDL fraction is analyzed by agarose electrophoresis, Albumin 2.3 3.5–5.0 g/dL
the particles will migrate in a broad ␤ region, rather than 2⫹
Ca 7.6 9.0–10.5 mg/dL
in the normal pre-␤ region. Definitive diagnosis requires a
determination of apo E isoforms by isoelectric focusing or Cholesterol 201 140–200 mg/dL
DNA typing, resulting in either apo E2/2 homozygosity or, Uric acid 15.4 3.5–7.9 mg/dL
rarely, apo E mutation or deficiency. Treatment does not Creatinine 4.5 0.5–1.2 mg/dL
totally rely on a diagnosis because these patients, as those
BUN 87 7–25 mg/dL
with other dyslipidemias, can be treated with niacin, gem-
fibrozil, HMG-CoA reductase inhibitors, or just a low-fat Glucose 88 75–105 mg/dL
diet. Because of the cholesterol-enriched composition of Total bilirubin 1.3 0.2–1.0 mg/dL
these particles, use of the Friedewald equation102 to calcu- Triglycerides 327 65–157 mg/dL
late LDL cholesterol levels will result in an underestima-
Lactate 200 90–190 IU/L
tion of VLDL cholesterol and, therefore, an overestimation
dehydrogenase
of LDL cholesterol, compared with beta-quantification.103
Aspartate 45 8–40 IU/L
Lipoprotein(a) Elevation transaminase
Amylase 380 76–375 IU/L
Elevations in the serum concentration of Lp(a), espe-
cially in conjunction with elevations of LDL, increase the
risk of CHD and CVD.104,105 Higher Lp(a) levels have
been observed more frequently in patients with CHD
than in normal control subjects,106 although prospective with the coagulation factor, plasminogen,109 it has been
studies have not conclusively determined this positive proposed that it competes with plasminogen for fibrin
association.107 Lp(a) are variants of LDL with an extra binding sites, thus increasing plaque formation.110,111
apolipoprotein, called apo (a); the size and serum con- Most LDL-lowering drugs have no effect on Lp(a) con-
centrations of Lp(a) are largely genetically deter- centration, even when LDL cholesterol is significantly
mined.108 Because apo (a) has a high degree of homology lowered. The two drugs shown to have some effect are

Selectivity within the apo B–containing lipoproteins, such
as removing VLDL while retaining LDL, can be obtained
by including antibodies to minor apolipoproteins. HDL
can be selectively bound using antibodies to apo A-I, the
major protein of HDL. As another example, immobilized
monoclonal antibodies have been used to separate a frac-
tion of remnant lipoproteins designated RLP (remnant-
like particles), shown to be particularly atherogenic.149
High-Density Lipoprotein Methods
The measurement of HDL cholesterol has assumed pro-
gressively greater importance in the NCEP treatment
guidelines. In the earliest guidelines, HDL cholesterol
was measured as a risk factor but otherwise was not con-
sidered in treatment decisions. Following recommenda-
tions of a National Institutes of Health–sponsored
consensus panel,74 the 1993 NCEP ATP II guidelines in-
cluded HDL cholesterol measurement with total choles-
terol in the first medical workup, which was reinforced
by the 2001 ATP III guidelines.66 Because the risk asso-
ciated with HDL cholesterol is expressed over a relatively
small concentration range, accuracy in the measurement
is especially important.
For routine diagnostic purposes, HDL for many years
was separated almost exclusively by chemical precipita-
tion, involving a two-step procedure with manual pre-
treatment. A precipitation reagent added to serum or
plasma aggregated non-HDL lipoproteins, which were
sedimented by centrifugation, at forces of approximately
1500g (gravity) with lengthy centrifugation times of 10
to 30 minutes or higher forces of 10,000 to 15,000g, de-
creasing centrifugation times to 3 minutes. HDL is then
quantified as cholesterol in the supernate, usually by one
of the enzymatic assays modified for the lower HDL cho-
lesterol range.
The earliest common precipitation method used he-
parin in combination with manganese to precipitate the
apo B–containing lipoproteins.150,151 Because manganese
interfered with enzymatic assays, alternative reagents
were developed.152 Sodium phosphotungstate153 with
magnesium became commonly used, but because of its
sensitivity to reaction conditions and greater variability,
it was largely replaced by dextran sulfate (a synthetic he-
parin) with magnesium.154 The earliest dextran sulfate
methods used material of 500 kD, which was replaced by
a 50-kD material, considered to be more specific.145
Polyethylene glycol also precipitates lipoproteins, but it
requires 100-fold higher reagent concentrations with
highly viscous reagents, which are difficult to pipet pre-
cisely.155 Numerous commercial versions of these pre-
cipitation reagents became available, which in earlier
years often gave quite different results but gradually be-
came more comparable as standardization programs
were implemented.
CHAPTER 14 ■ LIPIDS AND LIPOPROTEINS 347
CASE STUDY 14-5
A 49-year-old woman was referred for a lipid evalua-
tion by her dermatologist after she developed a
papular rash over her trunk and arms. The rash con-
sisted of multiple, red, raised lesions with yellow
centers. She had no previous history of such a rash
and no family history of lipid disorders or CHD. She
is postmenopausal, on standard estrogen replace-
ment therapy, and otherwise healthy.
Questions
1. What is the rash? What is the cause of her rash?
2. Is her oral estrogen contributing?
3. Is her glucose contributing?
4. What treatments are warranted, and what is her
most acute risk?
CASE STUDY TABLE 14-5.1
LABORATORY RESULTS
SERUM GROSSLY LIPEMIC
Triglycerides 6,200 mg/dL
Total cholesterol 458 mg/dL
Fasting glucose 160 mg/dL
Liver function tests Normal
and electrolytes
A significant problem with HDL precipitation meth-
ods is interference from elevated triglyceride levels.156
When triglyceride-rich VLDL and chylomicrons are pres-
ent, the low density of the aggregated lipoproteins may
prevent them from sedimenting or may even cause float-
ing during centrifugation. This incomplete sedimenta-
tion, indicated by cloudiness, turbidity, or particulate
matter floating in the supernate, results in overestima-
tion of HDL cholesterol. High-speed centrifugation re-
duces the proportion of turbid supernate. Predilution of
the specimen promotes clearing but may lead to errors in
the cholesterol analysis. Turbid supernates may also be
cleared by ultrafiltration, a method that is reliable but te-
dious and inefficient. Because of these drawbacks and the
fact that the laborious pretreatment step is not amenable
to full automation, the precipitation methods became in-
creasingly out of step with the modern automated clini-
cal laboratory.
The result has been development of a new class of di-
rect, sometimes termed homogeneous, methods, which
automate the HDL quantification, making them better
suited for the modern chemistry laboratory. Specific
350 PART 2 ■ CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES
CASE STUDY 14-6
Three patients are seen in clinic:
• Patient 1 is a 40-year-old man with hypertension,
who also smokes, but has not been previously diag-
nosed with CHD. His father developed CHD at age
53 years. He is fasting, and the results of his lipids
include a total cholesterol concentration of
210 mg/dL, triglycerides of 150 mg/dL, and an HDL
cholesterol value of 45 mg/dL. He has a fasting glu-
cose level of 98 mg/dL.
• Patient 2 is a 60-year-old woman with no family
history of CHD, who is normotensive and does not
smoke, with a total cholesterol concentration of
220 mg/dL, triglycerides of 85 mg/dL, and an HDL
cholesterol value of 80 mg/dL. Her fasting glucose
level is 85 mg/dL.
• Patient 3 is a 49-year-old man with no personal or
family history of CHD, and who is not hypertensive
and does not smoke. His fasting total cholesterol
level is 260 mg/dL, his triglycerides are 505 mg/dL,
his HDL cholesterol is 25 mg/dL, and his glucose
level is 134 mg/dL.
Questions
For each patient seen in clinic:
1. What is the LDL cholesterol level, as calculated
using the Friedewald calculation?
2. Which patient, if any, should have his or her LDL
cholesterol measured, rather than calculated?
Why?
3. How many known CHD risk factors does each
patient have?
4. Based on what is known, are these patients rec-
ommended for lipid therapy (diet or drug) and, if
so, on what basis?
weight, and smoking patterns can result in fluctuations in
the observed cholesterol and triglyceride values and the
distribution of the lipoproteins. Similarly, the presence of
clinical conditions, various diseases, or the medications
used in their treatment can affect the circulating lipopro-
teins. Conditions present during blood collection, such as
fasting status, posture, the choice of anticoagulant in the
collection tube, and storage conditions, can alter the
measurements. Typical observed biologic variation for
more than 1 year for total cholesterol averages approxi-
mately 6.1% CV. In the average patient, measurements
made over the course of a year would fall 66% of the time
within ⫾6.1% of the mean cholesterol concentration and
95% of the time within twice this range. Some patients
may exhibit substantially more biologic variation. Thus,
preanalytic variation generally is relatively large in rela-
tion to the usual analytic variation, which is typically less
than 3% CV, and must be considered in interpreting cho-
lesterol results. Some factors, such as posture and blood
collection, can be standardized to minimize the variation.
The NCEP guidelines recommend averaging at least two
successive measurements to reduce the effects of both
preanalytic and analytic sources.66 The use of stepped
cutpoints also reduces the practical effect of variation.
Accuracy
Accuracy or trueness is ensured by demonstrating trace-
ability or agreement through calibration to the respective
“gold standard” reference system. With cholesterol, the
reference system is advanced and complete, having served
as a model for standardization of other laboratory ana-
lytes.115,120 The definitive method at the National
Institute of Standards and Technology provides the ulti-
mate accuracy target but is too expensive and complicated
for frequent use.119 The reference method developed and
applied at the CDC, and calibrated by an approved pri-
mary reference standard to the definitive method, pro-
vides a transferable, practical reference link.118 The
reference method has been made conveniently accessible
through a network of standardized laboratories, the
Cholesterol Reference Method Laboratory Network. This
network was established in the United States and other
countries to extend standardization to manufacturers and
clinical laboratories.115 The network provides accuracy
comparisons leading to certification of performance using
fresh native serum specimens, necessary for reliable accu-
racy transfer because of analyte–matrix interaction prob-
lems on processed reference materials.171,172
Matrix Interactions
In the early stages of cholesterol standardization, which
were directed toward diagnostic manufacturers and rou-
tine laboratories, commercial lyophilized or freeze-dried
materials were used. These materials, made in large
quantities, often with spiking or artificial addition of an-
alytes, were assayed by the definitive and/or reference
methods and distributed widely for accuracy transfer.
Subsequently, biases were observed with some systems
on fresh patient specimens even though they appeared to
be accurate on the reference materials. Although such
manufactured reference materials are convenient, stable,
and amenable to shipment at ambient temperatures, the
manufacturing process, especially spiking and lyophiliza-
tion, altered the measurement properties in enzymatic
assays such that results were not representative of those
on patient specimens. To achieve reliable feedback on