- Email: [email protected]
Measurement of apolipoprotein B as a screening test for identifying children with elevated levels of low-density lipoprotein cholesterol
Measurement of apolipoprotein B as a screening test for identifying children with elevated levels of low-density lipoprotein cholesterol B a r b a r a A. Dennison, MD, David A. Kikuchi, PhD, S a t h a n u r R. Srinivasan, PhD, Larry S. W e b b e r , PhD, a n d G e r a l d S, Berenson, MD From the Departments of Pediatrics, Medicine, Biometry and Genetics, and Biochemistry, Louisiana State University Medical Center, New Orleans We c o m p a r e d the efficacy of two screening tests, measurement of a p o l i p o p r o tein B ( a p o B) levels a n d measurement of serum total cholesterol levels, in detecting e l e v a t e d low-density lipoprotein cholesterol (LDL-C) values in children. We studied 2850 children, a g e d 5 to 17 years, who had fasting lipid, lipoprotein, and a p o l i p o p r o t e i n levels measured as part of the Bogalusa Heart Study. The test characteristics of a p o B were superior to those of serum total cholesterol in screening children to d e t e c t e l e v a t e d levels of LDL-C (~95th percentile) and m o d e r a t e l y e l e v a t e d LDL-C levels (~80th percentile). Unusually high or low values of high-density lipoprotein cholesterol are responsible for most of the misclassification that occurs when measurement of total cholesterol is used as a screening test for identifying children with e l e v a t e d levels of LDL-C. This c o n f o u n d i n g effect of high-density lipoprotein cholesterol was eliminated when measurement of a p o B levels was used as a screening test. Because the a p o B test is more specific at a given sensitivity than the total cholesterol test, the a p o B test can cost more a n d still be less expensive as a screening strategy. As the methods for determining a p o l i p o p r o t e i n levels b e c o m e standardized a n d readily available, the measurement of apolipoproteins c o u l d be d e v e l o p e d into superior screening tests for the identification of patients with dyslipidemias. (J PEDIATR1990;117:358-63)
Elevated serum low-density lipoprotein cholesterol concentration is a major, potentially modifiable risk factor for cardiovascular disease in adults] and is associated with the early stages of atherosclerosis in adolescents and young adults. 2 Reduction of elevated LDL-C levels is associated with a reduction in the incidence of cardiovascular disease and death in adult men. 3, 4 The LDL-C levels track well
Supported by research grant No. HL-38844 from the National Heart, Lung, and Blood Institute, U.S. Public Health Service. Submitted for publication Nov. 16, 1989; accepted March 27, 1990. Reprint requests: Gerald S. Berenson, MD, Department of Medicine, Louisiana State University, Medical Center, 1542 Tulane Ave., New Orleans, LA 70112-2865. 9/20/21917 358
from childhood, beginning at age 1 to 2 years, into adulthood, s'6 Thus the screening procedures to identify children with elevated LDL-C levels appear justifiable. The Apo A-I Apo B HDL-C LDL-C VLDL-C
Apolipoprotein A-I Apolipoprotein B High-density lipoprotein cholesterol Low-density lipoprotein cholesterol Very low density lipoprotein cholesterol
National Institutes of Health Consensus Development Conference recommended using a total cholesterol measurement to screen all adults for elevated LDL-C levels] but the statement does not advocate universal screening of children for elevated LDL-C values, nor does that of the American Academy &Pediatrics Committee on Nutrition)
Volume 117 Number 3
Instead, both recommend screening only those children with a family history of premature cardiovascular disease or hyperlipidemia. Use of family history as an initial screening step is questionable because parental or grandparental history of early coronary artery disease or hypercholesterolemia, or both, identifies only half the children with elevated LDL-C values. 9' lO In adults the correlation between total cholesterol and LDL-C levels is very high; thus a blood total cholesterol value is an effective screening tool for elevated LDL-C values.ll However, because children have relatively higher high-density lipoprotein cholesterol and lower LDL-C levels than adults have, the correlation coefficient between total cholesterol and LDL-C levels is lower. 12 Thus the total cholesterol value is less efficient as a screening tool to identify children with elevated LDL-C levels. We explored an alternative strategy to screen children for elevated LDL-C Values. Levels of apolipoprotein B, the major protein moiety of LDL-C, are more highly correlated with LDL-C levels than are total cholesterol levels. 13 Moreover, previous studies have indicated that the apolipoproteins may be better indicators of cardiovascular disease risk than are the lipids or lipoproteins in adults with coronary artery disease and in children with a parental history of premature myocardial infarction. 1417 Thus we hypothesized that determination of apo B values would be a better screening test than determination of total cholesterol values to identify children with elevated LDL-C levels.
METHODS Study population. The Bogalusa Heart Study is a longterm population-based study of cardiovascular disease risk factors in children from birth through 26 years of age. 18 The study population includes all children residing in Ward 4, a political subdivision located in the southeast corner of Washington Parish (county), including the city of Bogalusa, La., with an approximate population of 22,000 (65% white, 35% black). In 1981-1982, a total of 3312 children, aged 5 to 17 years, were examined (80% of the eligible population). Children with missing lipid, lipoprotein, or apolipoprotein data (n = 78) and those who did not fast before the scheduled venipuncture (n = 384) were eliminated from analyses; the final sample size was 2850. Sixty-four percent were white and 36% were black; 50% were male. The Louisiana State University Medical Center Institutional Review Board approved these experiments. Written informed consent was obtained from a parent or guardian of each child. Confidentiality was stressed throughout the study; for data analyses, each subject was identified by a code number only. Collection of blood samples. Children were instructed to fast for 12 to 14 hours before venipuncture, with compliance determined by interview on the morning of examination.
M e a s u r e m e n t o f apo B level as screening test
359
Antecubital venous blood samples were drawn. Serum samples were prepared in tubes containing thimerosal (Aldrich Chemical Co., Inc., Milwaukee, Wis.), and kept at 4 ~ C until analysis the following day. Each screening day, on a random 10% subsample, a second blood sample was collected, labeled, and analyzed in a blind fashion to estimate the measurement error.
Serum lipid, lipoprotein cholesterol, and apolipoprotein analyses. Serum concentrations of total cholesterol and triglycerides were measured with an AutoAnalyzer II instrument (Technicon Instruments Corp., Tarrytown, N.Y.) with use of protocols developed by the Lipid Research Clinics. 19 Our core lipid laboratory has been standardized by the Centers for Disease Control (Atlanta, Ga.) and is monitored by a surveillance program. Serum levels of LDL-C, HDL-C, and VLDL-C were analyzed with a combination of heparin-calcium precipitation and agar-agarose gel electrophoresis. Details and reliability of the method have been reported. 2~Levels of apo B in serum were assayed by the electroimmunoassay procedure of Laurell. 13,21 In 377 pairs of blind duplicate determinations, intraclass correlation coefficients for the serum lipid and lipoprotein cholesterol assays ranged from 0.89 (HDL-C) to 0.99 (triglyceride); for apo B the reproducibility measurement was 0.96. Statistical analyses. The relationships between serum levels of total cholesterol, its components (VLDL-C, LDLC, and HDL-C), and apo B were examined, and the intraindividual Pearson product-moment correlation coefficients were calculated separately by race and gender. The screening characteristics of serum total cholesterol and apo B in predicting elevated LDL-C (defined as >_95th percentile) and moderately elevated LDL-C levels (defined as >~80th percentile) were determined for specific percentile cutoff points of serum total cholesterol and apo B levels. All percentiles were computed separately by race, gender, and age (2-year intervats). For screening tests based on continuous variables, the test characteristics will change as the cutoff point for labeling a test result as positive or negative changes. For specific percentile cutoff points, Sensitivity and specificity were computed. Sensitivity refers to the proportion of children with an elevated serum LDL-C value who had a screening test result (serum total cholesterol or apo B value) above a specified cutoff point. Specificity refers to the proportion of children without an elevated LDL-C value who had a screening test result (serum total cholesterol or apo B value) below the specified cutoff point. Positive predictive value refers to the proportion of children with a positive screening test result who had an elevated LDL-C value. Receiver operator characteristic curves are plots of the true positive rate (sensitivity) versus the false positive rate (1 - s p e cificity) at different cutoff points. 22 They are a way of visually explaining the trade-off between sensitivity and
360
Dennison et al.
The Journal of Pediatrics September 1990
lOO
100
80
80 .5
60
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o
Serum Total Cholesterol
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2
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Apolipoprotein B
o-4
Serum Total Cholesterol
20
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15
20
25
False positive rate (%) Fig. t. Characteristics of serum total cholesterol and apo B values for screening children for elevated LDL-C levels (>_95th percentile) are plotted as receiver-operator characteristic curves: true positive rate (sensitivity) versus false positive rate (1 - specificity). Apo B curve is much closer to idealized screening test result (100% true positive rate; 0% false positive rate) than is serum total cholesterol curve, demonstrating that measurement of apo B is superior to measurement of serum total cholesterol for identifying children with elevated LDL-C levels.
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False positive rate (%) Fig. 2. Screening test characteristics of serum total cholesterol and apo B values to identify children with moderately elevated LDL-C levels (>_80th percentile) are plotted as receiver-operator characteristic curves. Apo B curve is closer to perfect screening test result than is serum total cholesterol curve, demonstrating that measurement of apo B is better than measurement of total cholesterol for identifying children with moderately elevated LDL-C levels.
mean lipid and lipoprotein cholesterol values and apoli-
T a b l e I. Intraindividual Pearson correlation coefficients by race and gender
Total cholesterol White males White females Black males Black females Combined group VLDL-C White males White females Black males Black females Combined group LDL-C White males White females Black males Black females Combined group HDL-C White males White females Black males Black females Combined group
VLDL-C
LDL-C
HDL-C
Apo B
0.18 0.22 0.15 0.16 0.18
0.81 0.80 0.77 0.76 0.79
0.27 0.17 0.42 0.41 0.29
0.77 0.76 0.74 0.78 0.76
--
--
0.39 0.42 0.30 0.26 0.36
-0.64 -0.66 -0.41 -0.40 -0.58
0.48 0.54 0.39 0.39 0.47
-----
------
-0.31 -0.42 -0.24 -0.23 -0.32
0.93 0.93 0.92 0.93 0.93
poprotein levels of children classified correctly or incorrectly with each screening test were compared with the use of analysis of variance after adjustment for age by computing population marginal means. 23 All statistical analyses were p e r f o r m e d with the Statisti-
--
cal Analysis System ( S A S Institute Inc., Cary, N.C., 1982). Statistical significance was set at a two-tailed p value <0.05, unless otherwise stated. RESULTS Lipid correlations. The mean lipid, lipoprotein, and apolipoprotein values by age and gender have been reported.12, 13 The intraindividual Pearson correlation coefficients of serum levels of total cholesterol, its components, and apo B by race and gender are shown in Table I. Each of these correlation coefficients was significantly different from zero (p <0.001). S e r u m concentration of total cholesterol was positively correlated with all four variables, whereas H D L - C concentration was negatively correlated with the other three variables ( V L D L - C , LDL-C, and apo
. -.
.
.
.
-.
.
.
.
.
-.
.
--
0.32 -0.43 0.21 -0.16 0.31
Significance:all comparisons,p <0.001.
B concentration ). The H D L - C
correlation coefficients
showed the greatest racial variation. (Black children have higher H D L - C levels than white children, and H D L - C constitutes a greater proportion of the total cholesterol level; hence higher correlations of H D L - C with total cholesterol are expected in black children.) Conversely, black children
specificity as one changes the cutoff point, allowing one to
had lower correlations of H D L - C with V L D L - C , L D L - C , and apo B than white children had. For all four race-gen-
compare different screening tests with each other and with
der groups, L D L - C values were more strongly correlated
the ideal screening test (which would be 100% sensitive and 100% specific).
with apo B than with serum total cholesterol values (R --- 0.93 vs 0.76, respectively). A m o n g the four race-gen-
With the 95th percentiles of serum levels of total choles-
der groups, the correlation coefficients of apo B to L D L - C and of serum total cholesterol to L D L - C were similar.
terol and apo B used as cutoff points, children were classified as having either a positive or a negative test result. The
Therefore, in all subsequent analyses of apo B with L D L - C
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Measurement o f apo B level as screening test
361
Table II. Screening test characteristics of serum total cholesterol and apotipoprotein B for identifying children with elevated levels low-density lipoprotein cholesterol
Apolipoprotein B
Serum total cholesterol Specific Percentile* 75th 80th 85th 90th 95th
Sensitivity (%) 97 93 85 80 48
Specificity (%)
PPV (%)
79 84 89 94 98
18 22 27 41 50
Sensitivity (%) 100 100 98 91 71
Specificity (%)
PPV (%)
79 84 89 94 99
14 23 31 43 72
pPv, Positivepredictivevalue.
*Percentilesare age-, race-, and gender-specific.
and of serum total cholesterol with LDL-C, the results for the four race-gender groups were pooled. Sensitivity and specificity. The age-, race-, and genderspecifc percentiles of serum levels of total cholesterol and apo B were determined. The 75th to 95th percentiles were used as screening test cutoff points to identify children with elevated levels of LDL-C. Sensitivity was higher for apo B than for serum total cholesterol at all percentile cutoff points examined (Table II). These differences were accentuated at the higher percentile cutoff points. At the 85th percentiles, sensitivity was 85% for serum total cholesterol versus 98% for apo B; at the 95th percentiles, sensitivity was 48% for serum total cholesterol versus 71% for apo B. The specificities of each screening test, at the same percentile cutoff points, were comparable. The positive predictive values for the two tests were comparable between the 75th and 90th percentiles. At the 95th percentile, the positive predictive value of the apo B test was higher than that of the cholesterol test (72% vs 50%). The true positive rate (sensitivity) versus the false positive rate (1 - specificity) at specific percentile cutoff points of serum levels of total cholesterol and apo B were determined and are shown in Fig. 1 (receiver-operator characteristic curves). Measurement of apo B levels was superior to measurement of serum total cholesterol levels as a screening test to identify children with elevated LDL-C levels. Although few would argue that children with LDL-C values above the 95th percentile are at increased risk for premature cardiovascular disease, some have argued that children with more moderately elevated LDL-C levels (e.g., >_50th, 70th, or 90th percentiles) are also at increased risk. For illustrative purposes, we defined "moderately elevated" LDL-C values as those at or above the 80th percentile and constructed receiver-operator characteristic curves to compare measurement of total cholesterol values and measurement of apo B values as screening tests to identify these children (Fig. 2). Again, apo B values were superior to total cholesterol levels for screening children with moderately elevated LDL-C values.
Specific percentile cutoff points. Unusually high or low levels of HDL-C were responsiblc for most of the misclassification that occurred when measurement of total cholesterol was used as a screening test to identify children with high LDL-C levels. 12,24 When measurement of serum total cholesterol was used as a screening test, the children who wcrc missed (those with false negative results) had lower HDL-C levels and higher cholesterol ratios (serum total cholesterol/HDL-C ratio and LDL-C/HDL-C ratio) than those who were correctly classified (those with true positive test results) (Table III). Thus the children "missed" when mcasurcment of total cholesterol was used as a screening test might actually be at greatcr risk than the children who were idcntified. In contrast, when measurement of apo B was used as the screening test, HDL-C levels no longer acted as a confounder. Those missed (because of false negative tcst results) had higher HDL-C levels and the same cholesterol ratios as those with truc positive results. Thus, when measurement of apo B was used as a screening test, the children in whom elevated LDL-C values were detectcd were those at greatcst risk for cardiovascular disease. Cost analysis. Because of wide variation in costs of lipid, lipoprotein cholesterol, and apolipoprotein measurements, in combination with considerable variation in measurement error in different laboratories, a detailed cost analysis of screening costs is impossible, but some comparisons can be madc. To obtain a comparable sensitivity of 97% to 98% with each test, one needs to sct the cutoff point for serum total cholesterol levcls at the 75th percentile and the cutoff point for apo B levels at the 85th percentile. Thus 25% of patients screened with total cholcsterol versus 15% Of patients screened with apo B would require further evaluation. Table IV compares the costs of these two screcning strategies on the basis of total cholesterol and apo B measurements. For illustrative purposes, three different prices for the screening test and three priccs for the subsequent analysis have been used. The cost of further evaluation should include not only the costs of further laboratory tests and physician time but also the costs of contacting the pa-
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Dennison et al.
The Journal of Pediatrics September 1990
Table III. S e r u m lipid, liPoprotein cholesterol, and apolipoprotein B levels in children with elevated L D L - C levels classified by m e a s u r e m e n t of serum total cholesterol versus apo!ipoprotein B as a screening test
STC test' Positive result$ STC (mg/dl) VLDL-C (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) STC/HDL-C ratio LDL-C/HDL-C ratio Apo B (mg/dl)
230.6 14.7 166.2 49.8 7.4 5.7 145.7
+ 2.81J + 0.9 • 2.5 + 2.4 + 0.6 _+ 0.5 ___2.2
Negative resultr 196.0 _+ 2.7 16.0 + 0.9 149.1_ 2.4 30.5 + 2.3 9.1 _+ 0.6 7.2 _+ 0.5 127.9 _+ 2.T
Apo B test' pw <0.0001 NS <0.0001 <0.0001 <0.05 <0.05 <0.0001
Positive result$ 217.9 ~ 2.5 17.8 + 0.7 162.4 _+ 2.1 37.7 +_ 2.0 8.7 _+ 0.5 6.7 + 0.4 146.0 _ 1.7
Negative results 200.1 9.5 145.0 45.6 7.2 5.8 113.2
+ 3.9 + 1.1 _+ 3.2 _+ 3.1 + 0.7 __+_0.6 + 2.7
pw 0.0001 0.0001 0.0001 0105 NS NS 0.0001
Note that Whenmeasurement of serum total cholesterolis used as a screeningtest, the children who are missed (those with false negative results) have lowerHDLC levels, and higher STC/HDL-C andLDL-C/HDL-C ratios, than the identified children (those with true positive results). Thus the missed children may be those at greatest risk for cardiovascular disease. When measurement'of apo B is used as the screening test, the identified children are those at greatest risk; they have lower HDL-C values and tend toward higher cholesterol ratios. STC, Serum total cholesterol; NS, not significant. *Screening test results >95th age-, ra~ce-, and gender-specificpercentiles of serum total cholesterol or apo B values are classified as positive; results <95th percentiles are classified as negative. ~True positive result. :~False negative result. w lipid levels of children with a positive test result were compared with those with a negative test result by analysis of variance. flCholesterolvalues are adjusted for age, race, and gender. Values'listedare mean + SE. Units (except ratios) are expressedin milligrams per deciliter; to express cholesterol v~iluesin millimoles per liter, multiply by 0.02586.
Table IV. P r e l i m i n a r y cost analysis of screening strategies: S e r u m total cholesterol versus apolipoprotein B measurements
r u m total cholesterol test, the apo B test could cost more a n d yet still be a less expensive screening strategy. DISCUSSION
Costs ($)
Cost of screening per child ($)t
Screening test
Evaluation'
STC
Apo B
5.00 10.00 25.00 5.00 10.00 25.00 5.00 10.00 25.00
75.00 75.00 75.00 150.00 150.00 150.00 250.00 250.00 250.00
23.75 28.25 43.75 42.50 47.50 62.50 67.50 72.50 87.50
i 6.25 21.25 36.25 27.50 32.50 47.50 42.50 47.50 62.50
STC, Serum total cholesterol. *Evaluation costs should include the costs of evaluating each child who has a positive screening test result. tFor these calculations a serum total cholesterol >--_75thpercentile or apo B ~85th percentile was considered a positive screening test result.
tient, the costs associated with the additional time and inconvenience of a r e t u r n visit for the patient and his family, and any unnecessary worry for parents whose children are later found to have " n o r m a l " L D L - C values, (i.e., those with false positive test results). T h e cost differential between the two screening strategies increases as the cost of the subsequent evaluation increases (compare the screening costs when the evaluation costs $250 vs $75). Because the apo B test is more specific at a given sensitivity t h a n the se-
This study demonstrates t h a t serum L D L - C levels were more strongly correlated with apo B levels t h a n with serum total cholesterol levels in all four race-gender groups. T h e screening test characteristics of apo B were superior to those of serum total cholesterol in identifying children with both elevated a n d moderately elevated L D L - C levels. A t a given ievel of sensitivity, the specificity of the apo B test was higher t h a n t h a t of the total cholesterol test. This finding is i m p o r t a n t because a major expense in most screening strategies is the cost of the subsequent evaluation. As the cost of follow-up and f u r t h e r evaluation increases, strategies t h a t are more specific (i.e., have fewer false positive results) are more cost efficient. As a screening p r o g r a m becomes widespread, economies of scale would result in the tests' becoming less costly. W h e n m e a s u r e m e n t of total cholesterol concentration is used as a screening test to identify children with elevated levels of L D L - C , unusually high or low values of H D L - C are responsible for most of the misclassification t h a t occurs. T h e m e a s u r e m e n t of a p o B values as a screening test eliminated this confounding effect by H D L - C levels. Technical a d v a n c e m e n t s have resulted in the use of micromethods for measuring apolipoprotein levels in plasma, serum, and dried b l o o d samples collected on filter paper.25"27Additional studies are needed to d e t e r m i n e w h e t h e r the same screening characteristics will be obtained from measuring apolipoprotein levels in microsamples of
Volume 117 Number 3
blood from nonfasting subjects (the desired sampling method for screening programs). Currently, apo B measurements are not readily available; standardization is just beginning, and there is no quality control system. These problems should be overcome within a few years. Although the major focus of cholesterol screening programs has been identification of those with elevated L D L C values, 6' 7 decreased H D L - C and apolipoprotein A-I levels are also important in prediction of risk for atherosclerotic cardiovascular disease.14-15, 28 Thus screening strategies might be expanded to identify persons with decreased apo A-I levels or an elevated ratio of apo B to apo A-I, in addition to elevated apo B values. In a recent study in Denmark, the apo B / a p o A-I ratio was used as the screening strategy to identify schoolchildren at risk for familial hypercholesterolemia. 27,29 It is possible that this method could be developed and standardized in the same manner as neonatal screening for phenylketonuria or lead screening in toddlers. O f course, the costs and benefits of any universal screening strategy need to be evaluated before such a program is instituted. The Bogalusa Heart Study is a joint effort of numerous people. We thank Mrs. Bettye Seal for her work as community coordinator, the nurses of the Bogalusa project staff, teachers and staff of the Bogalusa school system, Bogalusa community volunteers, and, finally, the children of Bogalusa and their parents for making this study possible. REFERENCES 1. Kannel WB, McGee D, Gordon T. A general cardiovascular risk profile: Framingham Study. Am J Cardio11976;38:46-51. 2. Newman WP III, Freedman DS, Voors AW, et al. Relation of serum lipoprotein levels and systolic blood pressure to early atherosclerosis: the Bogalusa Heart Study. N Engl J Med 1986;314:t38-44. 3. Lipid Research Clinics Program. The Lipid Research Clinics Coronary Primary Prevention Trial results. II. The relation of reduction in incidence of coronary heart disease to cholesterol lowering. JAMA 1984;251:365-74. 4. Brensike JF, Levy RI, Kelsy SF, et al. Effects of therapy with cholestyramine on progression of coronary arteriosclerosis: resuits of the NHLBI Type II Coronary Intervention Study. Circulation 1984;69:313-24. 5. Freedman DS, Srinivasan SR, Cresanta JL, et al. Serum lipids and lipoproteins. Pediatrics 1987;80(suppl):789-96. 6. Webber LS, Srinivasan SR, Berenson GS. Tracking of serum lipids and lipoproteins over 12 years into young adulthood: the Bogalusa Heart Study [Abstract]. Circulation 1988:79:II-481. 7. National Institutes of Health Consensus Development Conference Statement. Lowering blood cholesterol to prevent heart disease. JAMA 1985;253:2080-6. 8. American Academy of Pediatrics Committee on Nutrition. Indications for cholesterol testing in children. Pediatrics 1989;1:141-2. 9. Dennison BA, Kikuchi DA, Srinivasan SR, et al. Parental history of cardiovascular disease as an indication for screening for lipoprotein abnormalities in children. J PEDIATR 1989; 115:186-94.
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10. Griffen TC, Christoffel KK, Binns H J, et al. Family history evaluation as a predictive screen for childhood hypereholesterolemia. Pediatrics 1989;84:365-73. 11. Kannel WB, Castelli WP, Gordon T. Cholesterol in the prediction of atherosclerotic disease. Ann Intern Med 1979; 90:85-91. 12. Dennison BA, Kikuchi DA, Srinivasan SR, et al. Efficacy of serum total cholesterol in detecting elevated LDL-C in children: the Bogalusa Heart Study. Pediatrics 1990;85:472-9. 13. Srinivasan SR, Freedman DS, Sharma C, et al. Serum apolipoproteins A-I and B in 2854 children from a biracial community: the Bogalusa Heart Study. Pediatrics 1986;78:189-200. 14. Kwiterovich PO Jr, Sniderman AD. Atherosclerosis and apoproteins B and A-I. Prev Med 1983;12:815-34. 15. Maciejko J J, Holmes DR, Kottke BA, et al. Apolipoprotein A-I as a marker of angiographically assessed coronary-artery disease. N Engl J Med 1983;309:385-9. 16. Cambien F, Warnet JM, Jacqueson A, et al. Relation of parental history of early myocardial infarction to the level of apoprotein B in men. Circulation 1987;76:266-71. 17. Freedman DS, Srinivasan SR, Shear CL, et al. The relation of apolipoproteins A-1 and B in children to parental myocardial infarction. N Engl J Med 1986;315:721-6. 18. Berenson GS, McMahan CA, Voors AW, et al. Cardiovascular risk factors in children: the early natural history of atherosclerosis and essential hypertension. New York: Oxford UniVersity Press, 1980. 19. Manual of laboratory operation, Lipid Research Clinics program; vol 1. Bethesda, Md.: National Institutes of Health, 1974; U.S. Department of Health, Education, and Welfare publication No. (NIH) 75-628. 20. Srinivasan SR, Berenson GS. Serum lipoproteins in children and methods for study. In: Lewis LA, ed. CRC handbook of electrophoresis, vol III. Lipoprotein methodology and human studies. Boca Raton, Fla.: CRC Press, 1983:185-204. 21. Laurell CB. Electroimmunoassay. Scand J Clin Lab Invest (Suppl) 1972;124:21-3. 22. Fletcher RH, Fletcher SW, Wagner MD. Clinical epidemiology. Baltimore: Williams & Wilkins, 1982:49-50. 23. SAS user's guide: statistics. Cary, N.C.: SAS Institute Inc., 1982:257-8. 24. Kwiterovich PO Jr, Heiss G, Johnson N, et al. Assessment of plasma total cholesterol as a test to detect elevated low-density (beta) lipoprotein cholesterol levels (type IIa hyperlipoproteinemia) in young subjects from a population-based sample. Am J Epidemiol 1982;115:192-204. 25. Dudman NPB, Blades BL, Wilchen DEL, Aitken JM. Radial immunodiffusion assay of apolipoprotein B in blood dried on filter paper: a potential screening method for familial type II hypercholesterolemia. Clin Chim Acta 1985;149:11%7. 26. Ohta T, Ninomiya N, Matsuda I. Enzyme-linked immunosorbent assay for apolipoprotein B on dried blood spot derived from newborn infant. In: Therrell BL, ed. Advances in neonatal screening. Amsterdam: Excerpta Medica, 1987:345-6. 27. Micic S, Arends J, Norgaard-Pedersen B, et al. Simultaneous quantification by double rocket immunoelectrophoresis of apolipoproteins A-I and B in blood spotted on filter paper. Clin Chem 1988;34:2452-5. 28. Kannel WB. High-density lipoproteins: epidemiologic profile and risks of coronary artery disease. Am J Cardiol 1983;52:9B12B. 29. Tegllund L, Skovby F, Andersen GE. Screening for familial hypercholesterolemia in schoolchildren [Abstract]. Pediatr Res 1989;25:204A.
Elevated serum low-density lipoprotein cholesterol concentration is a major, potentially modifiable risk factor for cardiovascular disease in adults] and is associated with the early stages of atherosclerosis in adolescents and young adults. 2 Reduction of elevated LDL-C levels is associated with a reduction in the incidence of cardiovascular disease and death in adult men. 3, 4 The LDL-C levels track well
Supported by research grant No. HL-38844 from the National Heart, Lung, and Blood Institute, U.S. Public Health Service. Submitted for publication Nov. 16, 1989; accepted March 27, 1990. Reprint requests: Gerald S. Berenson, MD, Department of Medicine, Louisiana State University, Medical Center, 1542 Tulane Ave., New Orleans, LA 70112-2865. 9/20/21917 358
from childhood, beginning at age 1 to 2 years, into adulthood, s'6 Thus the screening procedures to identify children with elevated LDL-C levels appear justifiable. The Apo A-I Apo B HDL-C LDL-C VLDL-C
Apolipoprotein A-I Apolipoprotein B High-density lipoprotein cholesterol Low-density lipoprotein cholesterol Very low density lipoprotein cholesterol
National Institutes of Health Consensus Development Conference recommended using a total cholesterol measurement to screen all adults for elevated LDL-C levels] but the statement does not advocate universal screening of children for elevated LDL-C values, nor does that of the American Academy &Pediatrics Committee on Nutrition)
Volume 117 Number 3
Instead, both recommend screening only those children with a family history of premature cardiovascular disease or hyperlipidemia. Use of family history as an initial screening step is questionable because parental or grandparental history of early coronary artery disease or hypercholesterolemia, or both, identifies only half the children with elevated LDL-C values. 9' lO In adults the correlation between total cholesterol and LDL-C levels is very high; thus a blood total cholesterol value is an effective screening tool for elevated LDL-C values.ll However, because children have relatively higher high-density lipoprotein cholesterol and lower LDL-C levels than adults have, the correlation coefficient between total cholesterol and LDL-C levels is lower. 12 Thus the total cholesterol value is less efficient as a screening tool to identify children with elevated LDL-C levels. We explored an alternative strategy to screen children for elevated LDL-C Values. Levels of apolipoprotein B, the major protein moiety of LDL-C, are more highly correlated with LDL-C levels than are total cholesterol levels. 13 Moreover, previous studies have indicated that the apolipoproteins may be better indicators of cardiovascular disease risk than are the lipids or lipoproteins in adults with coronary artery disease and in children with a parental history of premature myocardial infarction. 1417 Thus we hypothesized that determination of apo B values would be a better screening test than determination of total cholesterol values to identify children with elevated LDL-C levels.
METHODS Study population. The Bogalusa Heart Study is a longterm population-based study of cardiovascular disease risk factors in children from birth through 26 years of age. 18 The study population includes all children residing in Ward 4, a political subdivision located in the southeast corner of Washington Parish (county), including the city of Bogalusa, La., with an approximate population of 22,000 (65% white, 35% black). In 1981-1982, a total of 3312 children, aged 5 to 17 years, were examined (80% of the eligible population). Children with missing lipid, lipoprotein, or apolipoprotein data (n = 78) and those who did not fast before the scheduled venipuncture (n = 384) were eliminated from analyses; the final sample size was 2850. Sixty-four percent were white and 36% were black; 50% were male. The Louisiana State University Medical Center Institutional Review Board approved these experiments. Written informed consent was obtained from a parent or guardian of each child. Confidentiality was stressed throughout the study; for data analyses, each subject was identified by a code number only. Collection of blood samples. Children were instructed to fast for 12 to 14 hours before venipuncture, with compliance determined by interview on the morning of examination.
M e a s u r e m e n t o f apo B level as screening test
359
Antecubital venous blood samples were drawn. Serum samples were prepared in tubes containing thimerosal (Aldrich Chemical Co., Inc., Milwaukee, Wis.), and kept at 4 ~ C until analysis the following day. Each screening day, on a random 10% subsample, a second blood sample was collected, labeled, and analyzed in a blind fashion to estimate the measurement error.
Serum lipid, lipoprotein cholesterol, and apolipoprotein analyses. Serum concentrations of total cholesterol and triglycerides were measured with an AutoAnalyzer II instrument (Technicon Instruments Corp., Tarrytown, N.Y.) with use of protocols developed by the Lipid Research Clinics. 19 Our core lipid laboratory has been standardized by the Centers for Disease Control (Atlanta, Ga.) and is monitored by a surveillance program. Serum levels of LDL-C, HDL-C, and VLDL-C were analyzed with a combination of heparin-calcium precipitation and agar-agarose gel electrophoresis. Details and reliability of the method have been reported. 2~Levels of apo B in serum were assayed by the electroimmunoassay procedure of Laurell. 13,21 In 377 pairs of blind duplicate determinations, intraclass correlation coefficients for the serum lipid and lipoprotein cholesterol assays ranged from 0.89 (HDL-C) to 0.99 (triglyceride); for apo B the reproducibility measurement was 0.96. Statistical analyses. The relationships between serum levels of total cholesterol, its components (VLDL-C, LDLC, and HDL-C), and apo B were examined, and the intraindividual Pearson product-moment correlation coefficients were calculated separately by race and gender. The screening characteristics of serum total cholesterol and apo B in predicting elevated LDL-C (defined as >_95th percentile) and moderately elevated LDL-C levels (defined as >~80th percentile) were determined for specific percentile cutoff points of serum total cholesterol and apo B levels. All percentiles were computed separately by race, gender, and age (2-year intervats). For screening tests based on continuous variables, the test characteristics will change as the cutoff point for labeling a test result as positive or negative changes. For specific percentile cutoff points, Sensitivity and specificity were computed. Sensitivity refers to the proportion of children with an elevated serum LDL-C value who had a screening test result (serum total cholesterol or apo B value) above a specified cutoff point. Specificity refers to the proportion of children without an elevated LDL-C value who had a screening test result (serum total cholesterol or apo B value) below the specified cutoff point. Positive predictive value refers to the proportion of children with a positive screening test result who had an elevated LDL-C value. Receiver operator characteristic curves are plots of the true positive rate (sensitivity) versus the false positive rate (1 - s p e cificity) at different cutoff points. 22 They are a way of visually explaining the trade-off between sensitivity and
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Dennison et al.
The Journal of Pediatrics September 1990
lOO
100
80
80 .5
60
70 /
/
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o
Serum Total Cholesterol
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2
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2
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40 I(
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Apolipoprotein B
o-4
Serum Total Cholesterol
20
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I
I
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5
10
15
20
25
False positive rate (%) Fig. t. Characteristics of serum total cholesterol and apo B values for screening children for elevated LDL-C levels (>_95th percentile) are plotted as receiver-operator characteristic curves: true positive rate (sensitivity) versus false positive rate (1 - specificity). Apo B curve is much closer to idealized screening test result (100% true positive rate; 0% false positive rate) than is serum total cholesterol curve, demonstrating that measurement of apo B is superior to measurement of serum total cholesterol for identifying children with elevated LDL-C levels.
o
2;
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False positive rate (%) Fig. 2. Screening test characteristics of serum total cholesterol and apo B values to identify children with moderately elevated LDL-C levels (>_80th percentile) are plotted as receiver-operator characteristic curves. Apo B curve is closer to perfect screening test result than is serum total cholesterol curve, demonstrating that measurement of apo B is better than measurement of total cholesterol for identifying children with moderately elevated LDL-C levels.
mean lipid and lipoprotein cholesterol values and apoli-
T a b l e I. Intraindividual Pearson correlation coefficients by race and gender
Total cholesterol White males White females Black males Black females Combined group VLDL-C White males White females Black males Black females Combined group LDL-C White males White females Black males Black females Combined group HDL-C White males White females Black males Black females Combined group
VLDL-C
LDL-C
HDL-C
Apo B
0.18 0.22 0.15 0.16 0.18
0.81 0.80 0.77 0.76 0.79
0.27 0.17 0.42 0.41 0.29
0.77 0.76 0.74 0.78 0.76
--
--
0.39 0.42 0.30 0.26 0.36
-0.64 -0.66 -0.41 -0.40 -0.58
0.48 0.54 0.39 0.39 0.47
-----
------
-0.31 -0.42 -0.24 -0.23 -0.32
0.93 0.93 0.92 0.93 0.93
poprotein levels of children classified correctly or incorrectly with each screening test were compared with the use of analysis of variance after adjustment for age by computing population marginal means. 23 All statistical analyses were p e r f o r m e d with the Statisti-
--
cal Analysis System ( S A S Institute Inc., Cary, N.C., 1982). Statistical significance was set at a two-tailed p value <0.05, unless otherwise stated. RESULTS Lipid correlations. The mean lipid, lipoprotein, and apolipoprotein values by age and gender have been reported.12, 13 The intraindividual Pearson correlation coefficients of serum levels of total cholesterol, its components, and apo B by race and gender are shown in Table I. Each of these correlation coefficients was significantly different from zero (p <0.001). S e r u m concentration of total cholesterol was positively correlated with all four variables, whereas H D L - C concentration was negatively correlated with the other three variables ( V L D L - C , LDL-C, and apo
. -.
.
.
.
-.
.
.
.
.
-.
.
--
0.32 -0.43 0.21 -0.16 0.31
Significance:all comparisons,p <0.001.
B concentration ). The H D L - C
correlation coefficients
showed the greatest racial variation. (Black children have higher H D L - C levels than white children, and H D L - C constitutes a greater proportion of the total cholesterol level; hence higher correlations of H D L - C with total cholesterol are expected in black children.) Conversely, black children
specificity as one changes the cutoff point, allowing one to
had lower correlations of H D L - C with V L D L - C , L D L - C , and apo B than white children had. For all four race-gen-
compare different screening tests with each other and with
der groups, L D L - C values were more strongly correlated
the ideal screening test (which would be 100% sensitive and 100% specific).
with apo B than with serum total cholesterol values (R --- 0.93 vs 0.76, respectively). A m o n g the four race-gen-
With the 95th percentiles of serum levels of total choles-
der groups, the correlation coefficients of apo B to L D L - C and of serum total cholesterol to L D L - C were similar.
terol and apo B used as cutoff points, children were classified as having either a positive or a negative test result. The
Therefore, in all subsequent analyses of apo B with L D L - C
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Measurement o f apo B level as screening test
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Table II. Screening test characteristics of serum total cholesterol and apotipoprotein B for identifying children with elevated levels low-density lipoprotein cholesterol
Apolipoprotein B
Serum total cholesterol Specific Percentile* 75th 80th 85th 90th 95th
Sensitivity (%) 97 93 85 80 48
Specificity (%)
PPV (%)
79 84 89 94 98
18 22 27 41 50
Sensitivity (%) 100 100 98 91 71
Specificity (%)
PPV (%)
79 84 89 94 99
14 23 31 43 72
pPv, Positivepredictivevalue.
*Percentilesare age-, race-, and gender-specific.
and of serum total cholesterol with LDL-C, the results for the four race-gender groups were pooled. Sensitivity and specificity. The age-, race-, and genderspecifc percentiles of serum levels of total cholesterol and apo B were determined. The 75th to 95th percentiles were used as screening test cutoff points to identify children with elevated levels of LDL-C. Sensitivity was higher for apo B than for serum total cholesterol at all percentile cutoff points examined (Table II). These differences were accentuated at the higher percentile cutoff points. At the 85th percentiles, sensitivity was 85% for serum total cholesterol versus 98% for apo B; at the 95th percentiles, sensitivity was 48% for serum total cholesterol versus 71% for apo B. The specificities of each screening test, at the same percentile cutoff points, were comparable. The positive predictive values for the two tests were comparable between the 75th and 90th percentiles. At the 95th percentile, the positive predictive value of the apo B test was higher than that of the cholesterol test (72% vs 50%). The true positive rate (sensitivity) versus the false positive rate (1 - specificity) at specific percentile cutoff points of serum levels of total cholesterol and apo B were determined and are shown in Fig. 1 (receiver-operator characteristic curves). Measurement of apo B levels was superior to measurement of serum total cholesterol levels as a screening test to identify children with elevated LDL-C levels. Although few would argue that children with LDL-C values above the 95th percentile are at increased risk for premature cardiovascular disease, some have argued that children with more moderately elevated LDL-C levels (e.g., >_50th, 70th, or 90th percentiles) are also at increased risk. For illustrative purposes, we defined "moderately elevated" LDL-C values as those at or above the 80th percentile and constructed receiver-operator characteristic curves to compare measurement of total cholesterol values and measurement of apo B values as screening tests to identify these children (Fig. 2). Again, apo B values were superior to total cholesterol levels for screening children with moderately elevated LDL-C values.
Specific percentile cutoff points. Unusually high or low levels of HDL-C were responsiblc for most of the misclassification that occurred when measurement of total cholesterol was used as a screening test to identify children with high LDL-C levels. 12,24 When measurement of serum total cholesterol was used as a screening test, the children who wcrc missed (those with false negative results) had lower HDL-C levels and higher cholesterol ratios (serum total cholesterol/HDL-C ratio and LDL-C/HDL-C ratio) than those who were correctly classified (those with true positive test results) (Table III). Thus the children "missed" when mcasurcment of total cholesterol was used as a screening test might actually be at greatcr risk than the children who were idcntified. In contrast, when measurement of apo B was used as the screening test, HDL-C levels no longer acted as a confounder. Those missed (because of false negative tcst results) had higher HDL-C levels and the same cholesterol ratios as those with truc positive results. Thus, when measurement of apo B was used as a screening test, the children in whom elevated LDL-C values were detectcd were those at greatcst risk for cardiovascular disease. Cost analysis. Because of wide variation in costs of lipid, lipoprotein cholesterol, and apolipoprotein measurements, in combination with considerable variation in measurement error in different laboratories, a detailed cost analysis of screening costs is impossible, but some comparisons can be madc. To obtain a comparable sensitivity of 97% to 98% with each test, one needs to sct the cutoff point for serum total cholesterol levcls at the 75th percentile and the cutoff point for apo B levels at the 85th percentile. Thus 25% of patients screened with total cholcsterol versus 15% Of patients screened with apo B would require further evaluation. Table IV compares the costs of these two screcning strategies on the basis of total cholesterol and apo B measurements. For illustrative purposes, three different prices for the screening test and three priccs for the subsequent analysis have been used. The cost of further evaluation should include not only the costs of further laboratory tests and physician time but also the costs of contacting the pa-
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Dennison et al.
The Journal of Pediatrics September 1990
Table III. S e r u m lipid, liPoprotein cholesterol, and apolipoprotein B levels in children with elevated L D L - C levels classified by m e a s u r e m e n t of serum total cholesterol versus apo!ipoprotein B as a screening test
STC test' Positive result$ STC (mg/dl) VLDL-C (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) STC/HDL-C ratio LDL-C/HDL-C ratio Apo B (mg/dl)
230.6 14.7 166.2 49.8 7.4 5.7 145.7
+ 2.81J + 0.9 • 2.5 + 2.4 + 0.6 _+ 0.5 ___2.2
Negative resultr 196.0 _+ 2.7 16.0 + 0.9 149.1_ 2.4 30.5 + 2.3 9.1 _+ 0.6 7.2 _+ 0.5 127.9 _+ 2.T
Apo B test' pw <0.0001 NS <0.0001 <0.0001 <0.05 <0.05 <0.0001
Positive result$ 217.9 ~ 2.5 17.8 + 0.7 162.4 _+ 2.1 37.7 +_ 2.0 8.7 _+ 0.5 6.7 + 0.4 146.0 _ 1.7
Negative results 200.1 9.5 145.0 45.6 7.2 5.8 113.2
+ 3.9 + 1.1 _+ 3.2 _+ 3.1 + 0.7 __+_0.6 + 2.7
pw 0.0001 0.0001 0.0001 0105 NS NS 0.0001
Note that Whenmeasurement of serum total cholesterolis used as a screeningtest, the children who are missed (those with false negative results) have lowerHDLC levels, and higher STC/HDL-C andLDL-C/HDL-C ratios, than the identified children (those with true positive results). Thus the missed children may be those at greatest risk for cardiovascular disease. When measurement'of apo B is used as the screening test, the identified children are those at greatest risk; they have lower HDL-C values and tend toward higher cholesterol ratios. STC, Serum total cholesterol; NS, not significant. *Screening test results >95th age-, ra~ce-, and gender-specificpercentiles of serum total cholesterol or apo B values are classified as positive; results <95th percentiles are classified as negative. ~True positive result. :~False negative result. w lipid levels of children with a positive test result were compared with those with a negative test result by analysis of variance. flCholesterolvalues are adjusted for age, race, and gender. Values'listedare mean + SE. Units (except ratios) are expressedin milligrams per deciliter; to express cholesterol v~iluesin millimoles per liter, multiply by 0.02586.
Table IV. P r e l i m i n a r y cost analysis of screening strategies: S e r u m total cholesterol versus apolipoprotein B measurements
r u m total cholesterol test, the apo B test could cost more a n d yet still be a less expensive screening strategy. DISCUSSION
Costs ($)
Cost of screening per child ($)t
Screening test
Evaluation'
STC
Apo B
5.00 10.00 25.00 5.00 10.00 25.00 5.00 10.00 25.00
75.00 75.00 75.00 150.00 150.00 150.00 250.00 250.00 250.00
23.75 28.25 43.75 42.50 47.50 62.50 67.50 72.50 87.50
i 6.25 21.25 36.25 27.50 32.50 47.50 42.50 47.50 62.50
STC, Serum total cholesterol. *Evaluation costs should include the costs of evaluating each child who has a positive screening test result. tFor these calculations a serum total cholesterol >--_75thpercentile or apo B ~85th percentile was considered a positive screening test result.
tient, the costs associated with the additional time and inconvenience of a r e t u r n visit for the patient and his family, and any unnecessary worry for parents whose children are later found to have " n o r m a l " L D L - C values, (i.e., those with false positive test results). T h e cost differential between the two screening strategies increases as the cost of the subsequent evaluation increases (compare the screening costs when the evaluation costs $250 vs $75). Because the apo B test is more specific at a given sensitivity t h a n the se-
This study demonstrates t h a t serum L D L - C levels were more strongly correlated with apo B levels t h a n with serum total cholesterol levels in all four race-gender groups. T h e screening test characteristics of apo B were superior to those of serum total cholesterol in identifying children with both elevated a n d moderately elevated L D L - C levels. A t a given ievel of sensitivity, the specificity of the apo B test was higher t h a n t h a t of the total cholesterol test. This finding is i m p o r t a n t because a major expense in most screening strategies is the cost of the subsequent evaluation. As the cost of follow-up and f u r t h e r evaluation increases, strategies t h a t are more specific (i.e., have fewer false positive results) are more cost efficient. As a screening p r o g r a m becomes widespread, economies of scale would result in the tests' becoming less costly. W h e n m e a s u r e m e n t of total cholesterol concentration is used as a screening test to identify children with elevated levels of L D L - C , unusually high or low values of H D L - C are responsible for most of the misclassification t h a t occurs. T h e m e a s u r e m e n t of a p o B values as a screening test eliminated this confounding effect by H D L - C levels. Technical a d v a n c e m e n t s have resulted in the use of micromethods for measuring apolipoprotein levels in plasma, serum, and dried b l o o d samples collected on filter paper.25"27Additional studies are needed to d e t e r m i n e w h e t h e r the same screening characteristics will be obtained from measuring apolipoprotein levels in microsamples of
Volume 117 Number 3
blood from nonfasting subjects (the desired sampling method for screening programs). Currently, apo B measurements are not readily available; standardization is just beginning, and there is no quality control system. These problems should be overcome within a few years. Although the major focus of cholesterol screening programs has been identification of those with elevated L D L C values, 6' 7 decreased H D L - C and apolipoprotein A-I levels are also important in prediction of risk for atherosclerotic cardiovascular disease.14-15, 28 Thus screening strategies might be expanded to identify persons with decreased apo A-I levels or an elevated ratio of apo B to apo A-I, in addition to elevated apo B values. In a recent study in Denmark, the apo B / a p o A-I ratio was used as the screening strategy to identify schoolchildren at risk for familial hypercholesterolemia. 27,29 It is possible that this method could be developed and standardized in the same manner as neonatal screening for phenylketonuria or lead screening in toddlers. O f course, the costs and benefits of any universal screening strategy need to be evaluated before such a program is instituted. The Bogalusa Heart Study is a joint effort of numerous people. We thank Mrs. Bettye Seal for her work as community coordinator, the nurses of the Bogalusa project staff, teachers and staff of the Bogalusa school system, Bogalusa community volunteers, and, finally, the children of Bogalusa and their parents for making this study possible. REFERENCES 1. Kannel WB, McGee D, Gordon T. A general cardiovascular risk profile: Framingham Study. Am J Cardio11976;38:46-51. 2. Newman WP III, Freedman DS, Voors AW, et al. Relation of serum lipoprotein levels and systolic blood pressure to early atherosclerosis: the Bogalusa Heart Study. N Engl J Med 1986;314:t38-44. 3. Lipid Research Clinics Program. The Lipid Research Clinics Coronary Primary Prevention Trial results. II. The relation of reduction in incidence of coronary heart disease to cholesterol lowering. JAMA 1984;251:365-74. 4. Brensike JF, Levy RI, Kelsy SF, et al. Effects of therapy with cholestyramine on progression of coronary arteriosclerosis: resuits of the NHLBI Type II Coronary Intervention Study. Circulation 1984;69:313-24. 5. Freedman DS, Srinivasan SR, Cresanta JL, et al. Serum lipids and lipoproteins. Pediatrics 1987;80(suppl):789-96. 6. Webber LS, Srinivasan SR, Berenson GS. Tracking of serum lipids and lipoproteins over 12 years into young adulthood: the Bogalusa Heart Study [Abstract]. Circulation 1988:79:II-481. 7. National Institutes of Health Consensus Development Conference Statement. Lowering blood cholesterol to prevent heart disease. JAMA 1985;253:2080-6. 8. American Academy of Pediatrics Committee on Nutrition. Indications for cholesterol testing in children. Pediatrics 1989;1:141-2. 9. Dennison BA, Kikuchi DA, Srinivasan SR, et al. Parental history of cardiovascular disease as an indication for screening for lipoprotein abnormalities in children. J PEDIATR 1989; 115:186-94.
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10. Griffen TC, Christoffel KK, Binns H J, et al. Family history evaluation as a predictive screen for childhood hypereholesterolemia. Pediatrics 1989;84:365-73. 11. Kannel WB, Castelli WP, Gordon T. Cholesterol in the prediction of atherosclerotic disease. Ann Intern Med 1979; 90:85-91. 12. Dennison BA, Kikuchi DA, Srinivasan SR, et al. Efficacy of serum total cholesterol in detecting elevated LDL-C in children: the Bogalusa Heart Study. Pediatrics 1990;85:472-9. 13. Srinivasan SR, Freedman DS, Sharma C, et al. Serum apolipoproteins A-I and B in 2854 children from a biracial community: the Bogalusa Heart Study. Pediatrics 1986;78:189-200. 14. Kwiterovich PO Jr, Sniderman AD. Atherosclerosis and apoproteins B and A-I. Prev Med 1983;12:815-34. 15. Maciejko J J, Holmes DR, Kottke BA, et al. Apolipoprotein A-I as a marker of angiographically assessed coronary-artery disease. N Engl J Med 1983;309:385-9. 16. Cambien F, Warnet JM, Jacqueson A, et al. Relation of parental history of early myocardial infarction to the level of apoprotein B in men. Circulation 1987;76:266-71. 17. Freedman DS, Srinivasan SR, Shear CL, et al. The relation of apolipoproteins A-1 and B in children to parental myocardial infarction. N Engl J Med 1986;315:721-6. 18. Berenson GS, McMahan CA, Voors AW, et al. Cardiovascular risk factors in children: the early natural history of atherosclerosis and essential hypertension. New York: Oxford UniVersity Press, 1980. 19. Manual of laboratory operation, Lipid Research Clinics program; vol 1. Bethesda, Md.: National Institutes of Health, 1974; U.S. Department of Health, Education, and Welfare publication No. (NIH) 75-628. 20. Srinivasan SR, Berenson GS. Serum lipoproteins in children and methods for study. In: Lewis LA, ed. CRC handbook of electrophoresis, vol III. Lipoprotein methodology and human studies. Boca Raton, Fla.: CRC Press, 1983:185-204. 21. Laurell CB. Electroimmunoassay. Scand J Clin Lab Invest (Suppl) 1972;124:21-3. 22. Fletcher RH, Fletcher SW, Wagner MD. Clinical epidemiology. Baltimore: Williams & Wilkins, 1982:49-50. 23. SAS user's guide: statistics. Cary, N.C.: SAS Institute Inc., 1982:257-8. 24. Kwiterovich PO Jr, Heiss G, Johnson N, et al. Assessment of plasma total cholesterol as a test to detect elevated low-density (beta) lipoprotein cholesterol levels (type IIa hyperlipoproteinemia) in young subjects from a population-based sample. Am J Epidemiol 1982;115:192-204. 25. Dudman NPB, Blades BL, Wilchen DEL, Aitken JM. Radial immunodiffusion assay of apolipoprotein B in blood dried on filter paper: a potential screening method for familial type II hypercholesterolemia. Clin Chim Acta 1985;149:11%7. 26. Ohta T, Ninomiya N, Matsuda I. Enzyme-linked immunosorbent assay for apolipoprotein B on dried blood spot derived from newborn infant. In: Therrell BL, ed. Advances in neonatal screening. Amsterdam: Excerpta Medica, 1987:345-6. 27. Micic S, Arends J, Norgaard-Pedersen B, et al. Simultaneous quantification by double rocket immunoelectrophoresis of apolipoproteins A-I and B in blood spotted on filter paper. Clin Chem 1988;34:2452-5. 28. Kannel WB. High-density lipoproteins: epidemiologic profile and risks of coronary artery disease. Am J Cardiol 1983;52:9B12B. 29. Tegllund L, Skovby F, Andersen GE. Screening for familial hypercholesterolemia in schoolchildren [Abstract]. Pediatr Res 1989;25:204A.