Apolipoprotein screening in Australian children: feasibility and the effect of age, sex, and ethnicity

Judith F Lynch, Michelle D Marshall and Xing L Wang
Med J Aust 1998; 168 (2): 61-64.
Published online: 19 January 1998

Apolipoprotein screening in Australian children: feasibility and the effect of age, sex, and ethnicity

Judith F Lynch, Michelle D Marshall, Xing L Wang and David E L Wilcken

MJA 1998; 168: 61-64  

Abstract - Introduction - Methods - Results - Discussion - References - Authors' details
- - ©MJA1998



Objectives: (i) To evaluate the feasibility of detecting adverse lipid profiles in schoolchildren by measuring capillary dried blood spot apolipoprotein levels, and (ii) to assess the effect of age, sex and ethnicity on apolipoprotein levels.
Design: We measured capillary dried blood spot apolipoproteins B and A-I (apo B and apo A-I); assessed levels in relation to age, sex and ethnicity; and recalled children with elevated levels for a full lipid profile measurement.
Participants and setting: 6992 children (3501 boys and 3491 girls), aged 5-13 years, from schools in eastern Sydney, 1991-1995.
Main outcome measures: Capillary blood levels of apolipoproteins B and A-I, and serum total cholesterol level.
Results: Of the 6951 children who provided an adequate fingerprick blood sample, we recalled 1465 children (21.1%) (640 boys [43.7%] and 825 girls [56.3%]) with elevated apo B levels and/or apo B : apo A-I ratios for further testing, either by us or by their family doctor (overall estimated compliance rate up to 70%). Among the 458 children who returned to us, there was a 90% positive predictive value for a total cholesterol level of over 4.5 mmol/L in those with both elevated apo B levels and high apo B : apo A-I ratios. Girls had higher apo B levels and apo B : apo A-I ratios than boys (P < 0.00001 for both), and in both sexes there was a trend downwards for apo B and upwards for apo B : apo A-I ratio over the age range tested, but levels were relatively stable between the ages of 6 and 10 years. Indian children (1.5% of the screened population) had the highest apo B levels, followed by white children (71.1%); Asian children (9.2%) had the lowest (P < 0.00001 compared with Indian and white children).
Conclusions: The high positive predictive value of capillary blood apolipoprotein levels for an adverse lipid profile in children suggests that measuring apolipoprotein levels by this method is a useful initial approach to cardiovascular risk assessment.  


Despite a recent decline in cardiac deaths, coronary disease is still the largest single cause of premature death in Australia.1 It is known that atherogenesis, the underlying pathological process, may begin in childhood;2,3 that relevant risk factors may track from childhood to adulthood (particularly elevated levels of total and low-density lipoprotein [LDL] cholesterol and increased body weight); and that these factors tend to aggregate within families.4,5 With these considerations in mind, we explored the feasibility of screening for adverse lipid profiles in a target group of primary schoolchildren, with the aim of implementing family-based coronary prevention by secondarily identifying any parents at risk.

We measured levels of apolipoprotein B (apo B), the carrier protein for the atherogenic LDL cholesterol, and apolipoprotein A-I (apo A-I), the principal carrier protein for the antiatherogenic high density lipoprotein (HDL) cholesterol, in capillary dried blood spots from 6992 primary schoolchildren aged 5-13 years. There is increasing evidence that high apo B and low apo A-I levels are as reflective of cardiovascular risk as are their respective lipoproteins.6 We describe here the positive predictive value of high apo B and/or apo B : apo A-I ratio for elevated total cholesterol levels and the effects of age, sex and eth nicity on lipid levels in Australian children.  


School and population demographics
We received permission from 70 schools in eastern Sydney (public primary schools, 45%; Catholic primary schools, 29%; and private fee-paying schools, 26%) to contact parents and offer them the option of their child participating in a "Heart Health Screening Program". Children were given consent forms to take home to their parents. On the forms, we asked for the country of origin of each family, and this information was confirmed by a family history questionnaire (sent to all parents within a week of the testing at school) asking each parent to identify their country of origin. Only 1% of the total declined to answer this question (the "unknown" group) (Table 1).

Blood collection and apolipoprotein measurement
Testing took place between 1991 and 1995. From each child we obtained a capillary blood sample by fingerprick, which was spotted directly onto filter paper (No. 903: Schleicher & Schuell Inc., Keene, NH, USA), allowed to air dry and stored at 2 70¡C until analysis. Concentrations of dried blood spot apo B, apo A-I and lipoprotein (a) (Lp(a)) were measured within two weeks by our previously described enzyme-linked immunosorbent assay methods,7-9 and from these results an apo B : apo A-I ratio was also calculated. These methods allowed us to carry out all three measurements on one small (minimum, 20 µL sample) capillary dried blood spot. We report here the results obtained for measurement of apo B and apo A-I only.

In the first school tested, we established the frequency distribution for apo B levels and apo B : apo A-I ratios and set the level for recall for each of these as at or above the 90th percentile. These cut-off points were adjusted from the apo lipoprotein results obtained from each of the next seven schools. However, a stable cut-off level was reached by the third school. We defined three categories of children for recall: those with an ele vated apo B level only (Criterion 1), those with a combination of elevated apo B level and apo B : apo A-I ratio (Criterion 2), and finally those with an elevated apo B : apo A-I ratio only (Criterion 3).

We notified the parents of the children for recall by letter, explaining the results and suggesting that both parents, the child, and any other siblings might come to us for further lipid testing, or attend their family doctor. In those who returned to us, we measured total cholesterol level, as well as HDL cholesterol and triglyceride levels, in a venous blood sample by standard methods. LDL cholesterol level was calculated by the Friedewald formula,10 and we also measured serum apo B, apo A-I and Lp(a) by the methods outlined above.7-9

The parents of children with apo B levels and apo B : apo A-I ratios below the established cut-off points were also sent a letter explaining that their children's levels currently fell below the recall criteria. These families were sent a healthy lifestyle pamphlet specifically directed to maintaining a low level of cardiovascular risk; they were also asked if they would like to volunteer as a healthy control family and have complete lipid profiles measured as above.

Statistical analysis
As the distributions of age, apo B and apo A-I levels, and apo B : apo A-I ratios were normal, analyses by parametric methods were appropriate. We used one-way analysis of variance (ANOVA) for comparisons of measured quantitative variables when there were more than two groups; for example, when children were divided according to high apo B, high apo B : apo A-I ratio, or both, as categorical variables, and levels of total cholesterol and LDL cholesterol, and total cholesterol to HDL cholesterol ratio as the continuous outcome variables. When the comparison was between two groups -- for example, when lipid values were compared in girls and boys as categorical determinants -- Student's t test was used.11 We employed a chi-squared comparison for assessing associations between categorical variables, and used the SPSS statistical software package for all analyses.12

Ethical approval
The study was approved by the ethics committee of the University of New South Wales.  


Initial testing
The parental consent rate to initial testing was 63.9%. There were 6992 children aged 5-13 years available for testing, 3501 (50.1%) boys and 3491 (49.9%) girls. Only 41 children (0.6%) failed to provide an adequate finger-prick blood sample on the day, leaving a total population of 6951 children tested.

Recall population
The cut-off point for apo B level was set at 0.550 g/L or more and for the apo B : apo A-I ratio 1.200 or more. We recalled 1465 children (or 21.1% of the population tested). Of these recalls, 65.3% were for elevated apo B only (Criterion 1), 19.6% for elevated apo B and apo B : apo A-I ratio (Criterion 2), and 15.1% for elevated apo B : apo A-I ratio only (Criterion 3).

In all, 458 of the children recalled returned with their families to our laboratory for lipid studies (31.2% compliance rate). To obtain an assessment of how many may have been seen by their family doctor (the other option we suggested), we contacted 205 of the remaining 1007 families. Among these, 118 had seen their family doctor for follow-up, and 89 had either moved or were not interested in further testing. This suggested an overall compliance rate of about 70%, if this sample was representative.

Of the non-recalled "normal" population of 5486 (78.9%), only 151 families (2.7%) volunteered for lipid testing, an insufficient number for meaningful statistical analysis.

Predictive value
We used the results of the 458 children who returned for a full lipid profile to assess the positive predictive value of our screening method. The National Heart Foundation of Australia recommends a total cholesterol level of 4.5 mmol/L as the upper limit for children in this age range.13 With a level of over 4.5 mmol/L as the endpoint, Criterion 1 (elevated apo B only) had a positive predictive value of 84% (95% confidence interval [CI], 80%-88%), and with Criterion 2 (elevated apo B level and apo B : apo A-I ratio combined) the predictive value increased to 90% (95% CI, 85%-95%). However, Criterion 3 (elevated apo B : apo A-I ratio only) yielded only 66% (95% CI, 54%-77%), possibly reflecting children with low HDL cholesterol and normal total cholesterol levels.

Raising the cut-off point did not substantially improve the positive predictive value of our screening method. When the cut-off point for apo B was increased from 0.550 g/L to 0.700 g/L, for example, the positive predictive value of Criterion 2, the most strongly predictive, only increased to 94% (95% CI, 91%-97%).

ANOVA confirmed the association between high apo B levels from analysis of dried blood spot and an adverse lipid profile, and also that a combined high apo B level and apo B : apo A-I ratio was a stronger predictor. In this analysis, criteria established by high apo B, high apo B : apo A-I ratio and a combination of both were determinant categorical variables, and the total cholesterol and LDL cholesterol levels and total chol es terol to HDL cholesterol ratio were the continuous outcomes. Total (P < 0.0006) and LDL cholesterol (P < 0.0014) levels and the total cholesterol to HDL cholesterol ratio (P < 0.0006) were all significantly higher in Criterion 2 than in Criterion 1 or Criterion 3 (Table 2).

Sex, age and ethnicity
There was a highly significant difference in the apo B and apo B : apo A-I ratio levels between boys and girls (P < 0.00001 for each) (Figures 1 and 2), but no difference (P = 0.6) in apo A-I levels (data not shown). The girls had consistently higher apo B levels and apo B : apo A-I ratios over the age range studied (Figures 1 and 2). This resulted in a significant difference in the recall rate between girls and boys (chi-squared = 23.362; P < 0.00001). This sex difference was also reflected in the total cholesterol levels of children returning for further lipid tests (data not shown).

As shown in Figures 1 and 2, there was a downward trend in apo B level and an upward one in apo B : apo A-I ratio in boys over the age range screened, while the opposite was observed in girls; but levels were relatively consistent in both between the ages of 6 and 10 years.

Most of the 6951 children screened were white (71.1%); the remainder were of diverse ethnic origins (Table 1). The mixed-race group (8.3% of the total) included white/Asian (28.2%), white/Arabic (17.8%), white/South American (10.7%), and other combinations (56.7%).

As age was a significant contributor to apo B concentrations, we compared the "age adjusted apo B levels" for both boys and girls to assess potential differences between major ethnic groups (Figure 3). Indian children had the highest apo B levels, followed by white children. Asian children had the lowest levels and these were significantly different from those of the Indian, white, mixed-race and South American children (P < 0.00001 for each). We did not include black, Australian Aboriginal or Pacific Islander ethnic groups in this analysis because of small numbers and highly skewed age distributions. In all ethnic groups, girls had higher levels than boys (Figure 3).



We have established previously that apolipoprotein measurements provide a convenient and effective approach to the detection of dyslipidaemia,14 and that apo B levels in children are correlated with the occurrence of coronary events in their grandparents, which highlights the relevance of measurements in children to assessing risk of vascular disease in older family members.15 We have also established a clear-cut association between increased apo B levels and apo B : apo A-I ratios and body mass index in Australian children.16 Here, we extend these find ings by demonstrating the feasibility of screening of schoolchildren's capillary blood apolipoprotein levels as an approach to family-based primary coronary prevention.

Although we do not have adequate data to determine the sensitivity or specificity of our screening method, the presence of both an elevated apo B level and raised apo B : apo A-I ratios had a positive predictive value of 90% for the detection of elevated total serum cholesterol level. Raising the apolipoprotein cut-off points only improved the predictive value to 94%. This method identified elevated apolipoprotein levels in 21.1% of our population, of whom 90% had elevated total cholesterol levels. Screening for apolipoprotein levels may be more relevant than screening for total cholesterol levels alone, as total cholesterol level does not show the interrelations between levels of the atherogenic LDL and the antiatherogenic HDL cholesterol.17 In a small number of children (0.48%) apo B levels were elevated without elevation of total cholesterol level.18 These families were also provided with dietary and lifestyle advice as this may also be associated with increased cardio vascular risk.

Our findings clearly show highly significant differences between boys and girls at this age, with girls having higher apo B levels and apo B : apo A-I ratios than boys. We suspect this biological difference, which was evident before the onset of puberty in both boys and girls, to be hormonally based, although we have no data to support this. The vari ation in apolipoprotein levels within the age range studied could also be hormonally based. Whatever the mech anisms, these same age-related sex differences have been identified in several other studies in children.19,20 The consistency of these results clearly indicates a need to have different cut-off points in boys and girls for elevated apolipoprotein levels. Our results also define the age range (between 6 and 10 years) when levels are most stable and most appropriate for screening, findings which will be incorporated into future studies.

There were uniform sex-related differences in apolipoprotein levels in each of the racial groups in our population, as well as very significant differences in levels between the various ethnic groups, as has also been found in previous studies.21,22 In our population, Indian and white children had the highest apo B and apo B : apo A-I ratios and Asian children had the lowest. It is well established that Chinese and Japanese populations have much lower cholesterol levels than whites and a correspondingly lower prevalence of coronary artery disease. Indian populations have a higher prevalence of cardiovascular disease.23 While these variations may relate to the genetic background of each ethnic group, diet and lifestyle undoubtedly make major contributions, and this is particularly evident in non-Western groups who have moved to live in a Western society. The higher prevalence of coronary disease in Indians living in the United Kingdom, for example, is well documented.24 Ethnic differences may become blurred with time if dietary habits become more uniform, and this has occurred among Asians emigrating to the United States and the United Kingdom; within a generation they acquired local lifestyles and a correspondingly higher prevalence of coronary artery disease than that in the communities they had left.25,26

In conclusion, our study demonstrates the feasibility of conducting a program of measuring apolipoprotein levels in capillary blood samples to assess lipid profiles in children to facilitate family-based coronary prevention. It documents wide acceptance by both schools and parents. Implicit in such a study is the potential for a multiplier effect in that, if a child has an elevated apolipoprotein level and therefore an adverse lipid profile, it is very likely that this will also be seen in at least one parent and other siblings.27 Our ongoing screening program requires the establishment of a concurrent intervention program to improve dietary and lifestyle habits of affected children and their parents. This is of more immediate relevance to affected parents as they are approaching the age of overt coronary disease. However, the program provides an ideal opportunity to establish healthy lifestyles in young children at a time of easy acceptance, and with the potential for preventing a disorder that may have its origins in childhood.28  


  1. Heart and Stroke Facts. A report by the National Heart Foundation of Australia. Canberra: NHFA, 1996.
  2. Stary HC. Evolution and progression of atherosclerotic lesions in coronary arteries of children and young adults. Arteriosclerosis 1989; 9(1 Suppl): I19-I32.
  3. Tracy RE, Newman WP, Wattigney WA, Berenson GS. Risk factors and atherosclerosis in youth autopsy findings of the Bogalusa Heart Study. Am J Med Sci 1995; 310(Suppl 1): S37-S41.
  4. Berenson GS, Wattigney WA, Bao W, et al. Rationale to study the early natural history of heart disease: the Bogalusa Heart Study [review]. Am J Med Sci 1995; 310(Suppl 1): S22-S28.
  5. Sanchez-Bayle M, Gonzalez-Requjo A, Ruiz-Jarabo C, et al. Serum lipids and lipoproteins in Spanish children and adolescents: a 5 year follow-up. Acta Paediatr 1996; 85: 292-294.
  6. Bao W, Srinivassan SR, Berenson GS. Tracking of serum apolipoproteins A-I and B in children and young adults: the Bogalusa Heart Study. J Clin Epidemiol 1993; 46: 609-616.
  7. Wang XL, Dudman NP, Wilcken DE. Enzyme-linked immunosorbent assay of apolipoprotein B in blood spotted onto filter paper, suitable for neonatal screening. Clin Chem 1989; 35: 1000-1004.
  8. Wang XL, Dudman NP, Blades BL, Wilcken DE. Changes in the immunoreactivity of apo A-I during storage. Clin Chem 1989; 179: 285-293.
  9. Wang XL, Wilcken DE, Dudman NP. An indirect sandwich ELISA for Lp(a) in serum and dried blood spots. Clin Chim Acta 1992; 207: 73-86.
  10. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18: 499-502.
  11. Norusis MJ. General factorial analysis of variance. SPSS for Windows Advanced Statistics, release 6.0. Chicago: SPSS Inc, 1993: 31-56.
  12. McNeil D. Statistical methods II: one-way analysis of variance. In: McNeil D, editor. Epidemiological research methods. New York: John Wiley & Sons, 1996: 66-71.
  13. National Diet and Heart Disease Advisory Committee, National Heart Foundation of Australia. Guide to plasma lipids for doctors. Curr Therap 1992; 10(Suppl 1): S1-S8.
  14. Wang XL, Wilcken DE, Dudman NP. Apolipoprotein A- I and B and the B/A-I ratio in the first year of life. Pediatr Res 1991; 30: 544-549.
  15. Wilcken DEL, Wang XL, Greenwood J, Lynch JF. Lipoprotein (a) and apolipoproteins B and A-I in children and coronary vascular events in their grandparents. J Pediatr 1993; 123: 519-526.
  16. Wilcken DE, Lynch JF, Marshall MD, et al. Relevance of body weight to apolipoprotein levels in Australian children. Med J Aust 1996; 164: 22-25.
  17. Taylor CJ, Olpin S, Rattenbury J, et al. Familial hyper cholesterolaemia: pilot study to identify children at risk. J Clin Pathol 1993; 46: 730-733.
  18. Grundy S. Small LDL. Atherogenic dyslipidaemia and the metabolic syndrome. Circulation 1997; 95: 1-4.
  19. Resnicow K, Morley-Kotchen J, Wynder E. Plasma cholesterol levels of 6585 children in the United States: results of the know your body screening in five states. Pediatrics 1989; 84: 969-976.
  20. Christensen B, Glueck C, Kwiterovich P, et al. Plasma cholesterol and triglyceride distributions in 13,665 children and adolescents: the Prevalence Study of the Lipid Research Clinics Program. Pediatr Res 1980; 14: 194-202.
  21. Zhang W, Evans AE, Cambien F, et al. Distribution of lipid variables in subjects in Belfast, Northern Ireland and Taiyuan, PR China. Atherosclerosis 1993; 102: 175-180.
  22. Freedman DS, Lee SL, Byers T, et al. Serum cholesterol levels in a multiracial sample of 7,439 preschool children from Arizona. Prev Med 1992; 21: 162-176.
  23. Enas EA, Mehta J. Malignant coronary artery disease in young Asian Indians: thoughts on pathogenesis, prevention, and therapy. Coronary Artery Disease in Asian Indians (CADI) Study [review]. Clin Cardiol 1995; 18: 131-135.
  24. Bhatnagar D, Anand IS, Durrington PN, et al. Coronary risk factors in people from the Indian subcontinent living in west London and their siblings in India. Lancet 1995; 345: 405-409.
  25. Egusa G, Murakami F, Ito C, et al. Westernized food habits and concentration of serum lipids in the Japanese. Atherosclerosis 1993; 100: 249-255.
  26. Robinson D, Kawamura T, Hinohara S, Sakamoto Y. Levels of cardiovascular risk factors in Japanese people living in the UK. J Cardiovasc Risk 1995; 2: 449-458.
  27. Shaukat N, de Bono DP, Jones DR. Like father like son? Sons of patients of European or Indian origin with coronary artery disease reflect their parents' risk factor patterns. Br Heart J 1995; 74: 318-323.
  28. Resnicow K, Cross D, Lacosse J, Nichols P. Evaluation of a school-site cardiovascular risk factor screening intervention. Prev Med 1993; 22: 838-856.
(Received 15 Apr, accepted 15 Oct, 1997)  

Authors' details

Department of Cardiovascular Medicine, Prince Henry and Prince of Wales Hospitals, Sydney; and Community Health Services and Programs, South Eastern Sydney Area Health Service, Royal South Sydney Hospital, Sydney, NSW.
Judith F Lynch, HTech, Heart Health Education Program Co-ordinator;
Michelle D Marshall, BSc, Scientific Officer;
Xing L Wang, PhD, Research Fellow;
David E L Wilcken , MD, FRACP, Visiting Professor of Medicine.

Reprints will not be available from the authors.
Correspondence: Professor D E L Wilcken, Department of Cardiovascular Medicine, Room 163, Clinical Sciences Building, Prince Henry Hospital, Little Bay, Sydney, NSW 2036.
Readers may print a single copy for personal use. No further reproduction or distribution of the articles should proceed without the permission of the publisher. For permission, contact the Australasian Medical Publishing Company
Journalists are welcome to write news stories based on what they read here, but should acknowledge their source as "an article published on the Internet by The Medical Journal of Australia <>".


Received 19 September 2018, accepted 19 September 2018

  • Judith F Lynch
  • Michelle D Marshall
  • Xing L Wang



remove_circle_outline Delete Author
add_circle_outline Add Author

Do you have any competing interests to declare? *

I/we agree to assign copyright to the Medical Journal of Australia and agree to the Conditions of publication *
I/we agree to the Terms of use of the Medical Journal of Australia *
Email me when people comment on this article

Responses are now closed for this article.