Pathobiological determinants of atherosclerosis in youth (PBDAY Study), 1986-96.

Pathobiological determinants of atherosclerosis in youth (PBDAY Study), 1986±96 V N. H. Sternby, 1 J.E. Fernandez-Britto, 2 & P. Nordet 3 This article is a summary of the 10-year multinational collaborative WHO/ISFC Study of Pathobiological Determinants of Atherosclerosis in Youth (PBDAY Study). Details are provided of the study design, relevant results, conclusions, and recommendations, as formulated at a consultation of the heads of PBDAY Reference Centres, held in Budapest, Hungary, in October 1996. The WHO/ISFC study provides unique information about the determinants of athero- sclerosis and structural changes in the arteries, especially during their early stages, and their progression from early life in populations with vastly different lifestyles. The pilot study covered subjects aged 5±34 years, of both sexes, from 18 centres in 15 countries, while the main study covered 11 centres in 11 countries. Included were both developed and developing countries with different economic, sociocultural and nutritional patterns from five WHO regions. Collected was background epidemiological information, information about cases, and special studies of arteries using various morphometric methods and specialized techniques. Atherosclerotic lesions start to develop early in life independently of race, sex or geographical origin. The rate of fatty streak development is higher between 15 and 25 years of age, while raised lesions begin developing slowly during the second decade of life, progressing steadily during the third and more rapidly during the fourth. Fatty streaks are more prevalent among females and raised lesions among males. The prevalence and extent of raised lesions were greater in countries with a high prevalence of known risk factors and high mortality rates for cardiovascular diseases, coronary heart disease, and cerebrovascular diseases. Voir page 254 le re sume en francËais. En la pa gina 254 figura un resumen en espanÄ ol. Introduction Atherosclerosis, with its complications, is the pathological process that underlies most cases of coronary heart disease, cerebrovascular disease, and aortic and peripheral vascular disease. In developed countries it is thus a major cause of preventable morbidity, disability, and premature death, and is also emerging as a problem in developing countries. It is a lifelong disease process: the initial stages occurring among children and young people are silent and without symptoms, with clinical manifestations appearing in middle age or later, mainly as sudden cardiac death, myocardial infarction, angina pectoris, stroke, aortic aneurysm, renovascular hypertension, and intermittent claudication (1±13). This study, like the PDAY Study (14), a complements the International Atherosclerosis Pro- ject (13) and the WHO Study of Atherosclerosis of the Aorta and Coronary Arteries in Five Towns (9). Since 1982, many pathologists from different countries have been involved in the preparation and review of the PBDAY Study protocol (15±17). Objectives The objectives of the PBDAY Study are outlined below. . To explore the structural changes in arteries which may determine the development of atherosclero- sis, particularly its early stages and progression. . To study the topographical relationships of different atherosclerotic lesions and their relation to possible causal factors. . To explore the influence of contrasting socio- cultural settings and individual characteristics on the presence and progress of atherosclerotic lesions in youth. . To assess the extent to which changes in the arterial wall of young individuals can be detected by morphometry, atherometric analysis, histo- V Prepared by the authors for the PBDAY Principal investigators. A full description of the study appears in the following: Report of the Joint WHO/ISFC Study Pathobiological Determinants of Atherosclerosis in Youth (PBDAY), a ten-year multinational collaborative study (unpub- lished document WHO/CVD/97.1). 1 Professor, Department of Pathology, University of Lund, Lund, Sweden. 2 Professor, Instituto Superior de Ciancias Medicas de la Habana, Hospital C.J. Findlay, Havana, Cuba. 3 Medical Officer, Department of Noncommunicable Disease Preven- tion, World Health Organization, 1211 Geneva 27, Switzerland. Requests for reprints should be sent to Dr Nordet at this address. Reprint No. 5994 a PDAY Study: the North American multicentre cooperative research programme ``Pathobiological Determinants of Atherosclerosis in Youth''. 250 # World Health Organization 1999 Bulletin of the World Health Organization, 1999, 77 (3) Research chemistry, immunohistochemistry, biochemistry and ultrastructural studies before the appearance of grossly visible atherosclerotic lesions. . To study the differences in the lipid and/or lipoprotein composition and location of the arterial wall and its relation to pathobiological findings. Study design The management of the study was delegated to the PBDAY Steering Committee, which organized the administrative and technical work, as well as coordinating data collection and the receipt and distribution of specimens, in close collaboration with members of the PDAY Study Steering Committee (15±17). Special analyses using various morpho- metric methods and specialized techniques were developed at seven reference centres and used to examine specimens collected and forwarded from 18 Collaborating Centres to the Receiving, Processing and Distribution Centres (see Annex). The study included subjects of both sexes ( age range, 5±34 years), with a preponderance of those who had suffered fatal accidents. The pilot study covered populations from 18 centres in 15 countries, while the main study covered 11 centres in 11 coun- tries, including both developed and developing countries from five WHO regions, with different economic, sociocultural and nutritional patterns. The data required to fulfil the study's objectives were obtained from three sources (15): ± background epidemiological information; ± subject/individual information; and ± collection, preparation and analysis of specimens. The background epidemiological information was prepared at the management and data centre, using the most recent and reliable data from any available source. The subject/individual information was obtained from individual records, while family information was obtained from the questionnaire forms (forms 01 and 02) (16). Specimens were collected, prepared and shipped by the Collaborating Centres. The core procedure included a general autopsy, amore detailed examination of thoracic and abdominal organs, and collection of the main specimens, which were forwarded frozen or formalin-fixed in plastic contain- ers/bags to the Receiving, Processing and Distribu- tion Centres (16±19), as follows : ± aorta (Ao), abdominal (AA), descending thoracic (DTA); ± coronary arteries (CA) and branches, right (RC), left main (LMC), left circumflex (LC), left anterior descending (LAD); and ± postmortem blood, kidney tissue, and myocardial tissue. Specimens were analysed (17) using different morphometric methods in the following Reference Centres: Malmo (visual grading (9, 17±19)), Siena (semiautomatic macro- and microscopic computer- assisted grading (17, 20)) and Havana (quantitative automatic computer-assisted grading) ``atherometric system'' b (17, 21, 22); and also using special techni- ques in Budapest (histochemistry and immunohis- tochemistry (17, 23±26), Geneva (blood and subcellular analysis (17)), Heidelberg (ultrastructural techniques (17, 27±29)) and Moscow (biochemistry (17, 30±32)). All analyses of specimens were performed blind. The results were forwarded to the data centre, using the appropriate record form (forms 04±21) for data processing and elaboration. In 1987, a pilot study was undertaken in 18 centres to assess the feasibility of carrying out the PBDAY Study in each site and to perfect the data recording instruments. Results Background epidemiological information Atherosclerosis is a major cause of morbidity, disability and premature mortality in developed countries, and is becoming so in many developing countries. Age-standardized mortality rates for all cardio- vascular diseases, coronary heart disease, and cerebrovascular diseases were higher in Latvia, Lithuania and Hungary, and lower in Italy, Mexico, Hong Kong Special Administrative Region of China, and Sri Lanka. Clinical and epidemiological data were pro- cessed from 1277 cases (958 males and 319 females); 133 were aged 5±14 years, 509 were aged 15±24 years, and 635 were aged 25±34 years. The prevalence of cardiovascular risk factors (arterial hypertension, diabetes mellitus, and smok- ing) and alcohol consumptionwere, in general, higher in Lithuania, Hungary, andGermany. The prevalence of smoking and hypertension were also high in Cuba. Analysis by clinical and epidemiological char- acteristics of the pool of all cases of atherosclerosis indicates that the rate was high among people with high blood pressure, diabetes mellitus, a smoking habit and regular-to-heavy alcohol consumption; among individuals aged 25±34 years; and among males and in the WHO European Region and in the Region of the Americas. Morphometric studies Atherosclerotic lesions begin to develop early in life, independently of race, sex or geographical origin. The proportion of affected subjects and the mean percentage of intimal surface affected by athero- b This method also provides the ponderative atherometric indices Ð obstruction, stenosis, and benignity Ð which make it possible to estimate the severity of atherosclerosis. 251Bulletin of the World Health Organization, 1999, 77 (3) Pathobiological determinants of atherosclerosis in youth sclerotic lesions exhibit considerable inter-individual and inter-vessel variability (see Fig. 1±3). Although there was a good correlation between the results of the three methods used, the two computer-assisted methods proved more reliable and also more suitable for data elaboration. Morphometric visual grading (Malmo). Data from 134 cases were studied: 94 males (70.1%) and 40 females (29.9%); 16.4% in the age group 5±14 years, 41.1% in the age group 15±24 years and 42.5% in the age group 25±35 years. Fatty streaks were found in the Ao of all cases, being more prevalent for thoracic Ao (96%) and less for right coronary artery tissue. Raised lesions (fibrous plaques plus calcified and complicated plaques) were seen in a small proportion of cases (7.1% in the AA and 11.1% in the RC). Macroscopic and microscopic computer- assisted grading (Siena). Macroscopic study of Ao and RC. Fatty streak lesions in Ao were found in 100% of the 355 cases and in 68% of the 319 RC specimens. The occurrence of fatty streaks was low (0±33%) on the whole ventral surface and high (>50%) on the dorsal surface of Ao and was strictly related to branching regions, where fatty streaks are highly likely to occur. Raised lesions occurred in a low proportion of cases (7.05% of DTA specimens, 25.75% of AA specimens and 22.05% of RC specimens). Most fatty streaks and raised lesions completely overlapped. Histomorphometric study of Ao and CA. The prevalence of fatty streaks increased with age in both sexes until a plateau was reached (m <1), while the prevalence of raised lesions in males increased exponentially with age (m >1) and was higher in LAD and AA specimens. Mean intimal andmedial thickness were greater for males and in AA and LAD specimens. Both these thicknesses increased with age, with great variability among different vessels and by sex; and both were greater among smokers and those with hypertension. Atherometric system (Havana). Fatty streak lesions were observed in 99.6% of the 996 DTA specimens, in 95.8%of the 947AA specimens, and in 38.65% of the 958 RC specimens, with the mean proportion of intimal surface affected comprising 32.6% in DTA, 38.6% in AA and 12.05% in RC specimens. Raised lesions were found in 12.6% of DTA, 16.4% of AA and 2.6% of RC specimens, with themean proportion of intimal surface affected being 0.95% in DTA, 3.2% in AA and 2.6% in RC specimens (Fig. 1). Fatty streaks were found in all three age groups; although raised lesions were seen in a small proportion of cases, their development progresses slowly from the second decade of life and accelerates during the fourth. The thoracic aorta (TA) is themain site for fatty streaks during the first 15 years of life, but thereafter the AA is predominant (Fig. 2). Females were most affected by fatty streaks in all vessels. For all age groups, males developed fibrous plaques earlier than females in both aortas and in RC specimens. Themean percentages of subjects and the area affected by raised lesions were higher among smokers. Atherosclerotic lesions, mainly fatty streaks, were observed in cases from all participating countries, developed and developing (Fig. 3). The extent of the area affected by raised lesions and the ponderative atherometric indices for obstruction and stenosis were higher in countries with a high prevalence of well-known risk factors and high mortality rates for cardiovascular diseases, coronary heart disease and cerebrovascular diseases (Cuba, Germany, Hungary, and Lithuania). Special studies Morphometric quantitative lesion analysis (Heidel- berg); structure of coronary arteries and myocardial capillarization in youth; and nephrosclerosis index. The mean values of the most relevant variables in the cases studied (717 males and 241 females) are shown in Table 1. Cellular density gives an indication of the extent of the extracellular matrix and is more closely related to epidemiological data on cardiovascular mortality than intimal thickness. The nephrosclerosis index is positively corre- lated with blood pressure. Hypertension and smoking have a considerable influence on intimal thickness and intimal cellular density. Histology, histochemistry and immunohisto- chemistry of atherosclerotic lesions (Budapest). Histology and histochemistry of atherosclerotic lesions. The Fig. 1 Percentage of affected subjects and mean percentage of intimal surface affected by atherosclerotic lesions in the three arteries (DTA = descending thoracic artery, AA = abdominal artery, RC = right coronary). 252 Bulletin of the World Health Organization, 1999, 77 (3) Research mean intima/media (I/M) ratio of the 214 cases studied was greater in LAD specimens than in either AA or TA specimens. The prevalence of atheroma- tous lesions (type III±VI lesions) was the same in LAD and AA, but less common in TA specimens. Myoelastic layer formation was found in all arteries, in both early and atheromatous lesions. Immunohistochemistry of early atherosclero- tic lesions. The immunohistochemistry of intimal cells was studied using 36 standard aortic samples. The intensity of expression of HLA-DR in endothe- lial cells was the same for each type of lesion. The number of HLA-DR-positive cells was significantly greater in initial lesions and fatty streaks than in cases of diffuse intimal thickening, owing to the increased number of foam cells. Immunoreactions using different macrophage-specific markers suggested the presence of specific marker expression during the progression of early lesions. Chemistry of arterial wall. Lipid determina- tion of arterial wall (Moscow, Havana, Malmo). The lipid fractions of the intimal and medial wall of 191 TA specimens and 180 AA specimens were studied. Dried de-fatted mass is the major compo- nent of the arterial wall (919.7 mg per g of dried tissue). The three commonest lipid components were phospholipid (32.0 mg per g), esterified cholesterol (27.1 mg per g) and free fatty acids (15.1 mg per g), while free cholesterol and triglycerides were present in low concentrations. There was no difference in the lipid fractions by sex: the concentrations of free and esterified cholesterol in TA tend to increase with age. Fig. 2. Percentage of affected subjects and mean percentage of intimal surface affected by atherosclerotic lesions, by age group (FS = fatty streak, RS = raised lesions, A. sub = % affected subjects, A. area = % affected area). Fig. 3. Mean percentage of intimal surface affected by atherosclerotic lesions by country (see Annex codes). Table 1. Mean values of the key study variables Variable Male Female Density of intima cells 1 696.2 1 889.4 Density of media cell 942.0 1 082.8 Intimal thickness (mm) 336.6 247.1 Medial thickness (mm) 178.2 149.5 Nephrosclerosis index 28.8 28.7 Capillary length density of myocardium 193.3 185.7 253Bulletin of the World Health Organization, 1999, 77 (3) Pathobiological determinants of atherosclerosis in youth The planned special studies with frozen speci- mens (Geneva RC) could not be completed owing to inadequate specimen transportation and preservation. Conclusions . Atherosclerotic lesions and early arterial changes can be detected by various morphometric meth- ods before the appearance of grossly visible atherosclerotic lesions. . Atherosclerotic lesions begin their progressive development early in life, independently of an individual's race, sex or geographical origin. . Development of fatty streak is higher for the age group 15±25 years, while raised lesions begin developing slowly during the second decade of life, progress steadily during the third, and more rapidly during the fourth. Fatty streaks are more prevalent among females, and raised lesions among males and smokers. . Inmost cases there is overlap between localization of fatty streaks and raised lesions in the arterial wall. . The prevalence and extent of raised lesions were higher in countries that had a high prevalence of well-known risk factors and high mortality rates for cardiovascular diseases, coronary heart dis- ease, and cerebrovascular diseases. . There were marked differences in vascular structure, especially intimal cellular density and thickness, and myocardial capillarization in differ- ent populations and risk factors. . There were structural, histochemical and immu- nohistochemical variations in early atherosclerotic lesions in different populations. . These findings reiterate the need to promote a healthy lifestyle from very early childhood, as well as the primary and secondary prevention of cardiovascular diseases. n Acknowledgements We are grateful to Dr I. Gyarfas, Budapest, Hungary; Dr E.G.J. Olsen, London, England; Dr T. Strasser, Geneva, Switzerland; Dr J.P. Strong, New Orleans, LA, USA; andDr R.Wissler, Boston, MA, USA, who assisted WHO in the planning, implementation and follow-up of the study. The study was supported financially by the hospital and university administrations of the participating centres and by the International Society and Federation of Cardiology. Re sume De terminants physiopathologiques de l'athe roscle rose chez les jeunes (Etude PBDAY), 1986-1996 Cet article rend brieÁ vement compte de l'e tude collective multinationale conduite sur 10 ans par l'OMS et la Socie te et Fe de ration internationale de Cardiologie sur les de terminants physiopathologiques de l'athe roscle rose chez les jeunes. Il donne des indications de taille es sur le plan de l'e tude, ses re sultats, et les conclusions et recommandations qui ont e te formule es aÁ Budapest (Hongrie) en octobre 1996 lors d'une consultation des chefs des centres de re fe rence ayant participe aÁ l'e tude. Cette e tude a fourni des donne es uniques sur les de terminants de l'athe roscle rose et les modifications de la structure des arteÁ res, en particulier aÁ leurs premiers stades, et sur leur progression, depuis le plus jeune aà ge, au sein de populations caracte rise es par des modes de vie treÁ s diffe rents. L'e tude pilote a porte sur des sujets des deux sexes aà ge s de 5 aÁ 34 ans dans 18 centres de 15 pays cependant que l'e tude principale a couvert 11 centres de 11 pays, soit des pays de veloppe s et en de veloppement appartenant aux cinq Re gions de l'OMS et caracte rise s par des scheÂmas e conomiques, socioculturels et nutri- tionnels treÁ s divers. On a recueilli des donne es e pideÂmiologiques de base, des informations sur les cas et les re sultats d'e tudes spe ciales sur les arteÁ res faites aÁ l'aide de diverses me thodes anatomome triques et de techniques spe cialise es. Les le sions athe roscle rotiques apparaissent au de but de la vie inde pendamment de la race, du sexe ou de l'origine ge ographique. L'e paississement des fibres est plus rapide entre 15 et 25 ans cependant que l'athe rome commence aÁ se de velopper lentement entre 10 et 20 ans, progresse re gulieÁ rement entre 20 et 30 ans, puis plus rapidement apreÁ s 30 ans. L'e paississement des fibres est plus fre quent chez les sujets de sexe feÂminin et l'athe rome chez ceux de sexe masculin. La pre valence et l'e tendue des athe romes sont plus grandes dans les pays ouÁ la fre quence des facteurs de risque connus est e leve e, de meÃme que la mortalite due aux maladies cardio- vasculaires, aux cardiopathies coronariennes et aux maladies ce re brovasculaires. Resumen Determinantes biopatolo gicos de la aterosclerosis en la juventud (Estudio PBDAY), 1986-1996 En el presente artõÂculo se resume el Estudio OMS/ISFC sobre los Determinantes Biopatolo gicos de la Ateros- clerosis en la Juventud (Estudio PBDAY), estudio colaborativo multinacional de diez anÄ os de duracio n. Se informa sobre el disenÄ o del estudio, los resultados de mayor intere s, las conclusiones y las recomendaciones 254 Bulletin of the World Health Organization, 1999, 77 (3) Research formuladas en una reunio n consultiva celebrada por los jefes de los Centros de Referencia PBDAY en Budapest (HungrõÂa) en octubre de 1996. El estudio OMS/ISFC aporta informacio n singular sobre los determinantes de la aterosclerosis y de los cambios estructurales sufridos por las arterias, especial- mente en las primeras fases del proceso, asõ como sobre su evolucio n desde los primeros anÄ os de la existencia en poblaciones con muy distintos modos de vida. En el estudio piloto participaron sujetos de 5 a 34 anÄ os de ambos sexos de 18 centros de 15 paõÂses, mientras que el estudio principal abarco 11 centros de 11 paõÂses. Se incluyeron paõÂses tanto desarrollados como en desarrol- lo, con distinto perfil econo mico, sociocultural y nutricional, de cinco regiones de la OMS. Se procedio a reunir informacio n epidemiolo gica basal, informacio n sobre casos, y datos obtenidos mediante la aplicacio n de diversos me todos morfome tricos y te cnicas especializa- das al estudio de las arterias. Las lesiones aterosclero ticas empiezan a aparecer en fases tempranas de la vida, con independencia de la raza, el sexo o el origen geogra fico. El ritmo de aparicio n de las estrõÂas adiposas es mayor entre los 15 y los 25 anÄ os, y las lesiones protuberantes empiezan a desarro- llarse lentamente durante la segunda de cada de la vida, siguen progresando durante la tercera, y lo hacen au n ma s ra pidamente durante la cuarta. Las estrõÂas adiposas son ma s frecuentes en las mujeres, y las lesiones protuberantes en los varones. La prevalencia y extensio n de estas u ltimas era mayor en los paõÂses con alta prevalencia de factores de riesgo conocidos y altas tasas de mortalidad por enfermedades cardiovasculares, cardiopatõÂa coronaria y enfermedades cerebrovascu- lares. References 1. Prevention of coronary heart disease. Report of a WHO Expert Committee. Geneva, World Health Organization, 1982 (WHO Technical Report Series, No. 678). 2. World health statistics annual, 1989±94. Geneva, World Health Organization, 1990±95. 3. Prevention in childhood and youth of adult cardiovascular diseases: time for action. Report of a WHO Expert Committee. Geneva,World Health Organization, 1990 (WHO Technical Report Series, No. 792). 4. Pathobiological Determinants of Atherosclerosis in Youth (PDAY) ResearchGroup. Relationship of atherosclerosis in young men to serum lipoprotein cholesterol concentration and smoking. A preliminary report from the PDAY. Journal of the American Medical Association, 1990, 264: 3018±3024. 5. Enos WF et al. Pathogenesis of coronary disease in American soldiers killed in Korea. Journal of the American Medical Association, 1955, 158: 912±914. 6. McNamara JJ et al. Coronary artery disease in combat casualties in Vietnam. Journal of the American Medical Association, 1971, 216: 1185±1187. 7. Holman RL et al. The natural history of atherosclerosis: the early aortic lesions as seen in New Orleans in the middle of the 20th century. American journal of pathology, 1958, 34: 209±235. 8. Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group. Natural history of aortic and coronary atherosclerotic lesions in youth. Finding from the PDAY study. Arteriosclerosis and thrombosis, 1993, 13(9):1291±1298. 9. Kagan AR et al. Atherosclerosis of the aorta and coronary arteries in five towns. Bulletin of the World Health Organization, 1976, 53: 486±645. 10. Solberg LA, Strong JP. Risk factors and atherosclerosis lesions. A review of autopsy studies. Arteriosclerosis, 1983, 3: 187±198. 11. Wissler RW and the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group. An overview of the quantitative influence of several risk factors on progression of atherosclerosis in young people in the United States. American journal of the medical sciences, 1995, 310 (Suppl.1): S29±S36. 12. Freedman DS et al. Black±white differences in aortic fatty streaks in adolescence and early adulthood: the Bogalusa Heart Study. Circulation, 1988, 77: 856±864. 13. McGill HC et al. The geographic pathology of atherosclerosis. Laboratory investigation, 1968, 18(5): 463±653. 14. PDAY Study. Protocol and manual of procedures. Chicago, IL, University of Chicago, Department of Pathology, 1985. 15. WHO/ISFC PBDAY Study (protocol) . Unpublished document WHO/CVD/87.3 (available upon request from: Cardiovascular Diseases, World Health Organization, 1211 Geneva 27, Switzerland). 16. WHO/ISFC PBDAY Study manual of operation. Unpublished document WHO/CVD/87.4 (available upon request from: Cardio- vascular Diseases, World Health Organization, 1211 Geneva 27, Switzerland). 17. WHO/ISFC PBDAY Studymanual of operation, part II. Unpublished document WHO/CVD/PBDAY/88.2 (available upon request from: Cardiovascular Diseases, World Health Organization, 1211 Geneva 27, Switzerland). 18. Sternby NH. Atherosclerosis in a defined population. Acta pathologica et microbiologica scandinavica, 1968, Suppl. 194: 5. 19. Guzman MA et al. Selected methodologic aspects of interna- tional atherosclerosis project. Laboratory investigation, 1968, 18: 479±497. 20. Cornhill JF, Herderick EE, Stary HC. Topography of human aortic sudanophilic lesions In: Liepsch DW, ed. Blood flow in large arteries: applications to therogenesis and clinical medicine. (Monographs on Atherosclerosis, 1990, 15: 13±19). 21. Fernandez-Britto JE, Carlevaro PV. Atherometric system: morphometric standardized methodology to study atherosclerosis and its consequences. Gegenbaurs morphologisches Jahrbuch (Leipzig), 1989, 135(1): 2±12. 22. Carlevaro PV. An atherometric system to study the athero- sclerosis lesions. Pathological research and practice, 1987, 182: 488±489. 23. Gown AM, Tsukada T, Ross R. Human atherosclerosis. II. Immunocytochemical analysis of the cellular composition of human atherosclerotic lesions. American journal of pathology, 1986, 125: 191±207. 24. Emerson EE, Robertson AL. T lymphocytes in aortic and coronary intimas. Their potential role in atherogenesis. American journal of pathology, 1988, 130: 369±376. 25. Illye s G, Kadar A. [Immunohistochemistry of atherosclerotic lesions]. Lege artis medicinae, 1993, 3(6): 518±522 (in Hungarian). 26. Katsuda S et al. Human atherosclerosis. III. Immunocyto- chemical analysis of the cell composition of lesions of young adults. American journal of pathology, 1992, 140: 907. 27. Mattfeldt Y, Mall G. Estimation of length and surface of anisotropic capillaries. Journal of microscopy, 1984, 135: 181±190. 28. Tracy ER. Quantitative measures of the severity of hypertensive nephrosclerosis. American journal of microscopy, 1970, 91: 25. 29. Sterio DC. The unbiased estimation of number and sizes of arbitrary particles using the dissector. Journal of microscopy, 1984, 134: 127. 255Bulletin of the World Health Organization, 1999, 77 (3) Pathobiological determinants of atherosclerosis in youth 30. Folch J et al. A simple method for isolation and purification of total lipids from animal tissues. Journal of biological chemistry, 1957, 226(1): 497±509. 31. Frei RW. A chemical study of some parameters in quantitative investigations of thin-layer chromatography by absorption method. Journal of chromatography, 1972, 64(2): 285±295. 32. Kates M. Techniques of lipidology. New York, Elsevier, 1972. Annex PBDAY Study: Sites and key personnel PBDAY Collaborating Centres a 01. Beijing, China (P) Dr Wen-Ying Huang and Thao Peizhen Department of Pathology Cardiovascular Institute and Fu Wai Hospital Chinese Academy of Medical Sciences Beijing China 02. Berlin, Germany (P) Dr F. Vollmar Institute of Pathology Bezirkskrankenhaus Suhl Germany 03. Budapest, Hungary (P,M) Dr Anna Kadar and Dr G. Illye s Semmelweis Medical University 2nd Department of Pathology Budapest Hungary 05. Calabar, Nigeria (P) Dr Ed. B. Attahj Provost College of Medical Sciences University of Calabar Calabar Nigeria 07. Chandigarh, India (P,M) Dr B.N. Datta Professor of Pathology and Head Department of Morbid Anatomy Postgraduate Institute of Medical Education and Research Chandigarh India 11. Havana, Cuba (P,M) Dr J.E. Fernandez-Britto Instituto Superior de Ciencias Medicas de la Habana Hospital C.J. Finlay Havana Cuba 12. Heidelberg, Germany (P,M) Dr G. Mall Direktor des Pathologischen Instituts StaÈ dtische Kliniken Darmstadt Germany Dr I. Siemens Landesfachklinik Eisfelder Str 41/A1 Hildburghausen Germany 13. Hong Kong Special Administrative Region of China (P,M) Dr F.C.S. Ho Department of Pathology University of Hong Kong Queen Mary Hospital Compound Hong Kong Special Administrative Region of China 14. Shatin, Hong Kong Special Administrative Region of China (P,M) Dr L.J. McGuire Department of Morbid Anatomy Faculty of Medicine Chinese University of Hong Kong Shatin, New Territories, Hong Kong Special Administrative Region of China 15. Ibadan, Nigeria (P,M) Dr T. A. Junaid Department of Pathology College of Medicine University of Ibadan University College Hospital Ibadan Nigeria 16. Kaunas, Lithuania (P,M) Dr E. Stalioraityte and Dr Z. Januskevicius Research Institute of Cardiology Kaunas Lithuania 18. Mexico City, Mexico (P,M) Dr L. Cueto and Dr C. Posadas Instituto Nacional de Cardiologia ``Ignacio Chavez'' Mexico City Mexico 20. Peradeniya, Sri Lanka (P,M) Dr S. Mendis Senior Lecturer Department of Medicine Faculty of Medicine University of Peradeniya Peradeniya Sri Lanka 21. Riga, Latvia (P,M) Dr V.A. Volkov Chief Bureau of Pathology Latvian Ministry of Health Riga Latvia 23. Siena, Italy (P,M) Dr G. Weber Dr G. Bianciardi and Dr P. Tanganelli Atherosclerosis Research Centre Institute of Pathological Anatomy University of Siena Siena Italy a P = Pilot study; M = Main study. 256 Bulletin of the World Health Organization, 1999, 77 (3) Research 24. Tashkent, Uzbekistan (P) Dr M.S. Abdullakhodjaeva Pathology Department Medical Institute Tashkent Uzbekistan 25. Tokyo, Japan (P) Dr T. Ishii Department of Pathology Tokyo Medical College Tokyo Japan 26. Yaounde , Cameroon (P,M) Dr A. Mbakop De partement de pathologie Centre universitaire des sciences de la sante (C.U.S.S) Yaounde Cameroon PBDAY Coordinating Centre Responsible Officer: Dr N.H. Sternby Department of Pathology University of Lund Malmo Sweden PBDAY Management and Data Centre Responsible Officers: Dr I. Gyarfas, Dr P. Nordet , and Mrs M. Anker World Health Organization, Geneva; and Dr J.E. Fernandez-Britta Havana University Havana Cuba PBDAY Receiving, Processing and Distributing Centres Malmo Receiving, Processing and Distributing Centre for Fixed Specimens Responsible Officer: Dr N.H. Sternby Department of Pathology University of Lund Malmo Sweden Geneva Receiving, Processing and Distributing Centre for Frozen Specimens Responsible Officer: Dr J.N. Cox Department of Pathology Medical University of Geneva Geneva Switzerland PBDAY Reference Centres Budapest, Hungary Immunohistochemistry: Dr A. Kadar and Dr G. Illye s Geneva, Switzerland Blood and Subcellular Analysis: Dr J. Cox , Dr I. James and Dr G. Gabbiani Havana, Cuba Atherometric system analysis: Dr J.E. Fernandez-Britto Heidelberg, Germany Quantitative lesion analysis: Dr G. Mall and Dr I. Siemens Malmo, Sweden Morphometric visual grading: Dr N.H. Sternby Moscow, Russian Federation Biochemical analysis: Dr A.M. Vikhert Siena, Italy Morphometric quantitative analysis: Dr G. Weber, Dr P. Tanganelli and Dr G. Bianciardi PBDAY Steering Committee Members M. Anker, Geneva, Switzerland J. Cox, Geneva, Switzerland J.E. Fernandez-Britto, Havana, Cuba I. Gyarfas, Geneva, Switzerland A. Kadar, Budapest, Hungary G. Mall/I. Siemens, Heidelberg, Germany P. Nordet, Geneva, Switzerland N.H. Sternby, Malmo, Sweden A.M. Vikhert, Moscow, Russian Federation G. Weber, Siena, Italy Also invited E.G.J. Olsen, London, England M. Perry, St Louis, Washington, USA R. Wissler, Boston, USA J.P. Strong, New Orleans, USA 257Bulletin of the World Health Organization, 1999, 77 (3) Pathobiological determinants of atherosclerosis in youth