2.1 Population and sample ascertainment
Shuangcheng(Figure 1), a specific region of southwestern Harbin, northeast China, that was characterized by cold weather, low elevation, farming communities, and plains, was selected as the survey area. The population of the region is approximately 830,000, with 650,000 people living in rural areas (18 Xiang, 256 villages).
Geographically defined clusters based on village register census data were used as the study sampling frame within each county/district. In this study, a cluster random sampling method, similar to that used in the study by Doumen[18] was employed. The sample size was determined by RVA prevalence (age ≥ 40 years, 0.043)[14] within the allowable error boundary of 20% and a 95% confidence interval (CI)[19],along with assuming an examination response rate of 85% and a design effect of 1.5 to account for inefficiencies associated with the cluster sampling design. In brief, the sample design used village-based clusters of almost the same size (approximately 1000 people). Using streets as dividing lines, villages with populations of more than 1500 people were separated into two units, and villages with populations of fewer than 500 people were merged with the closest neighbouring village with populations of fewer than 800 people[20]. Then, 582 sampling units were obtained, from which 35 units were randomly selected. This study included individuals over 50 years old, who comprised approximately 20% of the total population[21]. Finally, 6849 people were eligible for this study.
2.2 Data collection methods
The research protocol was approved by the Medical Ethics Committee of the First Affiliated Hospital of Harbin Medical University (No:201532) and all the subjects provided informed consent, according to the Declaration of Helsinki. Households listings of names of residents ≥50 years of age were obtained from the village registers, followed by door-to-door visits conducted by the enumeration team. Individuals temporarily absent at the time of the household visit were included in the enumeration. Unregistered adults ≥50 years of age were enumerated and included in the study sample if they had been living in the household for at least six months. Refusal to provide informed consent was considered as an exclusion criterion.
All participants were administered a questionnaire[22], basic physical examination, and laboratory evaluation, which included basic demographic characteristics(name, age, gender, locality, etc.), medical histories (hypertension, hyperlipemia, diabetes, stroke, cardiovascular and cerebrovascular diseases, smoking status and alcohol consumption), blood pressure measurements, fasting blood glucose measurement and blood lipid measurements. All examinations were carried out in the villages, either in clinics or in the houses of the village committees. Those who did not appear at the examination site were revisited, repeatedly if necessary, by a member of the enumeration team to encourage participation.
2.3 Assessment of Hypertension, Hyperlipemia, CCVds and their Risk Factors
The methods for the assessment of hypertension, CCVds, and their risk factors are highlighted here.self-reported diagnosis histories of CCVds and smoking habits and alcohol consumption were obtained from a questionnaire. Sitting brachial blood pressure was measured three times by trained technicians with a random-zero sphygmomanometer after 5 minutes of rest. Blood samples were collected between 7:00 and 9:00 a.m. after at least an 8 h overnight fasting. Sterile vacuum tubes with and without ethylenediaminetetraacetic acid were used, and centrifugation was performed within 3-h of blood collection. Serum analysis was performed in the laboratory of the First Affiliated Hospital of Harbin Medical University (quality control of the laboratory was certified and monitored yearly by the Ministry of Health, China). Hypertension was diagnosed, if the systolic blood pressure was ≥ 140mmHg or diastolic blood pressure was ≥ 90mmHg, or a self-reported diagnosis history of hypertension and antihypertensive therapy was self-reported. Hyperlipemia was diagnosed, if Triglycerides were ≥ 2.0mmol/L and high-density lipoprotein was ≤ 1.0mmol/L, or a self-reported history of hyperlipemia and lipid-lowering therapy was self-reported. Diabetes mellitus was diagnosed, if the fasting glucose was ≥ 7.0 mmol/ L (≥126 mg/dl) or the use of insulin or oral hypoglycaemic medication was self-reported[22].
2.4 Assessment of retinal vascular abnormalities
To evaluate RVAs, two 40° non-mydriatic retinal photographs of one eye from each participant were taken using a fundus camera (Type. Classic, 3nethra, Indian). One photograph was centred on the optic disc and another on the fovea centre (Figure 2 follows the standard Fields 1 and 2 in the Early Treatment of Diabetic Retinopathy Study (ETDRS))[23].To achieve balance, if the identification numbers were even, the right eye was chosen;on the contrary, if the identification numbers were uneven, the left eye was chosen. The above retinal photography methods were principally followed the atherosclerosis risk in the community (ARIC) protocol[3].
The inclusion criteria for the study population was as follows: subjects who had clear fundus photographs that could be evaluated were included(including those with previous cataract surgery or previous history of vitrectomy, and intravitreal medications). The exclusion criteria of this study population were as follows: subjects whose fundus photographs were not clear and could not be evaluated; and subjects who lacked relevant data.
The colour photographs (image resolution: 2048*1536 24 bits per pixel, JPG) of each subject were evaluated, according to the evaluation criteria of the ARIC study[3] and ETDRS[23]. The photographs were assessed by two assessors who were trained at the Retinal Vascular Imaging Centre, University of Melbourne (the assessors were blinded to the participant characteristics) using a semi-quantitative manual grading approach for the digital images.
For the evaluation of RVAs, FAN, AVN, AS, retinopathy and GAN were assessed. The grader compared possible abnormalities with standard and example photographs to help determine their presence and severity. The ARIC grid[3](described in Figure 2) was applied to divide the retina into standard regions. The regions outside zone A region (zone B and distal regions), FAN, AVN, AS, and GAN in each quadrant were graded. Standard photographs for retinal microvascular signs were selected by a retinal specialist from a standard photographic set developed for the Modified Airlie House Classification of Diabetic Retinopathy[23].
FAN[3] was diagnosed if the artery with a diameter greater than or equal to 50 μm, appeared to narrow, with a diameter is equal to or smaller than 2/3 of its distal and proximal vessel segments. According to the total length of vascular stenosis in the quadrant, less than ½ DD, between ½DD and 2 DD, and 2 DD or more, indicated the different severity levels including mild, moderate and severe, respectively. AVN[3] was diagnosed if both sides of the venous blood column were gradually narrowed at the intersection of the arteries and veins. If the narrowing approached approximately ½ of the blood column, AVN was classified as “mild/moderate.” If the narrowing was equal to or less than ½ of the blood column, AVN was considered as “severe.” AS[3] was considered definite if white sheathing was observed on one side or both sides of retinal vessels. For the assessment of generalized narrowing of the retinal arterioles, the arterial diameter was compared with the corresponding veins, unless the veins were engorged and tortuous. If the arterioles appeared to be narrow in comparison with the veins, GAN was graded as “questionable.” If some arterioles in the eye were markedly narrowed or thready but appeared normal in other quadrants of the fundus, GAN was graded as “definite.” If the arterioles were small and thready throughout the entire eye, GAN was graded as “severe”[14].
For retinopathy, the following lesions were evaluated in each of the four quadrants of the retina: microaneurysms, hemorrhages, soft exudates or cotton wool spots, hard exudates, macular oedema, intraretinal microvascular abnormalities, venous beading, new vessels in the disc or elsewhere, and vitreous haemorrhage. Retinopathy was diagnosed if any of these lesions were definite or probable in any of the four quadrants. The severity of retinopathy was summarized according to the ETDRS severity scale. [23]
2.5 Quality control of RVAs assessment
To examine the reproducibility of the assessment of RVAs, 100 pictures were randomly selected and evaluated by two ophthalmologists respectively. Every observer repeated the work two weeks later.
2.6 Statistical analysis
The statistical analysis was carried out using SPSS 19.0 (Chicago, IL, USA). The five primary endpoints in the study were FAN, AVN, AS, retinopathy and GAN. We calculated the prevalence of each retinal outcome, according to gender and age group. The association between RVAs (FAN, AVN, AS, retinopathy and GAN) and CCVds and their risk factors was determined using logistic regressions. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. All the P-values were determined a 2-sided test and were regarded as significant when they were less than 0.05.
2.1 Population and sample ascertainment
Shuangcheng(Figure 1), a specific region of southwestern Harbin, northeast China, that was characterized by cold weather, low elevation, farming communities, and plains, was selected as the survey area. The population of the region is approximately 830,000, with 650,000 people living in rural areas (18 Xiang, 256 villages).
Geographically defined clusters based on village register census data were used as the study sampling frame within each county/district. In this study, a cluster random sampling method, similar to that used in the study by Doumen[18] was employed. The sample size was determined by RVA prevalence (age ≥ 40 years, 0.043)[14] within the allowable error boundary of 20% and a 95% confidence interval (CI)[19],along with assuming an examination response rate of 85% and a design effect of 1.5 to account for inefficiencies associated with the cluster sampling design. In brief, the sample design used village-based clusters of almost the same size (approximately 1000 people). Using streets as dividing lines, villages with populations of more than 1500 people were separated into two units, and villages with populations of fewer than 500 people were merged with the closest neighbouring village with populations of fewer than 800 people[20]. Then, 582 sampling units were obtained, from which 35 units were randomly selected. This study included individuals over 50 years old, who comprised approximately 20% of the total population[21]. Finally, 6849 people were eligible for this study.
2.2 Data collection methods
The research protocol was approved by the Medical Ethics Committee of the First Affiliated Hospital of Harbin Medical University (No:201532) and all the subjects provided informed consent, according to the Declaration of Helsinki. Households listings of names of residents ≥50 years of age were obtained from the village registers, followed by door-to-door visits conducted by the enumeration team. Individuals temporarily absent at the time of the household visit were included in the enumeration. Unregistered adults ≥50 years of age were enumerated and included in the study sample if they had been living in the household for at least six months. Refusal to provide informed consent was considered as an exclusion criterion.
All participants were administered a questionnaire[22], basic physical examination, and laboratory evaluation, which included basic demographic characteristics(name, age, gender, locality, etc.), medical histories (hypertension, hyperlipemia, diabetes, stroke, cardiovascular and cerebrovascular diseases, smoking status and alcohol consumption), blood pressure measurements, fasting blood glucose measurement and blood lipid measurements. All examinations were carried out in the villages, either in clinics or in the houses of the village committees. Those who did not appear at the examination site were revisited, repeatedly if necessary, by a member of the enumeration team to encourage participation.
2.3 Assessment of Hypertension, Hyperlipemia, CCVds and their Risk Factors
The methods for the assessment of hypertension, CCVds, and their risk factors are highlighted here.self-reported diagnosis histories of CCVds and smoking habits and alcohol consumption were obtained from a questionnaire. Sitting brachial blood pressure was measured three times by trained technicians with a random-zero sphygmomanometer after 5 minutes of rest. Blood samples were collected between 7:00 and 9:00 a.m. after at least an 8 h overnight fasting. Sterile vacuum tubes with and without ethylenediaminetetraacetic acid were used, and centrifugation was performed within 3-h of blood collection. Serum analysis was performed in the laboratory of the First Affiliated Hospital of Harbin Medical University (quality control of the laboratory was certified and monitored yearly by the Ministry of Health, China). Hypertension was diagnosed, if the systolic blood pressure was ≥ 140mmHg or diastolic blood pressure was ≥ 90mmHg, or a self-reported diagnosis history of hypertension and antihypertensive therapy was self-reported. Hyperlipemia was diagnosed, if Triglycerides were ≥ 2.0mmol/L and high-density lipoprotein was ≤ 1.0mmol/L, or a self-reported history of hyperlipemia and lipid-lowering therapy was self-reported. Diabetes mellitus was diagnosed, if the fasting glucose was ≥ 7.0 mmol/ L (≥126 mg/dl) or the use of insulin or oral hypoglycaemic medication was self-reported[22].
2.4 Assessment of retinal vascular abnormalities
To evaluate RVAs, two 40° non-mydriatic retinal photographs of one eye from each participant were taken using a fundus camera (Type. Classic, 3nethra, Indian). One photograph was centred on the optic disc and another on the fovea centre (Figure 2 follows the standard Fields 1 and 2 in the Early Treatment of Diabetic Retinopathy Study (ETDRS))[23].To achieve balance, if the identification numbers were even, the right eye was chosen;on the contrary, if the identification numbers were uneven, the left eye was chosen. The above retinal photography methods were principally followed the A protocol[3].
The inclusion criteria for the study population was as follows: subjects who had clear fundus photographs that could be evaluated were included(including those with previous cataract surgery or previous history of vitrectomy, and intravitreal medications). The exclusion criteria of this study population were as follows: subjects whose fundus photographs were not clear and could not be evaluated; and subjects who lacked relevant data.
The colour photographs (image resolution: 2048*1536 24 bits per pixel, JPG) of each subject were evaluated, according to the evaluation criteria of the ARIC study[3] and ETDRS[23]. The photographs were assessed by two assessors who were trained at the Retinal Vascular Imaging Centre, University of Melbourne (the assessors were blinded to the participant characteristics) using a semi-quantitative manual grading approach for the digital images.
For the evaluation of RVAs, FAN, AVN, AS, retinopathy and GAN were assessed. The grader compared possible abnormalities with standard and example photographs to help determine their presence and severity. The ARIC grid[3](described in Figure 2) was applied to divide the retina into standard regions. The regions outside zone A region (zone B and distal regions), FAN, AVN, AS, and GAN in each quadrant were graded. Standard photographs for retinal microvascular signs were selected by a retinal specialist from a standard photographic set developed for the Modified Airlie House Classification of Diabetic Retinopathy[23].
FAN[3] was diagnosed if the artery with a diameter greater than or equal to 50 μm, appeared to narrow, with a diameter is equal to or smaller than 2/3 of its distal and proximal vessel segments. According to the total length of vascular stenosis in the quadrant, less than ½ DD, between ½DD and 2 DD, and 2 DD or more, indicated the different severity levels including mild, moderate and severe, respectively. AVN[3] was diagnosed if both sides of the venous blood column were gradually narrowed at the intersection of the arteries and veins. If the narrowing approached approximately ½ of the blood column, AVN was classified as “mild/moderate.” If the narrowing was equal to or less than ½ of the blood column, AVN was considered as “severe.” AS[3] was considered definite if white sheathing was observed on one side or both sides of retinal vessels. For the assessment of generalized narrowing of the retinal arterioles, the arterial diameter was compared with the corresponding veins, unless the veins were engorged and tortuous. If the arterioles appeared to be narrow in comparison with the veins, GAN was graded as “questionable.” If some arterioles in the eye were markedly narrowed or thready but appeared normal in other quadrants of the fundus, GAN was graded as “definite.” If the arterioles were small and thready throughout the entire eye, GAN was graded as “severe”[14].
For retinopathy, the following lesions were evaluated in each of the four quadrants of the retina: microaneurysms, hemorrhages, soft exudates or cotton wool spots, hard exudates, macular oedema, intraretinal microvascular abnormalities, venous beading, new vessels in the disc or elsewhere, and vitreous haemorrhage. Retinopathy was diagnosed if any of these lesions were definite or probable in any of the four quadrants. The severity of retinopathy was summarized according to the ETDRS severity scale. [23]
2.5 Quality control of RVAs assessment
To examine the reproducibility of the assessment of RVAs, 100 pictures were randomly selected and evaluated by two ophthalmologists respectively. Every observer repeated the work two weeks later.
2.6 Statistical analysis
The statistical analysis was carried out using SPSS 19.0 (Chicago, IL, USA). The five primary endpoints in the study were FAN, AVN, AS, retinopathy and GAN. We calculated the prevalence of each retinal outcome, according to gender and age group. The association between RVAs (FAN, AVN, AS, retinopathy and GAN) and CCVds and their risk factors was determined using logistic regressions. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. All the P-values were determined a 2-sided test and were regarded as significant when they were less than 0.05.