This was a cross-sectional study of 97 treatment naïve patients who had been diagnosed with exudative AMD based on the definition of the Japanese AMD Study Group.30 In more detail, exudative AMD was diagnosed in patients who were over 50 years old with at least one following abnormalities (CNV, serous pigment epithelium detachment, subretinal hemorrhage, and fibrous scars) within an area of 6000 µm in diameter centered at the fovea. High myopia (over − 6.0 diopter), uveitis, trauma, and other degenerative diseases were excluded. All participants were patients of the Nara Medical University Hospital, Kashihara City, Nara Prefecture, Japan from 2014 to 2019. This study protocol was approved by the Institutional Review Board of the Nara Medical University and a signed informed consent was obtained from all the participants before the examinations. This study was performed in accordance with the Declaration of Helsinki. We confirm that all methods were performed in accordance with the relevant guidelines and regulations.
All patients underwent conventional ophthalmological examinations including slit-lamp examinations, fundus examinations, OCT (Spectralis, Heidelberg Engineering, Dossenheim, Germany), fundus photography, fluorescein angiography (FA), and indocyanine green angiography (ICGA) at the initial examination. The fundus photographs and OCT images were used to determine the presence of subretinal hemorrhages. After re-examination of all of the images, the patients were diagnosed with PNV or AMD.
According to the recent reports on PNV analysis,18,19,22,23,25,31 we diagnosed a patient as PNV when all following criteria were satisfied: Type1 CNV (including PCV), CVD ≥ 180 µm, CCT ≥ 220 µm, absence of drusen in the fundus photographs, and increased choroidal vascular permeability in the late phase of the ICGA images (Fig. 3). PCV was characterized by polypoidal lesions in ICGA. CVD was defined as the vertical diameter of the largest vessel in the Haller's layer. CVD and CCT were measured independently by two different researchers using a scale bar contained within the OCT system.23 The judgments were made independently by two retinal specialists, and in cases when the judgments were different, the final decision was made by a macular disease specialist (N.O.).
We collected blood samples by venipuncture at the initial examination. The whole blood samples were stored in tubes containing a 1:9 volume of 3.8% trisodium citrate. The plasma was separated and saved at -80º C. For the measurements, the plasma was thawed and maintained at 37º C before the measurements. The level of plasma VWF Antigen (VWF: Ag) was measured by sandwich enzyme-linked immunosorbent assay (ELISA) using a rabbit anti–human VWF polyclonal antiserum (DAKO, Glostrup, Denmark).13 We determined the activity of ADAMTS13 (ADAMTS13: AC) by a chromogenic ADAMTS13 activity ELISA kit (Kainos, Tokyo, Japan).32 The 100% reference value was defined as the amount of VWF: Ag and ADAMTS13: AC in the plasma of 20 normal volunteers (10 men and 10 women ages 20 to 40 years). We also measured the plasma vascular endothelial growth factor A (VEGF-A) by ELISA (Quantikine VEGF ELISA kit; R&D Systems, Minneapolis, Minnesota, USA). The VWF multimers were analyzed by the method of Ruggeri and Zimmerman33, modified by Warren et al.34 The measurements were made under the conditions described by Budde et al,35 and we defined the high molecular weight bands that were not detected in normal plasma as UL-VWFMs. Then, we performed densitometric analysis using ImageJ (National Institute of Health, Bethesda, Maryland, USA). UL-VWFMs were defined as being positive when the ratio of UL-VWFMs to total VWF was > 1% (Fig. 4).36
We extracted genomic DNA from the leukocytes by a genomic DNA kit (QIAmpDNA; Qiagen, Valencia, California, USA) and genotyped the SNPs of p.I62V (rs800292) and p.Y402H (rs1061170) in CFH. Polymerase chain reaction (PCR) with specific primers was used to amplify the polymorphic sites.37 The PCR products were used as the templates for direct DNA sequencing (Applied Biosystems, Foster City, California, USA) on an automated sequencer (3730xl DNA analyzer; Applied Biosystems). The genotypes in the CFH were classified based on the previous studies.38,39
Statistical analyses
Two-tailed Mann-Whitney U tests were used to compare the averages of continuous variables (such as age) and Fisher’s exact tests to compare the proportions of categorical variables (such as sex) between groups. Odds ratios and 95% confidence intervals were calculated by using Fisher’s exact tests. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria).40 Since both CCT and CVD followed a normal distribution, the correlation was evaluated at Pearson's correlation coefficient. On the other hand, the measured values of VEGF-A were not normally distributed, and the exact values were not partially shown because the values were below the minimum detectable dose of 9 pg/ml. Therefore, a non-parametric test was applied, and a conservative value of 8 pg/ml was input for each value below the MDD when performing the calculations. The threshold for statistically significance was P < 0.05.