Molecular Vision 2006; 12:1143-1147 <http://www.molvis.org/molvis/v12/a129/> Received 2 September 2006 | Accepted 21 September 2006 | Published 2 October 2006

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Application of multiplex cytometric bead array technology for the measurement of angiogenic factors in the vitreous

Richard Maier,¹, Martin Weger,¹ Eva-Maria Haller-Schober,¹ Yosuf El-Shabrawi,¹ Anna Theisl,¹ Alfred Barth,² Reingard Aigner,³ Anton Haas¹
 
 

¹Department of Ophthalmology, Medical University of Graz, Graz, Austria; ²Institute of Management Science, Division Ergonomics and Organization, Vienna University of Technology, Vienna, Austria; ³Division of Nuclear Medicine, Medical University of Graz, Graz, Austria

Correspondence to: Richard Maier, M.D., Department of Ophthalmology, Medical University Graz, Auenbruggerplatz 4, 8036 Graz, Austria; Phone: 0043-316-385-2394; FAX: 0043-316-385-3261; email: richardmaier@chello.at

Abstract

Purpose: This study was carried out to compare cytometric bead array (CBA) technology with conventional enzyme linked immunosorbent assay (ELISA) for the measurement of both vitreous and serum concentrations of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and angiogenin (ANG) in diabetic and non-diabetic patients.

Methods: Measurement of vitreous and serum concentrations of IL-8, VEGF, and ANG using both ELISA and CBA was performed in 26 probands (13 diabetics and 13 non-diabetic control subjects).

Results: Vitreous and serum concentrations of IL-8, VEGF, and ANG determined by CBA showed a strong correlation with those measured by ELISA. Vitreous levels of IL-8, VEGF, and ANG were significantly higher in diabetics compared to non-diabetic control subjects. No significant correlation between vitreous and serum levels of any of the investigated parameters were found in either diabetics or control individuals.

Conclusions: The present study is the first to utilize cytometric bead array technology for the measurement of angiogenic factors in the vitreous. Measurements obtained by ELISA and CBA technologies were highly correlated for IL-8, VEGF, and ANG in both vitreous and serum samples. Diabetic individuals showed significant elevation of IL-8, VEGF, and ANG in the vitreous but not in serum samples compared to control subjects. The most striking advantage of the CBA technology is the fact that numerous parameters can be measured in parallel using a comparatively small sample volume. It is therefore more rapid and cost effective than ELISA technology. CBA technology is a new, accurate method to measure IL-8, VEGF, and ANG in the vitreous.

Introduction

Diabetic retinopathy (DR) is one of the primary causes of visual loss worldwide [1]. DR is caused by alterations of the retinal microvasculature leading to the breakdown of the blood-retina barrier and pathological angiogenesis. Angiogenesis is a far-reaching event that is characterized by new blood vessels arising from pre-existing vasculature. The synthesis of various vascular growth factors, which are strongly stimulated by ischemia in retinal tissue, play a crucial role in the development of retinal neovascularization [2,3].

There is a large body of evidence indicating that interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and angiogenin (ANG) contribute to the pathogenesis of diabetic retinopathy [4-6]. Increased vascular permeability causing the breakdown of the blood-retina barrier leads to elevated levels of angiogenic factors in the vitreous [7,8]. Furthermore, the production of angiogenic factors within the vitreous itself including monocytes, macrophages, retinal pigmental epithelia, and glial cells is plausible [9]. The ability to aspirate vitreous is an ideal setting to monitor retinal angiogenesis and investigate the role of angiogenic factors in diabetic retinopathy.

Up to the present, measurement of vitreous angiogenic and inflammatory factors has been performed by ELISA-based technology. Even though ELISA assays are adequate for measuring the aforementioned factors in vitreous fluid, there are limitations imposed by small sample volume. The ELISA method is well suited to perform a single factor analysis but for the vitreous simultaneous quantification of numerous factors from a small sample volume is preferable. Flow cytometry is an analytical tool that allows discrimination of different particles on the basis of size and/or color. Cytometric bead array (CBA) technology employs a series of particles with discrete fluorescence intensities to simultaneously detect multiple soluble analytes from a single sample. This technology, combined with flow cytometry, is a powerful multiple analyte assay system. CBA multiplex beads simplify panel assays since just one sample is needed to detect and quantify several parameter, and the independent measurement for each bead population ensures high precision. The advantage of the CBA-technology is the requirement for much smaller sample volumes, a more rapid and cost-effective evaluation, and the possibility to measure numerous other parameters in parallel. Previous investigators have described measurement of cytokines by CBA technology in different body fluids such as serum, tears, nasal lavage and sputum [10].

To the best of our knowledge, the CBA-technology, however, has not yet been used for the measurement of cytokines and for angiogenic factors in the vitreous. In the present study IL-8, VEGF, and ANG, which are well-set angiogenic factors, were measured in serum and vitreous by established ELISA assays and were compared to those obtained by CBA applied to flow cytometry, in order to implement CBA technology in the quantification of these factors in the vitreous.

Methods

Study population

Thirteen patients with diabetes mellitus (five non-insulin dependent, eight insulin dependent) and 13 non-diabetic control subjects were included in the present case-control study. Informed consent was obtained from all probands prior to entering the study. For all probands the indications for pars plana vitrectomy were epiretinal membranes and macular holes. From 13 diabetic individuals, four had no diabetic retinopathy, three had non-proliferative diabetic retinopathy, and six had quiescent proliferative diabetic retinopathy. Exclusion criteria included previous intra-ocular surgery, acute ocular infection, uveitis, trauma, vitreous hemorrhage, and retinal detachment. All examinations were performed in accordance with the ethical standards of the Declaration of Helsinki.

Sample collection and preparation

Undiluted vitreous samples were obtained during a standard three-port pars plana vitrectomy. At the beginning of the surgery, vitreous fluid (approximately 1 ml) was collected via pars plana using a 20 gauge needle on a 2 ml syringe after the infusion port was made without running the fluid on. The obtained specimen was immediately transferred into a sterile plastic tube on ice. Subsequently the sample was centrifuged at 5,000 rpm for 10 min, aliquoted, and stored at -70 °C until assayed.

Blood samples were obtained by venous puncture prior to vitrectomy, and immediately placed on ice. Subsequently each sample was centrifuged at 3,000 rpm for 10 min, aliquoted, and stored at -20 °C until assayed.

The following angiogenic factors were determined in serum as well as in vitreal samples by both ELSIA and CBA: IL-8, VEGF, and ANG.

Cytometric bead array (CBA)

Concentrations of three different angiogenic factors were determined in both serum and vitreous samples of 26 probands (13 diabetics and 13 non-diabetes control subjects) using the BDTM Bead Array (CBA) Human Angiogenesis Kit (Catalog number 558014; BD Bioscience-Pharmingen, San Diego, CA). This kit allows the simultaneous measurement of IL-8, VEGF, and ANG levels in a single sample. The kit contains three bead populations with distinct fluorescence intensities. The beads were coated with capture antibodies specific to IL-8, VEGF, and ANG and mixed with phycoerythrin (PE) conjugated detection antibodies. These capture beads were incubated with recombinant standards or test samples to form sandwich complexes. The CBA was resolved in the FL-3 channel of a FACSCalibur flow cytometer (BD Bioscience-Pharmingen), and the results were generated in graphic and tabular formate using CBA analysis software (BD Bioscience-Pharmingen). The assay sensitivities for IL-8, VEGF, and ANG were 0.8, 12.6 and 3.4 pg/ml, respectively.

Serum and vitreous samples were first run without predilution. For the detection of VEGF in the vitreous 3 samples had to be prediluted at a ratio of 1:20. As for ANG, both serum and vitreous samples had to be prediluted (serum at a ratio of 1:500 and 1:640 [one sample] and vitreous at a ratio of 1:30 and 1:5 [one sample]), respectively.

The test was performed and analyzed according to the manufacturer's instructions. In brief, 50 μl of premixed capture beads were mixed with 50 μl PE-detection reagent. After addition of 50 μl of the provided standards or sample (serum, vitreous) the mixture was incubated in the dark for 3 h at room temperature. After incubation the mixture was washed, centrifuged (at 200x g for 5 min) and the pellet was resuspended in 300 μl of wash buffer. The BD FACSCalibur flow cytometer was calibrated with setup beads and 1,500 events were acquired for each sample. Individual analyte concentrations were indicated by their fluorescence intensities (FL-2) and were computed by using the rescpective standard reference curve and BD CBA software.

Enzyme-linked immuno assays (ELISA)

VEGF, IL-8, and ANG in serum and vitreous were also determined by ELISA using commercially available kits (human IL-8 Immunoassay and human VEGF, Quantikine^TM R & D Systems Minneapolis, MN and human angiogenin Bio® Ray Biotech, Inc., Nocross, GA). The minimum detectable concentrations in serum are less than 9.0 pg/ml (VEGF), 3.5 pg/ml (IL-8), and 1.5 pg/ml (ANG), respectively. The immunoassays were performed and analyzed according to the manufacturer's instructions. All samples were run in duplicate.

For IL-8 detection, 50 μl serum or vitreous were analyzed without predilution. For VEGF determination, 100 μl serum and vitreous were analyzed without predilution with the exception of five vitreous samples, which required a 10 fold dilution.

For detection of ANG, 100 μl serum or vitreous were analyzed. According to the manufacturer's instructions, the serum samples were tested after a 10,000 fold dilution, the vitreous samples were analyzed after a 500 fold and 1,000 fold dilution, respectively.

Concentrations of VEGF, IL-8, and ANG were calculated based on the respective standard curves. Serum and vitreous concentrations were expressed as pg/ml.

Statistical analysis

Data were analyzed using the statistical package for social sciences (SPSS Version 11.0) for Windows. Group differences between diabetic and non-diabetic samples were analyzed using a two tailed t-test or two tailed Mann-Whitney test depending on normality assumptions and homogeneity of variances. All tests were performed at an error level of 5% and due to multiple univariate testing the Bonferroni correction algorithm of error I level was applied to retain the global error level at 5%.

Correlations were calculated to investigate the association between the CBA and ELISA methods. Depending on normality assumptions, Pearson's correlation coefficient or Spearman's correlation coefficient were applied.

Results

Study population

The mean age of the 13 diabetic probands (6 men and 7 women) was 66.4 years (SD±9.4 years), in the non-diabetic control subjects (6 men and 7 women) 67.8 years (SD±8.6 years). With regard to age no significant difference between both groups was observed. The mean duration of diabetes mellitus was 9.4 years with a mean hemoglobin A1c (HbA1c) level of 6.9±0.9% compared to HbA1c 5.5±0.3% in control subjects (normal range HbA1c: 4.0-5.9%).

Method comparison

A strong correlation between vitreal IL-8, VEGF, an ANG concentrations measured by ELISA and those obtained by CBA technology was observed. This also applied to serum concentrations of the aforementioned angiogenic factors (see Table 1).

Diabetic versus non-diabetic samples

In both ELISA and CBA assays IL-8, VEGF, and ANG levels were found to be significantly higher in vitreal samples of diabetic patients compared to those of non-diabetic control subjects (Table 2 and Table 3). No significant difference in serum IL-8, VEGF, and ANG levels obtained by either ELISA and CBA assays was observed between cases and control subjects (Table 4 and Table 5).

For diabetic samples a significant positive correlation was obtained in both ELISA and CBA assays between vitreal IL-8 and vitreal VEGF levels. Samples with high vitreal IL-8 levels also showed significantly increased vitreal VEGF concentrations (p=0.001).

HbA1c levels in diabetic individuals were significantly correlated with vitreal levels of IL-8, VEGF, and ANG (p<0.05). No significant association between vitreous and serum levels for angiogenic factors IL-8, VEGF, and ANG was observed in both groups (p>0.05).

Discussion

To the best of our knowledge the present study is the first to utilize CBA technology for the measurement of angiogenic factors in the vitreous. CBA technology has already been used for the measurement of a large number of cytokines and growth factors in aqueous humor, plasma, and serum samples, as well as in tissue culture supernatants [11-14]. The determination of cytokines and growth factors in the vitreous by CBA promises to be of substantial benefit for ophthalmologic investigations. In contrast to ELISA this technology is not limited by small sample volumes.

This study was designed to implement the CBA technology for measurement of angiogenic factors in the vitreous and to determine the levels of IL-8, VEGF, and ANG in the vitreous and serum of diabetic individuals. In previous studies the measurement of several angiogenic factors by conventional ELISA in the vitreous has been reported [15,16]. It is hypothesized that the breakdown of the blood-retina barrier leads to raised levels of angiogenic factors in the vitreous of diabetic individuals [17,18]. Present data corroborates this hypothesis. IL-8, VEGF, and ANG levels were significantly higher in vitreal samples from diabetic probands compared to controls. Higher levels of IL-8, VEGF, and ANG in the vitreous may promote retinal vascular damage in diabetic patients and may lead to diabetic retinopathy. Therefore the measurement of angiogenic factors is important for monitoring the angiogenic process but also since new therapeutic approaches involve the neutralization of angiogenic factors in the vitreous.

Several studies have implicated IL-8 in inflammation-mediated angiogenesis. Retinal glial and endothelial cells release IL-8 in the ocular fluids of patients with retinal hypoxia [19,20]. IL-8 values measured in the vitreous and serum by ELISA and CBA assay showed significant correlation in diabetic individuals and controls. Both methods showed significantly higher vitreal levels of IL-8 in diabetic probands than controls. Furthermore vitreal IL-8 and VEGF levels were significantly correlated with each other. This supports the theory of an angiogenic and inflammatory element in the pathogenesis of diabetic retinopathy. Our data is in agreement with previous studies reporting that IL-8 and VEGF are implicated in the development of intraocular neovascularization [21]. IL-8 serum levels were not significantly higher in diabetic individuals compared to control subjects.

Retinal ischemia and hypoxia induce intraocular neovascularization by stimulating the release of angiogenic molecules, such as VEGF, which is not only the major mediator of retinal angiogenesis but also a potent inducer of vasopermeability [22]. The expression of VEGF is up-regulated in retinal pigmented epithelial cells, glial cells, and vitreal fibroblasts [23]. ELISA- as well CBA-determined VEGF levels in vitreous and serum were significantly correlated. In both methods, vitreal levels of diabetic probands were significantly higher than in controls while serum levels showed no significant elevation in diabetic individuals compared to control subjects.

ANG is a potent blood vessel-inducing protein. It is postulated that raised ANG levels reflect a breakdown of the blood-retina barrier referring to diabetic retinopathy. So far vitreal levels of ANG have been measured just once by ELISA, which were raised in diabetic individuals [24]. ANG levels in the present study support this previous observation. Vitreal levels of ANG showed significant elevation in diabetic probands in both ELISA and CBA assays. As for IL-8 and VEGF shown, serum levels of ANG showed no tendency of increase in diabetic individuals.

This study validates CBA as a new, efficient, and cost-effective method to measure IL-8, VEGF, and ANG in the vitreous. CBA results for IL-8, VEGF, and ANG in vitreous fluid and serum highly correlate with the results obtained by the conventional ELISA test so far used for routine diagnostic purposes. CBA correlates well with the ELISA assay but the absolute concentrations obtained by each assay differ with the kits provided by different manufacturers [25]. The sample volumes required for ELISA are four to six times greater than those needed for CBA. Therefore, CBA technology may be readily implemented into a clinical laboratory not only because flow cytometry is widely used but also because of its flexibility, cost-effectiveness, ease of manual operation, and potential to measure several parameters in parallel.

In conclusion, CBA technology is comparable with conventional ELISA assay, which is the "gold standard". It is a new, reliable and powerful tool for IL-8, VEGF and ANG measurements in the vitreous.

Acknowledgements

The authors thank Heintz Hans Joergen for his skillful technical assistance.

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