|Year : 2013 | Volume
| Issue : 1 | Page : 17-22
Platelet aggregation in various stages of diabetic retinopathy ( evaluation using the PFA-100)
Deena M.M. Habashy1, Mohamed R. Mohamed2
1 Department of Clinical Pathology, Hematology Unit, Ain Shams University, Cairo, Egypt
2 Department of Opthalmology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||01-Oct-2012|
|Date of Acceptance||18-Oct-2012|
|Date of Web Publication||20-Jun-2014|
Deena M.M. Habashy
Department of Clinical Pathology, Hematology Unit, Faculty of Medicine, Ain Shams University, 11566 Cairo
Source of Support: None, Conflict of Interest: None
Platelet hyperactivity has been reported in diabetic patients. Some evidence suggests that platelet hyperaggregation may participate in the pathogenesis of diabetic complications such as retinopathy.
We aimed to compare platelet aggregation (PA) in patients with type II diabetes with no apparent retinopathy (NAR), nonproliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR) patients, and healthy individuals to evaluate the possible role of PA in the pathogenesis and staging of DR.
Participants and methods
Blood samples from 30 patients (10 diabetics with NAR, 10 with NPDR, and 10 with PDR) and 10 healthy individuals were assayed for PA using a platelet function analyzer-100.
On comparing all the studied groups in terms of the demographic, clinical, and laboratory parameters, the mean age and the duration of diabetes were significantly higher in the PDR group (P<0.001). The PDR group also showed higher levels of platelet count, glycated hemoglobin, and shorter closure time (CT) (P<0.001). Both NPDR and PDR showed higher levels of fibrinogen (P<0.001). CT was correlated inversely with the fibrinogen level in the NAR group (P=0.000) and with the duration of diabetes in the NPDR group (P=0.04). No correlation was found between CT and any of the parameters studied in the PDR group (P>0.05).
PA is increased in the PDR stage in comparison with NAR, NPDR stages, and healthy individuals. This may provide a clue of its role in the pathogenesis and staging of DR. Also, this study highlights the crucial contribution of glycemic control and duration of diabetes in the progression of DR.
Keywords: diabetic retinopathy, platelet function analyzer-100, platelet aggregation
|How to cite this article:|
Habashy DM, Mohamed MR. Platelet aggregation in various stages of diabetic retinopathy ( evaluation using the PFA-100). Egypt J Haematol 2013;38:17-22
|How to cite this URL:|
Habashy DM, Mohamed MR. Platelet aggregation in various stages of diabetic retinopathy ( evaluation using the PFA-100). Egypt J Haematol [serial online] 2013 [cited 2020 Dec 5];38:17-22. Available from: http://www.ehj.eg.net/text.asp?2013/38/1/17/134798
| Introduction|| |
Diabetes mellitus (DM) is a major medical problem worldwide. Diabetes causes an array of long-term systemic complications that have a huge impact on the patient as well as the society, as the disease typically affects individuals in their most productive years 1. Patients with diabetes often develop ophthalmic complications, such as corneal abnormalities, glaucoma, iris neovascularization, cataracts, and neuropathies. The most common and potentially most blinding of these complications, however, is diabetic retinopathy (DR) 2.
DR is associated with an increased number and size of platelet-fibrin thrombi in the retinal capillaries compared with normal 3. These thrombi can contribute toward capillary obliteration and retinal ischemia. It has been reported that chronic hyperglycemia causes an increase in the diacylglycerol levels in the retina, which may activate protein kinase C 4. Through increased intracellular Ca2+, protein kinase C stimulates endothelial cells, leukocytes, and platelets to produce platelet-activating factor (PAF) 5. PAF, confined to membranes, stimulates PAF receptors 6 on platelets, inducing the activation of these platelets. Activated platelets produce a number of platelet-derived microparticles 7, which contribute toward thrombus formation by providing and expanding a catalytic surface for the coagulation cascade. Pathological levels of fluid shear stress in abnormal retinal blood vessels affected by hyperglycemia may cause both further platelet aggregation (PA) and shedding of more microparticles from the platelet plasma membrane 8.
In addition, elevated sorbitol in the retina and erythrocytes can reduce vascular prostacyclin accompanied by an increased synthesis of thromboxane through induction of ADP 9 or collagen 10 in whole blood. The imbalance of thromboxane and prostacylin enhances platelet hyperactivity 11. Adhesion proteins that are cofactors in the aggregation of human platelets and mediating the ADP-induced response of these cells are also increased significantly 12. Although increased PA has been shown in patients with DM, its possible correlation with the severity of DR remains to be elucidated 13.
We aimed to compare PA in patients with type II diabetes with no apparent retinopathy (NAR), nonproliferative DR (NPDR), proliferative DR (PDR) patients, and healthy individuals to evaluate the possible role of PA in the pathogenesis and staging of DR and also to examine the correlation between PA and some of the predisposing factors of DR.
| Patients and methods|| |
Thirty patients with type II diabetes were enrolled in this study. Patients were attending the Opthalmology Clinic of Ain Shams University Hospitals. They were classified into three groups: 10 with NAR, mean age 52.7±3.7 years. There were five men and five women, with a male to female ratio of 1 : 1, and 10 patients with NPDR, mean age 56.7±4.0 years. There were six men and four women, with a male to female ratio of 1.5 : 1, and 10 patients with PDR, mean age 60.2±1.8 years. There were six men and four women, with a male to female ratio of 1.5 : 1. Ten healthy individuals were studied as a control group, mean age 50.2±2.6 years. There were six men and four women, with a male to female ratio of 1.5 : 1. All patients were informed about the objectives and procedures of the study and provided written consent.
Three milliliters of venous blood was collected in EDTA. These anticoagulated blood samples were used for complete blood count. Another blood sample was drawn directly into an evacuated plastic tube containing 3.2% buffered sodium citrate (one part anticoagulant to nine parts blood) for PA. After sample collection, proper mixing of the anticoagulant was ensured by gently inverting the tube by hand three to four times. Hemolyzed blood samples were excluded. Samples were stored undisturbed at room temperature (+16 to +26°C) and were stable for up to 4 h. Plasma samples were used for the estimation of fibrinogen levels, fasting plasma glucose (FPG), and glycated hemoglobin (HbA1c).
Criteria for the diagnosis of diabetes mellitus 14
- Symptoms of diabetes plus casual plasma glucose concentration of at least 200 mg/dl (11.1 mmol/l). Casual is defined as any time of the day without taking into consideration time since the last meal. The classic symptoms of diabetes include polyuria, polydipsia, and unexplained weight loss.
- FPG of at least 126 mg/dl (7.0 mmol/l). Fasting is defined as no caloric intake for at least 8 h.
- Two hours postprandial blood glucose of at least 200 mg/dl (11.1 mmol/l) during an oral glucose tolerance test. The test should be performed as described by the WHO using a glucose load containing the equivalent of 75 g anhydrous glucose dissolved in water.
Diagnosis and staging of diabetic retinopathy 15
Complete ocular examination was carried out for all the patients, including best-corrected visual acuity using a high-contrast Snellen’s letter, slit-lamp examination (Topcon Corp., Tokyo, Japan) of the anterior segment, measurement of the ocular tension by a Goldman applanation tonometer (Haag-Streit, Berne, Switzerland). The pupils were dilated using tropicamide 1% eye drops and phenylephrine 2.5% eye drops for fundus examination using the indirect ophthalmoscope (Keeler, London, UK) with a +20 D double aspheric lens (Volk Optical Inc., Mentor, Ohio, USA) and slit-lamp biomicroscopy with the +90 D biconvex lens (Volk Optical Inc.). All the patients were subjected to fundus fluorescein angiography, where the pupils were dilated using tropicamide 1% eye drops and phenylephrine 2.5% eye drops. Fluorescein dye (10%) was used to perform the angiography using the Topcon TRX 50IX (Topcon Corp.) with the software version Imagenet 2000. The technique involved taking color pictures of the patient fundus, then taking a red-free image, followed by an intravenous injection of 5 ml of 10% sodium fluorescein, and taking the images at ∼1-s intervals.
Patients were divided into groups according to the staging of DR:
- No apparent diabetic retinopathy: no abnormalities.
- Mild NPDR: microaneurysms only.
- Moderate NPDR: more than just microaneurysms but less than severe NPDR.
- Severe NPDR: no signs of proliferative retinopathy, with any of the following: greater than 20 intraretinal hemorrhages in each of four quadrants; definite venous beading in two or more quadrants; prominent intraretinal microvascular abnormalities in one or more quadrants.
- PDR: optic disc or retinal neovascularization and/or vitreous or preretinal hemorrhage.
Platelet aggregation by platelet function analyzer
The Dade Behring PFA-100 (Marburg, Germany) is a microprocessor-controlled instrument that provides a quantitative measure of primary, platelet-related hemostasis at high shear stress 16. To perform the test, 0.8 ml of citrated whole blood was transferred into the reservoir of a disposable test cartridge within 2 h of blood sampling. The anticoagulated blood was warmed to 37°C and drawn under vacuum through a 200-pm-diameter stainless-steel capillary and a 150-pm-diameter aperture in a nitrocellulose membrane coated with collagen and adenosine diphosphate. In response to the high shear rates and the agonists, a platelet aggregate is formed that blocks blood flow through the aperture.
Interpretation of results
The time taken to occlude the aperture is reported as the closure time (CT) in seconds and is measured to a maximum of 300 s 17. The reference range was considered to be between 68 and 121 s 18.
Other laboratory tests
FPG, HbA1c, and fibrinogen levels were assessed for all the participants studied.
Statistical analysis of the data was carried out using the SPSS 15 software package (SPSS Inc., Chicago, Illinois, USA) under the Windows 7 operating system. Graphic presentation of data was carried out using EXCEL 2010 (Redmond, Washington, USA) software. Qualitative data parameters were presented in the form of frequency and percentage and were analyzed for group differences and parameter associations using χ2-test (χ2 value) according to the nature of the data. The central tendency of quantitative data parameters was presented in the form of mean and median, and the measure of spread was presented as SD, 25th, and 75th percentiles. Comparative analysis was carried out using analysis of variance (F value). Variables correlations were performed using the Pearson correlation test (r value).
The Box Plot (Box-and-Whisker Plot), the central box, represents the values from the lower to the upper quartile (25th to 75th percentile). The middle line represents the median. The rounded marker represents the mean. The upper whisker represents the highest of either the maximum value or 1.5 interquartile range distance from the 75th percentile. The lower whisker represents the lowest of either the minimum value or 1.5 interquartile range distance from the 25th percentile. Probability level (P value) was assumed to be significant if less than 0.05 and highly significant if the P value was less than 0.001. P value was considered nonsignificant if greater than or equal to 0.05.
| Results|| |
The demographic, clinical, and laboratory characteristics of the studied groups are shown in [Table 1].
|Table 1: Demographic data, duration of diabetes, and laboratory characteristics of the studied groups|
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On comparing all the groups studied in terms of demographic data, duration of diabetes, and laboratory parameters, the mean age and duration of diabetes were significantly higher in the PDR group (P<0.001). The PDR group also showed higher levels of platelet count and HbA1c (P<0.001) [Table 2]. Both NPDR and PDR showed higher levels of fibrinogen (P<0.001) [Table 2]. The PDR group showed the shortest median of CT (P<0.001) [Table 2] and [Figure 1].
|Table 2: Comparison of all studied groups in demographic data, duration of diabetes, and laboratory parameters|
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|Figure 1: Comparison of all studied groups in terms of CT. CT, closure time.|
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The correlation between CT and age, duration of diabetes, and laboratory parameters showed that CT was inversely correlated with the fibrinogen level in the NAR group (P=0.000) [Table 3]. Also, CT was inversely correlated with the duration of diabetes in the NPDR group (P=0.04) [Table 4]. No correlation was found between CT and any of the studied parameters in the PDR group (P>0.05) [Table 5].
|Table 3: Correlation between closure time and age, duration of diabetes, and laboratory data in the no apparent retinopathy group|
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|Table 4: Correlation between closure time and age, duration of diabetes, and laboratory data in the nonproliferative diabetic retinopathy group|
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|Table 5: Correlation between closure time and age, duration of diabetes, and laboratory data in the proliferative diabetic retinopathy group|
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| Discussion|| |
The pathophysiology of diabetic microangiopathy and macroangiopathy may depend on whether the patient has type I or II disease, but both types of diabetes are associated with a hypercoagulable state driven by platelet hyper-reactivity 19. Among the factors suggested to be involved in the development of DR is altered platelet function 20.
We studied PA in different stages of DR and in healthy individuals using a PFA-100 analyzer. PDR had the shortest CT (P<0.001) [Table 2] and [Figure 1], indicating a higher rate of PA than the control, NAR, and NPDR groups, which may imply a relation between the rate of increase in PA and stages of DR. Agardh et al. 20 found that patients with PDR had an increased PA in vitro after stimulation with collagen and ADP. It was shown before that low-dose thrombin (<0.5 µg/ml)-stimulated PA in the diabetic patients was considerably elevated compared with healthy control individuals 21.
Similarly, it was reported previously that platelets are activated in diabetes 19,22. PA was greater in patients with DR 23. A study found that the concentration of ADP that produced 50% maximum intensity of aggregation was 1.81 µmol/l in patients without DR, 0.92 µmol/l in patients with background DR, 0.85 µmol/l in patients with ischemic PDR, and 0.44 µmol/l in patients with edematous DR 24. It was shown before that light scattering can quantify spontaneous platelet aggregation. The total light intensities of aggregate size (small, medium, and large) were compared in different stages of DR. NDR showed a significant increase in small aggregates as compared with the control. Pre-proliferative diabetic retinopathy showed a tendency toward an increase in medium and large aggregates compared with the control. PDR showed a tendency toward an increase in large aggregates compared with the control. Small aggregates increased before DR became evident. Aggregate sizes increased with the progress of DR 25.
However, Tahira 26 did not find any significant difference between PA of diabetic patients with and without DR. Also, in a previous study, no significant correlation was found between PA and the severity of DR 27.
No correlation was found in this work between PA and age in any of the stages of DR [Table 3], [Table 4] and [Table 5] as mentioned before 28. In contrast, previously, it has been reported that platelet nitric oxide production and responsiveness decreases with age and this is reflected in increased circulating monocyte-platelet aggregates 29. In our study, the mean age was significantly higher in the PDR group (P<0.001) [Table 2]. It has been suggested before that the prevalence and severity of DR increase with age 30.
In this work, a significant inverse correlation was detected between CT and duration of diabetes in the NPDR group (P=0.04) [Table 4], which suggests a relation between the increase in PA and duration of disease. Contrary to our expectations, we did not find a correlation between CT and duration of diabetes in the PDR group (P=0.92) [Table 5]; this may be because of the limited number of cases studied. It has been reported before that platelet accumulation increased with the duration of diabetes 31.
The duration of diabetes in this study was significantly higher in the PDR group (P<0.001) [Table 2]. Previously, it has been reported that the severity of DR increases with the duration of diabetes 30 and that the longer the duration of diabetes, the higher the prevalence of DR 32,33.
No correlation was found in this study between CT and HbA1c in any of the stages of DR [Table 3], [Table 4] and [Table 5]. In agreement with this, Mandal et al. 34 reported that there was no relationship between PA and glycosylated Hb levels. However, Demirtunc et al. 35 studied 70 patients with type II DM and suggested a close relationship between poor glycemic control and increased platelet activity in those patients. The median HbA1c% in this study was higher in NPDR and PDR groups (7.7 and 8.7, respectively) than the NAR and control groups (P<0.001) [Table 2], which reflects the role of glycemic control in the progression of DR. A previous work showed that the frequency of DR was 46.6% (28.8% have NPDR and 17.8% have PDR). There was a statistically significant relationship between HbA1c levels and DR (both NPDR and PDR) (P<0.000). The frequency of retinopathy (both background and proliferative) was 4.8% in the group of diabetics with a mean HbA1c level less than 6%, 8.7% in those with a mean HbA1c level between 6.1 and 6.9%, 62.8% in those with a mean HbA1c level between 7 and 9.9%, and 82.2% in those with a mean HbA1c level exceeding 10% 33.
Also, HbA1c value greater than 8.0% was significantly related to sight-threatening diabetic retinopathy 36. In contrast, Singh et al. 37 reported that the mean value of HbA1c was higher in proliferative retinopathy than in background retinopathy, but with no statistical significance.
In this study, a significant inverse correlation was found between CT and fibrinogen level only in the NAR group (P=0.000) [Table 3], which may be because of our small sample size. It has been reported before that elevation of the fibrinogen level in diabetes may contribute toward fibrin clot formation and PA 38. In this study, both NPDR and PDR groups had higher levels of fibrinogen than the NAR and control groups (P<0.001) [Table 2] as reported previously 39. Previously, it was shown that the measured fibrinogen levels of patients with different stages of DR were in the pathological range and were significantly higher than those of healthy volunteers, but there was no significant difference in the fibrinogen level between the different stages of retinopathy 40.
The platelet count in this work did not correlate with PA in either stages of DR [Table 3], [Table 4] and [Table 5]. However, the PDR group showed higher levels of platelet count (P<0.001) [Table 2]. It has been shown previously that the mean platelet volume values of patients with PDR were significantly higher than the values of the control group (P<0.05). A significant correlation was found between the degree of retinopathy and the mean values of mean platelet volume in diabetic patients (P<0.05) 41. It has been found before that the mean platelet component was 26.9 g/dl in healthy individuals and 22.5 g/dl in diabetics. Patients with NPDR and PDR showed a similar decrease in the mean platelet component level, whereas those with no DR were at an intermediate position between normal controls and DR 23.
| Conclusion|| |
PA is increased in the PDR stage in comparison with NAR, NPDR stages, and healthy individuals. This may provide a clue of its role in the pathogenesis and staging of DR. Also, this study highlights the crucial contribution of glycemic control and duration of diabetes in the progression of DR. Studies assessing the correlation of PA with glycemic control and duration of disease are recommended to be carried out on a wider scale to verify its association with the most important predisposing factors of DR and direct clinicians’ attention toward this association; if proved in controlling DR patients.
The facilities offered by the Clinical Pathology Department, Hematology Unit, Faculty of Medicine, Ain Shams University, to carry out this work are greatly appreciated.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]