FP1395 : To study the role of haematological biomarkers in type2 diabetes patients with diabetic retinopathy.

Dr.Jolly Rohatgi,Dr. GUPTA VED PRAKASH

Abstract

Context: Purpose of this study is to evaluate the role of haematologicalbiomarkers in detection of diabetic retinopathy(DR) in type 2 diabetic patients(DM).

Aim: Comparison ofhaematological biomarkers in type2 DM withand without Retinopathyandcontrols.

Setting and design: This was an observational, cross sectional studycomprising of 3 groups, group A with Diabetic Retinopathy(DR,n=66), group B without Diabetic Retinopathy(NDR,n=33) &group C of controls(n=33).Group A was further divided on the basis of severity of retinopathy.

Methods: Detailed work up including HbA1c levels, platelet Indices (Mean Platelet Volume (MPV), Platelet Distribution Width (PDW), platelet count & plateletcrit) was done.Anticoagulated blood (ethylene diamine tetracetic acid) was collected and analyzed in an automated blood cell counter for platelet count and indices [MPV, PDW, platelet count & plateletcrit].

Statistical analysis: One way ANOVA/Kruskal Wallis test was used depending on normality conditions and post hoc tukey test/ Mann Whitney test was used to compare the values among the 3 groups. Chi square test was used for comparing the proportion among the 3 groups.

Result: Mean age was 54.27 ± 9.42 yearsyrs. Mean duration of DM was10.29 ± 7.2 and 3.83 ± 3.2 years in DR & NDR group. MPV values were significantly higher in DR group (10.77±1.2) than NDR (9.2±1.1) & least in controls (8.3±0.89) (p-0.00). A 6.13fold increase in risk of retinopathy development with increased MPV.MeanPDW was significantly higher in the DR group (19.22±2.5) than NDR (13.89±2.9) & least in controls (11.3±2.5) (p-0.00). Both MPV and PDWvalues increased significantly with increasing severity of retinopathy (p-0.04 &0.00 respectively).Other markers were not significant.

Conclusion:MPV & PDW are important biomarkers which can helppredict the development & severity of diabetic retinopathy.

Keywords: diabetes mellitus, diabetic retinopathy, mean platelet volume, platelet distribution width, platelet indices.

Key message: Platelet indices are important biomarkers for early detection of diabetic retinopathy and its severity. 

Introduction

Diabetic retinopathy is one the most common and severe microvascular complication of Diabetes Mellitus (DM) and the leading cause of irreversible blindness.¹The prevalence of DR is expected to increase and the number of people at risk of vision loss is predicted to double by 2030 with the increasing rate of diabetes epidemic.²

Despite advances, the prevalence of DR remains high around 40%.³ A number of interconnected biochemical mechanisms associated with hyperglycemia have been implicated in the pathogenesis of DR. Despite established screening programmes, early diagnosis and treatment of the condition, not all patients are benefitted or prevented from developing the disease or its consequences, reinforcing the fact that DR is a multifactorial disease.³

There are various established known risk factors of DR of which duration of diabetes isprobably the strongest predictor of the disease for development and progression of retinopathy.⁴ Others include degree of hyperglycemia, hypertension, deranged lipid profile etc.⁴The protective effect of glycemic control has also beenconfirmed in patients with type 2 diabetes by the U.K. Prospective Diabetes Study (UKPDS) which demonstrated that improved blood glucose control reduced the risk of developing retinopathy. The overall rate was decreased by 25% in patients receiving intensive therapy.⁵

Insulin resistance (IR) (i.e., resistance to insulin-stimulated glucose uptake) presents in a majority of individuals with type 2 diabetes; it appears to be a common precursor of both diabetes and macrovascular disease.⁶ IR is a multisystem disorder that is associated with multiple metabolic and cellular alterations. Factors that contribute to IR are genetics, obesity, physical inactivity, and advancing age.⁷ Metabolic disturbances that commonly occur in patients with IR are atherogenic dyslipidaemia, hypertension, glucose intolerance, and also a prothrombotic state.^6,7

People with the metabolic syndrome exhibit a pattern of coagulation factors that promote thrombosis or retard thrombolysis.^7,8 The prothrombotic state is characterized by increased fibrinogen levels,⁹ increased plasminogen activator inhibitor (PAI)-1,¹⁰ and different abnormalities in platelet function.^8,11

It has been postulated that abnormal insulin activation in diabetic patients leads to an increased platelet function and their microvascular complications.⁸

Few studies have shown positive correlation between Mean Platelet Volume (MPV) and presence of DR especially maculopathy. However other indices especially Platelet Distribution Width (PDW) which is more specific marker of platelet activity has shown varied results.^12–15

The purpose of this study is to evaluate the role and compare the various hematological biomarkers in diabetic patients with and without retinopathy and to associate these biomarkers with the stages of DR. This may help in identifying diabetic patients at risk of developing DR, such patients can be screened and diagnosed at early stages.

Subjects and methods

This study comprised of 3 groups divided as diabetics with retinopathy (group A), diabetics without retinopathy (group B) and healthy controls (group C). The retinopathy group was further divided into subgroups according to severity of retinopathy (A1, A2, A3, A4). Group A had 66 patients, group B and C had 33 patients each, so a total of 132 patients from the outpatient department of our hospital.

After a full, free and voluntary consent and ethical clearance all the patients underwent a detailed systemic and ocular history and complete ocular examination.Fundus examination was done with Direct Ophthalmoscopy, Indirect Ophthalmoscopy and 90D examination, optical coherence tomography (OCT), fundus photo/ fluorescein angiography wherever required and possible for staging of diabetic retinopathy.Systemic examination and investigations were done with special emphasis on blood Sugar levels (fasting and post prandial), HbA1c levels, blood pressure, platelet Indices (MPV, PDW, platelet count, plateletcrit), renal function test, urine analysis, lipid profile, neurological examination.For platelet indices around 2ml of blood sample was collected inan ethylene diamine tetracetic acid vacutainer in sitting position from antecubital vein and processed in the automated cell counter ABX micro-60 machineexactly within 2 hours of collection and the indices were noted thereafter. Normal value for MPV and PDW were taken as less than equal to 10 and 15 respectively.

Appropriate statistical tests were applied to assess the significance of difference between groups.One way ANOVA/Kruskal Wallis test was used depending on normality conditions and post hoc tukey test/ Mann Whitney test was used to compare the values among the 3 groups. Chi square test was used for comparing the proportion among the 3 groups.

Results

The age range of all patients was 20-75 years. The mean age of patients was 53.24 ± 9.68 years in control group, 54.64 ± 10.79 years in diabetics without retinopathy and 54.59 ± 8.63 years in diabetics with retinopathy which is not significantly different from each other (p value – 0.774).Most patients belonged to the age group of 51-65 years (n= 73, 56.1 %).

The duration of diabetes in our study group varied from 0.25 to 30 years with a mean of 8.1 ± 6.9 years. The mean duration in group A was 10.29 ± 7.2 years and group B was 3.83 ± 3.2 years. The difference in the duration of diabetes was highly significant in the two groups (p value – 0.00).In patients with non-proliferative retinopathy (group A1, A2, A3) duration was 0.25-5 years. But in patients with proliferative retinopathy (A4) maximum had duration of their disease between 6-10 years.No patient without retinopathy had a duration of more than 15 years and in our study, 25% of PDR patients had diabetes of more than 15 years duration. 

Mean platelet volume

Mean MPV values in the 3 groups is shown in fig 1. The MPV values were highest in the diabetic retinopathy group followed by diabetics without retinopathy group which was still higher than normal controls. This difference was highly significant p-value being 0.00. Mean MPV values of group A were significantly higher than group B(p value – 0.00). In group A, mean MPV values increased significantly (p-value – 0.04) with increasing severity of retinopathy shown in fig 2.

Only one patient in control group had high MPV value (0.03%) whereas it was 48 (72.7%) and 10 (30.3%) in group A and B. It was also observed that only the retinopathy group had MPV more than 12. Whereas in the retinopathy group, 50%, 61%, 87.5%, 88% patients had high MPV values in mild, moderate, severe NPDR and PDR groups respectively. Amongst the subjects with high MPV values in group A and B, 83% belonged to group A whereas 17% to group B (table 1).

Logistic regression analysisperformed to assess the effect of MPV values on the risk of developing diabetic retinopathy, showed a 6.13-fold increase in the risk of retinopathy development with increased MPV values (OR: 6.133; P = 0.000)(95% confidence interval). 

Platelet distribution width

The mean PDW values in the three groups is shown in fig 3 and it was highest in the diabetic retinopathy group followed by diabetics without retinopathy with and lowest in the normal controls with p-value of 0.00.Mean PDW values of group A were significantly higher than group B(p value – 0.00).The mean PDW value also increased significantly (p-value – 0.00) with increasing severity of diabetic retinopathy shown in fig 4.

Only 2 subjects (0.03%) in group A had normal PDW values compared to 27 (82%)in group C. Whereas 64 subjects (97%) in group A had high PDW values compared to only 6 (0.2%) in group C.  

Platelet count

The mean platelet counts in controls, diabetics without and with retinopathy was 1.91lacs±56.5, 1.92lacs±58.2 and 1.85lacs±51.7 (statistically insignificant p-value=0.818). Even the mean platelet count within the retinopathygroup was not significant. 

Plateletcrit

The mean plateletcrit value was notsignificantly different in the three groups. Although it increased progressively from controls to diabetic without retinopathy to diabetics with retinopathy and also increased with increasing severity of retinopathy in group A, it was not found to be statistically significant. 

Discussion

Diabetic retinopathy (DR) is one of the most common and severe complication of DM and is also the leading cause of blindness in adults aged 20-75yrs.¹⁶Pathogenesis of Diabetic retinopathyis mainly microvascular abnormalities although other factors like inflammation and neurodegeration have recently been suggested.^3,17 DR naturally progresses fromnon-proliferative abnormalities to proliferative diabetic retinopathy (PDR), characterized by the growth of new blood vessels on the retina and posterior surface of vitreous. Macular edema, characterized by retinal thickening from leaky blood vessels, can develop in all stages of retinopathy.¹⁸

Insulin resistance (IR)has been reported in a majority of individuals with type 2 diabetes. This results in an abnormal insulin activation which leads to increased platelet functions. Thus, a prothrombotic state is one of the common metabolic disturbances seen in these patients.^6,7 This has been postulated to be a cause of microvascular complications of diabetes.⁸Thus, we tried to isolate these indices which in future can help as markers of retinopathy.

There have been few studies in the available literature on platelet indices in patients with DM.

The most frequently studied platelet indices include MPV ^{12,14,19–21} and PDW^19,22. Others include plateletcrit, platelet count and P-LCR.^19,22We have studied MPV, PDW, plateletcrit and platelet count in our study.

The MPV values were significantly higherin the retinopathy group as compared to non-retinopathy group and both were significantly higher than controls (p value: 0.00). Similar observations have been given in other studies conductedby Buch et al ¹⁹ andAyhan Tuzcuet al.¹²On the other hand, Aydinli et al.²⁰and Jindal et al. ²²found no significant relationship between vascular complications and MPV values in cases with DM.When comparing the mean MPV values within the retinopathy group there was a significantly increasing trend with increasing severity of retinopathy. This result was in agreement to the study conducted by Ateş et al. ²¹who compared the mean MPV values of cases with non-proliferative, and proliferative diabetic retinopathy to those of healthy controls and found that MPV values were significantly higher in non-proliferative and proliferative retinopathy groups than in controls.

Study by Zhong et al., suggested that MPV values could be a risk factor in retinal neovascularization as they found MPV values were significantly higher in proliferative diabetic retinopathy patients than in healthy controls.¹⁴However, they did not compare MPV values in patients with and without retinopathy.

We also found a6.13-fold increase in the risk of retinopathy development with increased MPV values in Indian patients (OR: 6.133; P = 0.000) which is much higher than the reported risk of 1.4-fold in the study by Ayhan Tuzcuet al in the west.¹²

In our study,the mean PDW values were also significantly higher in the diabetic with and without retinopathy group as compared to the controls. While, in diabetic patients these values were significantly higher in retinopathy group as compared to non-retinopathy group.The values also significantly increased with increasing severity of retinopathy. This comparison has been done in only two studies^19,22 previously, bothshowing similar results. Jindal et al also performed a discriminant analysis including age, duration of diabetes, platelet count and the three platelet indices and could accurately classify approximately 78.6% of diabetic patients with complications. These results were encouraging as these factors may act as indicators of microvascular complications in diabetic patients.²² This was not possible in our study due to less number of patients.

There wasonly one study conducted previously which compared the platelet count between healthy controls, diabetics without retinopathy and with retinopathy by Buch et al, where the platelet count was significantly decreased in diabetic cases (P = 0.005).¹⁹In our study, there was no significant relationship established between the groups, p-value of 0.818.

Plateletcrit was also amongst the platelet indices which was not found to be statistically significant in the three groups although it increased from healthy controls to diabetics without retinopathy to diabetics with retinopathy.There are nostudies in literature which compared the plateletcrit between healthy controls, diabetics without retinopathy and with retinopathy.

These results suggest that platelet indices (MPV, PDW) are important indicators of retinopathy in diabetic patients.^19,22 Also, their importance lies in the fact that these indices are conveniently obtained from automated cell counters. As diabetic retinopathy is a significant causes of ocular morbidity in diabetics and is usually detected quite late in the course of disease, early detection will help in reducing the morbidity, blindness and health care costs in patients with diabetes.²²

In conclusion, the present study highlights the significant increase in platelet indices like MPV and PDW, in diabeticscompared to control subjects. It is suggested that platelet indices be routinely done in diabetic workup as these may help in identifying patients likely to have diabetic retinopathy.Logistic regression analysis showed a 6.13-fold increase in the risk of retinopathy development with increased MPV values.However, a larger diabetic population needs to be screened to confirm the role of platelet indices in identifying patients likely to have severe diabetic retinopathy.

References

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12. Ayhan Tuzcu E, Arıca S, Ilhan N, Daglioglu M, Coskun M, Ilhan O, et al. Relationship between mean platelet volume and retinopathy in patients with type 2 diabetes mellitus. Graefe’s Arch Clin Exp Ophthalmol. Springer Berlin Heidelberg; 2014 Feb 17;252(2):237–40.

13. Liu J, Liu X, Li Y, Quan J, Wei S, An S, et al. The association of neutrophil-to-lymphocyte ratio, mean platelet volume and platelet distribution width with diabetic retinopathy and nephropathy: a meta-analysis. Biosci Rep. 2018 Mar 26;BSR20180172.

14. Zhong Z-L, Han M, Chen S. Risk factors associated with retinal neovascularization of diabetic retinopathy in type 2 diabetes mellitus. Int J Ophthalmol. 2011;4(2).

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Table 1: Incidence of normal and raised MPV in group A and B

Normal MPV	Raised MPV	Total	p-value
Group A %within group %within MPV	18 27.3% 43.9%	48 72.7% %	66
Group B %within group %within MPV	23 69.7%56.1%	10 30.3% 17.2%	33
	41	58	99