Study of Platelet Indices in Type 2 Diabetic Patients and Its Correlation with Vascular Complications

Background: Diabetes Mellitus is a metabolic syndrome characterized by hyperglycemia resulting in macrovascular and microvascular complications. Altered platelet morphology and functions have been linked with the pathological processes and high risk of vascular disease. Platelet indices (Mean platelet volume-MPV, Platelet distribution width-PDW, and Platelet large cell ratio-PLCR) are determinants of platelet functionality. Methods: The aim of this study was to study the platelet indices (MPV, PDW, P-LCR) in type 2 diabetic patients with vascular complications and compare them in diabetic patients without vascular complications. The present study was conducted on 70 diabetic patients with vascular complications and 70 diabetic patients without vascular complications for a period of one year and eight months in department of pathology, JSS hospital, Mysore. Patients were divided into cases and controls depending on the presence or absence of macrovascular complications (Myocardial infarction , stroke, peripheral arterial disease) and microvascular complications (retinopathy, nephropathy and neuropathy). Platelet indices (MPV, PDW, P-LCR) were measured using an Automated Blood Counter. FBS and HbA1C levels were collected from the clinical proforma. Result: Platelet indices were significantly higher in diabetic patients with vascular complications compared to those without complications [11.37±1.19 fL vs 10.17±0.71 fL (P=0.0001), 13.90±2.99 fL vs 11.28±1.55 fL (P=0.0001), 35.61±9.35% vs 26.14±5.79% (P=0.0001) respectively]. Conclusion: The present study showed a significantly higher MPV, PDW and P-LCR in diabetic patients with vascular complications compared to those without complications. This indicates that elevated platelet indices could be the cause for vascular complications. Hence MPV, PDW, P-LCR can be used as simple and cost effective predictive parameters of platelet activation to monitor and predict the risk of vascular complications.


Introduction
Diabetes Mellitus is characterized by hyperglycemia resulting in micro and macrovascular complications affecting the nerves, kidneys, eyes, CVS etc. [1] It is associated with varying degree of hyperglycemia accompanied with the biochemical alterations in carbohydrate, protein and lipid metabolism. [2] The injurious effects of hyperglycemia are characterized as macrovascular and microvascular complications. Altered platelet morphology and functions have been linked with the pathological processes and high risk of vascular disease. [3] The platelet indices -(Platelet -PLT, Mean platelet volume -MPV, Platelet distribution width -PDW and Platelet large cell ratio -PLCR) are the determinants of platelet functionality, among which increased mean platelet volume (MPV) and platelet distribution width (PDW) were found to be attributed in the causation of thromboembolic complications. [4,5] It is also noted that the platelets with increased number and size possibly affect the platelet distribution width contributing in the pathogenesis of vascular complications. [6] Hyperactivity of platelets have an important role in the initiation of thrombosis and atherosclerotic lesions. Larger platelets are more active enzymatically and metabolically and have a higher thrombotic ability as compared to the small sized platelets. [7] Sustained hyperglycemia leads to alterations in the vessel wall leading to endothelial dysfunction and vascular lesions in diabetic complications. [8 ] Formation of advanced glycation end products, activation of protein kinase C and disturbances in polyol pathways are the possible mechanisms by which increased glucose induces vascular abnormalities. [9] Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 5, September-October, 2017 progression of vascular lesions. [11] Increased platelet size may be one of the factor causing increased risk of atherosclerosis associated with diabetes mellitus and vascular complications. [12] The aim of our study was to determine the hyperactivity of platelets in type 2 diabetic patients and its association with vascular complications. This was done by comparing the platelet indices-MPV, PDW and P-LCR, FBS and HbA1C levels among diabetic patients with vascular complications and diabetic patients without vascular complications.

Materials and Methods
This was an analytical study carried out on 70 type 2 diabetic patients having vascular complications and 70 type 2 diabetic patients without vascular complications for a period of one year and eight months in the department of pathology, JSS hospital, mysore. Data was collected fulfilling the inclusion and exclusion criteria.

Inclusion Criteria: Diabetic patients with vascular complications and diabetic patients without vascular
Exclusion Criteria : 1.) Non-diabetic patients with vascular complications & 2.) Diabetics on antiplatelet drugs such as aspirin and clopidogrel.
Patients were divided into cases and controls depending on the presence or absence of macrovascular complications (Myocardial infarction , stroke, peripheral arterial disease) and microvascular complications (retinopathy, nephropathy and neuropathy). Platelet indices like MPV, PDW, P-LCR were measured in the above target groups using Automatic Blood Counter (SYSMEX , XN-1000). Venous blood samples collected in a vacutainer containing di-potassium EDTA were used. Samples were processed within one hour of collection and were maintained at room temperature. Plasma glucose levels and HbA1c levels of the patient were collected from the clinical data.
Statistical analysis was done using Statistical package for social sciences (SPSS version 22) software. Descriptive statistics such as numbers and percentages were used to describe categorical variables. Mean and standard deviations were used to describe continuous variables like MPV, PDW and P-LCR. Independent sample t-test was applied to find out the significant difference in MPV, PDW and P-LCR between the cases and controls. Pearsons correlation was used to analyse association between different variables. Statistical significance was determined at 5% level of significance (ie. < 0.05 is significant). Microsoft word and Excel have been used to generate graphs, tables etc. Among the controls, a positive statistical Pearson correlation was seen between MPV and HbA1c (P -0.047), PDW and HbA1c (P -0.003), P-LCR and HbA1c (P -0.026) and FBS and HbA1c levels (P -0.0001) (Graph 3). However, no statistical correlation was noted between MPV and age, duration of DM, FBS; PDW and age, duration of DM, FBS; P-LCR and age, duration of DM, FBS.    (21) 11±2.2 7.8±1.3 0.0001 Khandekar MM et al (15) 10.43±1.03 9.2±0.91 0.001 Sharma M et al (22) 17.60±2.04 9.93±0.64 <0.001 Khode V et al (23) 9.54 ± 0.9 9.21 ± 0.6 0.018 Present study 11.377±1.1969 10.173±0.7134 0.0001

Discussion
Diabetes Mellitus is a metabolic syndrome characterized by hyperglycemia resulting in macrovascular and microvascular complications. Platelet hyperactivity has been linked with the pathological processes and high risk of vascular disease.
MPV is an indicator of average size and activity of platelets. Larger platelets are more active enzymatically and metabolically and have higher thrombotic ability as compared to small sized platelets which are depicted by increased MPV. Normal range -7.5-11.5 fL. P-LCR is the increased percentage of large platelets. It is the ratio of large platelets from the 12 fL discriminator or larger. Normal range -11.9-66.9%. PDW reflects how uniform the platelets are in size. Activated Platelets with increased number and size of pseudopodia differ in size, leading to alterations in platelet distribution width. Normal range of PDW is 8.3-25.0 fL.
Platelets play a pivotal role in atherothrombosis. [13] Central to the pathogenesis of occlusive arterial disease is the activation of platelets at sites of vascular injury via pathologically exaggerated and dysregulated protective mechanisms of hemostasis. [14] Platelets secrete and express a large number of substances that are crucial mediators of coagulation, inflammation, thrombosis and atherosclerosis.
Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 5, September-October, 2017 [15,16] Inadequate glycemic control, protein glycation and oxidative stress cause endothelial injury and platelet activation with altered platelet morphology and function leading to chronic complications in diabetics. [17] The present study was done to determine if platelets are activated in diabetes and in its association with micro and macro-vascular complications.
Demographic Data: In the present study, the patients age ranged from 40 years to 90 years. The mean age of diabetic patients with vascular complications (cases) was higher compared to those without complications (controls) [63.30±10.04 vs 61.29±8.88] which correlated with the studies conducted by Jabeen F et al (2) on various population. The maximum number of cases in this study were seen between the age of 50-69 years. Among the cases, 44 out of 70 (62.9%) patients were males and 26 out of 70 (37.1%) were females with male to female ratio of 1.7 : 1. This indicates that there was male preponderance in our study which correlated with the study conducted by Bath P et al [16] .

Duration of Diabetes:
Duration of diabetes mellitus ranged from 5 years to >20 years in both the groups (cases and controls) in the present study with the mean duration being 15.97±4.42. However other studies conducted by Alex kodiatte T et al [1] and Dindar S et al [14] showed a lesser mean duration.  (20) and Ozder A et al. (18) . Among controls, 58 out of 70 (82.8%) had HbA1C levels of ≥ 6.5% and 12 out of 70 (17.1%) had HbA1C levels of < 6.5% with a mean HbA1C levels of 8.31±2.22. In the present study the HbA1C levels were significantly higher in cases compared to that of controls.  (21) . Other studies showed a mean MPV lesser than the present study whereas one study conducted by Sharma M et al (22) showed higher MPV than our study (  (15) . However, study conducted by Khode V et al (23) showed PDW values lower than the present study ( Pearsons Correlation: A positive statistical Pearson correlation was seen among cases between PDW and Duration of diabetes mellitus (P -0.035) and FBS and HbA1c levels (P -0.004). However, no statistical correlation was noted between other parameters. Among the controls, a positive statistical Pearson correlation was seen between MPV and HbA1c (P -0.047), PDW and HbA1c (P -0.003), P-LCR and HbA1c (P -0.026) and FBS and HbA1c levels (P-0.0001) (table 9).

Conclusion
Mean platelet volume (MPV), platelet distribution width (PDW) and platelet large cell ratio (P-LCR) are considered the important markers of platelet activation which can be easily measured as part of whole blood count. Hence MPV, PDW, P-LCR can be used as a simple and cost effective predictive parameters of platelet activation to monitor and predict the risk of vascular complications.