Platelet Parameters in Diabetes Mellitus in Relation to HbA1c Levels

Introduction

Immature Platelet Fraction (IPF) is a reflection of bone marrow rebound and represents the young reticulated platelet number in the circulation. They are also richer in RNA than mature platelets.¹

The IPF measured with automated hematology analyzers has been increasingly used as a marker of thrombopoiesis and platelet activity. We evaluated IPF in diabetic patients at high risk for both cardiovascular disease (CVD) and diabetes-related complications. Elevated IPF levels in diabetic patients have been associated with poor glycaemic control and cardiovascular complications.

In patients with poorly controlled diabetes mellitus (DM), platelet-endothelial interaction plays a key role in atherothrombotic events. The immature platelet fraction, measured by an automated hematologic analyzer, represents the size and cytoplasmic RNA contents of platelets. It serves as an indicator of thrombopoiesis and is frequently used as a marker of platelet activity.²

IPF is a new hematological parameter that reflects the rate of thrombopoiesis. In patients with thrombocytopenia, IPF has been proposed as a marker of platelet recovery. The aim of this study was to investigate the association between IPF and platelet count in patients with diabetic mellitus and concurrent thrombocytopenia. Immature platelets were first described in 1969 as reticulated platelets. On microscopic analysis, RNA condensates within platelets resemble pattern of immature red blood cells (IRBC) after staining of the reticulum. Over the past two decades, various methods involving flow cytometry have been developed, and the significance of reticulated platelet has become increasingly recognized. However, performing reticulated platelet assay with a standard flow cytometer is time-consuming, requires a trained technologist, and lacks an established standardized protocol.

With the development of an easy to apply, rapid and reliable routine test based on the Fluorescence flow cytometry (FFC) technique, the role of the immature platelet fraction (IPF) can be reexamined. IPF can be measured with the XE-IPF Master optional software module on the XE-2100 and is automatically included in the more advanced XE-5000 analyzer.³

Megakaryocytes pinch off reticulated platelets, which mature into thrombocytes within 1-2 days. The proportion of reticulated platelets in peripheral blood reflects the rate of thrombopoiesis. When bone marrow is passive, the reticulated platelet will also be active. Platelet RNA is related to megakaryocyte function.

This information helps distinguish between a bone marrow disorder and increased platelet destruction or loss.

Assessing the IPF% provides an additional way to interpret platelet counts and may help reduce the need for a bone marrow test.⁴

Materials & Methods

The present study was conducted at Padmashree Diagnostics, Bangalore, over a six-month period with 60 patients.

Written informed consents were obtained from the patients, and their diabetic status was evaluated. The platelet indices were documented with special reference to immature platelet fraction.

All known cases of type 2 diabetes mellitus, including both male and female patients, were included. In contrast, samples with haemolysis or icterus, patients who did not consent to the study, patients with other medical conditions, and patients on anticoagulant therapy were excluded.

Procedure

A total of 60 patient samples were collected and analyzed using the Mindray haematology analyzer. Corresponding blood glucose levels and HbA1c levels were recorded. The obtained results were tabulated and analysed using SPSS software.

Results

Of the 60 patients who participated in the study, 25 were male (41.7%) and 35 were female (58.3%).

Among the 60 patients, 28 had HbA1c levels below 6%, 21 had levels between 6% and 8%, and 11 levels above 8%. The mean HbA1c in this study was 8.067% (SD 1.435), ranging from 6.0% to 11.2%. A total of 72.1% of subjects (31 patients) demonstrated poor glycaemic control (Table 1).

Table 2 shows the variaton of platelet parameters accoring to HbA1c levels. Platelet count increased as HbA1c values increased. Patients with HbA1c values less than 6% had a mean platelet count of 2.83, platelet distribution width (PDW) of 14.35, plateletcrit (PCT) of 0.24, mean platelet volume (MPV) of 8.72, and immature platelet fraction (IPF) of 4.34. In patients with HbA1c levels between 6-8%, the mean platelet count was 2.96, PDW was 15.92, PCT was 0.28, MPV was 9.98, and IPF was 3.99. In patients with HbA1c levels more than 8%, the mean platelet count was 3.25, PDW was 15.94, PCT was 0.31, MPV was 10.08, and IPF was 4.77.

Figure 1 illustrates the trend of platelet parameters in relation to HbA1c levels. Platelet count, PDW, PCT, MPV, and IPF, all showed an increase with rising HbA1c levels. Although these results did not reach statistical significance, notable variations in platelet parameters were observed across the diabetic subgroups.

Discussion

A total of 72.1% subjects (31 patients) demonstrated poor glycaemic control. This proportion was higher than that reported by Hekimsoy et al. (mean HbA1c=7.49%; SD=1.95) and lower than that reported by Kodiatte et al., in 2012 (mean HbA1c=9.13%; SD=2.5).^5-6 The mean MPV in this study was 10.36 fL (SD=0.84), with values ranging from 8.5 to 12.3 fL. This value was within the analyzer’s reference range (9.2 to 12 fL). Shah et al. examined MPV as a distinguisher between the presence and absence of diabetes and identified 8.20 fL as the cut off (P=0.0073).⁷ All subjects in the present study had MPV values above this cut-off. The mean MPV observed here was consistent with that reported by Hekimsoy et al. (mean MPV=10.62 fL; SD=1.71) and Lee et al. (median MPV=10.35 fL; range=9.79-11.0), but higher than that reported by Kodiatte et al. (mean MPV=8.29 fL; SD=0.735).^5,6,8,9

The mean MPV in patients with poor glycaemic control was higher than in those with good glycaemic control; however, the difference was not statistically significant (P=0.494). This finding is consistent with Lee et al., who also reported no significant difference in MPV values (P>0.05).8,9 In contrast, several other studies have shown significant associations between MPV and glycaemic control. Demirtunc et al. found significantly higher MPV values in patients with HbA1c >7% compared to those with HbA1c ≤7% (9.0±0.7 vs 8.4±0.8 fL; P=0.01). Moreover, MPV significantly decreased following improvement in glycaemic control (from 9.0±0.7 to 8.4±0.8 fL; P=0.003), accompanied by reduction in HbA1c values (from 8.4±1.2% vs 6.3±1.2%; P=0.0001). Their study involved 70 patients with DM and 40 healthy controls, excluded patients with thrombosis and malignancy, and used citrate as the anticoagulant with platelet analysis performed on the Coulter Gen-S system. MPV was assessed twice in the subgroup with HbA1c >7% (35 people), at baseline and three months after treatment. Kodiatte et al., and Shah et al., also reported a significant association between MPV and HbA1c levels.^6,7

Increased platelet reactivity in type 2 diabetes is influenced by several factors, such as metabolic abnormalities, resistance and insulin deficiency, oxidative stress, and inflammation. Metabolic abnormalities include hyperglycaemia and dyslipidaemia. Hyperglycaemia may lead to increased platelet reactivity through several mechanisms. Dyslipidaemia can cause an increase in platelet reactivity.^7,10 Khemka et al., reported that MPV was significantly higher in individuals with hyperlipidaemia compared to those with a normal lipid profile.¹¹

Type 2 diabetes is also associated with systemic inflammation and oxidative stress that can contribute to increased platelet reactivity. Oxidative stress disturbs endothelial function and reduces the production of NO. The impaired endothelial function interferes with the synthesis of prostacyclin. In type 2 diabetes, oxidative stress contributes to increased platelet reactivity through direct effects on platelets and endothelial dysfunction. Comorbidities are common in type 2 diabetes. Platelet reactivity may be enhanced in patients with hypertension or heart disease. This heightened reactivity is influence by sympathetic nervous system activation, the reninangiotensin system, shear stress, increased production of reactive oxygen species (ROS), regulatory changes in calcium signalling, endothelial dysfunction, and decreased availability of NO.¹² 

Platelets are also more active in malignancy, where tumour cells, such as those in breast cancer, induce platelet aggregation. In addition, platelets play an important role in tumour metastasis.¹³ Mean platelet volume and IPF are markers of platelet activity and bone marrow thrombopoiesis.^8,10,12 Platelet activity in type 2 diabetes is not only influenced by hyperglycaemia and insulin resistance, but also by dyslipidaemia, inflammation, ROS production, and comorbid conditions. The coexistence of diabetes mellitus with comorbidities further enhances platelet activation, leading to higher MPV and IPF values, compared with hyperglycaemia alone.

The subjects in this study had heterogeneous clinical conditions, despite meeting the inclusion and exclusion criteria. Thirteen subjects had heart disease, three had malignancy, and two had pulmonary tuberculosis. The coexistence of DM with these conditions could contribute to additional platelet activation, resulting in higher MPV and IPF values. This heterogeneity may have been one of the factors contributing to statistical insignificance, even though the average MPV and IPF values tended to be higher in subjects with poor glycaemic control.

The life span of erythrocytes and platelets may be other contributing factors in this study. HbA1c is influenced by the lifespan of erythrocytes, which typically is 120 days, and therefore reflects glycaemic control over the previous 2-3 months. Similarly, MPV and IPF are affected by the lifespan of platelets in circulation, which normally is 7-10 days. A high HbA1c value does not necessarily indicate elevated blood glucose level at the time of measurement, as blood glucose level can be influenced by intake before an examination. In this study, HbA1c was affected by blood glucose levels, particularly hyperglycaemia.

The measurement of HbA1c reflects the rate of glycation in haemoglobin, while MPV and IPF do not assess glycation in platelets. Glycation of platelet membranes can lead to platelet activation and increased turnover, resulting in enhanced thrombopoiesis. Platelet activation is influenced by multiple factors, such as hyperglycaemia, dyslipidaemia, insulin resistance, and endothelial conditions. The heterogeneous and contradictory results reported in previous studies may be attributable to these conditions. Additional variables affecting the results of this study included the reference values for MPV and IPF. Variations in these values complicated the interpretation of whether MPV and IPF were truly elevated.

This study had several limitations. First, there were no non-diabetic patients included as controls. Second, observations were conducted only once, making it difficult to assess the effect of glycaemic control on changes in MPV and IPF values, as well as their relationship with the risk of complications in type 2 diabetes mellitus. Additionally, this study did not account for body mass index (BMI) and did not analyze the relationship between blood glucose levels, medication use, comorbidities, and diabetes-related complications.

Conclusion

This study found no significant differences in platelet parameters according to glycaemic control, although the mean IPF value was elevated above the normal range, while the mean MPV remained within normal limits. Additionally, no correlation was observed between MPV or IPF and HbA1c. Future studies involving more homogeneous populations, including both diabetic and non-diabetic subjects or healthy controls, may help determine differences in MPV and IPF values between these groups. Prospective studies with multiple observations could also be conducted to evaluate the prognostic value of MPV and IPF complications associated with type 2 diabetes mellitus.

Conflict of interests

None