Evaluation of Platelet-Neutrophil and Platelet - Monocyte Ratio in Healthy Subjects and Chronic Periodontitis Patients - A Clinical and Biochemical Analysis

Introduction

The unparalleled significance of blood as the ultimate bodily fluid for indicating an on-going disease process has been highlighted in numerous studies. The total white blood cell count in peripheral blood plays a crucial role in diagnosing inflammatory conditions or infections, which can act as risk factors for various systemic diseases.¹

Higher number of leukocytes make blood more viscous and decrease the blood flow by adhering to the endothelial cells lining the blood vessels. Reduction in blood flow especially in narrow or blocked arteries due to atherosclerotic plaque formation can be an important etiologic factor for various cardiovascular diseases.^2,3

An elevation in number of active thrombocytes can be seen in inflammatory and infectious process and this is known as “Reactive Thrombocytosis”.⁴ Platelet leukocyte complexes, which include platelet-neutrophil and platelet-monocyte, are crucial components of inflammation and thrombosis and are sensitive indicators of platelet activation. Numerous inflammatory conditions, including arterial thrombosis, asthma, rheumatoid arthritis, and ensuing coronary artery and cerebrovascular disorders, might activate platelets.^5,6

Periodontitis is an infectious, inflammatory disease of chronic and multifactorial nature characterized by destruction of supporting periodontium of the teeth which might eventually lead to loss of both hard and soft tissue supporting structures.⁷

Platelets showed an increased activation status in patients with periodontitis compared with platelets from healthy controls. Cytokines and chemokines released by activated platelets uncover proinflammatory receptors which enables their binding to leukocytes and endothelial cells.⁸ Treatment of periodontal diseases could be a key strategy to alter peripheral blood indicators and lower the risk of cardiovascular diseases. Therefore evaluation of peripheral blood cell levels can provide valuable information about the presence and intensity of inflammatory process.⁹

Hence, this study was carried out to evaluate platelet-neutrophil and platelet- monocyte ratios in periodontally healthy subjects and patients with chronic periodontitis before and two weeks after non-surgical periodontal therapy to assess whether amount of periodontal inflammation in these subjects can influence the peripheral blood cell levels.

Materials and Methods

Study population

Random selection of patients was carried out in the outpatient section of department of periodontics, Rajarajeswari Dental College and Hospital, Bangalore. Sample size was determined based on power analysis at a confidence interval of 90% (P <0.05) and a total of 20 patients were selected. The subjects were divided into two groups:

• Group A: 10 healthy individuals without any clinical signs of periodontal inflammation belonging to age group of 25-45 years with periodontal pocket depth (PPD) ≤ 3 mm, clinical attachment level (CAL) < 2 mm.
• Group B: 10 chronic periodontitis patients of age between 30-45 years with PPD ≥ 4 mm and CAL ≥ 2 mm in more than 30% of the teeth.

Inclusion criteria

1. Patients with at least 20 functioning teeth without undergoing any periodontal surgical procedure.
2. Patients not under any medication within last six months which may modify the periodontal status.

Exclusion criteria

1. Subjects taking any antibiotics, systemic steroids or anti-inflammatory drugs during the study or six months prior to the period of study. 
2. Alcoholics, smokers, subjects suffering from cardiovascular diseases, diabetes and having any form of ongoing acute/chronic viral, fungal or bacterial infections.
3. Patients who declined to accept the study's terms and conditions.

All the subjects selected for the study gave a written informed consent. Ethical clearance for the study was obtained from Institutional Ethical Review Board (Ethical clearance number: RRDCHET/02PERIO/2020).

Estimation of peripheral blood

Collection of samples was carried out after obtaining the consent of the subject before and two weeks after non-surgical periodontal therapy. Using a 5ml syringe, 3 ml of blood was collected from anticubital fossa by venipuncture using 20 gauge needles as per the WHO guidelines.¹⁰ The blood was transferred to EDTA containing test tubes and was sent for routine blood investigation.

Non-surgical periodontal therapy

Non-surgical periodontal therapy was performed in group B. Complete supragingival scaling was carried out using ultrasonic scalers, subgingival scaling and root planing was done using Gracey curettes. The blood samples were collected prior to the procedure. All the patients were given proper oral hygiene instructions and 0.12% of chlorhexidine digluconate mouth rinse twice a day for two weeks as an adjunctive oral hygiene measures and was recalled after two weeks for the collection of blood sample.

Statistical analysis

SPSS Software was used to perform statistical analysis of the collected information. One-way ANOVA (Analysis of Variance) was applied to compare the mean values among two groups. Pair wise comparison between the groups was carried out using Tukey test. The results were denoted in text and tables.

Results

In the present study, the platelet neutrophil ratio in group B (23.12±2.40) was greater than group A (19.57±2.49) at baseline and the difference was statistically significant (Mean diff -3.55, P value 0.009). In contrast, the ratio in group B reduced following non-surgical periodontal therapy from baseline (23.12±2.40) to two weeks (21.25±2.49); however the difference was not statistically significant (Mean diff. 1.87, P value 0.222).

The reduced ratio correlated with that of the healthy group (Table 1 and 2).

In case of the platelet-monocyte ratio, group A (1.10±0.83) demonstrated a higher ratio than group B (0.66±0.60) at baseline. Following non-surgical periodontal therapy after two weeks, the ratio was shown to have decreased (0.50±0.28). All the changes were not statistically significant (Table 3 and 4).

Discussion

The present study showed that the high grade inflammatory component associated with periodontitis can impact the peripheral circulation leading to increase in the platelet- leucocyte complexes. The mechanism behind the development of platelet-leucocyte complexes is that, as platelets become stimulated, they express CD62P on their surfaces and engage their receptor PSGL-1 on leukocytes, forming aggregates of platelet– leukocytes.¹¹ Aggregates of platelet-leukocytes are important for functional variations of these cells. Unlike the uncomplexed cells, formed aggregates are found to produce proinflammatory cytokines in larger amounts.¹²

Platelet-leukocyte aggregates are of importance, not only in homing leukocytes to inflammatory sites but also in better bacterial clearance.^13,14 Neutrophils and monocytes were activated by all species of oral bacteria tested, but this study observed no differences between patients and controls. Interestingly, it was observed that platelets from periodontitis patients demonstrate heightened sensitivity to activation triggered by oral inflammation.

Studies have shown that endotoxin-treated platelets activate neutrophils, leading to the release of proteases that are capable of damaging the underlying endothelium, resulting in increased exposure of subendothelial tissues to platelets. Adhesion of platelets to the damaged vessel wall is recognized to add to the pathogenesis of atherosclerosis.⁶ Platelet-monocyte complexes are of importance, not only in homing to inflammatory sites, but also for functional alterations of these cells. Formed Platelet Monocyte Complexes create monocyte chemotactic protein-1 (MCP-1) and IL-8 in greater amounts than uncomplexed monocytes; both these proinflammatory cytokines are related to progression of atherosclerosis.^15,12 Moreover, platelets provide cholesterol to monocytes for cholesteryl ester synthesis,¹⁶ and these monocytes may then separate into foam cells seen in atherosclerotic lesions.¹⁷ On the contrary, the present study showed that the platelet- monocyte ratio was not elevated in the chronic periodontitis patients. Moreover, it was found higher in the healthy controls.

In addition, other potential risk factors causing atherosclerosis have been suggested, including traits related to inflammation, obesity, and infection.¹⁸ An increased total white blood cell (WBC) count poses as a risk factor for the progression of atherosclerosis and has been directly linked to a higher threat for coronary artery disease, incidence of stroke, and mortality from cardiovascular disease.^19,20 Several prospective studies have demonstrated a positive association between WBC count and the incidence of coronary heart disease. This association, along with the presence of mononuclear cells in atheromatous plaques, suggest that atherosclerosis may be an inflammatory disease linked to either distant or local infections or inflammatory conditions.^21,22

The study findings indicate that in comparison to the healthy group, the group with a persistent periodontal infection has higher platelet neutrophil ratio. There were statistically significant differences in the observed values. Moreover, the non-surgical periodontal therapy and improvement in the periodontal status reduced the counts of platelets and leucocytes. Numerous prior studies have demonstrated elevated platelet levels and their activation in patients with periodontitis,^5,23 can be well controlled by periodontal treatment.²⁴ Our study findings are also consistent with this conclusion.

Conclusion

The results obtained from the present study suggest that chronic periodontal disease may modify the leucocyte and platelet counts when compared to healthy controls and could be regarded as one of the mechanisms that describe how periodontitis is interrelated to the onset of atherosclerosis and cardiovascular disease. Management of periodontitis may be an important intervention to modify peripheral blood markers, thereby reducing the risk of cardiovascular diseases. Therefore evaluation of peripheral blood levels can provide valuable information about the presence and intensity of inflammatory process and additional research utilising biomarkers may yield more comprehensive understanding of the correlation between systemic diseases and periodontitis.

Funding

The study was self-financed

Conflict of interest

Nil