Quantitative evaluation of modified advanced platelet-rich fibrin buffy coat among diabetic patients and tobacco smokers with chronic periodontitis.

BACKGROUND: The use of biofuel like platelet-rich fibrin (PRF) may enhance the healing potential of the tissues in tobacco smokers and diabetic patients. AIM: To evaluate the modified advanced PRF (A-PRF) buffy coat quantitatively in uncontrolled type-2 diabetic patients and tobacco smokers with chronic periodontitis by determining the size outcome of the buffy coat, the platelet and leukocyte concentration, and also to find out the influence of gender on these parameters.
MATERIALS AND METHODS: In this cross-sectional study, 180 generalized chronic periodontitis patients (46-55 years) were enrolled, Group 1 (Control group): 60 systemically healthy subjects; Group 2 (Test group): 60 Heavy tobacco smokers; and Group 3 (Test group): 60 uncontrolled type 2 diabetic patients. 15 ml of blood was drawn from all subjects to assess the size outcome, platelet, and leukocyte concentration also to find out the influence of gender on the various parameters of the prepared modified (A-PRF) membrane.
RESULTS: Uncontrolled type-2 diabetic patients had maximum height (23.39 ± 1.69 mm) and width (7.26 ± 0.16 mm) of modified (A-PRF) clot when compared to healthy individuals and tobacco smokers. The total number of platelet (245.38 ± 40.72 1000/ul) and leukocyte count (6.11 ± 0.60 1000/ul) and their percentage of concentration were 95.21 ± 2.16% and 77.25% ±1.98%, respectively, and is significantly higher in uncontrolled type-2 diabetic patients (P ≤ 0.05). Females were associated with the larger sized modified (A-PRF) clot with more number of platelets and leukocytes count than males.
CONCLUSIONS: Size outcome of modified (A-PRF) clot was found to be comparatively larger in uncontrolled type-2 diabetic patients particularly in females of all the groups when compared to males. Among the hematological parameters, the total count as well as the percentage of platelet and leukocyte in modified (A-PRF) membrane was found to be higher in uncontrolled type-2 diabetic patients. Female subjects exhibited higher platelet and leukocyte concentration than males. Copyright:

INTRODUCTION

One of the most interesting recent breakthroughs in the field of regenerative dentistry is understanding about the physiological roles of platelets and leukocytes in wound healing. After tissue injury, the platelet concentrates which hold on to the growth factors enmeshed in the fibrin network make their sustained release over a period of time, thus accelerating the wound healing.[1] It was assumed that platelets and leukocytes optimized wound healing by enhancing the amount of growth factors that were necessary for tissue growth. The most common type of platelet concentrate used in these procedures was platelet-rich fibrin (PRF).[2] In addition, a modification of the preparation setting based on the low-speed centrifugation concept was introduced. Modified Advanced PRF (A-PRF) (Modified A-PRF PROCESS©, France) represents the latest evolution of the original PRF protocol whereby two specific protocol parameters, i.e., centrifugation speed (G-force) reduction and centrifugation time, increased to enhance growth factors, platelets and leukocytes contents release from the formed modified (A-PRF) clot.[3]

It is now well established from a large body of epidemiologic evidence that type-2 DM and tobacco smoking are considered risk factors for periodontal disease.[45] Delayed wound healing might be a detrimental factor in the success of regeneration in tobacco smokers and diabetic patients. The use of biofuel like PRF may enhance the healing potential of the tissues in these patients. However, the regeneration of the periodontium in diabetic patients and tobacco smokers is a challenging task to the periodontist because of the existing variation in the concentration of platelets and leukocytes, their state of activation can be an important predictor for assessing how much success to expect in terms of regeneration.

Hence, this study was undertaken with the aim to evaluate the modified A-PRF buffy coat quantitatively in uncontrolled type-2 diabetic patients and tobacco smokers with chronic periodontitis by determining the size outcome of the buffy coat, the platelet and leukocytes concentration, and also to find out the influence of gender on these parameters.

MATERIALS AND METHODS

Study population and design

The study comprised three groups with 180 chronic periodontitis patients aged between 46 and 55 years, out of these, two were test groups and the other one was a control group with equal distribution of male and female subjects in all the study groups. All subjects were informed about the study and written consent was obtained. Ethical approval was obtained by the institutional ethics committee.

Group 1 (Control group): 60 Systemically healthy individuals with generalized chronic periodontitis

Group 2 (Test group): 60 Heavy tobacco smokers with generalized chronic periodontitis

Group 3 (Test group): 60 Uncontrolled type 2 diabetic patients with generalized chronic periodontitis.

Subjects who have fulfilled the following inclusion criteria were included in the study: (1) patients with generalized chronic periodontitis with minimum 20 teeth having probing pocket depth (PPD) of ≥6 mm, Clinical Attachment Loss (CAL) of ≥5 mm, and Radiographic bone Loss extending to mid-third of root and beyond; (2) subjects who are smoking more than 25 cigarettes per day for the past 5 years or more; (3) subjects with a history of uncontrolled Type 2 diabetes for more than 6 months (with random blood sugar level ≥200 mg/dl or ≥11.1 mmol/l or HbA1c ≥7%) on medications like metformin and troglitazone; and (4) patients who have not undergone systemic or periodontal surgery and the exclusion criteria were (1) subjects with bleeding disorders or any systemic disorders; (2) subjects with any diagnosed malignancy; (3) subjects with decrease or increase in platelet count; (4) pregnant and lactating mothers; (5) subjects on antibiotics, anticoagulants, immunosuppressive, or cytotoxic medications from the past 6 months; and (6) subjects with adverse habits such as pan chewing and alcohol.

Periodontal examination

It was done by using mouth mirror and UNC-15 Periodontal Probe to assess the following indices and clinical parameters, they were Plaque Index by Silness and Loe in 1964,[6] Gingival Bleeding Index by Ainamo and Bay in 1975,[7] and PPD by Grant et al. in 1965.[8] The probe was passed within the gingival sulcus along the circumference of the tooth. CAL was measured from the cemento–enamel junction to the base of the pocket using UNC-15 Periodontal Probe.[6] Three measurements were made on the buccal aspect and three on the lingual aspect of each tooth – a total of six sites per tooth (mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual) were recorded for PPD and CAL.

Blood sample collection and preparation of modified advanced platelet-rich fibrin samples

The fasting blood sample was collected from each participant to prepare modified (A-PRF) for hematologic analysis. A 15 ml disposable syringe and tourniquet were used to draw 15 ml intravenous blood from antecubital vein and slides were prepared for the spreading analysis test [Figure 1a and b].

Figure 1

Preparation of modified (advanced platelet-rich fibrin): (a) Collection of blood sample. (b) Transfer of blood into test tube. (c) Nonsilica coated test tubes in centrifugation machine. (d) Centrifuged at 1300 rpm for 14 min. (e) Modified (advanced platelet-rich fibrin)

Baseline platelet and leukocyte count were done by separation of 3 ml of unprocessed blood from 15 ml of blood sample. About 3 ml of venous blood was transferred into EDTA containing vacutainer tubes for whole blood analysis by using Sysmex Automated Hematology Analyzer to estimate baseline platelet and total leukocyte count [Figure 2].

Figure 2

Procedure of total platelet and leukocyte concentration present in modified (advanced platelet-rich fibrin) membrane: (a) Sysmex automated hematology analyzer. (b) Test tube in automated cell counter. (c) Blood cell count

Preparation of modified (A-PRF) for platelet and leukocyte count – The remaining 12 ml of blood was immediately (within 1 min) transferred into the sterile glass tubes (non-silica coated) (Borosilicate glass, New Delhi, India) for preparing modified (A-PRF) by centrifugation (Remi R-8C, New Delhi, India) in a standard benchtop centrifuge (208 g, 1300 rpm for 14 min) [Figure 1c and d] due to the difference in densities, it resulted in the separation of three basic fractions: a base of red blood cells (RBCs) at the bottom, acellular plasma on the surface, and finally, a platelet concentrate clot between these two [Figure 1e]. The platelet concentrate clot thus formed is obtained with the help of sterile tweezer and was segmented by scissor in such a manner as to preserve a small RBC layer hence, the most potential regenerative area, i.e., platelets and white blood cells (WBCs) concentrated in an intermediate layer located between RBC's and modified (A-PRF) clot [Figure 3a].

Figure 3

Procedure for measurement of size outcome of modified (advanced platelet-rich fibrin) clot: (a) Separation of modified (advanced platelet-rich fibrin) clot. (b) Width of modified (advanced platelet-rich fibrin) clot. (c) Height of Modified (advanced platelet-rich fibrin) clot

Quantification of Platelets and Leukocytes in modified (A-PRF) Membrane Indirectly by Using the Following Formula-As direct measurement of platelet and leukocyte concentration in modified (A-PRF) was not possible, the calculation of the number of platelets and leukocytes in modified (A-PRF) for each group was done indirectly, by determining the numerical difference between the baseline platelet and leukocyte concentration (non-centrifuged) and the residual platelet and leukocyte concentration (centrifuged) from the serum remaining after removal of modified (A-PRF).

Platelet and WBC concentration in fully clotted modified (A-PRF) membrane were determined by the following formula:

(PLTs/WBCs in fully clotted Modified [A-PRF] preparations) = (PLTs/WBCs in Whole Blood/Baseline) - (PLTs/WBCs in Residual Blood)

Residual blood = ([PLT/WBCs in semi-clotted RBC fraction] + [PLTs/WBCs in liquid serum] + [PLTs/WBCs in liquid exudate fraction])

Percentage of Platelets and WBC's in fully clotted modified (A-PRF) membrane was determined by the following formula:

(Percentage of PLTs/WBCs in fully clotted Modified [A-PRF] preparations) = (Total PLTs/WBCs concentration present in A-PRF membrane) ÷ (PLTs/WBCs in Whole Blood/Baseline) × 100

Measurement of The Size Outcome of Modified (A-PRF) Clot – Following centrifugation of 12 ml of blood, the caps of the centrifugation tubes were removed and exposed to air for 5 min to induce further clot. Thereafter, the clots were removed. The red clot attached to the yellow modified (A-PRF) clot was separated,[9] and the final modified (A-PRF) clot was measured both in width and height by the help of electronic Vernier caliper. A total surface area of clot was calculated by multiplying the width and height measurements of each individual modified (A-PRF) clot and comparison of clot dimensions sizes were done [Figure 3b and c].

Standardized and Efficient Preparation of Modified (A-PRF) Membrane – The modified (A-PRF) clots were made into membranes using the modified (A-PRF) Box (Box grid, Sialkot, Punjab) which allows the adequate preparation of homogeneous modified (A-PRF) membranes with higher growth factor content, and prevents shrinkage of the fibrin matrix architecture was shown in Figure 4a and b.

Figure 4

Preparation of Modified (advanced platelet-rich fibrin) Membrane: (a) Modified (advanced platelet-rich fibrin) clot. (b) Modified (advanced platelet-rich fibrin) membrane

Statistical analysis

The IBM Statistical Package for the Social Sciences (SPSS) Statistics 21.0, United states, was used to analyze the data collected. Statistical analyses were performed by one-way ANOVA, pairwise comparisons by Tukey's multiple post hoc procedures, and independent/Student’s/unpaired t-test. Mean, standard deviation, standard error, and percentage of mean were estimated for different variables in all the groups. Comparisons and difference between the test and control groups were carried out using one-way ANOVA, Tukey's multiple post hoc procedures were performed to evaluate pairwise difference if ANOVA was found to be significant. Comparisons within the group with gender were carried out using repeated measures independent/unpaired t-test. To achieve 85% power (instituted by G * power, version 3.0.1; Franz Faul universitat, Kiel, Germany) and detect significant differences with effect size of 0.47 (P ≤ 0.05), a total of 60 participants were required in each group.

RESULTS

This cross-sectional cytology study was undertaken with the aim of evaluating the modified A-PRF buffy coat quantitatively by determining the size outcome of the buffy coat, the platelet and leukocytes concentration, and also to find out the influence of gender on these parameters.

Size outcome of modified (advanced platelet-rich fibrin) clot

The mean average height and width of modified (A-PRF) clot for all 3 study groups are shown in Tables 1 and 2. Statistically significant differences were seen among the three groups. Hence, size outcomes obtained from uncontrolled type-2 diabetes patients showed maximum height and width of modified (A-PRF) clot when compared to tobacco smokers in test group.

Table 1

Comparison of average height of Modified (A- PRF) clot between healthy individuals, tobacco smokers and type-2 diabetic patients with chronic periodontitis

Groups	n	Mean	SD	SE
Healthy individuals	60	30.33	2.45	0.32
Tobacco smokers	60	17.12	1.72	0.22
Type-2 diabetes	60	23.39	1.69	0.22
Total	180	23.61	5.76	0.43
F	665.0207
P	0.0001*
Pair wise comparisons by Tukeys multiple post hoc procedures
Healthy individuals vs Tobacco smokers	P=0.0001*
Healthy individuals vs Type-2 diabetes	P=0.0001*
Tobacco smokers vs Type-2 diabetes	P=0.0001*

*Statistically significant (P<0.05); SD-standard deviation; SE- Standard error; P-Probability Value; F value- F test; Turkeys multiple prosthoc; one way Anova; A-Prf – Modified advanced platelet rich fibrin

Table 2

Comparison of average width of Modified (A- PRF) clot between healthy individuals, tobacco smokers and type-2 diabetic patients with chronic periodontitis

Groups	n	Mean	SD	SE
Healthy individuals	60	7.93	0.59	0.08
Tobacco smokers	60	6.66	0.58	0.08
Type-2 diabetes	60	7.26	0.16	0.02
Total	180	7.29	0.71	0.05
F	102.6981
P	0.0001*
Pair wise comparisons by Tukeys multiple post hoc procedures
Healthy individuals vs Tobacco smokers	P=0.0001*
Healthy individuals vs Type-2 diabetes	P=0.0001*
Tobacco smokers vs Type-2 diabetes	P=0.0001*

*Statistically significant (P<0.05); SD-standard deviation; SE- Standard error; P-Probability Value; F value- F test; Turkeys multiple prosthoc; one way Anova; A-Prf – Modified advanced platelet rich fibrin

Platelet concentration in modified (advanced platelet-rich fibrin) membrane

The mean baseline platelet concentration, platelet concentration of residual blood, total number of platelet concentration present in modified (A-PRF) membrane, and Percentage of platelet concentration present in modified (A-PRF) membrane are shown in Tables 3-6. Statistically significant differences were seen among the three groups, whereas baseline platelet concentration, total number of platelet concentration present in modified (A-PRF) membrane, and percentage of platelet concentration present in modified (A-PRF) membrane obtained from uncontrolled type-2 diabetes patients showed significantly higher.

Table 3

Comparison of baseline Platelet concentration (non-centrifuged) present in blood between healthy individuals, tobacco smokers and type-2 diabetic patients with chronic periodontitis

Groups	n	Mean	SD	SE
Healthy individuals	60	245.22	74.49	9.62
Tobacco smokers	60	243.03	37.65	4.86
Type-2 diabetes	60	256.93	37.39	4.83
Total	180	248.39	52.86	3.94
F	1.2026
P	0.0001*
Pair wise comparisons by Tukeys multiple posthoc procedures
Healthy individuals vs Tobacco smokers	P=0.0001*
Healthy individuals vs Type-2 diabetes	P=0.0001*
Tobacco smokers vs Type-2 diabetes	P=0.0001*

*Statistically significant (P<0.05); SD-standard deviation; SE- Standard error; P-Probability Value; F value- F test; Turkeys multiple prosthoc; one way Anova; n – Total number of participants (Sample size)

Table 6

Comparison of percentage of platelet concentration present in Modified (A-PRF) membrane between healthy individuals, tobacco smokers and type-2 diabetic patients with chronic periodontitis

Groups	n	Mean	SD	SE
Healthy individuals	60	94.55	4.23	0.55
Tobacco smokers	60	93.23	2.30	0.30
Type-2 diabetes	60	95.21	2.16	0.28
Total	180	96.33	3.14	0.23
F	6.5492
P	0.0018*
Pair wise comparisons by Tukeys multiple posthoc procedures
Healthy individuals periodontitis vs Tobacco smokers	P=0.0467*
Healthy individuals periodontitis vs Type-2 diabetes	P=0.0456*
Tobacco smokers vs Type-2 diabetes	P=0.0011*

*Statistically significant (P<0.05); SD-standard deviation; SE- Standard error; P-Probability Value; F value- F test; Turkeys multiple prosthoc; one way Anova; n – Total number of participants (Sample size); A-Prf – Modified advanced platelet rich fibrin

Leukocyte concentration in modified (advanced platelet-rich fibrin) membrane

The mean baseline leukocyte concentration, leukocyte concentration of residual blood, total number of leukocyte concentration present in modified (A-PRF) membrane, and percentage of Leukocyte concentration present in modified (A-PRF) membrane ar shown in Tables 7-10. Statistically significant differences were seen among the three groups, whereas, baseline leukocyte concentration, total number of leukocyte concentration present in modified (A-PRF) membrane, and percentage of leukocyte concentration present in modified (A-PRF) membrane obtained from uncontrolled type-2 diabetes patients showed significantly higher.

Table 7

Comparison of baseline leukocyte concentration (non-centrifuged) present in blood between healthy individuals, tobacco smokers and type-2 diabetic patients with chronic periodontitis

Groups	n	Mean	SD	SE
Healthy individuals	60	7.81	1.29	0.17
Tobacco smokers	60	7.41	0.40	0.05
Type-2 diabetes	60	7.89	0.60	0.08
Total	180	7.70	0.88	0.07
F	5.4587
P	0.0050*
Pair wise comparisons by Tukeys multiple posthoc procedures
Healthy individuals vs Tobacco smokers	P=0.0290*
Healthy individuals vs Type-2 diabetes	P=0.0849*
Tobacco smokers vs Type-2 diabetes	P=0.0056*

*Statistically significant (P<0.05); SD-standard deviation; SE- Standard error; P-Probability Value; F value- F test; Turkeys multiple prosthoc; one way Anova; n – Total number of participants (Sample size)

Table 10

Comparison of Percentage of leukocyte concentration in Modified (A- PRF) membrane between healthy individuals, tobacco smokers and type-2 diabetic patients with chronic periodontitis

Groups	n	Mean	SD	SE
Healthy individuals	60	75.34	5.13	0.66
Tobacco smokers	60	73.75	9.06	1.17
Type-2 diabetes	60	77.25	1.98	0.26
Total	180	75.45	6.25	0.47
F	4.9119
P	0.0084*
Pair wise comparisons by Tukeys multiple posthoc procedures
Healthy individuals vs Tobacco smokers	P=0.0328*
Healthy individuals vs Type-2 diabetes	P=0.0203*
Tobacco smokers vs Type-2 diabetes	P=0.0050*

*Statistically significant (P<0.05); SD-standard deviation; SE- Standard error; P-Probability Value; F value- F test; Turkeys multiple prosthoc; one way Anova; n – Total number of participants (Sample size)

Gender-based comparison of all different parameters in healthy group

The mean differences of all different parameters between healthy males and females were statistically significant. Hence, size outcomes obtained from healthy females were shown maximum height and width of modified (A-PRF) clot, and the baseline platelet and leukocyte concentration in blood, total number of platelet and leukocyte concentration present in modified (A-PRF) membrane, and percentage of platelet and leukocyte concentration present in modified (A-PRF) membrane obtained from healthy females showed maximum value when compared to males in each parameter, as shown in Table 11.

Table 11

Comparison of males and females with different parameters in healthy individuals

Parameters	Male		Female		t	P
	Mean	SD	Mean	SD
Average height of Modified (A- PRF) clot	28.25	1.10	32.40	1.46	-12.4321	0.0001*
Average width of Modified (A- PRF) clot	7.64	0.30	8.23	0.66	-4.4435	0.0001*
Baseline Platelet concentration (non-centrifuged) in blood	177.40	14.98	313.03	39.34	-17.6456	0.0001*
Platelet concentration of residual blood after removal of Choukroun’s PRF clot	16.97	1.75	3.91	2.09	26.2184	0.0001*
Total number of platelet concentration present in Modified (A-PRF) membrane	161.07	14.52	309.13	39.33	-19.3433	0.0001*
Percentage of platelet concentration present in Modified (A-PRF) membrane	90.41	0.67	98.70	0.67	-47.9982	0.0001*
Baseline Leukocyte concentration (non-centrifuged) in blood	6.59	0.21	9.02	0.56	-22.2736	0.0001*
Leukocyte concentration of residual blood after removal of Choukroun’s PRF clot	1.94	0.09	1.77	0.12	6.3091	0.0001*
Total number of leukocyte concentration present in Modified (A-PRF) membrane	4.65	0.15	7.25	0.47	-28.6375	0.0001*
Percentage of leukocyte concentration in Modified (A- PRF) membrane	70.32	0.79	80.36	0.94	-44.7285	0.0001*

*Statistically significant (P<0.05); SD-standard deviation; P-Probability Value; Turkeys multiple prosthoc; one way Anova; A-Prf – Modified advanced platelet rich fibrin; t – Independent t-test

Gender-based comparison of all different parameters in tobacco smokers group

The mean differences of all different parameters between tobacco smokers males and females were statistically significant. Hence, size outcomes obtained from female tobacco smokers were shown maximum height and width of modified (A-PRF) clot, and the baseline platelet and leukocyte concentration in blood, total number of platelet and leukocyte concentration present in modified (A-PRF) membrane, and percentage of platelet and leukocyte concentration present in modified (A-PRF) membrane obtained from female tobacco smokers showed maximum value when compared to male tobacco smokers in each parameter, as shown in Table 12.

Table 12

Comparison of male and females with different parameters in Tobacco smokers with periodontitis

Parameters	Male		Female		t	P
	Mean	SD	Mean	SD
Average height of Modified (A- PRF) clot	15.66	0.94	18.58	0.83	-12.7458	0.0001*
Average width of Modified (A- PRF) clot	6.30	0.60	7.02	0.25	-6.0432	0.0001*
Baseline Platelet concentration (non-centrifuged) in blood	220.50	29.64	265.57	30.89	-5.7658	0.0001*
Platelet concentration of residual blood after removal of Choukroun’s PRF	19.83	3.00	11.97	1.57	12.7297	0.0001*
Total number of platelet concentration present in Modified (A-PRF) membrane	200.67	26.99	253.59	30.48	-7.1212	0.0001*
Percentage of platelet concentration present in Modified (A-PRF) membrane	91.04	0.63	95.42	0.66	-26.4554	0.0001*
Baseline Leukocyte concentration (non-centrifuged) in blood	7.04	0.14	7.77	0.18	-17.8363	0.0001*
Leukocyte concentration of residual blood after removal of Choukroun’s PRF	1.93	0.11	1.77	0.07	6.5461	0.0001*
Total number of leukocyte concentration present in Modified (A-PRF) membrane	5.11	0.17	6.00	0.14	-21.5469	0.0001*
Percentage of leukocyte concentration in Modified (A- PRF) membrane	72.63	1.56	74.87	12.72	-0.9601	0.0341*

*Statistically significant (P<0.05); SD-standard deviation; P-Probability Value; Turkeys multiple prosthoc; one way Anova; A-Prf – Modified advanced platelet rich fibrin; t – Independent t-test

Gender-based comparison of all different parameters in uncontrolled type-2 diabetic group

The mean differences of all different parameters between uncontrolled type-2 diabetes males and females were statistically significant. Hence, size outcomes obtained from diabetic females were shown maximum height and width, and the baseline platelet and leukocyte concentration in blood, total number of platelet and leukocyte concentration present in modified (A-PRF) membrane, and percentage of platelet and leukocyte concentration present in modified (A-PRF) membrane obtained from diabetic females showed maximum value when compared to males in each parameter, as shown in Table 13.

Table 13

Comparison of male and females with different parameters in uncontrolled Type-2 diabetes with periodontitis

Parameters	Male		Female		t	P
	Mean	SD	Mean	SD
Average height of Modified (A- PRF) clot	21.94	0.88	24.85	0.80	-13.4253	0.0001*
Average width of Modified (A- PRF) clot	7.18	0.06	7.35	0.18	-4.8061	0.0001*
Baseline Platelet concentration (non-centrifuged) in blood	228.87	12.43	285.00	32.55	-8.8234	0.0001*
Platelet concentration of residual blood after removal of Choukroun’s PRF	15.60	1.10	7.51	1.62	22.5890	0.0001*
Total number of platelet concentration present in Modified (A-PRF) membrane	213.27	12.32	277.49	32.98	-9.9903	0.0001*
Percentage of platelet concentration present in Modified (A-PRF) membrane	93.17	0.58	97.25	0.69	-24.6867	0.0001*
Baseline leukocyte concentration (non-centrifuged) in blood	7.32	0.15	8.46	0.20	-24.8656	0.0001*
Leukocyte concentration of residual blood after removal of Choukroun’s PRF	1.79	0.06	1.78	0.09	0.1695	0.0866*
Total number of leukocyte concentration present in Modified (A-PRF) membrane	5.54	0.15	6.68	0.17	-27.5934	0.0001*
Percentage of leukocyte concentration in Modified (A- PRF) membrane	75.46	0.59	79.03	1.02	-16.6196	0.0001*

*Statistically significant (P<0.05); SD-standard deviation; P-Probability Value; Turkeys multiple prosthoc; one way Anova; A-Prf – Modified advanced platelet rich fibrin; t – Independent t-test

DISCUSSION

As per our knowledge of the literature, this study was first of its own kind which deviated the thinking of understanding the size outcome of the modified (A-PRF) buffy coat, the platelet and leukocytes concentration in a compromised individuals, i.e., uncontrolled diabetic subjects and Heavy tobacco smokers. Delayed wound healing might be a detrimental factor in the success of regeneration in a heavy tobacco smokers and uncontrolled diabetic patients. As we know that platelet and leukocytes help in wound healing. Platelet and leukocyte concentration in modified (A-PRF) membrane decide any discrepancy and alteration in regeneration process.

In the present study, the size outcome of modified (A-PRF) clots was found to be larger in type-2 diabetic patients particularly in female subjects. This was in accordance with Richard J. Miron et al.[10] where, they were found that female membranes were on average 17% larger than those of male patients. This could be because of these reasons, the role of centrifugation was to separate blood layers transitionally over time, these differences were thought to be observed due to females generally containing lower hematocrit levels in their peripheral blood compared to males.[1112] As a result, the layer separation between plasma layers was increasingly more difficult in male patients with higher hematocrit levels. Second, in elderly age, the concentration of RBCs tends to decrease. As a result, the separation of plasma layers was also shown to be more easily separated, producing larger modified (A-PRF) clot in elderly females. Based on these findings, it may be anticipated that as a general rule that females and older patients typically produce larger modified (A-PRF) clot compared to males and younger patients.

Among the hematological parameters, the total number and percentage of platelet and leukocyte concentration in modified (A-PRF) membrane were higher in the female type-2 diabetic patients. This finding was similar to the observations found in the studies of Kodiatte et al.,[13] Demirtunc et al.,[14] Zuberi et al.[15] this may be related to osmotic effect resulting from increased glucose levels and the presence of some of its metabolites in blood[16] and also because of higher levels of larger and more reactive platelets in diabetic subjects. Contrary to this finding, Hekimsoy et al.[17] showed an increased number of platelets in a group of nondiabetic patients compared with diabetic patients, this was explained maybe because of the presence of other factors such as the mean platelet survival, the platelet production, and the turnover rate in Type-2 DM.

We also found the lowest platelet and leukocyte count in tobacco smokers it was because smoking causes alterations in various hematological parameters. This was in accordance with the studies of Chow CK,[18] Muhammad[19] and Bellizzi et al.[20] However, Contrary to the present study, Salamzadeh[21] showed higher platelet and WBC count among smokers. In this study female subjects showed higher platelet and leukocyte concentration. The possible reason could be because of higher platelet and leukocyte concentration in females compared to males. The reasons for the higher concentration in females were not clear, although clues may be sought in cyclical variations in platelet and leukocyte concentration in females. Despite the fact that the platelet concentration has been described as being extremely constant in humans,[22] in the healthy female it appears to decline just before the onset of the menses and was highest at the follicular and luteal phase[23] which would coincide with the peak estrogen levels. Our findings were also in accordance with Cho et al.[24] who found that correlation between high platelet and leukocyte concentration and female gender. Secondly, female smokers reach early menopause thereby reduced levels of estrogen which causes reduced platelet activation resulting in higher average platelet count. This was in accordance with the findings of Manfreds Greene et al.[25] The reduced platelet concentration observed in males suggests that sex differences in the hemostatic system and the effects of smoking on this system may be reflected in platelet concentration.

CONCLUSIONS

The observations of present study were concluded as among the hematological parameters the total number as well as the percentage of platelet and leukocyte concentrations in modified (A-PRF) membrane was found to be higher in uncontrolled type-2 diabetic patients. And the female subjects exhibited higher platelet and leukocyte concentration than males. This study showed that with increased blood sugar levels there were changes in the concentration and distribution of platelets and leukocytes. But still, the modified (A-PRF) membrane in uncontrolled type-2 diabetics contained moderate platelets and leukocytes which might act as a potential adjunct in the future regenerative procedures. Furthermore, females may have better regenerative capacity compared to males. Therefore, it is suggested for future a long-term, randomized controlled clinical trials using larger sample sizes to clinically evaluate the efficacy of modified (A-PRF) membrane as a regenerative material in periodontal defects of uncontrolled type-2 diabetics and tobacco smokers of both genders with different age groups. This research work emphasized the influence of systemic and behavioral conditions which could alter the fibrin clot formation and its interaction of cellular components which further decide any progress in the future periodontal regeneration process.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Table 4

Comparison of Platelet concentration of residual blood after removal of Modified (A-PRF) clot between healthy individuals, tobacco smokers and diabetic patients with chronic periodontitis

Groups	n	Mean	SD	SE
Healthy individuals	60	11.56	4.30	0.89
Tobacco smokers	60	15.90	4.62	0.60
Type-2 diabetes	60	10.44	6.86	0.56
Total	180	12.63	5.85	0.44
F	17.2784
P	0.0001*
Pair wise comparisons by Tukeys multiple posthoc procedures
Healthy individuals vs Tobacco smokers	P=0.0001*
Healthy individuals vs Type-2 diabetes	P=0.0001*
Tobacco smokers vs Type-2 diabetes	P=0.0001*

*Statistically significant (P<0.05); SD-standard deviation; SE- Standard error; P-Probability Value; F value- F test; Turkeys multiple prosthoc; one way Anova; n – Total number of participants (Sample size)

Table 5

Comparison of total number of platelet concentration present in Modified (A-PRF) membrane between healthy individuals, tobacco smokers and type-2 diabetic patients with chronic periodontitis

Groups	n	Mean	SD	SE
Healthy individuals	60	235.10	80.23	10.36
Tobacco smokers	60	227.13	39.07	5.04
Type-2 diabetes	60	245.38	40.72	5.26
Total	180	235.87	56.81	4.23
F	1.5655
P	0.0211*
Pair wise comparisons by Tukeys multiple posthoc procedures
Healthy individuals vs Tobacco smokers	P=0.0557*
Healthy individuals vs Type-2 diabetes	P=0.0467*
Tobacco smokers individuals vs Type-2 diabetes	P=0.0181*

*Statistically significant (P<0.05); SD-standard deviation; SE- Standard error; P-Probability Value; F value- F test; Turkeys multiple prosthoc; one way Anova; n – Total number of participants (Sample size); A-Prf – Modified advanced platelet rich fibrin

Table 8

Comparison of Leukocyte concentration of residual blood after removal of Modified (A-PRF) clot between healthy individuals, tobacco smokers and type-2 diabetic patients with chronic periodontitis

Groups	n	Mean	SD	SE
Healthy individuals	60	1.85	0.12	0.02
Tobacco smokers	60	1.86	0.14	0.02
Type-2 diabetes	60	1.79	0.08	0.01
Total	180	1.83	0.12	0.01
F	7.3215
P	0.0009*
Pair wise comparisons by Tukeys multiple posthoc procedures
Healthy individuals vs Tobacco smokers	P=0.0944*
Healthy individuals vs Type-2 diabetes	P=0.0016*
Tobacco smokers vs Type-2 diabetes	P=0.0048*

*Statistically significant (P<0.05); SD-standard deviation; SE- Standard error; P-Probability Value; F value- F test; Turkeys multiple prosthoc; one way Anova; n – Total number of participants (Sample size)

Table 9

Comparison of total number of leukocyte concentration present in Modified (A-PRF) membrane between healthy individuals, tobacco smokers and type-2 diabetic patients with chronic periodontitis

Groups	n	Mean	SD	SE
Healthy individuals	60	5.95	1.36	0.18
Tobacco smokers	60	5.56	0.48	0.06
Type-2 diabetes	60	6.11	0.60	0.08
Total	180	5.87	0.92	0.07
F	5.9893
P	0.0030*
Pair wise comparisons by Tukeys multiple posthoc procedures
Healthy individuals vs Tobacco smokers	P=0.0463*
Healthy individuals vs Type-2 diabetes	P=0.0580*
Tobacco smokers vs Type-2 diabetes	P=0.0022*

*Statistically significant (P<0.05); SD-standard deviation; SE- Standard error; P-Probability Value; F value- F test; Turkeys multiple prosthoc; one way Anova; n – Total number of participants (Sample size)