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ORIGINAL ARTICLE
Year : 2019  |  Volume : 32  |  Issue : 1  |  Page : 261-266

Platelet-rich plasma in lower limb ulcers


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shebein El-Kom, Menoufia Governorate, Egypt
2 Department of General Surgery, Faculty of Medicine, Menoufia University, Shebein El-Kom, Menoufia Governorate, Egypt

Date of Submission18-Jul-2017
Date of Acceptance27-Aug-2017
Date of Web Publication17-Apr-2019

Correspondence Address:
Abeer M Basiony
Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shebein El-Kom 32511, Menoufia Governorate
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_495_17

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  Abstract 


Objective
The aim of this study was to evaluate the role of platelet-rich plasma gel in patient with lower limb ulcers: diabetic and varicose ulcers.
Background
Diabetic foot ulcer is a major complication of diabetes mellitus. Platelet-rich plasma (PRP) is used for managing diabetic foot ulcers. PRP contains alpha- granules that release growth factors on activation such as platelet-derived growth factor (PDGF) which are important for wound healing by being angiogenic and helping stem cell differentiation and maturation.
Patients and methods
The study included 30 patients with chronic ulcers who were selected from vascular surgery department of Menoufia University Hospitals in the duration between February 2016 and March 2017. Patients were categorized into group I (15 patients with diabetic ulcers) and group II (15 patients with varicose ulcers). PRP gel was applied every 2 weeks for a maximum of 6 weeks. PRP was evaluated for concentration of PDGF-AB before and after activation by immunoassay.
Results
There was significant increase in PRP platelets count and PDGF-AB concentration after activation (P < 0.001). PRP platelets count and PDGF-AB concentration after activation were significantly higher in patients with completely healed ulcers than those with incomplete healing (P = 0.04 and 0.03, respectively). Healing rate percent showed significant positive correlation with activated PRP platelets count and PDGF concentration (r = 0.514, P = 0.004 and r = 0.525, P = 0.003, respectively).
Conclusion
Autologous PRP gel is an efficient and a safe source of growth factors for managing lower limb ulcers.

Keywords: diabetic foot, platelet-derived growth factor, platelet-rich plasma, varicose ulcer


How to cite this article:
El-Edel RH, Noreldin RI, Basiony AM, El-Khateep YM. Platelet-rich plasma in lower limb ulcers. Menoufia Med J 2019;32:261-6

How to cite this URL:
El-Edel RH, Noreldin RI, Basiony AM, El-Khateep YM. Platelet-rich plasma in lower limb ulcers. Menoufia Med J [serial online] 2019 [cited 2019 May 27];32:261-6. Available from: http://www.mmj.eg.net/text.asp?2019/32/1/261/256120




  Introduction Top


Diabetes mellitus (DM) is a multifactorial, metabolic disease characterized by deficient production and/or action of insulin [1]. Diabetic foot ulcer (DFU) is a major drawback of DM and usually fails to heal in spite of standard care [2].

DFU results from combination of neuropathy, peripheral vascular disease, and foot deformity. Peripheral neuropathy, being the greatest risk of foot ulceration, presents in 80% of DFUs [3]. It is characterized by variable degrees of impairment in autonomic, sensory, and motor functions [4]. Sensory neuropathy leads to repetitive trauma to the foot; autonomic neuropathy changes skin consistency, accelerating ulcer formation; and motor neuropathy causes atrophy of the foot muscles, resulting in deformity in toes [5], which usually leads to developing atypical walking patterns and finally ends with ulceration at the points of increased pressure [6]. Ischemic manifestations present in 40–50% of patients with diabetic foot, with the most having neuro-ischemic ulcers [7].

Actively applied appropriate treatment is mandatory for managing DFU including antimicrobial and physical therapy, offloading and debridement, biological therapy applying growth factors, and finally regenerative medicine applying human skin equivalent, recently derived from stem cells, to the ulcer site. Platelet concentrates are considered as an appropriate method to deliver growth factors from platelets to ulcer site, helping in wound healing [8].

Platelet concentrates are pooled from whole blood or from platelet apheresis to make a therapeutic dose. Being an autologous method, PRP is biocompatible and safe procedure for treating nonhealing ulcers [9] providing the required growth factors needed for wound healing [10]. Platelets have α granules, which on activation release transforming growth factor β, stimulating deposition of extracellular matrix and platelet-derived growth factor (PDGF), a potent chemotactic agent [11], being the most important factor involved in wound healing. They are important in regulating mesenchymal stem cells, macrophages, fibroblasts, and smooth muscle cells attraction and recruitment to the wound site and extracellular matrix synthesis during the healing process [12].

Our study aims to evaluate the therapeutic role of applying autologous platelet-rich plasma gel for nonhealing DFUs and varicose ulcers.


  Patients and Methods Top


This study was conducted on 30 subjects: 15 with DFUs as group I and 15 with varicose vein ulcers as group II was included as a comparative group. The present study included varicose vein group as a comparative group and did not use a control group such as use of saline gel or sterile gauze like in other studies that have examined the effect of PRP gel application on lower limb ulcers of different etiologies. Patients with superficial clean ulcers not extending to deep soft tissues were included in our study, whereas patients with thrombocytopenia (platelets count <100 000/μl) or deep or infected ulcers or at risk for amputation were excluded from the present study. Small sample size in the present study was because of difficulty to find cases with the all provided inclusion criteria in Egyptian society and as we did not involve different hospitals or medical centers in our study. The study protocol was approved by the ethical committee of the Faculty of Medicine, Menoufia University, and informed consents were taken from the patients before start of the study. For all patients, the followings were done: history taking, clinical evaluation of the ulcers, complete blood count (CBC), preparation of PRP and quantification of platelet count in it, preparation and application of PRP gel on ulcers and quantification of platelet-derived growth factor (PDGF-AB) in PRP prepared before and after activation, using calcium chloride (CaCl2), by sandwich-based enzyme-linked immunosorbent assay kits.

Sampling

Overall, 25 ml of venous blood was aspirated from cubital vein by wide-bore cannula after sterilization and divided into three parts: 20 ml of blood collected in four sterile tubes containing 750 μl acid citrate dextrose (Terumo BCT, Lakewood, Colorado, USA) for preparing PRP, 3 ml blood was collected in plain vacutainer for preparation of autologous thrombin and random blood sugar estimation by automated AU680 analyzer (Beckman Coulter Inc; Brea, California, USA), and 2 ml blood was collected in EDTA tube for CBC and glycated hemoglobin estimation (Stanbio Laboratory Inc., San Antonio, Texas, USA).

Methodology

Complete blood count

CBC was done for quantifying baseline platelets, using Automated Sysmex XN-10Hematology Analyzer (Sysmex, Kobe, Japan). PRP preparation included centrifugation of blood collected on acid citrate dextrose at 200g for 10 min to separate plasma that was collected in sterile plain tubes. Then separated plasma was centrifuged at 1200g for 10 min. The supernatant containing platelet-poor plasma was discarded from each tube. Platelet pellet at the bottom of tubes was mixed well with plasma to form PRP. Autologous thrombin, used for gelling PRP, was prepared by centrifuging blood sample collected in plain tube after being clotted for 5 min at speed 2000 g. PRP gel was prepared by adding CaCl210%, to release growth factors, and thrombin in sterilized Petridish in a proportion of PRP: thrombin: CaCl2= 5: 2: 1. The formed gel was applied to the ulcer after being debrided, and then ulcer was covered by sterile nonabsorbable dressing followed by sterile absorbable one for 4 days. PRP gel was applied every 2 weeks until healing or maximum of 6 weeks.

Patient follow-up

Patients were clinically evaluated before treatment and every 2 weeks from first application of PRP gel for a maximum of 6 weeks. Healing rate percent was calculated for each patient: Healing rate (%) = [(First surface area − last surface area)/( first surface area)] × 100] [13].

Quantitation of platelet-derived growth factor

PRP samples were stored at −80°C until time of assay. Samples were brought to room temperature before use. During assay, each sample was divided into two parts: one for assessing PDGF-AB before activation and the other part used after adding CaCl2 to assess PDGF-AB after activation. Calculation of growth factor was done by using indirect sandwich enzyme-linked immunosorbent assay by the provided protocol through kits supplied from Ray Biotech (Norcross, Georgia, USA). A capture antibody traps the target antigen on solid surface after incubation and then biotin-labeled detection antibody is added. The formed complex can be visualized through adding enzyme-linked antibody (streptavidin–horseradish peroxidase). The reading was done at wavelength of 450 nm.

Statistical analysis

The data collected were tabulated and analyzed by statistical package for social science, version 22.0 (IBM Corp., Armonk, New York, USA) on IBM compatible computer. Two types of statistics were done:

  1. Descriptive statistics: for example: percentage, mean, median, and SD
  2. Analytic statistics: Student's t-test, Mann–Whitney test (U) (nonparametric test), paired t-test, Wilcoxon signed-rank test (nonparametric test), and Spearman's correlation.



  Results Top


Diabetic patients (group I) included 60% males and 40% females. Their mean age was 52.3 ± 9.9 years, with 53.33% of them having comorbidities involving hypertension, varicose veins, systemic lupus erythromatosis, and chronic renal failure. Varicose vein patients (group II) included 73.3% males and 26.7% females. Their mean age was 35.3 ± 11.4 years, with 26.67% of them having comorbidities including hypertension and chronic renal failure [Table 1].
Table 1: Demographic and clinical characteristics of studied groups

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Among all cases, there was significant increase in PRP platelets count above baseline whole blood count, which was 2.7 times for diabetic ulcers group and 3.3 times for varicose ulcers group (P < 0.001). Moreover, PDGF-AB concentration significantly increased after activation (P < 0.001) [Table 2].
Table 2: Statistical comparison between baseline platelet and platelet-rich plasma platelet count, platelet-derived growth factor before and after activation within studied groups

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Regarding healing progress in studied patients, there was significant change in mean surface area in between follow-up visits for group I, being 9.6 cm 2 in the first visit to be 3.9 cm 2 in the third visit (P = 0.001), and for group II, being 3.9 cm 2 in the first visit to be 1.8 cm 2 in the third visit (P = 0.001) [Table 3] and [Figure 1]e.
Table 3: Comparison of surface area of ulcers at different visits among studied groups

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Figure 1: Reduction in surface area over follow-up period (a-e), patient with incomplete healing (f and g) and patient with complete healing (h and i).

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Among all cases, there was 63.1 ± 35.4% reduction in surface area, with 33.3% of all cases achieving complete healing, being more in diabetic group (40%) than in varicose group (26.7%) within the follow-up period (100% reduction in surface area, final surface area = 0 cm 2), and 66.7% of them achieving incomplete healing (final surface area >0 cm 2) [Figure 1]a, [Figure 1]b, [Figure 1]c, [Figure 1]d, [Figure 1]e, [Figure 1]f, [Figure 1]g, [Figure 1]h, [Figure 1]i.

Our study showed that healing rate percent had significant negative correlation with duration of original disease [DM or varicose vein (P < 0.001 and P = 0.002, respectively; r = −0.85 and −0.74, respectively], whereas a nonsignificant negative correlation with duration of ulcers in diabetic group and varicose group was observed [Table 4].
Table 4: Correlation between healing rate percent and either of; duration of disease, duration of ulcers platelet-rich plasma platelet count, platelet-derived growth factor after activation, mean random blood sugar, and glycated hemoglobin levels within studied groups

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Healing rate percent had significant positive correlation with PRP platelets count and PDGF concentration after activation for all cases (P = 0.004, r = 0.514 and P = 0.003, r = 0.525, respectively) [Table 4].

Regarding glycemic control in diabetic group, glycated hemoglobin percentage and mean random blood sugar for the three visits of follow-up showed significant negative correlation with healing rate percent (P = 0.006, r = −0.766 and P = 0.001, r = −0.670, respectively) [Table 4].

Among all cases, PRP platelets count and PDGF concentration after activation in patients with complete healing were significantly higher than patients with incomplete healing (P = 0.04 and 0.03, respectively) [Table 5].
Table 5: Statistical comparison between complete and incomplete healing in all cases regarding platelet-rich plasma platelet count and platelet-derived growth factor concentration after activation

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  Discussion Top


Knighton et al. [14] performed the first clinical study that demonstrated the promising role of locally acting factors derived from autologous blood in promoting healing of chronic cutaneous ulcers.

Successful PRP application is well known to minimize the risk of limb amputation, and if there are associated critical situations, critical limb ischemia or infection in nonhealing chronic ulcers. Our study included patients with noncritical situations (superficial clean ulcers without critical limb ischemia) and that agreed with the studies by Frykberg et al. [15] and Margolis et al. [16] whereas Slesaczeck et al. [17] included cases with critical situations and showed significant reduction in risk of amputation of lower limb owing to DFUs. Therefore, the present study did not comment on therapeutic effect of PRP on minimizing amputation risk.

Our study showed significant increase in PRP platelets count above baseline level for all cases after two spins centrifugation: first spin at 200g and second spin at 1200g. Nagataa et al. [18] used 10 ml blood centrifuged twice: first spin at 160 g for 20 min and then second spin at 400 g for 15 min, concentrating platelets 4.5 times above baseline, which was close to our results.

Araki et al. [19] centrifuged 50 ml whole blood twice: first spin at 1600 rpm for 10 min and second spin at 4600 rpm for 10 min and produced six times increase above baseline level, which was higher than our results.

Sarvajnamurthy et al. [20] prepared PRP in two-step centrifugation process: first centrifuged 20 ml blood at 500 rpm for 15 min and then 2000 rpm for 5–10 min. This method increased platelets 1.5 times above baseline, which was lesser than ours.

There was significant increase in PDGF-AB concentration after activation. The same was indicated by Sundman et al. [21] as well as Marx [22]; however, Weibrich et al. [23] found that there was no significant correlation between the platelet count in PRP and whole blood.

The present study showed reduction in surface area (63.1 ± 35.4%) among all cases of different etiologies, with 64.2 ± 35.2% for diabetic group, which was more than that of varicose group, 62.0 ± 36.8%, with significant change in surface area over follow-up visits. For all etiologies, 93.3% of wounds showed positive response, and 33.3% of all cases achieved complete healing within three treatments over 6 weeks. Frykberg et al. [15] showed 97% of wounds of different etiologies, without using control group, improved over 3 weeks of reduction in wound area (51 ± 43.1%) with application of three times. Our results were better because of longer duration of follow-up and smaller surface area of ulcers in our study.

De Leon et al. [24] showed that positive response occurred in 86.5% of ulcers of different etiologies within 2.2 weeks and 2.8 applications with 47.5% area reduction occurred. Our results were better owing to longer follow-up duration.

Regarding duration of original disease (DM or varicose vein), the present study showed significant negative correlation with healing rate percent for diabetic group as well as varicose group. That was may be explained by the longer duration of disease, the more pathological changes had occurred related to pathogenesis of the disease which by its personal way contribute to occurrence of ulcers in patients. Oyibo et al. [25] as well as Musa and Ahmed [26] and Margolis et al. [27] found that there was no significant correlation between duration of diabetes or varicose vein and healing rate.

Regarding effect of duration of ulcers on healing rate, the present study showed nonsignificant negative correlation between healing rate percent and duration of ulcers for both groups. That may be because of short duration of all ulcers (<12 months). The studies conducted by Patel et al. [28], Marilia et al. [29], and Margolis et al. [16] showed that ulcer healing rate was higher in cases whose duration of ulcer was within 1 year than ulcers longer than 1 year, and it was explained by time-dependent changes occurring in the ulcer microenvironment making healing process difficult to be achieved.

Regarding the effect of platelet count in PRP and PDGF on healing rate, the present study showed that the more the platelets count in prepared PRP, there will be more PDGF concentration and the better the healing rate, which was agreed with Wieman et al. [30] as well as Loay et al. [31].


  Conclusion Top


The present study showed 93.3% response with variable degrees of surface area reduction. Among all cases, reduction in surface area was 63.1% with 33.3% of them achieving complete healing over follow-up period. There was significant increase in platelets count after PRP preparation and in PDGF concentration after activation. Healing rate had significant positive correlation with platelet count in PRP and PDGF concentration after activation, whereas significant negative correlation with duration of original disease. From the previously mentioned results of the present study, it can be concluded that application of PRP after activation is an enriched source of PDGF-AB and an efficient procedure for treating ulcers especially if there is consequent good control of original cause of ulcers and if it is applied frequently.

Acknowledgements

This study is partially funded by Clinical Pathology Department, Faculty of Medicine, Menoufia University.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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