Blood Cell Count and the Presence or Absence of Infection in Venous Ulcers Treated With Platelet-Rich Plasma

Introduction. In general, chronic wounds are colonized by bacteria; however, when microorganisms start to multiply at higher levels, wounds can become infected, causing prolongation of the inflammatory phase and retardation of collagen synthesis and epithelialization. Objective. The objective of this study was to evaluate the presence of infection in venous ulcers after 12 weeks of treatment with autologous platelet-rich plasma (PRP) and determine global white blood cell counts. Materials and Methods. This case series study involved a sequential sample of 17 patients with venous ulcers treated with PRP for 12 weeks. Descriptive and inferential statistical analysis was performed using the McNemar test and χ^² test. Results. At baseline, 10 patients (58.8%) had wound infection. During the sixth week of treatment with PRP, only 3 patients (17.6%) continued to exhibit wound infection. After 12 weeks of PRP treatment, only 1 patient (5.9%) continued to exhibit wound infection. McNemar and χ^²tests used to assess the presence of infection in the intervention group produced a P value of .0039 for a comparison of baseline and week 6 and a P value of .0078 for a comparison of baseline and week 12. These results demonstrated significant differences from baseline at both 6 weeks and 12 weeks of treatment, with greater significance at 12 weeks. There was no relationship between global white blood cell count and the presence of infection. Conclusion. After intervention with PRP, 94% of patients experienced improvement concerning the infection of ulcers.

Introduction

Chronic wounds, defined as wounds that present with delayed healing, are considered to be a serious problem worldwide and are responsible for significant morbidity and mortality.¹ Also, these wounds have a considerable economic impact,¹ accounting for expenditures of approximately $25 billion per year in the United States.² The presence of wound infection is one of the factors responsible for increased medical expenses due to the resulting use of antimicrobial agents, hospitalization, and other complications.²

In general, chronic wounds are colonized by bacteria; however, low levels of such colonization do not delay the healing process because neutrophils, monocytes, lymphocytes, and macrophages increase as part of the physiological response, thereby increasing prostaglandin E₂ levels and collagen formation.³ However, when microorganisms start to multiply at higher levels, wounds can become infected, causing prolongation of the inflammatory phase and retardation of collagen synthesis and epithelialization, resulting in tissue damage.^3,4

In addition to the classic signs of pain, edema, erythema, and heat, clinical signs and symptoms of infection in chronic lesions can also include the presence of serous exudate with simultaneous inflammation, delayed healing, discoloration in granulation tissue, friable granulation tissue, increased exudate level, odor, wound decompensation, increased pain, increased temperature in the perilesional area, erythema, and edema.⁵

One prominent challenge in wound treatment is the selection of an appropriate material for wound coverage. A potential catalyzing agent in the process of wound repair, platelet-rich plasma (PRP) has been presented as an innovation for promoting tissue regeneration and acceleration of the healing process.^6-9 In several case series, autologous PRP produced promising results in the treatment of chronic ulcers, both with and without comparison with conventional treatments^10-12; all these studies recommended cost-benefit analyses and consideration of its accessibility, biocompatibility, safety, and efficacy.

To the authors’ knowledge, few studies have evaluated the action of PRP on infected wounds. It is believed that white blood cells (WBCs) and innate immune defense peptides present in PRP may act as agents that decrease the proliferation of microorganisms.¹³ On the other hand, the formation of fibrin due to the activation of the coagulation cascade and the presence of growth factors of PRP can favor microbial growth, especially of Pseudomonas aeruginosa 48 hours after its action on the wound.¹³ However, it was not found in the literature how this microorganism uses growth factors in its cell replication process.

Further research with autologous PRP is needed to determine precise indications for clinical protocols and resolve questions regarding both the use of PRP and clinical signs of infection in a wound. The objectives of the current research were to evaluate the presence of infection in venous ulcers after 12 weeks of treatment with autologous PRP and determine WBC counts.

Materials and Methods

This case series study involved a sequential sample of 17 patients with venous ulcers treated with topically applied autologous PRP in combination with petrolatum gauze (ADAPTIC; 3M)and compression therapy. The follow-up time was 12 weeks. The study site was the outpatient wound repair clinic of a federal and university-affiliated public hospital in the state of Rio de Janeiro, Brazil. The data collection period was from August 2016 to December 2017. The research protocol complied with the Declaration of Helsinki, was approved by the research ethics committee of the university’s faculty of medicine, and respected the principles established by the Ministry of Health of Brazil.

Before study participation, all participants provided informed consent; afterwards, the eligibility criteria (inclusion/exclusion) were assessed. The patients who met the inclusion criteria were subjected to an initial visit to start the treatment with PRP. The patients included in the study had not used PRP before.

Inclusion criteria consisted of the following: 18 years and older, without distinctions based on sex; a venous ulcer greater than 2 cm² but less than 100 cm²; the presence of wounds with an evolution time of more than 12 weeks; hematocrit greater than 34%; hemoglobin greater than 11 g/dL; and platelet count greater than 150000/mm³.

Exclusion criteria included the following: if the patient was pregnant or breastfeeding; alterations in prothrombin activation time or partial thromboplastin time; current use of corticosteroids; current immunosuppressive treatment or presence of an immunosuppressive disease; suspected malignancy of the ulcer; nonadherence to the proposed treatment plan; if the patient received a blood transfusion in the preceding 3 months; and presence of a circular ulcer.

All patients satisfied the inclusion criteria; there were no instances of discontinuation of treatment nor any treatment-related adverse events.

The patients included in the study were assessed by the dermatologist of the research team, and the patients had no indication for antibiotic therapy. The patients attended the outpatient clinic weekly for nursing consultation and dressing changes, with PRP application administered every 15 days.

Platelet-rich plasma dressing application

Each wound was first irrigated with 0.9% saline. The dressing application of PRP on the wound bed created a thin layer (1 mm–2 mm).¹⁴ After 5 minutes to 8 minutes, the wound was covered with dry sterile gauze; periwound hydration was kept with NDERM cream (Viemed). The dressing was fixed using cotton bands; compression therapy was the last layer of the bandage fixing.

Every 24 hours, the dressing was changed according to the home protocol. The wound was irrigated with 0.9% saline, dressed in sterile petrolatum gauze and ADAPTIC TOUCH Non-Adhering Silicone Dressing(3M),and supported by dry sterile gauze. The hydrating cream was used for periwound hydration once every 24 hours. The dressing was fixed using cotton bands and finished with compression therapy.

Mechanical debridement was performed only at the outpatient clinic, as necessary. All patients received kits containing dressing materials (ie, the sterile petrolatum gauze, nonadherent silicone dressing, sterile dry gauze, hydrating cream, cotton bands, and compression materials) in addition to written guidelines regarding daily dressing application at home.

Clinical specimens were collected a total of 3 times (once each on week 1, week 6, and week 12) by the nurses on the present research team by swabbing the venous ulcer using the quantitative swab culture and smear as described by Levine et al.¹⁵

For the microbial analyses, swabs were collected and added to Stuart transport medium, placed in 2.5 mL of sterile saline (0.9%), and then vortexed. Aliquots of approximately 0.5 mL were added to 2.0 mL of 2X tryptic soy broth (TSB), then incubated at 35°C (± 2°C) for 24 hours to 48 hours. After incubation, tubes showing a turbid culture medium were seeded on mannitol salt agar and cetrimide agar to determine the presence of Staphylococcus aureus and P aeruginosa, respectively. After incubating at 35°C (± 2°C) for 24 hours to 48 hours—ideal time for bacterial growth—the plates were analyzed for colony growth and characteristics. Saline and TSB samples were frozen in cryoprotective medium.

Suspected colonies were removed using an inoculation loop for identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Microflex LT; Bruker Daltonics).

The McNemar test and χ^² test with a significance threshold of 5% were performed to evaluate infection improvement during the 12 weeks of treatment. Clinical signs of infection were identified using an outpatient wound protocol involving evaluation of the presence of pain, odor, purulent exudate, edema, erythema, and heat (Table 1). In accordance with the Infectious Diseases Society of America¹⁶ guidelines for defining infection of diabetic wounds and wounds associated with other chronic diseases, the presence of infection in a wound was determined based on the presence of purulent exudate or 2 or more classic signs of inflammation (ie, heat, pain, erythema, and/or edema).

White blood cell counts were obtained during the screening visit and on the last day of the study (ie, before and after 12 weeks of treatment with autologous PRP). Minimum and maximum leukocyte counts of 4500/mm³ and 10500/mm³, respectively, were used to define the normal range; these values were the reference values utilized at the institution where the study was performed.

Results

Of the 17 included patients, 10 (59%) were male. With regard to age, 10 patients (59%) were aged 60 years to 80 years, followed by 6 patients (35.4%) aged 50 years to 59 years and 1 patient (5.9%) between 40 years and 49 years. Only 3 patients (18%) reported alcohol misuse, and 4 patients (23.5%) reported being smokers. Wounds were predominantly located in the malleolar region (72.2%), followed by the dorsum of the foot (27.7%). For all patients, current lesion onset had started more than 5 years ago. The average area of ulcers was 29.6 ± 23.9 cm².

At baseline, 10 patients (58.8%) had wound infection. Wound infection was detected in 3 patients (17.6%) and 1 patient (5.9%)during week 6 and week 12 of treatment, respectively. Complete wound healing was achieved after 12 weeks of treatment in 3 patients (18%).

The McNemar test and χ^² test revealed significant differences in the presence of infection between baseline and the sixth week of treatment (P = .0039) and between baseline and the 12th week of treatment (P = .0078).

Three microbial collections were performed (on week 1, week 6, and week 12) on the 17 included patients, yielding 48 total samples; 5 patients' ulcers healed before completing the 90 days of treatment and thus were not swabbed after complete closure of wound. Of the total samples, 22 were positive for P aeruginosa. Of the P aeruginosa samples, 6 were positive from the first collection, 8 from the second collection, and 8 from the third collection. Regarding S aureus, only 6 samples (10%) were positive for this microorganism. For patients with S aureus, 2 samples were positive for this bacterium from the first, second, and third collections.

In the context of leukocyte cells, no differences were observed between the numbers of different leukocyte cells detected in WBC counts before and after treatment. White blood cell counts produced normal values for most of the patients, as indicated in Table 2A and Table 2B.

Only 1 (6%) of the 2 (12%) patients who had elevated leukocytes before treatment endured wound infection; for this patient, infection was no longer present by week 6 of treatment, and leukocyte counts normalized. For the other patient, leukocytes remained above the reference range, even after treatment when the wound had already healed (Table 2A and Table 2B).

Discussion

Upon tissue injury, an inflammatory process immediately begins. Initially, there is acute inflammation, which consists of the body’s reaction to harmful agents such as microorganisms and damaged cells and entails the triggering of vascular responses, leukocyte migration/activation, and systemic reactions¹⁷. When the acute inflammatory response does not resolve in a timely manner, typically due to persistence of the noxious agent or interference with the normal healing process, a transition to the chronic inflammatory response occurs.¹⁸ Chronic inflammation is also referred to as slow long-term inflammation, lasting for prolonged periods of several months to years.¹⁸

The proliferation of microorganisms will trigger a response that involves the entire body, with clinical manifestations such as heat, erythema, edema, purulent exudate, and pain and/or systemic signs, including fever and leukocytosis, although such signs are often absent. In cases involving infected chronic wounds, including venous ulcers, the infection is usually localized to the wound; therefore, there are no systemic signs. In such cases, evaluations need to be targeted to local clinical characteristics.^3-5

Blood changes (qualitative and quantitative) occur secondary to local or systemic pathological processes. Hemograms can be used to assess peripheral blood and reflect a patient’s status at the moment of sample collection. Analyses of changes in leukocytes refer to production, release, and transit of these cells through the peripheral blood toward the target tissue.¹⁷ However, an increased level of leukocytes in the blood does not necessarily indicate the presence of infection; therefore, the clinical signs and symptoms that a patient presents should be considered when interpreting test results.¹⁷ In this sense, a hemogram should be used as a complementary datapoint to clinical evaluation that may aid evaluation of the intensity of a pathological process. Notably, good characterization of a wound is the first step in decision-making regarding the type and continuation of treatment.

Platelet-rich plasma presents itself as an intervention that allows the local application of growth factors in the wound, which stimulate the production of collagen and extracellular matrix through small amounts of plasma; PRP is a promising alternative in cases where conventional treatments were not successful.¹⁹The conventional treatments considered in this study were wound irrigation with saline followed by the application of a nonadherent dressing to provide a moist healing environment and use of compression therapy.¹²

Growth factors are members of a large group of polypeptides secreted by various regulatory molecules in the body. They act as mediators in cell maturation and are responsible for tissue damage repair processes.²⁰ Among the growth factors released by the platelets contained in plasma, transforming growth factor beta, vascular endothelial growth factor, growth factor of fibroblasts, platelet-derived growth factor, and epidermal growth factor can aid in tissue repair.

Growth factors can favor the repair of the injuries and enable the quickest return to functionality, possibly by stimulating neovascularization, which improves the blood supply and provides necessary nutrients for tissue regeneration.^21,22 The promising impact of application of platelet-derived growth factors to chronic wounds has been indicated in the literature,²³ which at the present time remains limited.

In the present study, the use of autologous PRP improved the signs of infection. It is believed that WBCs and innate immune defense peptides present in PRP may act as agents that decrease the proliferation of microorganisms.¹³ Thus, by reducing the microbial load in wounds, PRP treatment may reduce wound colonization and infection, favoring the process of physiological tissue repair. According to Silva et al,²⁴ purulent exudate differed from the exudate produced from debridement.

It is noteworthy that studies have identified PRP as a protective factor against wound infection; in particular, in vitro experiments have demonstrated antimicrobial effects of PRP on methicillin-resistant Staphylococcus aureus, methicillin-sensitive S aureus,^25,26Escherichia coli, Enterobacter cloacae, Bacillus cereus, and Bacillus subtilis.¹³ Thus, for wounds colonized by these microorganisms, microbial load could be reduced via the topical application of PRP.

Limitations

The main limitation of the study is related to the fact that it is a series of cases that make some inferences impossible.

Conclusions

Topically applied autologous PRP in chronic venous leg ulcers improved the wound infection between the first and sixth weeks and the first and 12th weeks of treatment and did not stimulate the growth of microorganisms. Leukocyte counts higher than the reference range were not related to wound infection. The authors suggest conducting further research with a larger number of patients.

Acknowledgments

Authors: Beatriz Guitton Renaud Baptista de Oliveira, DN, RN¹; Joyce Beatriz de Abreu Castro, MSN, RN²; Bruna Maiara Ferreira Barreto Pires, RN, PhD¹; Márcia de Assunção Ferreira, DN, RN²; Jane Marcy Neffá Pinto, PhD, MD¹; and Lenise Arneiro Teixeira, DSC, BF¹

Affiliations: ¹Universidade Federal Fluminense, Rio de Janeiro, Brazil; ²Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Correspondence: Joyce Beatriz de Abreu Castro, MD, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; joycebeatrizcastro@hotmail.com

Disclosure: This study was supported financially by the National Council for Scientific and Technological Development of Brazil. The authors disclose no financial or other conflicts of interest.

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Abstract