his study was conducted under the supervision and approval of the Health Science Ethics Review Board at Western University, London, Ontario, Canada.
Inclusion and exclusion criteria. Study participants were recruited from the Spinal Cord Injury and Chronic Wound Management Clinics in the outpatient department of a Rehabilitation Hospital. The medical records of individuals with nonhealing ulcers (present for >3 months) as a result of SCI were screened for ACD, defined as hemoglobin <110 g/L with normal red cell indices and normal or elevated ferritin. Persons with 1 or more nonhealing ulcers and low hemoglobin levels were contacted by a member of that patient’s direct medical care team and informed about the study. Potential participants had to be able to provide informed consent. Exclusion criteria included current use of rhuEPO or a medical condition that contraindicates the use of rhuEPO, including uncontrolled hypertension, a history of developing pure red cell aplasia (PRCA) following treatment with any erythropoiesis-regulating hormone, known hypersensitivity to mammalian cell-derived products such as human albumin, or inability for any reason to receive adequate antithrombotic treatment. Some versions of rhuEPO are formulated with human albumin, and cases of erythropoietin-induced hypertension have been reported,10 presenting an increased risk factor even though the type of rhuEPO used in this study did not contain any human albumin. PRCA complications as a result of rhuEPO have been known to occur. These complications are considered to be the result of using prefilled syringes with uncoated rubber stoppers that produced leachates and potentiated the immunogenicity of the product; modern reformulations of rhuEPO have correlated with a steady decrease in PRCA incidence.11 As rhuEPO stimulates red blood cell production, risk of clotting is potentially increased, necessitating treatment with an antithrombotic treatment.
Because this was a prospective, descriptive pilot case series, no calculation or expectation was placed on population size.
Study design. Patients recruited into the study underwent an initial baseline assessment of hematological status, wound characteristics, and level of inflammatory mediators. Prevalence data were estimated from the screening data. Following baseline assessment, a 2-week standardization period was initiated to optimize wound dressings, improve nutritional status, maximize pressure redistribution strategies, and stabilize comorbid medical conditions. All patients involved in this study were seen in the Regional Spinal Cord Injury Program and provided occupational therapy and physiotherapy assessments, which included a seating clinic. All recommendations made by the authors in this study were done in accordance with the Canadian Association of Wound Care Best Practice Recommendations, which are based on the Registered Nurses Association of Ontario Clinical Practice Guidelines.12
Each participant received a dietary assessment by the study personnel following the aforementioned guidelines. In addition to providing iron supplementation, determined based on laboratory results, the study staff consulted with the Regional Spinal Cord Injury Program dietitian who worked with them to maximize protein and calorie status consistent with participant wishes. Upon completion of the standardization period, blood values and wound status were assessed again to establish pre-rhuEPO treatment levels of ACD, inflammatory markers, and wound status. If hemoglobin rose above 110 g/L, the participant was considered a screen failure. Wound status was assessed using the Photographic Wound Assessment Tool13 (PWAT) and by measuring size and depth. These measurements were taken at 2 weeks and 5 weeks after commencement of rhuEPO treatments and at 3 months after rhuEPO treatments were completed. Tissue swabs were taken from the wound bed using a swab-collection technique described by Wyffels et al14 at each data point for analysis of VEGF and inflammatory mediator levels of IL-1, IL-6, and TNF-α. A minimum of 4 swabs was taken for each time point, covering all areas of the wound bed post-dressing removal and before wound bed irrigation and debridement.
Treatment. All patients received regular care (physiotherapy, occupational therapy, and seating assessments) in addition to the rhuEPO treatments. All ulcers were debrided and bandaged to usual treatment standards.12 Patients were treated by their regular nurses in a home care setting. These nurses provided regular wound care and dressing changes and administered the rhuEPO injections. Patients enrolled into treatment were given a course of rhuEPO injections of 75 IU/kg, 3 times per week, for 6 weeks. Study participants were required to visit the clinic 6 times: once for the initial screening, once for enrollment into the study, twice during the treatment phase, and twice post-treatment (1 week and 10 weeks post-treatment). Depending on the requirements of the patient, an iron supplement may have been prescribed. Because this study was designed to investigate the possible effects of rhuEPO on the healing of chronic wounds and these patients already were involved in a program to help support their regular health care needs, additional comorbid conditions were not investigated in this study.
Outcome measures. Outcomes were measured in 3 different domains: laboratory testing, changes in wound size and appearance, and protein analysis of wound fluid.
Laboratory testing. In order to determine anemia, complete blood count, serum ferritin, serum iron, total iron binding capacity, serum vitamin B12, and serum folate levels of circulating blood all were measured and observed. C-reactive protein (CRP), an acute phase reactant that is elevated in the presence of inflammation or infection, also was measured as an additional assessment of the possible change in the patient’s inflammatory status. Prealbumin, creatinine, and electrolytes also were assessed to monitor nutritional status.
Wound size and appearance. Changes in wound surface area were obtained using acetate tracings and calculated with a digitizing tablet (Visitrak™, Smith and Nephew Canada), which calculates wound area using an acetate tracing of the wound bed. Wound depth was determined by measuring the maximum depth a sterile cotton-tipped swab could be inserted into the wound and make contact with the deepest point. Wound bed appearance was assessed using the PWAT.14
Protein analysis. In order to monitor possible changes in cytokine and growth factors in the wound bed, IL-1, IL-6, TNF-α, and VEGF present in the wound fluid at the time of the assessments were measured using swabs taken each visit at the base and edges of the wound.
Data collection and analysis. Participants enrolled in the study were under observation for 20 weeks; data were collected at week 0 (baseline) and at weeks 2, 5, 8, and 20 throughout the study. All study data were anonymized and patients were given study numbers upon enrollment. Laboratory data and wound measurement data (including the PWAT) were collected and stored onsite in a secure location. PWAT measurements were taken onsite using previously established guidelines,13 and wound measurements were taken using the aforementioned measurement system. Analyses of the wound measurements, including averages and standard deviations, were performed using Microsoft Excel™. Laboratory samples were evaluated and assessed by the London Laboratory Services (London, Ontario). Wound fluid swabs for cytokine data were collected and stored at -80˚ C in buffered solution according to previously established guidelines.13
Cytokines were assessed in a multiplex protein; analyte assay was performed by an offsite facility for these specific targets. Concentrations of each protein were standardized using total sample protein concentration. Analysis was performed using the R statistical software package, version 3.0.2 (R Foundation for Statistical Computing, Vienna, Austria).
Comparisons of analyte concentrations are presented as relative quantities (ratios) for each time point (weeks 2, 5, 8, and 20) relative to week 0. Ratio calculations and graphs were developed using Microsoft Excel™.