Study Design
Clinical trial of superiority, randomized, double-blind, placebo-controlled, parallel, with 3 groups with a 1:1:1 allocation ratio. We have compared two active treatment groups, one receiving PRP and the other receiving plasma only, with a control group that received saline. Two ultrasound-guided joint injections were performed with a 2-week interval and outcomes were evaluated at week 24, with intermediate assessments at weeks 6 and 12. We have followed the Osteoarthritis Research Society International (OARSI) recommendations for KOA trials in the conduct of this study(21)ClinicalTrials.gov, NCT03138317, with first date of registration 03/05/2017.
Sample
The study was carried out at the Hospital das Clinicas Rheumatology Department of the University of São Paulo (HC-FMUSP), a tertiary hospital in São Paulo, Brazil. The sample consisted of participants diagnosed with KOA in rheumatology outpatient clinics and external participants advertised by word of mouth from the community and screened by telephone and then personally at the clinic. All participants were instructed about the study and signed an informed consent form.
The following criteria were used for inclusion: 1) men and women aged 45 to 80 years; 2) fulfill criteria for KOA of the American College of Rheumatology(22); 3) radiographic grade 2 or 3 scored by the Kellgren and Lawrence (KL) (23) in at least one knee; 4) pain from 3 to 8 cm in the visual analogue scale 0-10 cm (VAS) in at least one knee in the last week. Knee x-rays obtained within 6 months before allocation were accepted. The knee selected for treatment was the one reported with higher pain score as reported by the participant.
Exclusion criteria were: 1) use of analgesics, non-steroidal anti-inflammatory drugs, myorelaxants and systemic glucocorticoids within one week to allocation; 2) use of slow acting drugs for OA (such as chondroitin, glucosamine, diacerein) started within 8 weeks to allocation. For participants using these drugs for longer than 8 weeks, they could be maintained until the end of the study; 3) corticosteroids or HA intra-articular injection in the index knee within 6 months to allocation; 4) intra-articular injection of any drug in any other joint within 1 month to allocation; 5) introduction of any medical or physical intervention for the locomotor system within the last 3 months (exercise, acupuncture, cane, orthotics etc.); 6) KL 4 in any of the knees; 7) body-mass index (BMI) ≥ 35 kg/m2; 8) fibromyalgia and inflammatory arthropathies such as rheumatoid arthritis, connective tissue diseases, microcrystalline arthropathies, spondyloarthropathies and infectious arthropathies; 9) symptomatic OA of hips or feet; 10) previous surgery in the index knee; 11) difference in length of lower limbs > 1 cm; 12) skin lesion on index knee surface; 13) any blood dyscrasia (including thrombocytopenia) or use of anticoagulants; 14) other diseases: severe depression, non-controlled diabetes, decompensated cardiovascular disease, infection, immunosuppression (methotrexate up to 10 mg/week was allowed), systemic infectious disease, symptomatic lower limb vascular disease, neurological diseases, cancer or any other conditions believed to interfere with results; 15) any sick leave or similar due to KOA.
Procedures
All clinical assessments and intervention procedures were undertaken in a private laboratory. Clinical assessment consisted of self-reported questionnaires, physical examination and physical tests, and collection of blood for full blood count. Participants have also underwent ultrasonographic assessment(24, 25) of the index knee at baseline before treatment administration (Appendix 1). A single investigator (MD) performed all clinical assessments and a single radiologist with more than 20 years of experience in ultrasonography of the musculoskeletal system and interventional procedures (AGBL) performed all ultrasound evaluations and guided injections.
The knee injections were ultrasound-guided, performed using the superolateral approach, 2 cm above and laterally to the superolateral angle of the patella using a 22G needle, with administration of local anesthetic (2% lidocaine without vasoconstrictor). Using the same needle, participants received one of the three treatments (PRP, plasma or saline) according to allocation group. A bandage (Blood Stop®) was applied with orientation to be removed the day after the procedure. Participants were instructed to avoid exercises within 48 hours after the procedure.
Throughout the study, participants were allowed to use paracetamol in case of pain, at maximum dosage of 3g/day, and to avoid the use of other medications, mainly anti-inflammatory and dipyrone (metamizole).
Randomization, allocation and blinding
A randomization list was generated in blocks of 3 by a person not involved in the study, using the version 2015a of the MATLAB Program® and the allocation generation ratio was 1:1:1. For allocation concealment, correspondent letters for each treatment group were placed into sequentially numbered opaque envelopes according to the randomization list and then were sealed. These procedures were also done by a person not involved in the study. Each participant who has consented to enter the study was destined an envelope in the sequence. Allocation of participants occurred from March 2017 to October 2017 and the assessments were completed in March 2018.
All participants were submitted to blood collection to maintain blinding. The envelopes were opened sequentially by the laboratory pharmacist who prepared treatments, not involved in the study. After the preparation of the treatment injections by the pharmacist, the syringes were covered completely (including the needle base) with a non-transparent white adhesive label only with the participant's identification and delivered to the blinded physician who performed the guided injections. All researchers involved in the study including the statistician who performed the analysis were blinded. All participants were blinded. The revelation of the treatment groups occurred only after statistical analysis and production of the main results.
Preparation of PRP and plasma
Participants were submitted to collection of 40 ml of autologous blood by the antecubital vein in vacuum tubes (BD Vacutainer®) of 10 ml containing as anticoagulant the sodium citrate solution at 3.8% (Figure 1). Tubes were submitted to double centrifugation (Cel LS3 Plus® centrifuge). The first spin was performed at 1500 rpm (395 g) lasting 12 minutes. After 10 minutes of resting, an inferior layer consisting of erythrocytes, an intermediate layer with buffy coat and a superior layer of plasma were obtained. This plasma was submitted to a second spin at 2300 rpm (930 g) lasting 10 minutes(26) with separation of a pellet of platelets from plasma (without platelets). At this stage, plasma was obtained for use as treatment injections; and redilution of the pellet of platelets was undertaken to obtain a PRP with approximately 1 x 106 platelets/mm3 (approximately 3 times the basal platelet count) also for use as treatment injections(26). No activation was added to PRP.
Pre-centrifugation platelet count in peripheral blood was used to estimate the final volume of PRP to maintain the concentration of 1 x 106 platelets/mm3 in the injections. The final volume of injected PRP ranged from 1.4 to 5 ml for each participant. The volume of plasma and saline were also calculated according to the count of platelets in peripheral blood for each participant to maintain the blinding of the physician who did the injections (1.4 to 5 ml). Platelets, leukocytes and erythrocytes were counted after centrifugations in some samples of both PRP and plasma to confirm their expected concentration. Both PRP and plasma had approximately zero leucocytes and erythrocytes. The plasma used for treatment also had approximately zero platelets.
All procedures were performed in biological-protector hoods (Veco Bioprotector®), according to the recommendations of the Cell Medicine Society(27). The treatments were prepared in the laboratory RDO by a single pharmacist, who works in the laboratory, not involved in the study.
Outcomes
Primary outcome was change in VAS for overall pain in the index knee at week 24. Secondary outcomes were: 1) VAS for pain at rest; 2) VAS for pain at movement; 3) VAS for physician global assessment (PhGA); 4) participant’s global assessment (PGA) for improvement, 0-100%; 5) Western Ontario McMaster Universities Osteoarthritis Index (WOMAC) (28, 29); 6) Knee Injury and Osteoarthritis Outcome Score(KOOS)(30, 31); 7) Likert scale for improvement: no improvement, mild improvement, moderate improvement, good improvement, excellent improvement; 8) OMERACT-OARSI criteria(32) defined as A) improvement in pain or function ≥ 50% and absolute improvement ≥ 20 or B) improvement in at least 2 of the following 3 criteria: i) pain ≥ 20% and absolute improvement ≥ 10, ii) function ≥ 20% and absolute improvement ≥ 10; iii) PGA ≥ 20% and absolute improvement ≥ 10. For these criteria, pain was assessed by VAS for overall pain and function was assessed by WOMAC function subscale; 9) timed up and go test (TUGT)(33); 10) analgesic consumption, assessed by a self-reported diary.
Statistical analysis
Sample size was calculated for the primary outcome at week 24. A 2 cm reduction in VAS for overall pain was considered as the minimum difference to be detected between the groups with a standard deviation (SD) of 1.5, based on the study by Patel et al(16). An alpha of 5% and a power of study (1 - beta) of 80% were used. Fifteen participants were required in each of the three groups. To allow a loss of follow-up of 20%, the minimum number of participants in each group was 18. The calculation was performed by the Action Stat Pro® software.
Mean and SD were obtained for continuous variables and distribution was presented for categorical variables. To compare groups at baseline, the Chi-square or Fisher's exact tests were used for the categorical variables and ANOVA or Kruskal-Wallis for continuous variables. For data collected at 6, 12 and 24 weeks, the ANOVA test with repeated measures for continuous variables. Non-parametric tests were used for evaluation of Likert scale (Friedman test for paired data) and OMERACT-OARSI criteria (Cochran's Q test for paired data). We performed a post-hoc subanalysis to investigate potential predictors of improvement with PRP or plasma treatment. The analyses were conducted using the software SPSS version 20. A statistical significance of 5% was considered for all tests. Missing data were treated with multiple imputation.