Efficacy of intra-articular injection of allogeneic platelet-rich plasma for knee osteoarthritis combined with hematologic blood dyscrasias with platelet dysfunction: protocol of a randomized, double-blind, placebo-controlled trial

The allogeneic PRP injection for knee OA study is a prospective, randomized, double-blind, placebo-controlled trial design. This study protocol follows the recommendations of the SPIRIT Statement (supplement 1) [13] for clinical trial protocols elaboration and is reported according to the CONSORT Statement (supplement 2) [14]. The trial will be conducted at The First Affiliated Hospital of Soochow University, which is one of the three national clinical research centers for hematologic disease in China. The primary endpoint for the analysis of outcomes is 12 months after baseline assessment. The trial is registered at the Chinese Clinical Trial Registry with a number of ChiCTR2100048624 and follows the World Health Organization’s guidelines.

We will recruit 100 participants with painful knee OA combined with hematologic blood dyscrasias with platelet dysfunction from outpatient department. Participants are eligible for the study if they meet all inclusion criteria below:

The exclusion criteria are as follows:

Figure 1 and Table 1 outline the trial phases and the schedule for enrollment, screening, interventions and assessments. Participants will complete an onsite survey in the outpatient department to determine initial qualifications, and then a coordinating researcher will screen through a questionnaire. Participants deemed appropriate from the questionnaire screening will be allowed to undergo conventional radiographs (weight-bearing long-leg, A/P and lateral views) and blood screening. The screening will be performed in the outpatient department, where two experienced physicians evaluated patients’ eligibility for enrollment in this study through history taking, imaging examination, and laboratory tests. These steps could diminish possible bias about the inclusion criteria of age range. Participants who meet the inclusion and exclusion criteria will then be deemed suitable for the study. Eligible participants will complete the baseline clinical outcome scores, paper-based questionnaires and baseline knee MRI scans. Participants will also be asked to discontinue non-steroidal anti-inflammatory drugs and other analgesics for knee pain from 3 weeks before the baseline assessment until the 12-month follow-up assessment.

Paper-based clinical outcome scores will be distributed to participants at 1-, 3-, 6-, and 12-month to complete primary and secondary outcomes at the outpatient department. Knee MRI scans will be conducted at 12-month at the radiology center of the First Affiliated Hospital of Soochow University. At the last follow-up, patients will be asked if they would like to receive allogeneic PRP injection again. This will be registered accordingly.

The trial will be monitored by the clinical research monitoring committee of the First Affiliated Hospital of Soochow University. The monitoring committee aims to improve the quality of clinical research and ultimately, patient care. Before the trial commenced, a monitoring plan was set up. Monitors will be required to verify that:

Monitoring will be performed at the initiation of the study until the results of the study are published.

Randomization is centralized, and participants will be randomly allocated in a 1:1 ratio. An independent statistician will provide a computer-generated randomization list, using a permuted block design with block sizes of 4 and 6. The sequence is concealed by the use of a computer interface implemented in the electronic case report form. The patients, treating physicians and coordinating researchers will all be blinded to the allocation of the intervention. A study nurse will have access to the randomization result and the allocated intervention.

Participants will not be told until the end of the study which group they were allocated to. The study nurse will prepare the injection in a separate room and place an opaque label around the syringe and needle base to mask the contents. The nurse will then give the syringe to the injecting treating physician who will not know or be able to tell whether the syringe has allogeneic PRP or saline. All clinical and MRI assessments will be conducted by two radiologists with over 10 years of clinical work experience. All evaluators are blinded to treatment allocation.

Participants in both the allogeneic PRP and saline groups will receive one intra-articular knee injection at the same time after signing written informed consent (supplement 3), in line with commonly used injection protocols for this condition [6, 15]. The study nurse will give the treating physician an occluded syringe with a needle containing 4 ml of either normal saline or allogeneic PRP, and the contents will be injected into the knee joint. After the injection, passive knee flexion and extension will be performed six times with the participant observed resting for 20 min thereafter.

After injection, participants will be asked which group they believe they are in, and separately the injecting treating physician will also be asked which group they believe the participant is in to assess the success of blinding. This will be registered accordingly. The study nurse will ask the participant if they experienced any adverse events following the injection.

One healthy volunteer donor will donate about 300–400 ml (according to the definite number of participants) of peripheral venous blood. To assess the safety of the allogeneic PRP, tests for hepatitis B, hepatitis C, human immunodeficiency virus, and syphilis will be performed and confirmed before application. Every 8 ml of venous blood for each participant (as recommended by the company) is drawn manually using butterfly safety locks and emptied into 10-ml standard tubes (Regenlab PRP Kit-RegenACR®, Le Mont-sur-Lausanne, Switzerland) and are then put in RegenLab PRP Centrifuges. The centrifugation settings are set at 1500 g for 5 minutes. After centrifugation, the blood is fractionated. By gently inverting the Regen BCT tubes several times and process for resuspension of the cellular deposit in the supernatant, approximately 4 ml of PRP will be obtained from each tube. The PRP used for injection will not be activated before application. About 4 ml of PRP will be injected for each patient in the PRP group. In addition, one tube of PRP will be sent to the laboratory for platelet and leukocyte counting, and growth factor (VEGF and TGF-β) analysis. The whole process will be carried out by a study nurse in a separate room and the syringe and needle base will be placed an opaque label to mask the contents.

The study nurse will prepare a syringe with a saline solution (4 mL), attach a needle and place an opaque label around the syringe and needle base to mask the contents in a separate room. The nurse will then deliver the syringe to the treating physician for injection.

Study outcome measures are presented in Table 1. The primary endpoint measure are 12-month change in knee pain and function scores.

The following secondary outcome measures will also be collected:

MRI osteoarthritis knee score (MOAKS): an OA-specific semi-quantitative tool evaluating multi-feature joint changes associated with OA [22]. We will assess the following subscores:

Meniscal morphology: any region worsening at 12-month; scored as yes or no.

Intercondylar synovitis incorporating synovitis and effusion: worsening at 12-month; scored as yes or no.

Whole knee effusion: categorized as worsened, no change, or improved.

Progression of medial distal femur and proximal tibia bone marrow lesion size: scored as 0–3 per region, with higher scores indicating greater size.

Cartilage defects: score in the medial distal femur and the proximal tibia at baseline and 12-month using categorical scoring (range 0–4 per region, with higher scores indicating greater cartilage defects).

Progression of medial cartilage defects (yes/no) will be defined as an increase in score by at least 1 from baseline to follow-up in either the medial tibial or medial femoral compartment [23].

Participants will be asked to avoid the use of non-steroidal anti-inflammatory drugs and other analgesics for knee pain from 3 weeks before the baseline assessment until the 12-month follow-up assessment. Throughout the trial, key treatment (exercise therapy and healthy lifestyle advice) and co-interventions used by patients will be registered, such as non-steroidal anti-inflammatory drugs, other analgesic drugs, intra-articular injections or inlays.

All adverse events are reported spontaneously by the patient or observed by the study nurse. This will include questions about any adverse events that participants believe may be related to the trial intervention, including their nature, how long they lasted for and what action if any, they took. To date, no serious adverse events have been reported in the literature concerning PRP intra-articular injections of the knee. The local Medical Ethical Commission will be notified of any serious adverse events. In the event this happens, the advice of the Medical Ethical Commission will be followed accordingly.

The coordinating researchers will attempt to reduce loss to follow-up as much as possible by contacting every patient and being present at every patient visit. Each patient will be assisted by a coordinating researcher to ensure that each paper-based questionnaire is filled in. If the patient is lost to follow-up, an analysis of demographic and prognostic characteristics will be performed on these cases and the remaining patients. The patient eligible for and compliant with each follow-up will be documented.

If the proportion of missing data exceeds 10%, missing outcome data will be imputed using multiple imputation methodology and the sensitivity of the missing at random assumption will be investigated [24].

The sample size calculation is based on the primary outcome measure, the WOMAC score at 12-month. Based on previous studies, [5, 16, 17, 25] a 11.5 points reduction in WOMAC total score was considered as the minimum difference to be detected between the groups with a standard deviation (SD) of 16. Accepting a false-positive rate of 5% (α = 0.05) and a power of at least 90% (β = 0.10), we determined a theoretical minimum sample size of 84 patients. Considering the estimated 15% dropout rate, a total of 50 patients per group were required. Patient recruitment will be terminated in both groups when the required minimum number of patients is attained.

Paper-based questionnaires will be the primary data collection tools for the study. The questionnaires will be completed at the outpatient clinic at baseline and each follow-up time point. On receipt of the questionnaire forms, the coordinating researchers will make a visual check of the responses and query missing data when possible. The paper-based questionnaire will be securely kept by a study nurse.

A study nurse, blinded to the group allocation, will store the forms into an electronic database by double data entry to minimise typing errors. The coordinating researchers, blinded to the group allocation, will perform a visual check of the data in the electronic database and then query all missing, implausible and inconsistent data. Patient records in the participating hospitals will be used when collecting missing data or interpreting inconsistent or implausible data.

After 12-month follow-up visits are completed and all data stored, a research assistant, not involved in the trial, will aggregate all the data into a separate database, which will be the source for the final data analysis. Participant files will be maintained in storage (both in electronic and paper formats) at the coordinating center for a period of 10 years after completion of the study (10-year follow-up visits).

Baseline characteristics will be analyzed between groups using descriptive statistics. Continuous variables will be expressed in terms of the means and the standard deviations, and compared using the student t test (normally distributed) or Mann-Whitney U test (nonnormally distributed). The categorical data will be expressed as frequency and compared using the Chi-square test or Fisher’s exact test. Normality will be confirmed using the Kolmogorov-Smirnov test.

To test for the effect of treatment on the between-group difference in the primary outcome (WOMAC score), we will use a repeated measurement general linear model (GLM) with Sidak test for multiple comparisons. Changes from baseline to all follow-up time points (1-, 3-, 6- and 12-month) will be included in the model to assess the differences in the primary outcome at different follow-up times. The GLM will also be used as a multivariate analysis to assess the influence of the treatment on the follow-up evolution of all the clinical scores and objective measures performed. The mixed linear regression model (MLM) will be used to assess whether PRP effects on the primary outcome at 12 months will be moderated by age, body mass index, and Kellgren and Lawrence grade. The between-group differences of continuous, normally distributed and homoscedastic data will be compared for their means using the analysis of variance (ANOVA). Nonnormal data will be compared using the Mann-Whitney U test.

To test for the effect of treatment on between-group differences in secondary outcomes, we will also use the repeated measurement general linear model (GLM) with the Sidak test for multiple comparisons. Changes from baseline to all follow-up time points will also be included in the model. The statistical analysis method is similar to the primary analysis. MRI-derived measurements will be compared between groups using appropriate models, adjusting for age, sex, body mass index, and the stratifying variables. The success of blinding will be assessed using the James Blinding Index [26].

The trial will be conducted according to the principles of the Declaration of Helsinki. Ethical approval has been obtained from the National Clinical Research Center for Hematologic Disease, the First Affiliated Hospital of Soochow University Ethics Committee (No.2021–356). The trial has been registered in the Chinese Clinical Trial Registry (ChiCTR2100048624) and any revisions about the protocol are documented in this registry. All participants will provide written informed consent before any study-related procedures.

The results of the trial will be reported first to trial collaborators. The main report will be drafted by the trial coordinating team, and the final version will be agreed upon by the Trial Steering Committee before submission for publication, on behalf of the collaboration.

The results of this trial will substantially inform clinical practice on the clinical effectiveness and safety of the treatment in these patients. The results of this project will be disseminated through peer-reviewed journals, conference presentations, the National Library for Health and through local mechanisms at the participating center.