Population
All participants were drawn from the list of professional rugby players from one professional rugby club within the Irish Rugby Football Union (IRFU). Randomisation was not conducted as there was no intervention arm in the study. During biochemical analysis only the participant number will be known and there is no difference in technique used between participants. Initial contact was made via the IRFU chief medical officer for permission to run the study over an initial three-year period, which has been granted. Following this, permission was sought and granted from the head coach and medical staff of the identified professional rugby team. Each year, all professional rugby players on the team are invited to participate in the study. Sample size is dependent on the number of players per team per year.
Age-matched controls will be from a consenting cohort of healthy volunteers. The inclusion criteria for these controls are that they are male and match the ages of the participant from the professional sports player cohort. The exclusion criteria for the control cohort is if they play the sport of rugby either at a professional or amateur level within the previous 12 months. Further, the age-matched control participants must not have had a head injury or central nervous system illness in the previous 12 months to the blood draw.
Inclusion criteria
Participants are eligible to participate if they (1) are part of the professional rugby team; (2) aged 18 or over; (3) consent to take part in the study over the whole year.
Exclusion criteria
Participants are excluded if they (1) are unable to attend the preseason baseline draw; (2) unable to give informed consent.
Data collection/investigations
Informed consent is obtained from each participant before commencing testing and assessments (see online supplemental material). Baseline testing is conducted for each consenting player (ie, participant) during the preseason period and further testing is carried out during the season in the event of a SRC. Participants adhere to the normal clinical assessment for a SRC carried out by the team’s medical officer. This clinical assessment is in line with the World Rugby’s head injury assessment (HIA) protocol and a validated computer-based neuropsychometric assessment (ImPACT) for concussion. The participants also adhere to the gradated return to play (GRTP) protocol as set out by World Rugby61 and the IRFU.62
Baseline questionnaire
Participants complete a baseline questionnaire (see online supplemental material) which includes questions regarding their concussion history including symptoms associated with, length of, and outcome of previous SRC injuries. Players are not included or excluded based on their SRC history. The questionnaire was developed in line with previous sports-based concussion studies.63–66
Time frames
The study has been designed to span a minimum of 3 years to capture three playing seasons (1 playing season per year). Each year, during the preseason period, a baseline blood sample is acquired following exercise and a HIA and neuropsychometric assessment is completed. The exercise routine is part of the pre-season training programme within the first week of training. All participants carry out the same cardio training routine prior to the blood draw. Subsequently, over the course of the season, if any participant is suspected of suffering a SRC during a professional match or training session, that participant enters the HIA process, in line with current regulations. As part of this study, any participant that enters the standard HIA process will undergo a HIA, a neuropsychometric assessment, and blood sampling at the following time points:
Within 72 hours of injury,
6 days post-injury (±1 day); this aligns with the minimum GRTP protocol for a professional rugby player, no player will return to play before 6 days,1
13 days post-injury (±1 day) to examine if biomarkers are still present despite the results of the HIA and the neuropsychometric assessment. This time point is to capture if the blood biomarkers are still present after a period of recovery of SRC which has been suggested to be approximately 10–13 days.67 68
The final time point for sample collection at 13 days post-injury was chosen as it was most common timepoint for players to have returned to play after the initial 7-day GRTP, based on clinical assessment. This allows for the clinical and biochemical assessment of players who returned prior to 14 days and to compare any alterations in blood biomarkers or if biochemical recovery had occurred in conjunction with clinical recovery. Further, the players that enter HIA process in year 1 are followed up in the baseline blood sample in year 2 (similar in year 3). Furthermore, the majority of players who do not enter the HIA process will have baseline blood samples taken over the course of the 3 pre-season blood draws that can be measured and compared over the three seasons.
Serial head injury assessment
Participants that suffer a suspected SRC will complete the HIA protocol (see online supplemental material) at the time points outlined above. This is a form of the SCAT5 which has been modified for professional rugby and GRTP.61 This assessment will be carried out by the medical officer of the professional rugby club.
Neuropsychometric assessment
During preseason, all players in the club complete a baseline computer-based neuropsychometric assessment—ImPACT [https://impacttest.com/]. This assessment measures different cognitive domains including visual memory, visual processing speed, reaction times, working memory and attention.69 70 Post-injury, participants are re-assessed once symptom free to determine recovery of these domains and to assess any persistent patterns of deficit. This assessment forms part of the overall concussion assessment and management plan.
Blood draw and storage
The medical officer and/or clinical research nurse take blood samples through venepuncture, according to local policy guidelines, at the time points outlined above. A total of five vials of blood are collected: three 7.5 mL plasma vials (K3EDTA collection tubes; Sarstedt 01.1605.004) and two 4.9 mL serum vials (serum gel with clotting activator collection tubes; Sarstedt 04.1935).
Following blood sample acquisition, the sample is anonymised with a unique participant identifier, which ensures participant confidentiality. Furthermore, study team members carrying out blood sample analysis will not be involved in the consenting process of study participants and, thus, are blind to their identity, thereby minimising potential bias. The blood samples are transported in a sealed transport box on ice to a biochemistry lab located near the blood draw location. Approximately 1.5 mL aliquots of whole blood are prepared immediately from one of the two K3EDTA tubes. The second K3EDTA tube and the three serum tubes are allowed to stand at room temperature for 30 min to facilitate separation of the blood components. The vials are then centrifuged at 2000 xg for 10 min at 4°C. The serum and plasma samples are aliquoted into cryovials with a minimum volume of 400 µL per cryovial.
All aliquoted cryovials are placed within a −80°C freezer for long-term storage. Each aliquot can be removed to probe for different biomarkers without multiple freeze-thaw cycles of a core sample if the samples were not aliquoted into multiple vials.
Blood biomarkers: targeted assessment
Serum or plasma samples are analysed, at different SRC time points, using commercial immunosorbent assays, to determine the levels of different blood-based biomarkers. The targeted biomarkers to be investigated are S100β, GFAP, UCH-L1, BDNF.
Blood biomarkers: untargeted assessment
Serum or plasma samples will be analysed, at different SRC time points, using mass spectrometry analysis, in a discovery-based approach to identify any new candidate blood-based biomarkers for further evaluation. Here, samples undergo plasma immunoaffinity fractionation to deplete the most abundant plasma proteins, due to their dominating concentration, thereby increasing the overall coverage for detection of proteins present at lower concentrations. Biomarkers will be considered for further assessment if they are detected post-SRC or during recovery, and they were not detected in the blood of control samples or the concentration has deviated significantly from baseline levels. A sub-panel of suitable candidate biomarkers will be further evaluated through direct assessment of the associated participants’ blood via immunoblotting and/or ELISA analysis for discovery and verification purposes towards investigation in a larger cohort.
Outcomes
Each participant that is believed to have an SRC enters the HIA process. These participants have clinical assessments conducted at pre-defined time points, in line with the World Rugby guidelines. Blood samples are also acquired at these time points to quantify the levels of blood-based biomarkers. Biomarker levels are correlated to the results of the HIA and neuropsychometric assessments. This facilitates preliminary investigation of the correlation between clinical assessments and biomarker levels to determine if the biomarkers can be used to objectively assess SRC recovery, in a professional sports club setting.
Statistical analysis
This pilot study has been developed to determine the logistics of including blood draws in the current clinical process for HIA within a professional sports setting and to assist with the calculation of sample size needed for an expanded study in a larger cohort study. To determine the power size from the data obtained in year 3 of the study, a Cohen’s f2 test with an f value of 0.25 (medium effect size) will be used to determine the effect size of participants needed, assuming a power of 80% and a significance level of 5% for comparing the different groups.