TALLAS feasibility study design and patients
The “Tranexamic Acid in Lower Limb Arthroplasty” (TALLAS) pilot trial was a feasibility study for a larger multi-centre trial to investigate the efficacy and safety of TXA in patients over 45 years of age, undergoing lower-limb arthroplasty [4]. In this double-blind randomised controlled trial, TXA was administered at a dose of 15 mg/kg and rate of 50 mg/min intravenously at skin incision (for total hip replacement) or just prior to tourniquet release (for total knee replacement) and repeated at 8 and 16 h post-operatively. EDTA- and citrate-collected blood was obtained from 9 patients (placebo n=4, TXA n=9) before the start of surgery (preOP), at the end of surgery (EOS) as well as on post-operative day 1 (POD-1) and 3 (POD-3). The TALLAS pilot trial and this sub-study were both approved by the hospital’s ethics committee (Alfred Hospital Research Ethics Committee) and each participant provided written informed consent. Samples from additional 10 non-randomised patients were collected after study recruitment had ended, who all received TXA equivalent to the randomised patients. Hence, the dose and duration of TXA treatment was identical across all individuals treated with TXA in this study.
Sample collection and laboratory analysis
The 9 consecutive patients of the study population were analysed for immunological changes in blood and plasma; 4 were given placebo, and 5 were administered TXA. Blood was drawn into a 10-mL K2-EDTA tube and a 2.7 mL citrate vacutainer tube (BD Biosciences) and samples processed. The same procedure was performed for the 10 additional patients who were not randomised, all receiving TXA.
Full blood examination
Differential white blood cell counts, red blood cells (RBC) and platelets were assessed in citrated whole blood using the Hemavet 950FS analyser (Drew Scientific, USA) or the Cell-Dyn Emerald analysing system (Abbott, USA).
Flowcytometry
PBMCs were thawed and stained for flowcytometry according to our published protocol [5]. The assessed myeloid and lymphoid cell populations as well as functional markers were selected as they represent key cells/markers involved in inflammation and immune stimulation as well as immunosuppression.
In brief, up to 2 million freshly thawed PBMCs were washed twice and transferred to a 96-well round-bottom plate (Corning, USA). The staining was then performed with two separate antibody panels.
Panel I was used to identify myeloid cell populations and included: CD33-FITC, HLA-DR-APC/Cy7, CD11c-BV421, CD141-APC, CD1c-PECy7, CD14-AF700, CD16-BV510, CD123-BV650, CD83-PE/Dazzle 594, CD86-PerCP/Cy5.5, PD-L1-BV711, CCR4-BV605, CCR7-BV785, TNFR2-PE, and dead-cell-staining Zombie Yellow (all BioLegend, USA). After incubation for 20 min on ice, cells were washed and resuspended in 100 uL of PBS+2% FBS+1%PFA for fixation.
Panel II was used to identify lymphoid cell populations and included: CD3-BV510, CD4-BV650, CD8-AF700, CD56-APC/Cy7, CD25-PerCP/Cy5.5, CD95-APC, CD45R0-BV711, LAP-BV421, CTLA4-PE/Cy7, PD-1-FITC, CCR4-BV605, CCR7-BV785, TNFR2-PE, and dead-cell-staining Zombie Yellow (all BioLegend, USA). Cells were incubated for 20 min on ice, followed by a washing step before they were permeabilized in 200 uL of “Fix/Perm buffer” (eBioscience, USA). After incubation for 30–60 min at room temperature cells were washed using “Perm buffer” (eBioscience) before 50 mL of FoxP3-PE/Dazzle594 antibody diluted in Perm buffer was added for intracellular staining of cells. Samples were incubated for 30 min at room temperature, and then washed again with Perm buffer, and finally resuspended in 100 uL of PBS.
Stained samples were analyzed using a 4-laser LSR Fortessa (BD Biosciences) with BD FACSDiva software (BD Biosciences) for data acquisition. Single stain controls for compensation were generated utilizing UltraComp eBeads (eBioscience) and the analysis of acquired samples was performed in FlowJo (FlowJo, LLC, Ashland, CA) data analysis software.
For the analysis, debris was excluded based on size (FSC) and complexity (SSC). For phenotypic identification of myeloid cell populations, we used SSC as well as the markers CD33, CD123, CD14, CD16, HLA-DR, CD11c, CD141, and CD1c. For the evaluation of cell activation, migration, function, and cell viability, the additional markers CD83, CD86, CCR4, CCR7, PD-L1, TNFR2, and Zombie Yellow for live/dead discrimination were used. The gating strategy for myeloid cells is demonstrated in Suppl. Fig. 1.
Lymphocyte populations were identified using SSC in combination with CD3, CD4, CD8, CD56, CD25, and FoxP3. Cell activation, migration, function, and cell viability were determined with CD45R0, CCR4, CCR7, PD-1, CD95, TNFR2, LAP, CTLA4, and Zombie Yellow for live/dead discrimination. Technical issues with the CD3 stain led to a complete loss in signal for this marker, critical for T cell identification, which is why T cells were excluded from the flowcytometric analysis. The gating strategy for NK cells is illustrated in Suppl. Fig. 2.
For the assessment of absolute cell counts we first determined the frequencies of the individual cell populations identified within the analysed PBMC sample. We then used the combined counts of the lymphocyte and monocyte populations from the corresponding full blood examination (FBE) result to calculate the absolute number of circulating cells (please note that the granulocyte populations are lost during the PBMC isolation process and therefore have to be excluded from the analysis).
The expression strength of the described surface markers was assessed and described as the mean fluorescence intensity (MFI) on the individual cell subsets.
B cells were identified through negative gating as HLA-DR+ CD14- CD11c- CD123- cells, as previously described [6]. The gating strategy for B cells as an addition to our previously established protocol is demonstrated in Suppl. Fig. 3.
IL-6 ELISA
IL-6 levels were evaluated using an elisakit.com IL-6 ELISA Kit (elisakit.com, Australia) according to the manufacturer’s instructions.
IL-10 ELISA
IL-10 levels were quantified harnessing a BD OptEIA IL-10 ELISA Kit (BD Biosciences, USA) according to the manufacturer’s instructions.
IFN-y ELISA
The plasma levels of IFN-y were determined using a JOMAR LIFE RESEARCH RAY-ELH-IFNg-2 ELSIA Kit (Jomar Life Research, Australia) according to the manufacturer’s instructions.
Plasmin-antiplasmin (PAP) ELISA
Plasma PAP complex levels were quantified using a DRG PAP micro ELISA Kit (DRG Instruments, Germany) according to the manufacturer’s instructions.
Statistics
All statistical analyses were performed within Prism 7, Graphpad software (La Jolla, USA). The data were compared with preOP levels using a repeated measure one-way ANOVA with Dunnett’s multiple comparisons test. For this analysis, patients for which a time point was missing (FBE results for one patient in the placebo group and flowcytometry results for one patient in the TXA group) had to be entirely excluded from the analysis. For comparisons between the placebo and TXA groups at an individual time point, data were normalised and postoperative time points presented as fold change of preOP level. Results were then analysed using a 2-tailed Student t test. Differences were considered statistically significant if p < 0.05. The data sets were assessed for outliers with a ROUT test (Q=1%). A maximum of two outliers was detected for some of the evaluated parameters in which case the participant was entirely removed from the analysis. Normal distribution of data was confirmed using a Shapiro-Wilk test.