Ethical clearance for the study was obtained from the Health Research Ethics Committee (HREC) of Stellenbosch University (South Africa) (N19/03/043, project ID #9521). For the volunteers who provided blood samples, the experimental objectives, risks, and details were explained to volunteers and informed consent were obtained prior to blood collection. Strict compliance to ethical guidelines and principles of the Declaration of Helsinki, South African Guidelines for Good Clinical Practice, and Medical Research Council Ethical Guidelines for Research were kept for the duration of the study and for all research protocols.
Sample Demographics and Blood Collection
Blood samples were obtained from healthy individuals (n=15; 9 females, 6 males) to serve as controls for comparison. Healthy volunteers were only included if they did not smoke, did not suffer from cardiovascular disease or any coagulopathies, not pregnant or taking contraceptives, or did not suffer from Long COVID. ME/CFS patients (n=25; 20 females; 5 males) were recruited via the ME/CFS Foundation of South Africa, and only included in this study if they had not previously been infected with the COVID-19 virus. Participants had to have been diagnosed for longer than 6 months, and were still asked to complete the International Consensus Criteria (ICC) questionnaire 48 to gain an understanding of their perspective of disease severity. Blood was collected in citrated tubes. Whole blood (WB) was used for viscoelastic studies, after which the samples were centrifuged at 3000×g for 15 min at room temperature to collect platelet poor plasma (PPP). Platelets were identified in the hematocrit, after PPP was removed and a stored at −80 °C for later analysis.
Clotting properties of both WB and PPP samples were measured by using the Thrombelastograph® (TEG®) 5000 Hemostasis Analyzer (Haemoscope Corp). Analysing WB can allow for the detection of clotting abnormalities influenced by blood as a whole, while TEG® of PPP allows one to assess the contribution of only the clotting proteins without cellular components (such as erythrocytes and platelets) 49. 20μL of 0.01M calcium chloride (required to initiate coagulation in blood drawn within citrate tubes) was added to the TEG® cup, followed by 340μL of either WB or PPP. The test was promptly started and left to run until the maximal amplitude of the clot had been reached.
Fibrinaloid Microclot Analysis inside Platelet Poor Plasma (PPP)
PPP was used to study microclots presence in participants with ME/CFS, and compared to those present in healthy participants. PPP samples were incubated with the fluorescent probe, Thioflavin T (ThT) (Sigma-Aldrich, St. Louis, MO, USA), at a final concentration of 5 μM and for a period of 30 min, prior to viewing with a fluorescence microscope. ThT binds to open hydrophobic areas on fibrinogen that is indicative of amyloid protein changes 50, 51, 52, 53, Samples were viewed on the Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63×/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany), with the excitation wavelength set at 450-488nm and the emission wavelength set at 499-529nm. The % area of the fibrinaloid microclot presence was determined by analysing micrographs using ImageJ 1.53e. After setting the scale, images are converted to 8-bit. The threshold, using the Huang setting, was then set by increasing the white background intensity to 255 and the black (fluorescence) signal intensity to 13-17. Next, we used the ‘analyse particles’ assessment and set particle size at 1-infinity. Three representative images were chosen per subject. The data generated were then analysed with GraphPad Prism 8.4.3.
Fibrinaloid Microclot Analysis after addition of thrombin to create extensive fibrin clots
ThT was used again to identify amyloid presence and load within clot networks. 49μL of PPP was incubated with ThT (again at a final exposurevconcentration of 5 μM) for 30 minutes at room temperature. 5μL of the sample was then transferred to a glass slide, followed by 2,5μL of thrombin (7 U.ml−1, South African National Blood Service). The sample was left to stand for 2 minutes in order for fibrin networks to form, after which a coverslip was placed on top of the clot. Samples were viewed on the Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63×/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany), with ThT’s excitation wavelength set at 450-488nm and the emission wavelength set at 499-529nm. Fluorescent intensity of fibrin clot micrographs was calculated using ImageJ 1.53e. ‘Mean Gray Value’ and ‘area’ were chosen as measurement settings.
Platelet Assessment Using Fluorescence Microscopy
Two fluorescent markers, PAC-1 (FITC-conjugated) (340507, BD Biosciences, San Jose, CA, USA) and CD62P (PE-conjugated) (IM1759U, Beckman Coulter, Brea, CA, USA), were obtained to assess the state of platelets by using the Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63×/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany). After centrifugation of the blood tubes and removal of the plasma, 20μL of haematocrit was slowly (due to its viscosity) pipetted and transferred to an Eppendorf microcentrifuge tube. After allowing the fluorescent markers to reach room temperature, 4μL of both PAC-1 and CD62P was added to the microcentrifuge tube containing 20μL of haematocrit. The incubation period lasted for 30 minutes, in a dark room, at room temperature. The excitation wavelength for PAC-1 was set at 450 to 488 nm and the emission wavelength at 499 to 529 nm; and the excitation wavelength for CD62P was set at 540 to 570 nm and the emission wavelength at 577 nm to 607 nm. Platelet phenotype is assessed with the grading system we have recently implemented 47, where platelet spreading and clumping scores were used and allocated a score of 1 to 4 for severity of spreading and clumping.
Statistics were completed on GraphPad Prism 9.3.1. Data were subjected normality tests (Sharpiro-Wilks). Parametric data were then subject to unpaired t-tests, and non-parametric data were analysed with the unpaired Mann-Whitney test. Data are represented as mean ± standard deviation, or median [Q1-Q3]. Graphical data is represented as mean ± SEM.