Ethical approval for blood collection and analysis of the patients with COVID-19, T2DM and healthy individuals, was given by the Health Research Ethics Committee (HREC) of Stellenbosch University (reference number: 9521). This laboratory study was carried out in strict adherence to the International Declaration of Helsinki, South African Guidelines for Good Clinical Practice and the South African Medical Research Council (SAMRC), Ethical Guidelines for research. Oral consent was obtained from COVID-19 patients to participate in the study. Written consent was obtained from T2DM patients and healthy participants.
20 COVID-19-positive samples (11 males and 9 females) were obtained and blood samples collected before treatment was embarked upon. Blood samples were collected by JS. Platelet poor plasma (PPP) prepared and stored at -80°C, until fluorescent microscopy analysis.
Type 2 Diabetes Mellitus (T2DM)
Stored Platelet poor plasma samples were randomly selected from our Laboratory’s stored sample repository. 10 age-matched T2DM (6 Males and 4 females), collected in 2018, were used in this analysis.
Our healthy sample was 10 age-matched controls (4 males and 6 females), previously collected and stored in our plasma repository. They were non-smokers, with CRP levels within healthy ranges, and not on any anti-inflammatory medication.
Lung CT scans
Amongst the COVID-19 patient sample 10 patients were admitted, but stabilized and blood drawn and sent home for observation. Where patients were clinically deemed as moderate or severely ill, CT scans of the patients were performed to determine the severity of the lung pathology. We divided our sample into mild disease (no CT scan) and moderate to severely ill. The CT scan and severity score  confirmed moderate to severely ill patients according to the ‘ground glass’ opacities in the lungs.
Fluorescent Microscopy of patient whole blood and platelet poor plasma (PPP)
A simple fluorescence assay was developed by comparing fluorescent (anomalous) amyloid signals present in PPP from COVID-19 patients, T2DM and those from healthy age-matched individuals, all of whom were studied using PPP that had been stored at -80°C. On the day of analysis, PPP was thawed and incubated with the dye thioflavin T (ThT; 5 µM final concentration), which detects amyloid-like structures . Following this, the sample was incubated for 30 min (protected from light) at room temperature. PPP smears were then created by transferring a small volume (5 µl) of the stained PPP sample to a microscope slide (similar methods were followed to create a blood smear). A cover slip was placed over the prepared smear and viewed using a Zeiss AxioObserver 7 fluorescent microscope with a Plan-Apochromat 63x/1.4 Oil DIC M27 objective.
For ThT quantification, the excitation was set at 406 to 440 nm and emission at 546 to 564 nm. Unstained samples were also prepared with both healthy and COVID-19 PPP, to assess any autofluorescence. Micrograph analysis was done using ImageJ (version 2.0.0-rc-34/1.5a). The % area of amyloid were calculated using the thresholding method. This method allows a measurement of area of amyloid signal. The RGB images are opened in ImageJ, each image is calibrated by setting the scale (calculated using the image pixel size and the known size of the scale bar). Each image is then converted to black and white ((8 bit, this is adjusted under the image type setting). The next step is to threshold the image by adjusting the background intensity to white (255) and then thresholding the now black amyloid signal (in these images between 11 and 15). We used the Huang setting during thresholding. Huang’s method is an optimization method which finds the optimal threshold value by minimizing the measures of fuzziness. The black amyloid area is then analyzed using the analyze particle setting where we use the particle size that is measured from 1 to infinity. The particle size setting allows us to exclude any background signal that might not be true amyloid signal. The area per data per particle size that is generated is then copied into a spreadsheet (see our raw data). Statistical analysis was done using GraphPad Prism 8 (version 8.4.3). Sensitivity and specificity of the data were calculated according to the following calculations: