Rapid Flow Cytometric Analysis of Fibrin Amyloid Microclots in Long COVID

Abstract

Long COVID has become a significant global health and economic burden, yet there are currently no established diagnostic tools to identify which patients might benefit from specific treatments. One of the major pathophysiological factors contributing to Long COVID is the presence of hypercoagulability; this results in insoluble amyloid microclots that are resistant to fibrinolysis. Our previous research using fluorescence microscopy has demonstrated a significant amyloid microclot load in Long COVID patients. However, this approach lacked statistical robustness, objectivity, and rapid throughput. In the current study, we have used imaging flow cytometry for the first time to show significantly increased concentration and size of these microclots. We identified notable variations in size and fluorescence between microclots in Long COVID and those of controls even using a 20x objective. By combining cell imaging and the high-event-rate nature of a conventional flow cytometer, imaging flow cytometry can eliminate erroneous results and increase accuracy in gating and analysis beyond what pure quantitative measurements from conventional flow cytometry can provide. Although imaging flow cytometry was used in our study, our results suggest that the signals indicating the presence of microclots should be easily detectable using a conventional flow cytometer. Flow cytometry is a more widely available technique which has been used in pathology laboratories for decades, rendering it a potentially more suitable and accessible method for detecting microclots in individuals suffering from both Long COVID and other conditions with similar pathology, such as myalgic encephalomyelitis.

Note:
Funding Information: DBK thanks the Novo Nordisk Foundation (grant NNF20CC0035580) for financial support. EP acknowledges the South African Medical Research Council and the NRF (grant number: 142142) for consumables. We thank KERNLS (https://kernls.com/), Balvi Research Foundation and Polybio Research Foundation for funding of the Luminex Flow Cytometer.

Declaration of Interests: EP is a director of Biocode Technologies; ST is the COO at Biocode Technologies. The other authors have no competing interests to declare.

Ethics Approval Statement: Ethical clearance for the study was obtained from the Health Research Ethics Committee (HREC) of Stellenbosch University (South Africa) (references: B21/03/001_COVID-19, project ID: 21911 (long COVID registry) and N19/03/043, project ID 9521 with yearly re-approval). The experimental objectives, risks, and details were explained to volunteers and informed consent was obtained prior to blood collection. Strict compliance to ethical guidelines and the 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.

Keywords: Long COVID, amyloid microclots, imaging flow cytometry, fluorescence