Digital holo-tomographic 3D maps of COVID-19 microclots in blood to assess disease severity, 2023, Bergaglio

[EndME]

Digital holo-tomographic 3D maps of COVID-19 microclots in blood to assess disease severity

Abstract
The coronavirus disease 2019 (COVID-19) has impacted health globally. Cumulative evidence points to long-term effects of COVID-19 such as cardiovascular and cognitive disorders diagnosed in patients even after the recovery period.

In particular, micrometer-sized blood clots and hyperactivated platelets have been identified as potential indicators of long COVID. Here we resolve individual microclot structures in platelet-rich plasma of donors with different subphenotypes of COVID-19 in a label-free manner, using 3D digital holo-tomographic microscopy (DHTM). Based on 3D refractive index (RI) tomograms, the size, dry mass, and prevalence of microclot composites were quantified and then parametrically differentiated from fibrin-rich microclots and platelet aggregates in the plasma of COVID-19 donors. Importantly, fewer microclots and platelet aggregates were detected in the plasma of healthy controls when compared to COVID-19 donors.

Our work highlights the utility of integrating DHTM in clinical settings that may allow the detection of individuals at risk of developing microvascular thrombotic disorders and for monitoring the efficiency of prescribed treatments by screening plasma samples.

https://www.biorxiv.org/content/10.1101/2023.09.12.557318v1

 

[EndME]

A Swiss team using a different method to Pretorius et al to assess "microclots" and platelet hyperactivation. This study is for acute Covid-19, but the authors are more than aware of Long Covid.

 

[SNT Gatchaman]

Selected quotes from introduction —

To date, evidence for the presence of microclots in the plasma of COVID-19 and long COVID donors has been mainly obtained using in vitro ThT staining and fluorescent microscopy

it would be highly desirable to devise a methodology that would allow for direct determination of microclot morphology, composition, and prevalence in blood in a label-free manner compatible with operating even in resource-limited clinical settings. To address this pending need, we have developed an imaging methodology based on digital holo-tomographic microscopy (DHTM)

First, the refractive index (RI) maps of synthetically prepared blood clot fragments in aqueous solution from healthy individuals and donors with COVID-19 were registered using DHTM

Raman spectroscopy measurements were also conducted to further characterize the blood clot structure and composition. Next, we extended the imaging and analysis protocols to directly examine the platelet-rich plasma (PRP) of individuals with different COVID-19 subphenotypes, including COVID-19 positive and recovered.

Microclots with varying structure and composition were detected in plasma samples from all the donors with COVID-19, regardless of symptom severity. Microclots were classified based on their composition as either microclot composites, fibrin-rich microclots or platelet aggregates.

Our findings highlight that label-free high-throughput microscopy can be used as a point-of-care technique to visualize and quantify the presence of microclots directly in plasma without the need for complicated sample preparation techniques.

 

[SNT Gatchaman]

Section: Label-free digital holo-tomographic microscopy of synthetically prepared blood clots

Methods —

Healthy and COVID-19 fixed blood clot samples were commercially obtained from a single donor, respectively. Whole blood was collected via venipuncture, added to an empty tube, and allowed to clot. The clot was mechanically disrupted and passed through a 30 μm filter in order to generate smaller clot fragments. Finally, the clots were fixed in 4% paraformaldehyde (PFA) and they were provided as 1 mL aliquots and stored at 4°C. For DHTM imaging, 200 μL of blood clots in aqueous solution was transferred in a 35-mm Ibidi ibiTreat μ-Dish for DHTM imaging.

Results: —

To further understand the compositional differences within a microclot structure, we performed Raman spectroscopy [...] Spectrochemical analysis revealed the presence of signal peaks in the spectral regions associated with the presence of fibrin [...], hemoglobin [...] and lipids

The averaged spectral signature of blood clots from a donor with COVID-19 presented spectral similarities to the healthy blood clots in the hemoglobin and lipid regions.

In the fibrin regions, a maximum peak at 976 cm -1 was detected in the COVID-19 but not in the healthy blood clots. Here, the presence of a peak at 937 cm-1 may be indicative of a shift of the fibrin band in the healthy blood clots. In contrast, both blood clot samples presented a maximum peak in a secondary fibrin region at 1342 cm-1 , with a lower signal intensity in the COVID-19 blood clots. Such differences in the spectra between the healthy and COVID-19 blood clot samples may be indicative of changes occurring in the overall structure and composition of blood clots.

The structural parameters of healthy and COVID-19 blood clot fragments were extracted and quantified, including the overall size, the fibrin fibril diameter, and the dry mass.

A two-sample t-test revealed a significant increase in clot length (healthy: 50.1 ± 18.9 μm; COVID-19: 66.2 ± 21.5 μm) and width (healthy: 31.0 ± 14.5 μm; COVID-19: 49.1 ± 19.8 μm) in COVID-19 compared to healthy blood clots, suggesting an overall difference in the size of the clot fragments. In addition, the diameter of the fibrin fibrils was significantly larger in the COVID-19 compared to the healthy samples (healthy: 0.64 ± 0.13 μm; COVID-19: 0.77 ± 0.14 μm).

 

[SNT Gatchaman]

Section: Structural analysis of microclots in COVID-19 platelet-rich plasma

Methods —

Platelet-rich plasma (PRP) was commercially obtained for healthy and convalescent COVID-19 donors [US/UK]. Whole blood was collected in K2EDTA vacutainers and centrifuged in order to separate plasma from the cell pallet. PRP samples were transported and stored at -20°C. For all PRP samples, 50 μL of PRP was diluted in 200 μL of Alsever's solution and 250 μL was transferred in a 35-mm Ibidi ibiTreat μ-Dish for DHTM imaging.

Label-free holo-tomographic imaging was performed using a 3D Cell Explorer microscope. Before each measurement, the petri dish containing [...] the plasma solution was placed in the microscope sample holder, and blood clots were allowed to sediment to the bottom of the petri dish for 10 minutes before imaging. DHTM was operated under standard laboratory conditions.

Results —

Microclots were found in all the analyzed COVID-19 plasma samples, regardless of age, symptomatology, and IgG and IgM antibody levels.

27-year-old female donor [...] with a COVID-19 positive and mild symptomatology subphenotype

A total of ∼4 microclot composite structures, ∼5 fibrin-rich microclots, and ∼1570 platelet aggregates were observed in 75 μL of plasma, indicating extensive platelet pathology characterized by aggregated platelets in plasma

26-year-old female donor [...] with a COVID-19 positive and moderate symptomatology subphenotype

A total of ~20 microclots were detected in 75 μL of PRP, classified as either microclot composite or fibrin-rich microclots. The segmented and classified 3D RI tomograms show the spatially magnified structure of a microclot composite (Fig. 3b) and reveal the presence of single fibrin fibrils in the plasma

23-year-old female [...] with a recovered COVID-19 and mild symptomatology subphenotype

Microclot composites (n = 16) presented varying morphologies, including more dense structures with fibrin fibrils and net-like structures intertwined with platelets. Platelet clumping was also observed (n = 83) [...] a few large microclot composite structures were detected, measuring up to ~80 μm in size

Interestingly, microclots were detected in plasma from [COVID-19 positive female donor with no symptomatology] even in the absence of clinical features

 

[SNT Gatchaman]

Selected quotes from Discussion —

Blood clot structure can be described by parameters such as the fibrin fibril diameter, fibrin length, clot composition, clot density, and clot porosity

Similar to fluorescent markers in immunofluorescence, digital staining allows for specific labeling of multiple structures based on the refractive index values, but without altering the inherent features of the sample

Taken together, DHTM analysis of micrometer-sized blood clot fragments reveals remarkable details of clot structure and composition in a label-free manner, which can be quantified and further classified into different blood clot types. The morphological parameters that we have identified in the first part of our study were then further extended to directly characterize microclots in plasma.

Microclot composites were found in all COVID-19 plasma samples, with the highest prevalence in the COVID-19 positive donor with moderate symptomatology. These composites structurally resemble the microclots previously reported by Pretorius and colleagues using fluorescence microscopy

In conclusion, the label-free DHTM-based approach demonstrated in our study could serve as a powerful tool for screening microclots in plasma. Information obtained in a label-free manner from such an approach, with high spatial resolution, could provide deeper insights into understanding the role of microclots in health and disease.

 

[SNT Gatchaman]

Wikipedia or more than you ever wanted to know about the physics at —

Principles and techniques of digital holographic microscopy (2010, SPIE Reviews)

 

[EndME]

Now published as 3D Holo-tomographic Mapping of COVID-19 Microclots in Blood to Assess Disease Severity.