**4. Discussion**

In vitro and in vivo aggregation conditions are, of course, different due to the channel shape, presence, or absence of an endothelium layer, surrounding medium, etc. [21]. Moreover, in vivo aggregation in big vessels differs from the aggregation in capillaries [3]. In this study, we investigated in vivo RBC aggregation in nailfold capillaries, therefore excluding effects that can be observed in big vessels, such as axial migration of RBC, etc. [3]. Hemorheological parameters (including blood flow in capillaries) show alterations in pathophysiological processes in a complex way [22]. That is why our data could have provided contradictory results if we had not used criteria for exclusion (mentioned above in Section 2. Materials and Methods 2.1. Patients).

We found correlations between blood flow parameters measured in vitro and in vivo, as well as significant differences between the control group and CHD patients with and without T2DM. Firstly, the RBC aggregation in CHD patients was enhanced compared to the healthy volunteers (see Table 2). The increase in RBC aggregation has already been linked to cardiovascular diseases, including CHD and arterial hypertension using different techniques [23–25]. However, the relationship between aggregation and pathologies is ye<sup>t</sup> to be established—article [26] suggests that aggregation and pathologies are not linked directly but rather share the same factors, such as obesity and cigarette smoking, among others. We found only a weak correlation for these factors and aggregation in CHD patients.

Secondly, the presence of T2DM had a significant effect on aggregation properties: Both AI and T1/2 were higher (*p* < 0.05) compared to the patients without it (see Table 3). Of course, the effect of T2DM on RBC aggregation is already established [27], but being able to detect it specifically for CHD patients opens many doors in terms of diagnostics and monitoring the discussed pathologies. Two other parameters (Tagg and CBV) did not show significant differences; this could be in part due to the methodology. Both LT and capillaroscopy study a limited number of RBCs, whereas laser aggregometry analyzes ensembles of tens of thousands. A high uncertainty in Tagg and CBV demonstrated a grea<sup>t</sup> variation of RBC properties for each individual patient, which could have been caused by his or her health status. It could also mean that the effect of elevated aggregation was clearer during the later stages of aggregate formation (3D aggregate structures) than in the initial doublet formation.

Higher CBV means lower friction in the vessels and therefore weaker RBC aggregation [2]. This was clearly manifested for patients with T2DM in Figure 3 by the negative correlation with AI (r = −0.81) and positive correlation of T1/2 (r = 0.82). For patients with a high CBV it was more likely that their RBC aggregation be more numerous and take less time for a large ensemble. Tagg measured with LT did not correlate with CBV for all groups.

Additionally, it is important to mention the differences of blood temperature during measurement of different in vitro parameters. RheoScan parameters (AI and T1/2) were measured at 37 ◦C, whereas the LT parameter (Tagg) was measured at room temperature at 22 ◦C. Because the aggregation depends on the temperature, these different conditions might have influenced the obtained data [3,28]. For example, RheoScan parameters (AI and T1/2) greatly depend on temperature [28]. However, in our previous work [29] it was shown that for LT measurements the temperature-dependent change of the RBC aggregation was nearly absent for the temperatures of 20 ◦C and 38 ◦C. This means that we could compare all in vitro parameters between each other as if they were measured at the same temperatures.

The novelty of the presented work consists of a complex analysis of in vitro and in vivo parameters for different pathologies. The results of studies performed by alternative methods do not contradict our conclusions and show increased aggregation of RBCs in patients with CHD (including various complications) compared to healthy donors [3,30–32].

One of the limitations of the study is the small number of patients with both CHD and T2DM; in the future, we plan to increase this number. This will allow for grouping the patients by specific medication used, such as antiaggregants and anticoagulants. Another point that can be improved is the observation of additional factors that influence the blood flow, for example plasma components [3,33]. In addition, BMI and other factors influence platelet activation and aggregation [34], which can indirectly affect the aggregation of RBCs; this was not accounted for in this article.
