## 4 Discussion  
Plasma jet technology has attracted widespread attention in biomedical applications. It has been reported that cold plasma and PTS can selectively induce cell death in various tumor cells, typically by producing a wide range of ROS/RNS [34, 35]. Enhanced basal RNS, especially nitric oxide (NO), might play a role in the prevention and progression of cancer. The selective nature of cold plasma is hypothesized to rely on ill-fated metabolism and  
Figure 5: Investigation of hemolysis of RBCs isolated from CLL patients and healthy individuals after exposure to either cold plasma (60 s or 120 s) or PTS (120 s) after 24 h culture on agar plates (**a**). The quantitative spectrophotometric experiment was performed at 6 h (**b**). Deionized water (dH\({}_{2}\)O) and SDS 10% served as positive controls. Data are represented as mean + SEM of four donors each; *** denotes \(p<0.001\). baseline ROS/RNS in cancer cells compared to non-malignant counterparts, suggesting the former to be more sensitive to external ROS/RNS stressors and eventually leading to cell death [36; 37]. PTS also contains high levels of H2O2 and NO2- capable of inducing programmed cell death at higher concentrations in malignant cells [38; 39]. Using a plasma device that can operate in RNS production mode, this study provides information about the effects of cold-plasma-produced RNS on the blood of leukemia patients, as we reported decreased cell viability in the whole blood of patients with hematologic malignancies, namely CLL. In our ex vivo study protocol of patient and healthy donor blood, we aimed to identify the effect of cold plasma or PTS-mediated effects on hematological parameters such as CBC, WBC, RBC morphology, PT, PTT, cytotoxicity in WBC, RNS, lipid peroxidation, and hemolytic activity. Our findings showed that cold plasma exposure and PTS treatment increased blood plasma RNS and MDA levels in both CLL patients and healthy individuals without significantly altering complete blood count, WBC, RBC morphology, or PT and PTT time. Our data suggest a selective effect on reduced metabolic activity in cells of CLL blood compared to healthy donor blood. Therefore, considering that the plasma therapy does not affect non-malignant leukocyte cells to a significant extent, the cold plasma treatment and the PTS exposure using the current plasma setup seem to be cytotoxic to mature malignant B cells known to be accumulated in the blood of CLL patients [40]. A range of previous in vitro studies on leukemia cells of different phenotypes, such as acute myeloid leukemia (AML) and acute T-cell leukemia, have provided evidence of cold-plasma-induced toxicity. This includes THP-1 and Jurkat cells [41; 42; 43], TK.6 cells [44], MOLM13 [45; 46], U937 [41; 42; 47; 48; 49; 50], and Molt-4 [41; 50], which are among the previously in vitro studied cell lines. However, a study directly comparing the sensitivity of Jurkat and THP-1 leukemia cells with their non-malignant counterparts, T-helper cells and monocytes, revealed that the leukemia cells were much more resistant to cold-plasma-induced toxicity compared to the healthy donor cells [51]. Although the cells in that study were cultured in a cell culture medium and not derived from whole donor or patient blood, further research is needed to assess the selectivity of the plasma approach based on single-cell data. To the best of our knowledge, our report is the first study to show that both cold plasma treatment and PTS exposure reduced the metabolic activity of whole blood cells in CLL patients. However, further cellular and molecular studies are needed to determine which cells in CLL patients are targeted by cold plasma and PTS. The direct plasma treatment induced greater MDA levels than PTS (indirect) exposure. This might be due to the delivery of relatively short-lived reactive species, such as peroxonitrile, shown to be generated by plasma jets [52] that can induce lipid peroxidation and MDA formation.