Morphological changes in leukocytes are valuable markers for diseases and immune responses. In our earlier work, we presented Cellytics, a device that uses lens-free shadow imaging technology (LSIT) to monitor natural killer cell activity. Here, we present an improved Cellytics system that has been upgraded to a four-channel configuration to achieve higher throughput while maintaining robust reproducibility for rapid and cost-effective leukocyte analysis. The performance of this multi-channel Cellytics system was improved through refinements to the micro-pinhole chip. Etched pinholes provided better image resolution and clarity compared to drilled pinholes. To stimulate leukocytes, we used an activation stimulator cocktail (ASC) and quantified the resulting morphological changes using shadow-based metrics, including peak-to-peak distance (PPD) and maxima-to-minima standard deviation (MMD-SD). In addition, we developed a new leukocyte activation parameter (LAP) to specifically assess these activation-induced morphological changes. After ASC stimulation, leukocytes showed significantly increased PPD and LAP values and decreased MMD-SD compared to non-activated leukocytes. These results are consistent with the results of the flow cytometric analysis. These results emphasize the potential of Cellytics for the rapid and accurate assessment of leukocyte activation and provide a valuable tool for both clinical diagnostics and basic immunological research.

Natural killer (NK) cells are important effectors of innate immunity that eliminate virus-infected and malignant cells. Monitoring NK cell activity is essential for assessing immune function. However, conventional methods, such as flow cytometry, are labor-intensive and time-consuming. This protocol describes a label-free technique for the rapid analysis of natural killer (NK) cell activity using lens-free shadow imaging technology (LSIT). NK cells isolated from peripheral blood and stimulated with a proprietary activator were analyzed using the LSIT platform within 5 min. The LSIT platform captures holographic images, known as shadow images, created by the interference of light with NK cells, and monitors their activation status by analyzing changes in shadow parameters. Key parameters derived from the shadow images, including the peak-to-peak distance (PPD) and standard deviation of the secondary maxima width (WSM-SD), quantitatively reflected the cell morphology and internal complexity. These parameters were integrated into the combined shadow parameter (CSP) and innate immunity index (I3) to provide a comprehensive assessment and quantification of NK cell activity based on morphological changes in the cells. I3, calculated as the percentage change in CSP after activation, served as a direct measure of NK cell activity. NK cells from healthy individuals consistently showed significantly higher I3 values than those from immunocompromised individuals. The LSIT platform enables fast and cost-effective immune profiling that is suitable for point-of-care diagnostics.

Morphological changes in white blood cells (WBCs) are valuable markers of disease and immune response. This study introduces a multi-channel Lens-free Shadow Imaging Technology (LSIT) device for rapid, cost-effective leukocyte analysis. Device performance was optimized by evaluating micro-pinhole chip parameters, with etched pinholes demonstrating superior image quality and sharpness compared to drilled pinholes. Using an activation stimulator cocktail (ASC), WBC activation was induced, and resulting morphological changes were quantified using shadow parameters, including peak-to-peak distance (PPD) and maxima-to-minima standard deviation (MMD-SD). A novel leukocyte activation parameter (LAP) was developed to measure these activation-induced changes comprehensively. Results showed that ASC-stimulated WBCs exhibited increased PPD and LAP values and decreased MMD-SD, consistent with flow cytometry findings. These results highlight the potential of LSIT and LAP for rapid and accurate assessment of leukocyte activation, offering a valuable tool for clinical diagnostics and immunological research.