Field-Deployable Lens-Free Imaging Platform for Rapid Label-Free Analysis of Natural Killer Cell Activation

Our goal is to rapidly assess immune function by quantifying NK cell activation in real time, using label-free, lens-free imaging to enhance immune monitoring. We have shown that the innate immunity index, or I3, reliably distinguishes between the NK cell activity of healthy and immunocompromised donors and correlates strongly with the cytokines and flow cytometry markers. The existing NK cell assays requires labels, bulky instruments, and long incubation times.

Our LSIT platform fill this gap by enabling fast, affordable, and label-free profiling using simple optoelectronics. Our label-free LSIT platform differentiates NK cell activation at a single cell level within 30 seconds using shadow parameters, eliminating the need for staining and costly flow cytometry. We will extend LSIT to monitor T and B cell activation and integrate deep learning algorithms for high-throughput classification of different immune cell states.

To begin, remove the kits for natural killer, or NK, cell isolation from the refrigerator and allow them to come to room temperature, between 15 and 28 degrees Celsius. Prepare the required equipment by placing a magnetic separator, pipettes with sterile tips, and set an incubator or heating block to 37 degrees Celsius. Using a pipette, add 0.5 milliliters of whole blood to the green-capped reaction tube containing the antibody cocktail.

Gently mix the contents by pipetting up and down the tube five to six times. Then, incubate the tube for five minutes. Transfer the entire contents of the green-capped tube into the red-capped separation tube one using a pipette, and gently mix by pipetting the tube five to six times.

Place the red-capped tube on the magnetic separator and incubate at room temperature for 10 minutes. While keeping the magnet in place, use a pipette to transfer approximately 1.5 milliliters of the supernatant into the purple-capped separation tube two. Gently mix the contents by pipetting the tube five to six times.

Now, place the purple-capped tube on the magnetic separator and incubate for 10 minutes. While the magnet is still in place, transfer approximately 1 milliliter of the supernatant into a recovery tube with a gray lid using a pipette, and carefully mix the contents by pipetting the tube. Next, add 100 microliters of the isolated natural killer cell suspension into the vehicle tube and another 100 microliters into the activation stimulator cocktail, or ASC, tube.

Gently mix the contents of both tubes using a vortex mixer to ensure even distribution. Incubate the tubes in the heating block or incubator set at 37 degrees Celsius for one hour. Switch on the LSIT platform.

Launch the LSIT capture software. Log in by entering your ID and password, then select Calibrate to initiate calibration mode. Press the Open button on the touchscreen or use the physical sliding door button to open the drawer.

When the drawer opens, remove the calibration slide and store it. Now, click Set Background to calibrate the optical intensity. Replace the calibration slide in the drawer and close the drawer.

Then, click Start Calibration to complete the process. Remove an assay slide from its pouch. Label it with the sample data before placing it on a clean, flat surface.

Pipette 10 microliters of the vehicle sample into channels A and B, and then pipette 10 microliters of the ASC-stimulated sample into channels C and D.Now, press Open to eject the drawer. Remove the calibration slide and place it in a storage box. Insert the prepared assay slide into the drawer and press Close to secure it.

Go back to the main menu and select the NK Cell Activity and Test from the main screen. Enter the sample ID or scan the barcode to load the sample profile. Then, press Capture to begin image recording.

After capturing the image, click on Analyze to begin processing. Review the NK cell activity percentage and cell count displayed on the screen. Download the results to a USB drive or print them using the touchscreen menu.

Select Cell Counting and Test on the LSIT main interface. Load 10 microliters of the new sample onto a fresh assay slide channel using a pipette. Insert the slide into the drawer.

Close the drawer using the touchscreen or button and enter the sample ID on the system. Enter the dilution factor on the touchscreen, if applicable, and press Counting to begin cell quantification. View the results displayed as cells per microliter on the screen.

After analysis, open the drawer by pressing the Open button or toggling the physical switch. Remove the used slide from the drawer using gloved hands. Finally, place the calibration slide back into the drawer to protect the sensor and close the drawer using the touchscreen or the physical toggle switch.

The LSIT platform with integrated software quantitatively assessed NK cell activation by comparing the CSP values of activated and unstimulated control cells. It provided the immunity report with a summarized statistical table. ASC stimulation induced clear morphological changes in NK cells, including increased size and cytoplasmic complexity, as seen in Hema 3-stained Cytospins after 30 minutes and two hours.

Shadow images showed ASC-stimulated cells had visibly more complex diffraction patterns compared to vehicle controls, with extracted parameters indicating increased peak-to-peak distance, cytoplasmic granularity, and combined shadow parameter. Combined shadow parameter values allowed clear classification between healthy donors and cancer patients, with minimal overlap in single cell distributions of activated and nonactivated NK cells. Healthy donors showed significantly higher innate immunity index values than cancer patients following ASC stimulation.