Preparation of blood smears

The study was approved by the School of Automation and Information Ethics Committee, Xi’an University of Technology. Participants had provided their verbal informed consent to participate in this study, and the procedure had approved by the School of Automation and Information Ethics Committee. The participants were the clinical patients of Xi’an University of Technology Hospital, and the clinicians had verbal informed the participants that their whole blood would be used for this study after routine blood tests, all participants had agreed. All blood samples had already been made routine blood tests for disease diagnoses, and the blood samples we used was the residual whole blood and would discard. In order to facilitate participants, the clinicians only obtained their verbal informed consents and recorded them on the patient records. For participants’ privacy, the blood samples and the test reports had hidden the participants’ name, age and other private information.

The specimens were treated with Wright’s stain [16], and different types of WBCs had different characteristics in the images. Patient blood samples had approximately 4 × 10⁶~5 × 10⁶ cells/mL, which was a very high concentration for the shadow imaging system. In our system, the shadow images were blurred by diffraction, and the images of the cells would be polluted if another cell were too close. Therefore, if the two cells were too close, the individual images (Fig 2D) would be mixed together. Due with this problem, the whole blood was diluted with PBS (Saline 1X, PO₄ 0.0067M), 20 μL human whole blood was mixed with 60 μL PBS.

To identify the WBCs, the whole blood was stained using the Wright-Giemsa method [17]. A drop of blood was taken from the patient and smeared on a glass slide. To obtain a smear with proper thickness, the spreading slide was held at an angle greater than 45°. The blood smear was stained with Wright-Giemsa stain for 3 min, and then large amounts of distilled water were added to the stain. The solution was mixed by gentle blowing every few minutes, and then the stain was gently washed off under running water for 30 s. To dry, the slide was shaken off and air-dried for an additional 5 min. Finally, the blood smear was used for shadow image capture.