Label-free identification and classification of leukocyte subpopulations in peripheral blood smears by optical photothermal infrared spectroscopy

Abstract

The differential count of leukocyte subpopulations from peripheral blood smears remains a cornerstone of clinical hematology but relies on staining protocols or flow cytometry, that introduce variability and obscure native biochemistry. This study presents label-free workflows that distinguish leukocytes from erythrocytes in full blood smears and classifies the leukocyte subtypes as granulocytes, lymphocytes and monocytes using their single cell optical photothermal infrared (OPTIR) spectra. The first approach uses discrete wavenumber images using two key bands at 1546 and 2922 cm-1 to screen leukocytes. The second approach using intrinsic fluorescence imaging of leukocytes offers approximately 1000 times faster screening. Multiple OPTIR spectra are acquired per leukocyte followed by post-acquisition Giemsa staining for cytological cell verification. Full blood smears were prepared on calcium fluoride and standard glass slides to compare substrate effects, and OPTIR spectra were collected with 532 and 785 nm probe lasers to assess probe wavelengths effects. Principal component-linear discriminant analysis separated granulocytes from mononuclear subtypes under all substrate-probe laser combinations tested on the basis of lipid, nucleic-acid and protein bands in OPTIR spectra. Lymphocytes and monocytes were better distinguished on calcium fluoride than on glass. The OPTIR spectra stayed stable over 14 days of storage on both substrates, whereas the intrinsic fluorescence used for cell localization was weak in fresh smears and increased with storage. The non-destructive, fixation-free measurement preserves native protein and lipid composition and allows subsequent cytological staining, supporting OPTIR on routine glass slides for future label-free hematological screening.