Impact of sample preparation on holographic imaging of leukocytes

Abstract

The label-free analysis of leukocytes by holographic microscopy has the potential to overcome the extensive labeling procedures of conventional analysis by automated hematology analyzers and manual blood smear analysis. For the enrichment of leukocytes, selective lysis of erythrocytes is applied in flow cytometry to minimize the background. In the case of quantitative phase measurements, lysis procedures may affect the morphology of leukocytes inducing refractive index changes, which can deteriorate the specificity and sensitivity of the method. To study the effects of selective erythrocyte lysis on phase measurements of leukocytes, we compared two widespread methods: lysis by hypotonic shock followed by magnetic depletion of erythrocytes and lysis with a commercial lysis buffer containing ammonium chloride and potassium bicarbonate. We observed that lymphocytes were less affected than granulocytes and monocytes by both methods and that the intracellular changes increased with increasing incubation times. These findings indicate a significant effect of erythrocyte lysis on leukocytes and need to be studied for future hematology applications.