Label‐Free High‐Throughput Leukemia Detection by Holographic Microscopy

Abstract

Complete blood count and differentiation of leukocytes (DIFF) belong to the most frequently performed laboratory diagnostic tests. Here, a flow cytometry‐based method for label‐free DIFF of untouched leukocytes by digital holographic microscopy on the rich phase contrast of peripheral leukocyte images, using highly controlled 2D hydrodynamic focusing conditions is reported. Principal component analysis of morphological characteristics of the reconstructed images allows classification of nine leukocyte types, in addition to different types of leukemia and demonstrates disappearance of acute myeloid leukemia cells in remission. To exclude confounding effects, the classification strategy is tested by the analysis of 20 blinded clinical samples. Here, 70% of the specimens are correctly classified with further 20% classifications close to a correct diagnosis. Taken together, the findings indicate a broad clinical applicability of the cytometry method for automated and reagent‐free diagnosis of hematological disorders.

Quantitative phase imaging of cells in a flow cytometry arrangement utilizing Michelson interferometer‐based off‐axis digital holographic microscopy

“…Our approach is not only capable to detect automated and reliable absolute biophysical parameters such as cell radius, refractive index and dry mass of single cells in flow, but also provides the morphology of individual single cells and even permits to trace their position in the flow by evaluation of single exposure off‐axis holograms. This minimizes the amount of required calibration procedures and allows compared to earlier approaches , due to the feature of automated refocusing, also the consideration of cells that are not imaged in focus onto the hologram recording device. Improvements for practical applications and enhanced throughput are expected by using high speed camera hardware and graphics processing unit (GPU)‐based acceleration of the numerical hologram evaluation procedures, for example, following approaches as described in ref.…”

Label‐Free Leukemia Monitoring by Computer Vision

“…The approach was not label‐free, required smearing samples, and unlike our presented approach it relied on subjective inputs from experts rather than biomarker labeling for training. Ugele et al used label‐free digital holographic microscopy to achieve convincing classification of nine leukocyte types, as well as different leukemia subtypes principally at diagnosis . This is unfortunately not suited to minimal residual disease detection because a high throughput modality is required (such as imaging flow cytometry)—large numbers of cells need to be measured to find the rare leukemic cells.…”

Optical Investigation of Individual Red Blood Cells for Determining Cell Count and Cellular Hemoglobin Concentration in a Microfluidic Channel

“…The channel design comprised of three inlets having identical dimensions in width and height (h = 144 μm), which merge into a 2000 μm wide and 1.56 cm long channel, and a single outlet reservoir. A neMESYS Base120 pump system with three modules (CETONI GmbH, Korbussen, Germany) was equipped with 10 mL gas tight syringes (VWR, Darmstadt, Germany) and used to control flow velocity and hydrodynamic focusing of cells [ 24 ].…”