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.
Highlights
Complete blood count and differentiation of leukocytes (DIFF) belong to the analyses are required, which still show high interobserver variation.[2,3] Addimost frequently performed laboratory diagnostic tests
Based on principal component analysis (PCA) and morphological parameters using training data sets of reconstructed, native leukemic cells, we developed a gating strategy for the differentiation of nine leukocyte subtypes, which enabled us to extend the DIFF to pathological samples, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), and myeloproliferative neoplasms (MPN)
The 2D sheath flow was favored to avoid any contacts of leukocytes with the channel walls, which could possibly lead to activation of cells or apoptosis and could bias the label-free differentiation of leukocytes
Summary
We chose a customized differential holographic microscope (Ovizio Imaging Systems), comprising a 528 nm super-LED Koehler illumination and a 40× objective (NA 0.55, Nikon), for high-throughput (105 fps, acquisition time 5 μs), labelfree in-flow imaging of leukocytes To test the applicability of our setup for the main leukocyte types, we first examined the label-free five-part DIFF of highly enriched basophils, eosinophils, lymphocytes, monocytes, and neutrophils with population purities of 91–99% from multiple healthy donors (Figure S2i–o, Supporting Information). We trained a support vector machine (SVM, radial kernel) on the training data and received an overall accuracy of 92% on the test data set, with sensitivities ≥80% for each of the five leukocyte types (basophils = 80%, eosinophils = 89%, lymphocytes = 89%, monocytes = 91%, neutrophils = 95%; Figure S3a, Supporting Information). We concluded that a DOF of ±2.3 μm was preferably for leukocyte differentiation
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