Label-free imaging and identification of typical cells of acute myeloid leukaemia and myelodysplastic syndrome by Raman microspectroscopy

Abstract

In clinical practice, the diagnosis and classification of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) start from the manual examination of stained smears of bone marrow (BM) and peripheral blood (PB) by using an optical microscope. This step is subjective and scarcely reproducible. Therefore, the development of subjective and potentially automatable methods for the recognition of typical AML/MDS cells is necessary. Here we have used Raman spectroscopy for distinguishing myeloblasts, promyelocytes, abnormal promyelocytes and erhytroblasts, which have to be counted for a correct diagnosis and morphological classification of AML and MDS. BM samples from patients affected by four different AML subtypes, mostly characterized by the presence of the four subpopulations selected for this study, were analyzed. First, each cell was scanned by acquiring 4096 spectra, thus obtaining Raman images which demonstrate an accurate description of morphological features characteristic of each subpopulation. Raman imaging coupled with hierarchical cluster analysis permitted the automatic discrimination and localization of the nucleus, the cytoplasm, myeloperoxidase containing granules and haemoglobin. Second, the averaged Raman fingerprint of each cell was analysed by multivariate analysis (principal component analysis and linear discriminant analysis) in order to study the typical vibrational features of each subpopulation and also for the automatic recognition of cells. The leave-one-out cross validation of a Raman-based classification model demonstrated the correct classification of myeloblasts, promyelocytes (normal/abnormal) and erhytroblasts with an accuracy of 100%. Normal and abnormal promyelocytes were distinguished with 95% accuracy. The overall classification accuracy considering the four subpopulations was 98%. This proof-of-concept study shows that Raman micro-spectroscopy could be a valid approach for developing label-free, objective and automatic methods for the morphological classification and counting of cells from AML/MDS patients, in substitution of the manual examination of BM and PB stained smears.

Leukemia cells detection based on electroporation assisted surface-enhanced Raman scattering

“…was found in leukemia cells as compared to BMC, suggesting a decrease in the percentage of phenylalanine relative to the total SERS-active constituents in cells. This is consistent with R. Vanna's report on lower Raman signal of phenylalanine in myeloid leukemia cells [4]. In addition, compared with normal cells (BMC), the SERS peaks at 670, 802 and 896 cm −1 , related to thymine/guanine, uracil and ribose-phosphate respectively, exhibited lower signal in HL60 cells and K562 cells, suggesting that there was abnormal proliferation of DNA or RNA in leukemia cells.…”

“…Since more than a decade ago, Raman microscopy has been a promising analytical tool for researchers working in the field of biomedical research, primarily because it is capable of detecting molecular vibrations that provide molecular information, including its structure and its environment [2]. Raman spectroscopy combined with statistical methods has been widely applied in disease diagnostics, including leukemia, oesophagus cancer, breast cancer, colorectal cancer, bladder cancer, lung cancer, and skin cancer [3][4][5]. However, the conventional Raman spectroscopy technique has many disadvantages.…”

Ions-modified nanoparticles affect functional remineralization and energy dissipation through the resin-dentin interface

“…Similarly, the increases of peaks at 1340 cm -1 (α-helices), after Raman analysis, denoted a greater sensitivity of collagen to molecular orientation, at dentin, in order to enhance further crystallization ). Furthermore, the ratio amide I/amide III raised and Raman CH2 signal diminished at dentin when samples were treated with Ca-NPs after 21 d of SBFS storage, indicating better organization of collagen with improved conformation of the polypeptide chains and lower immature stage of the dentin substrate, respectively (Vanna et al, 2015;Wang et al, 2009;Xu and Wang, 2011) (Table 4). Nevertheless, FWHMC of the carbonate band decreased after 21 d, though MMRC increased (Tables 3a, 3b).…”

“…The decrease of amide I peak indicates damage or removal of collagen fibrils (Xu and Wang, 2012). 1450 cm -1 (CH2 deformation bands) is generally assigned to proteins, lipids and carbohydrates; it is in accordance with the immature stage (Vanna et al, 2015).…”

Probing the action of a novel anti-leukaemic drug therapy at the single cell level using modern vibrational spectroscopy techniques

“…Probing drug-cell interactions with spectroscopic techniques has become increasingly popular and can contribute to the understanding of the mode of action of the drug at a cellular level 25, 26 . The majority of spectroscopic cellular studies reported to date are on cells that have been chemically fixed and are therefore often in a dehydrated state 17, 18, 23, 27–29 . Fixation aims to preserve the structural and biochemical constituents of cells in as close to in vivo conditions as possible and is widely accepted in the field of spectroscopy 29 .…”