Raman classification of selected subtypes of acute lymphoblastic leukemia (ALL).

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with chromosome translocations like KMT2A gene rearrangement (KMT2A-r) and BCR-ABL1 fusion gene have been recognized as crucial drivers in both BCP-ALL leukemogenesis and treatment management. Standard diagnostic protocols for proliferative diseases of the hematopoietic system, like KMT2A-r-ALL, are genetically based and strongly molecularly oriented. Therefore, an efficient diagnostic procedure requires not only experienced and multidisciplinary laboratory staff but also considerable instrumentation and material costs. In recent years, a Raman spectroscopy method has been increasingly used to detect subtle chemical changes in individual cells resulting from stress or disease. Therefore, the objective of this study was to identify Raman signatures for the molecular subtypes and to develop a classification method based on the unique spectroscopic profile of in vitro models that represent specific aberrations aimed at KMT2A-r (RS4;11, and SEM) and the BCR-ABL1 fusion gene (SUP-B15, BV-173, and SD-1). Data analysis was based on chemometric methods, i.e. principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and support vector machine (SVM). The PCA-based multivariate model was used for pattern recognition of each investigated group of cells while PLS-DA and SVM were used to build models for the discrimination of spectra from the studied BCP-ALL molecular subtypes. The results showed that the studied molecular subtypes of ALL have characteristic spectroscopic profiles reflecting their peculiar biochemical state. The content of lipids (1600 cm-1), nucleic acids (789 cm-1), and haemoproteins (754, 1130, and 1315 cm-1), which are crucial in cell metabolism, was indicated as the main source of differentiation between subtypes. Identification of spectroscopic markers of cells with BCR-ABL1 or KMT2A-r may be useful in pharmacological studies to monitor the effectiveness of chemotherapy and further to understand differences in molecular responses between leukemia primary cells and cell lines.

Reliable cell preparation protocol for Raman imaging to effectively differentiate normal leukocytes and leukemic blasts.

Leukemias are a remarkably diverse group of malignancies originating from abnormal progenitor cells in the bone marrow. Leukemia subtypes are classified according to the cell type that has undergone neoplastic transformation using demanding and time-consuming methods. Alternative is Raman imaging that can be used both for living and fixed cells. However, considering the diversity of leukemic cell types and normal leukocytes, and the availability of different sample preparation protocols, the main objective of this work was to verify them for leukemia and normal blood cell samples for Raman imaging. The effect of glutaraldehyde (GA) fixation in a concentration gradient (0.1 %, 0.5 %, and 2.5 % GA) on the molecular structure of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral blood mononuclear cells (PBMCs) was verified. Changes in the secondary structure of proteins within cells were indicated as the main effect of fixation, as shown by an increase in band intensity at 1041 cm-1, characteristic for in-plane δ(CH) deformation in phenylalanine (Phe). Different sensitivity of mononuclear and leukemic cells to fixation was observed. While the 0.1 % concentration of GA was too low to preserve the cell structure for an extended period of time, a GA concentration of 0.5 % seemed optimal for both normal and malignant cells. Chemical changes in PBMCs samples stored for 11 days were also investigated, which manifested in numerous modifications in the secondary structure of proteins and the content of nucleic acids. The impact of cell preculturing for 72 h after unbanking was verified, and there was no significant effect on the molecular structure of cells fixed with 0.5 % GA. In summary, the developed protocol for the preparation of samples for Raman imaging allows for the effective differentiation of fixed normal leukocytes from malignant T lymphoblasts.

Raman-based spectrophenotyping of the most important cells of the immune system.

INTRODUCTION: Human peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of cells that includes T and B lymphocytes. The total number of lymphocytes and their percentage in the blood can be a marker for the diagnosis of several human diseases. Currently, cytometric methods are widely used to distinguish subtypes of leukocytes and quantify their number. These techniques use cell immunophenotyping, which is limited by the number of fluorochrome-labeled antibodies that can be applied simultaneously. OBJECTIVE: B and T lymphocytes were isolated from peripheral blood obtained from healthy human donors. METHODS: The immunomagnetic negative selection was used for the enrichment of B and T cells fractions, and their purity was assessed by flow cytometry. Isolated cells were fixed with 0.5% glutaraldehyde and measured using confocal Raman imaging. K-means cluster analysis, principal component analysis and partial least squares discriminant methods were applied for the identification of spectroscopic markers to distinguish B and T cells. HPLC was the reference method for identifying carotene in T cells. RESULTS: Reliable discrimination between T and B lymphocytes based on their spectral profile has been demonstrated using label-free Raman imaging and chemometric analysis. The presence of carotene in T lymphocytes (in addition to the previously reported in plasma) was confirmed and for the first time unequivocally identified as ß-carotene. In addition, the molecular features of the lymphocytes nuclei were found to support the discriminant analysis. It has been shown that although the presence of carotenoids in T cells depends on individual donor variability, the reliable differentiation between lymphocytes is possible based on Raman spectra collected from individual cells. CONCLUSIONS: This proves the potential of Raman spectroscopy in clinical diagnostics to automatically differentiate between cells that are an important component of our immune system.

Towards Raman-Based Screening of Acute Lymphoblastic Leukemia-Type B (B-ALL) Subtypes.

Acute lymphoblastic leukemia (ALL) is the most common type of malignant neoplasms in the pediatric population. B-cell precursor ALLs (BCP-ALLs) are derived from the progenitors of B lymphocytes. Traditionally, risk factors stratifying therapy in ALL patients included age at diagnosis, initial leukocytosis, and the response to chemotherapy. Currently, treatment intensity is modified according to the presence of specific gene alterations in the leukemic genome. Raman imaging is a promising diagnostic tool, which enables the molecular characterization of cells and differentiation of subtypes of leukemia in clinical samples. This study aimed to characterize and distinguish cells isolated from the bone marrow of patients suffering from three subtypes of BCP-ALL, defined by gene rearrangements, i.e., BCR-ABL1 (Philadelphia-positive, t(9;22)), TEL-AML1 (t(12;21)) and TCF3-PBX1 (t(1;19)), using single-cell Raman imaging combined with multivariate statistical analysis. Spectra collected from clinical samples were compared with single-cell spectra of B-cells collected from healthy donors, constituting the control group. We demonstrated that Raman spectra of normal B cells strongly differ from spectra of their malignant counterparts, especially in the intensity of bands, which can be assigned to nucleic acids. We also showed that the identification of leukemia subtypes could be automated with the use of chemometric methods. Results prove the clinical suitability of Raman imaging for the identification of spectroscopic markers characterizing leukemia cells.

Toward Raman Subcellular Imaging of Endothelial Dysfunction.

Multiple diseases are at some point associated with altered endothelial function, and endothelial dysfunction (ED) contributes to their pathophysiology. Biochemical changes of the dysfunctional endothelium are linked to various cellular organelles, including the mitochondria, endoplasmic reticulum, and nucleus, so organelle-specific insight is needed for better understanding of endothelial pathobiology. Raman imaging, which combines chemical specificity with microscopic resolution, has proved to be useful in detecting biochemical changes in ED at the cellular level. However, the detection of spectroscopic markers associated with specific cell organelles, while desirable, cannot easily be achieved by Raman imaging without labeling. This critical review summarizes the current advances in Raman-based analysis of ED, with a focus on a new approach involving molecular Raman reporters that could facilitate the study of biochemical changes in cellular organelles. Finally, imaging techniques based on both conventional spontaneous Raman scattering and the emerging technique of stimulated Raman scattering are discussed.

Multiplex Raman imaging of organelles in endothelial cells.

Raman imaging using molecular reporters is a relatively new approach in subcellular investigations. It enables the visualization of organelles in cells with better selectivity and sensitivity compared to the label-free approach. Essentially Raman reporters possess in their structure an alkyne molecular group that can be selectively identified in the spectral region silent for biomolecules, hence facilitate the localization of individual organelles. The aim of this work is to visualize the main cell organelles in endothelial cells (HMEC-1) using established reporters (EdU and MitoBADY), but also to test a new one, namely falcarinol, which exhibits lipophilic properties. Moreover, we tested the possibility to use Raman reporters as a probe to detect changes in distribution of certain organelles after induced endothelial dysfunction (ED) in in vitro models. In both cases, induced ED is characterized by the formation of lipid droplets in the cells, which is why a good tool for the detection of lipid-rich organelles is so important in these studies. Two-dimensional Raman images were obtained, visualizing the distribution of selected organic compounds in the cell, such as proteins, lipids, and nucleic acids. Additionally, the distribution of EdU, MitoBADY and falcarinol in endothelial cells (ECs) was determined. Moreover, we highlight some drawback of established Raman reporter and the need for testing them in various physiological state of the cell.

Astaxanthin as a new Raman probe for biosensing of specific subcellular lipidic structures: can we detect lipids in cells under resonance conditions?

Here we report a new Raman probe for cellular studies on lipids detection and distribution. It is (3S, 3'S)-astaxanthin (AXT), a natural xanthophyll of hydrophobic properties and high solubility in lipids. It contains a chromophore group, a long polyene chain of eleven conjugated C=C bonds including two in the terminal rings, absorbing light in the visible range that coincides with the excitation of lasers commonly used in Raman spectroscopy for studying of biological samples. Depending on the laser, resonance (excitation in the visible range) or pre-resonance (the near infrared range) Raman spectrum of astaxanthin is dominated by bands at ca. 1008, 1158, and 1520 cm-1 that now can be also a marker of lipids distribution in the cells. We showed that AXT accumulates in lipidic structures of endothelial cells in time-dependent manner that provides possibility to visualize e.g. endoplasmic reticulum, as well as nuclear envelope. As a non-toxic reporter, it has a potential in the future studies on e.g. nucleus membranes damage in live cells in a very short measuring time.

Labeled vs. Label-Free Raman Imaging of Lipids in Endothelial Cells of Various Origins.

Endothelial cells (EC) constitute a single layer of the lining of blood vessels and play an important role in maintaining cardiovascular homeostasis. Endothelial dysfunction has been recognized as a primary or secondary cause of many diseases and it manifests itself, among others, by increased lipid content or a change in the lipid composition in the EC. Therefore, the analysis of cellular lipids is crucial to understand the mechanisms of disease development. Tumor necrosis factor alpha (TNF-α)-induced inflammation of EC alters the lipid content of cells, which can be detected by Raman spectroscopy. By default, lipid detection is carried out in a label-free manner, and these compounds are recognized based on their spectral profile characteristics. We consider (3S,3'S)-astaxanthin (AXT), a natural dye with a characteristic resonance spectrum, as a new Raman probe for the detection of lipids in the EC of various vascular beds, i.e., the aorta, brain and heart. AXT colocalizes with lipids in cells, enabling imaging of lipid-rich cellular components in a time-dependent manner using laser power 10 times lower than that commonly used to measure biological samples. The results show that AXT can be used to study lipids distribution in EC at various locations, suggesting its use as a universal probe for studying cellular lipids using Raman spectroscopy. The use of labeled Raman imaging of lipids in the EC of various organs could contribute to their easier identification and to a better understanding of the development and progression of various vascular diseases, and it could also potentially improve their diagnosis and treatment.