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A reliable Raman-spectroscopy-based approach for diagnosis, classification and follow-up of B-cell acute lymphoblastic leukemia.

Managò S, Valente C, Mirabelli P, Circolo D, Basile F, Corda D, De Luca AC - Sci Rep (2016)

Bottom Line: B-ALL diagnosis requires identification and classification of the leukemia cells.In combination with immunofluorescence and Western blotting, we show that these Raman markers reflect the relative changes in the potential biological markers from cell surface antigens, cytoplasmic proteins, and DNA content and correlate with the lymphoblastic B-cell maturation/differentiation stages.Our study demonstrates the potential of this technique for classification of B-leukemia cells into the different differentiation/maturation stages, as well as for the identification of key biochemical changes under chemotherapeutic treatments.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Biochemistry, National Research Council, Via P. Castellino 111, 80131 Naples, Italy.

ABSTRACT
Acute lymphoblastic leukemia type B (B-ALL) is a neoplastic disorder that shows high mortality rates due to immature lymphocyte B-cell proliferation. B-ALL diagnosis requires identification and classification of the leukemia cells. Here, we demonstrate the use of Raman spectroscopy to discriminate normal lymphocytic B-cells from three different B-leukemia transformed cell lines (i.e., RS4;11, REH, MN60 cells) based on their biochemical features. In combination with immunofluorescence and Western blotting, we show that these Raman markers reflect the relative changes in the potential biological markers from cell surface antigens, cytoplasmic proteins, and DNA content and correlate with the lymphoblastic B-cell maturation/differentiation stages. Our study demonstrates the potential of this technique for classification of B-leukemia cells into the different differentiation/maturation stages, as well as for the identification of key biochemical changes under chemotherapeutic treatments. Finally, preliminary results from clinical samples indicate high consistency of, and potential applications for, this Raman spectroscopy approach.

No MeSH data available.


Related in: MedlinePlus

Identification and classification of acute lymphocytic leukemia cell.(a) Representative confocal microscopy images of RS4;11, REH and MN60 B-leukemia cells fixed and processed for immunofluorescence analysis with anti-CD38, anti-CD19, anti-CD10 and anti-CD20 monoclonal antibodies (red), to monitor their expression levels. Gray, Hoechst-33258 nucleic-acid staining. Scale bar: 10 μm. (b) Representative immunoblotting of RS4;11, REH and MN60 cells with antibodies against CD10, CD19, CD20 and CD38 (as indicated). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown for the internal protein levels and molecular weight standards (kDa) are indicated on the left of each panel. The blots have been run under the same experimental conditions. (c) Mean Raman spectra of normal B-lymphocytes and the three analyzed B-leukemia cell lines (as indicated). Each spectrum is an average of 300 cells. (d) Raman spectral classification and assignment2223. Abbreviations: def, deformation; str, stretching; bk, vibration of backbone; sym, symmetric; A, adenine; C, cytosine; G, guanine; T, thymidine; U, uracil (ring breathing modes of DNA/RNA bases); Phe, phenylalanine; Tyr, tyrosine; Trp, tryptophan; NA, nucleic acids, P, proteins; L, lipids. (e) Difference spectra obtained by subtracting the normal B cell mean Raman spectrum from the RS4;11, REH and MN60 B-leukemia cell mean Raman spectra (as indicated). (f) PCA scatter plots comparing control B-lymphocytes and B-leukemia cells. (g) Confusion matrix for the classification of the control B-lymphocytes and the B-leukemia cells.
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f2: Identification and classification of acute lymphocytic leukemia cell.(a) Representative confocal microscopy images of RS4;11, REH and MN60 B-leukemia cells fixed and processed for immunofluorescence analysis with anti-CD38, anti-CD19, anti-CD10 and anti-CD20 monoclonal antibodies (red), to monitor their expression levels. Gray, Hoechst-33258 nucleic-acid staining. Scale bar: 10 μm. (b) Representative immunoblotting of RS4;11, REH and MN60 cells with antibodies against CD10, CD19, CD20 and CD38 (as indicated). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown for the internal protein levels and molecular weight standards (kDa) are indicated on the left of each panel. The blots have been run under the same experimental conditions. (c) Mean Raman spectra of normal B-lymphocytes and the three analyzed B-leukemia cell lines (as indicated). Each spectrum is an average of 300 cells. (d) Raman spectral classification and assignment2223. Abbreviations: def, deformation; str, stretching; bk, vibration of backbone; sym, symmetric; A, adenine; C, cytosine; G, guanine; T, thymidine; U, uracil (ring breathing modes of DNA/RNA bases); Phe, phenylalanine; Tyr, tyrosine; Trp, tryptophan; NA, nucleic acids, P, proteins; L, lipids. (e) Difference spectra obtained by subtracting the normal B cell mean Raman spectrum from the RS4;11, REH and MN60 B-leukemia cell mean Raman spectra (as indicated). (f) PCA scatter plots comparing control B-lymphocytes and B-leukemia cells. (g) Confusion matrix for the classification of the control B-lymphocytes and the B-leukemia cells.

Mentions: Using the well-defined quadruple CD38/CD19/CD20/CD10 staining for leukemia cells4950, we first examined these cells under confocal fluorescence microscopy and by Western blotting (Fig. 2a,b). CD38 and CD19 were expressed on the surface of the plasma membranes of all of these three cell lines, and their expression increased for the different differentiation stages from RS4;11 and REH cells to MN60 cells. CD20 was expressed only on the plasma membranes of the more differentiated MN60 cells1 (Fig. 1a,b). Finally, CD10 expression increased for the differentiation from RS4;11 to REH, and then decreased in MN60 cells, as already described for the L2-blast and L3-blast subtypes1. These immunophenotypic analyses validated the RS4;11 and REH cells as a model for the L2 subtype and the MN60 cells as a model for the L3 subtype of B-ALL cells.


A reliable Raman-spectroscopy-based approach for diagnosis, classification and follow-up of B-cell acute lymphoblastic leukemia.

Managò S, Valente C, Mirabelli P, Circolo D, Basile F, Corda D, De Luca AC - Sci Rep (2016)

Identification and classification of acute lymphocytic leukemia cell.(a) Representative confocal microscopy images of RS4;11, REH and MN60 B-leukemia cells fixed and processed for immunofluorescence analysis with anti-CD38, anti-CD19, anti-CD10 and anti-CD20 monoclonal antibodies (red), to monitor their expression levels. Gray, Hoechst-33258 nucleic-acid staining. Scale bar: 10 μm. (b) Representative immunoblotting of RS4;11, REH and MN60 cells with antibodies against CD10, CD19, CD20 and CD38 (as indicated). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown for the internal protein levels and molecular weight standards (kDa) are indicated on the left of each panel. The blots have been run under the same experimental conditions. (c) Mean Raman spectra of normal B-lymphocytes and the three analyzed B-leukemia cell lines (as indicated). Each spectrum is an average of 300 cells. (d) Raman spectral classification and assignment2223. Abbreviations: def, deformation; str, stretching; bk, vibration of backbone; sym, symmetric; A, adenine; C, cytosine; G, guanine; T, thymidine; U, uracil (ring breathing modes of DNA/RNA bases); Phe, phenylalanine; Tyr, tyrosine; Trp, tryptophan; NA, nucleic acids, P, proteins; L, lipids. (e) Difference spectra obtained by subtracting the normal B cell mean Raman spectrum from the RS4;11, REH and MN60 B-leukemia cell mean Raman spectra (as indicated). (f) PCA scatter plots comparing control B-lymphocytes and B-leukemia cells. (g) Confusion matrix for the classification of the control B-lymphocytes and the B-leukemia cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835730&req=5

f2: Identification and classification of acute lymphocytic leukemia cell.(a) Representative confocal microscopy images of RS4;11, REH and MN60 B-leukemia cells fixed and processed for immunofluorescence analysis with anti-CD38, anti-CD19, anti-CD10 and anti-CD20 monoclonal antibodies (red), to monitor their expression levels. Gray, Hoechst-33258 nucleic-acid staining. Scale bar: 10 μm. (b) Representative immunoblotting of RS4;11, REH and MN60 cells with antibodies against CD10, CD19, CD20 and CD38 (as indicated). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown for the internal protein levels and molecular weight standards (kDa) are indicated on the left of each panel. The blots have been run under the same experimental conditions. (c) Mean Raman spectra of normal B-lymphocytes and the three analyzed B-leukemia cell lines (as indicated). Each spectrum is an average of 300 cells. (d) Raman spectral classification and assignment2223. Abbreviations: def, deformation; str, stretching; bk, vibration of backbone; sym, symmetric; A, adenine; C, cytosine; G, guanine; T, thymidine; U, uracil (ring breathing modes of DNA/RNA bases); Phe, phenylalanine; Tyr, tyrosine; Trp, tryptophan; NA, nucleic acids, P, proteins; L, lipids. (e) Difference spectra obtained by subtracting the normal B cell mean Raman spectrum from the RS4;11, REH and MN60 B-leukemia cell mean Raman spectra (as indicated). (f) PCA scatter plots comparing control B-lymphocytes and B-leukemia cells. (g) Confusion matrix for the classification of the control B-lymphocytes and the B-leukemia cells.
Mentions: Using the well-defined quadruple CD38/CD19/CD20/CD10 staining for leukemia cells4950, we first examined these cells under confocal fluorescence microscopy and by Western blotting (Fig. 2a,b). CD38 and CD19 were expressed on the surface of the plasma membranes of all of these three cell lines, and their expression increased for the different differentiation stages from RS4;11 and REH cells to MN60 cells. CD20 was expressed only on the plasma membranes of the more differentiated MN60 cells1 (Fig. 1a,b). Finally, CD10 expression increased for the differentiation from RS4;11 to REH, and then decreased in MN60 cells, as already described for the L2-blast and L3-blast subtypes1. These immunophenotypic analyses validated the RS4;11 and REH cells as a model for the L2 subtype and the MN60 cells as a model for the L3 subtype of B-ALL cells.

Bottom Line: B-ALL diagnosis requires identification and classification of the leukemia cells.In combination with immunofluorescence and Western blotting, we show that these Raman markers reflect the relative changes in the potential biological markers from cell surface antigens, cytoplasmic proteins, and DNA content and correlate with the lymphoblastic B-cell maturation/differentiation stages.Our study demonstrates the potential of this technique for classification of B-leukemia cells into the different differentiation/maturation stages, as well as for the identification of key biochemical changes under chemotherapeutic treatments.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Biochemistry, National Research Council, Via P. Castellino 111, 80131 Naples, Italy.

ABSTRACT
Acute lymphoblastic leukemia type B (B-ALL) is a neoplastic disorder that shows high mortality rates due to immature lymphocyte B-cell proliferation. B-ALL diagnosis requires identification and classification of the leukemia cells. Here, we demonstrate the use of Raman spectroscopy to discriminate normal lymphocytic B-cells from three different B-leukemia transformed cell lines (i.e., RS4;11, REH, MN60 cells) based on their biochemical features. In combination with immunofluorescence and Western blotting, we show that these Raman markers reflect the relative changes in the potential biological markers from cell surface antigens, cytoplasmic proteins, and DNA content and correlate with the lymphoblastic B-cell maturation/differentiation stages. Our study demonstrates the potential of this technique for classification of B-leukemia cells into the different differentiation/maturation stages, as well as for the identification of key biochemical changes under chemotherapeutic treatments. Finally, preliminary results from clinical samples indicate high consistency of, and potential applications for, this Raman spectroscopy approach.

No MeSH data available.


Related in: MedlinePlus