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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

Proof of principle with clinical samples.(a) Immunophenotypes of the B-ALL cells from patients. The Pt-1 and Pt-2 B-ALL cells show a ‘common’ B-ALL immunoprofile due to the surface expression of CD19, CD10, CD38, CD45 (intermediate expression level, dim) and HLA-DR, with low surface expression of CD34 (low) and absence of CD20, and surface Immunoglobulins (SmIg). The Pt-3 B-ALL cells were more differentiated than those from Pt-1 and Pt-2, due to the surface membrane presence of CD20 with CD19, CD10, CD38 and HLA-DR, as well as bright intensity of expression for CD45. (b) Mean Raman spectra from 300 acquisitions of normal B-lymphocytes, REH B-leukemia cells, and the three clinical B-ALL cell samples. (c) Difference spectra obtained by subtracting the normal B-lymphocytes Raman spectrum from the clinical B-ALL samples and the REH B-leukemia cell spectra (black line). (d) Plot of the intensity ratios of the Raman signals at 1447 cm−1 and 785 cm−1 (I_1447/I_785), as indicated. (e) PCA scatter plots comparing normal B B-lymphocytes, REH B-leukemia cells, and the three clinical B-ALL cell samples.
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f5: Proof of principle with clinical samples.(a) Immunophenotypes of the B-ALL cells from patients. The Pt-1 and Pt-2 B-ALL cells show a ‘common’ B-ALL immunoprofile due to the surface expression of CD19, CD10, CD38, CD45 (intermediate expression level, dim) and HLA-DR, with low surface expression of CD34 (low) and absence of CD20, and surface Immunoglobulins (SmIg). The Pt-3 B-ALL cells were more differentiated than those from Pt-1 and Pt-2, due to the surface membrane presence of CD20 with CD19, CD10, CD38 and HLA-DR, as well as bright intensity of expression for CD45. (b) Mean Raman spectra from 300 acquisitions of normal B-lymphocytes, REH B-leukemia cells, and the three clinical B-ALL cell samples. (c) Difference spectra obtained by subtracting the normal B-lymphocytes Raman spectrum from the clinical B-ALL samples and the REH B-leukemia cell spectra (black line). (d) Plot of the intensity ratios of the Raman signals at 1447 cm−1 and 785 cm−1 (I_1447/I_785), as indicated. (e) PCA scatter plots comparing normal B B-lymphocytes, REH B-leukemia cells, and the three clinical B-ALL cell samples.

Mentions: First, we analyzed these leukemic cells using standard multi-parametric flow cytometry to characterize them based on the immunophenotypic classification (Fig. 5a). These leukemic cells from patients 1 (Pt-1) and 2 (Pt-2) showed very similar immunological features, including: plasma-membrane expression of CD19, CD10, CD38, and CD45 (intermediate expression level), and of HLA-DR antigens, with low surface expression of CD34 and no surface expression of CD20, and of immunoglobulins (SmIg) (Fig. 5a). According to this antigenic profile, these leukemia cells of Pt-1 and Pt-2 can both be classified as ‘common B-ALL’ and thus they should have similar Raman spectra to the REH B-leukemia transformed cell line. Conversely, for the Pt-3–derived cells, in addition to positivity for CD19, CD10, CD38, CD45, and HLA-DR, there was also plasma-membrane expression of CD20, which indicated that their B-ALL was of a more differentiated type than for the Pt-1 and Pt-2 cells (Fig. 5a). However, these Pt-3–derived cells did not express surface or cytoplasm immunoglobulins (Fig. 5a), which indicated that they might have originated from B-cell differentiation/maturation blocked at an intermediate process between the pro-B and the pre-B maturation stages. Thus, according to this immunological feature, the leukemic cells from Pt-3 should have a Raman spectrum with a profile in-between that of the B-leukemia REH and MN60 transformed cell lines.


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)

Proof of principle with clinical samples.(a) Immunophenotypes of the B-ALL cells from patients. The Pt-1 and Pt-2 B-ALL cells show a ‘common’ B-ALL immunoprofile due to the surface expression of CD19, CD10, CD38, CD45 (intermediate expression level, dim) and HLA-DR, with low surface expression of CD34 (low) and absence of CD20, and surface Immunoglobulins (SmIg). The Pt-3 B-ALL cells were more differentiated than those from Pt-1 and Pt-2, due to the surface membrane presence of CD20 with CD19, CD10, CD38 and HLA-DR, as well as bright intensity of expression for CD45. (b) Mean Raman spectra from 300 acquisitions of normal B-lymphocytes, REH B-leukemia cells, and the three clinical B-ALL cell samples. (c) Difference spectra obtained by subtracting the normal B-lymphocytes Raman spectrum from the clinical B-ALL samples and the REH B-leukemia cell spectra (black line). (d) Plot of the intensity ratios of the Raman signals at 1447 cm−1 and 785 cm−1 (I_1447/I_785), as indicated. (e) PCA scatter plots comparing normal B B-lymphocytes, REH B-leukemia cells, and the three clinical B-ALL cell samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Proof of principle with clinical samples.(a) Immunophenotypes of the B-ALL cells from patients. The Pt-1 and Pt-2 B-ALL cells show a ‘common’ B-ALL immunoprofile due to the surface expression of CD19, CD10, CD38, CD45 (intermediate expression level, dim) and HLA-DR, with low surface expression of CD34 (low) and absence of CD20, and surface Immunoglobulins (SmIg). The Pt-3 B-ALL cells were more differentiated than those from Pt-1 and Pt-2, due to the surface membrane presence of CD20 with CD19, CD10, CD38 and HLA-DR, as well as bright intensity of expression for CD45. (b) Mean Raman spectra from 300 acquisitions of normal B-lymphocytes, REH B-leukemia cells, and the three clinical B-ALL cell samples. (c) Difference spectra obtained by subtracting the normal B-lymphocytes Raman spectrum from the clinical B-ALL samples and the REH B-leukemia cell spectra (black line). (d) Plot of the intensity ratios of the Raman signals at 1447 cm−1 and 785 cm−1 (I_1447/I_785), as indicated. (e) PCA scatter plots comparing normal B B-lymphocytes, REH B-leukemia cells, and the three clinical B-ALL cell samples.
Mentions: First, we analyzed these leukemic cells using standard multi-parametric flow cytometry to characterize them based on the immunophenotypic classification (Fig. 5a). These leukemic cells from patients 1 (Pt-1) and 2 (Pt-2) showed very similar immunological features, including: plasma-membrane expression of CD19, CD10, CD38, and CD45 (intermediate expression level), and of HLA-DR antigens, with low surface expression of CD34 and no surface expression of CD20, and of immunoglobulins (SmIg) (Fig. 5a). According to this antigenic profile, these leukemia cells of Pt-1 and Pt-2 can both be classified as ‘common B-ALL’ and thus they should have similar Raman spectra to the REH B-leukemia transformed cell line. Conversely, for the Pt-3–derived cells, in addition to positivity for CD19, CD10, CD38, CD45, and HLA-DR, there was also plasma-membrane expression of CD20, which indicated that their B-ALL was of a more differentiated type than for the Pt-1 and Pt-2 cells (Fig. 5a). However, these Pt-3–derived cells did not express surface or cytoplasm immunoglobulins (Fig. 5a), which indicated that they might have originated from B-cell differentiation/maturation blocked at an intermediate process between the pro-B and the pre-B maturation stages. Thus, according to this immunological feature, the leukemic cells from Pt-3 should have a Raman spectrum with a profile in-between that of the B-leukemia REH and MN60 transformed cell lines.

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