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Automated assessment of β-cell area and density per islet and patient using TMEM27 and BACE2 immunofluorescence staining in human pancreatic β-cells.

Rechsteiner MP, Floros X, Boehm BO, Marselli L, Marchetti P, Stoffel M, Moch H, Spinas GA - PLoS ONE (2014)

Bottom Line: The output of the automated pipeline was first compared to a previously developed manual area scoring system which takes into account the intensity of the staining as well as the percentage of cells which are stained within an islet.Furthermore, the median area scores of TMEM27, BACE2 and insulin calculated from all T2D were significantly lower compared to the one of all ND.In summary, automated quantification outperforms manual scoring by reducing time and individual bias.

View Article: PubMed Central - PubMed

Affiliation: Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland.

ABSTRACT
In this study we aimed to establish an unbiased automatic quantification pipeline to assess islet specific features such as β-cell area and density per islet based on immunofluorescence stainings. To determine these parameters, the in vivo protein expression levels of TMEM27 and BACE2 in pancreatic islets of 32 patients with type 2 diabetes (T2D) and in 28 non-diabetic individuals (ND) were used as input for the automated pipeline. The output of the automated pipeline was first compared to a previously developed manual area scoring system which takes into account the intensity of the staining as well as the percentage of cells which are stained within an islet. The median TMEM27 and BACE2 area scores of all islets investigated per patient correlated significantly with the manual scoring and with the median area score of insulin. Furthermore, the median area scores of TMEM27, BACE2 and insulin calculated from all T2D were significantly lower compared to the one of all ND. TMEM27, BACE2, and insulin area scores correlated as well in each individual tissue specimen. Moreover, islet size determined by costaining of glucagon and either TMEM27 or BACE2 and β-cell density based either on TMEM27 or BACE2 positive cells correlated significantly. Finally, the TMEM27 area score showed a positive correlation with BMI in ND and an inverse pattern in T2D. In summary, automated quantification outperforms manual scoring by reducing time and individual bias. The simultaneous changes of TMEM27, BACE2, and insulin in the majority of the β-cells suggest that these proteins reflect the total number of functional insulin producing β-cells. Additionally, β-cell subpopulations may be identified which are positive for TMEM27, BACE2 or insulin only. Thus, the cumulative assessment of all three markers may provide further information about the real β-cell number per islet.

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Extraction of islet specific features per individual patient.Correlation of TMEM27 and BACE2 (A), TMEM27 and insulin (B), and BACE2 and insulin area scores per patient (C). Correlation of islet size assessed either by TMEM27, BACE2, and insulin per patient (D-F). Correlation of β-cell density assessed either by TMEM27 or BACE2 positive cells per patient (G). Values in Figure A-G were assessed by the automated pipeline and points represent the median values per parameter and patient.
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pone-0098932-g003: Extraction of islet specific features per individual patient.Correlation of TMEM27 and BACE2 (A), TMEM27 and insulin (B), and BACE2 and insulin area scores per patient (C). Correlation of islet size assessed either by TMEM27, BACE2, and insulin per patient (D-F). Correlation of β-cell density assessed either by TMEM27 or BACE2 positive cells per patient (G). Values in Figure A-G were assessed by the automated pipeline and points represent the median values per parameter and patient.

Mentions: The median of automatically assessed TMEM27 and BACE2 area scores correlated significantly in each individual patient (R2 = 0.27, p<0.001, Figure 3A). The insulin area score, which was assessed by IHC, correlated as well with TMEM27 (R2 = 0.02, p<0.05) and BACE2 (R2 = 0.16, p<0.01) as depicted in Figures 3B-C. Costaining of β-cells and α-cells using immunofluorescence revealed a significant correlation between islet size defined either by TMEM27 (β-cells) and glucagon (α-cells) staining or by BACE2 (β-cells) and glucagon staining. Moreover, islet size defined immunohistochemically by insulin staining and by the morphological separation of endocrine and exocrine tissue correlated as well with the islet sizes described above (R2 = 0.36, p<0.001; R2 = 0.19, p<0.001; R2 = 0.3, p<0.001; Figure 3D-F). Furthermore, β-cell density based on either TMEM27 or BACE2 positive cells correlated significantly (R2 = 0.17, p<0.001; Figure 3G).


Automated assessment of β-cell area and density per islet and patient using TMEM27 and BACE2 immunofluorescence staining in human pancreatic β-cells.

Rechsteiner MP, Floros X, Boehm BO, Marselli L, Marchetti P, Stoffel M, Moch H, Spinas GA - PLoS ONE (2014)

Extraction of islet specific features per individual patient.Correlation of TMEM27 and BACE2 (A), TMEM27 and insulin (B), and BACE2 and insulin area scores per patient (C). Correlation of islet size assessed either by TMEM27, BACE2, and insulin per patient (D-F). Correlation of β-cell density assessed either by TMEM27 or BACE2 positive cells per patient (G). Values in Figure A-G were assessed by the automated pipeline and points represent the median values per parameter and patient.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048234&req=5

pone-0098932-g003: Extraction of islet specific features per individual patient.Correlation of TMEM27 and BACE2 (A), TMEM27 and insulin (B), and BACE2 and insulin area scores per patient (C). Correlation of islet size assessed either by TMEM27, BACE2, and insulin per patient (D-F). Correlation of β-cell density assessed either by TMEM27 or BACE2 positive cells per patient (G). Values in Figure A-G were assessed by the automated pipeline and points represent the median values per parameter and patient.
Mentions: The median of automatically assessed TMEM27 and BACE2 area scores correlated significantly in each individual patient (R2 = 0.27, p<0.001, Figure 3A). The insulin area score, which was assessed by IHC, correlated as well with TMEM27 (R2 = 0.02, p<0.05) and BACE2 (R2 = 0.16, p<0.01) as depicted in Figures 3B-C. Costaining of β-cells and α-cells using immunofluorescence revealed a significant correlation between islet size defined either by TMEM27 (β-cells) and glucagon (α-cells) staining or by BACE2 (β-cells) and glucagon staining. Moreover, islet size defined immunohistochemically by insulin staining and by the morphological separation of endocrine and exocrine tissue correlated as well with the islet sizes described above (R2 = 0.36, p<0.001; R2 = 0.19, p<0.001; R2 = 0.3, p<0.001; Figure 3D-F). Furthermore, β-cell density based on either TMEM27 or BACE2 positive cells correlated significantly (R2 = 0.17, p<0.001; Figure 3G).

Bottom Line: The output of the automated pipeline was first compared to a previously developed manual area scoring system which takes into account the intensity of the staining as well as the percentage of cells which are stained within an islet.Furthermore, the median area scores of TMEM27, BACE2 and insulin calculated from all T2D were significantly lower compared to the one of all ND.In summary, automated quantification outperforms manual scoring by reducing time and individual bias.

View Article: PubMed Central - PubMed

Affiliation: Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland.

ABSTRACT
In this study we aimed to establish an unbiased automatic quantification pipeline to assess islet specific features such as β-cell area and density per islet based on immunofluorescence stainings. To determine these parameters, the in vivo protein expression levels of TMEM27 and BACE2 in pancreatic islets of 32 patients with type 2 diabetes (T2D) and in 28 non-diabetic individuals (ND) were used as input for the automated pipeline. The output of the automated pipeline was first compared to a previously developed manual area scoring system which takes into account the intensity of the staining as well as the percentage of cells which are stained within an islet. The median TMEM27 and BACE2 area scores of all islets investigated per patient correlated significantly with the manual scoring and with the median area score of insulin. Furthermore, the median area scores of TMEM27, BACE2 and insulin calculated from all T2D were significantly lower compared to the one of all ND. TMEM27, BACE2, and insulin area scores correlated as well in each individual tissue specimen. Moreover, islet size determined by costaining of glucagon and either TMEM27 or BACE2 and β-cell density based either on TMEM27 or BACE2 positive cells correlated significantly. Finally, the TMEM27 area score showed a positive correlation with BMI in ND and an inverse pattern in T2D. In summary, automated quantification outperforms manual scoring by reducing time and individual bias. The simultaneous changes of TMEM27, BACE2, and insulin in the majority of the β-cells suggest that these proteins reflect the total number of functional insulin producing β-cells. Additionally, β-cell subpopulations may be identified which are positive for TMEM27, BACE2 or insulin only. Thus, the cumulative assessment of all three markers may provide further information about the real β-cell number per islet.

Show MeSH
Related in: MedlinePlus