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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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Related in: MedlinePlus

Expression of TMEM27 and BACE2 in pancreatic human islets.Colocalization of insulin and TMEM27 was found in pancreatic β-cells, whereas no TMEM27 was detected in glucagon positive α-cells (A). BACE2 and insulin are coexpressed in pancreatic β-cells (B).
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pone-0098932-g001: Expression of TMEM27 and BACE2 in pancreatic human islets.Colocalization of insulin and TMEM27 was found in pancreatic β-cells, whereas no TMEM27 was detected in glucagon positive α-cells (A). BACE2 and insulin are coexpressed in pancreatic β-cells (B).

Mentions: Antibodies and procedures were the same as described [1]. Primary antibodies used were rabbit anti-mouse TMEM27/Collectrin4 (used in Figure 1A, upper panel), mouse anti-human TMEM27 9/28 (Roche, Switzerland; used in Figure 1A, lower panel), mouse anti-human BACE2 1/9 (Roche, Switzerland), guinea pig anti-human insulin (Linco Research, USA), and rabbit anti-human glucagon (Novocastra Laboratories Ltd, UK). Fluorescence pictures were taken with a resolution of 1376×1032×3 pixels and 20× magnification. Raw unedited material was used for the automated analysis pipeline. The stainings were done on serial cuts of the same tissue block.


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)

Expression of TMEM27 and BACE2 in pancreatic human islets.Colocalization of insulin and TMEM27 was found in pancreatic β-cells, whereas no TMEM27 was detected in glucagon positive α-cells (A). BACE2 and insulin are coexpressed in pancreatic β-cells (B).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098932-g001: Expression of TMEM27 and BACE2 in pancreatic human islets.Colocalization of insulin and TMEM27 was found in pancreatic β-cells, whereas no TMEM27 was detected in glucagon positive α-cells (A). BACE2 and insulin are coexpressed in pancreatic β-cells (B).
Mentions: Antibodies and procedures were the same as described [1]. Primary antibodies used were rabbit anti-mouse TMEM27/Collectrin4 (used in Figure 1A, upper panel), mouse anti-human TMEM27 9/28 (Roche, Switzerland; used in Figure 1A, lower panel), mouse anti-human BACE2 1/9 (Roche, Switzerland), guinea pig anti-human insulin (Linco Research, USA), and rabbit anti-human glucagon (Novocastra Laboratories Ltd, UK). Fluorescence pictures were taken with a resolution of 1376×1032×3 pixels and 20× magnification. Raw unedited material was used for the automated analysis pipeline. The stainings were done on serial cuts of the same tissue block.

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