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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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Assessment of β-cell area scores, islet size, and β-cell density.Correlation between the median of manually obtained area scores and the median of automated area scores per patient in the whole cohort (non-diabetic patients and type 2 diabetic patients) for TMEM27 (A), BACE2 (B), and insulin (C). Median area scores of all non-diabetic patients vs. all type 2 diabetic patients for TMEM27 (D), BACE2 (E), and insulin (F). Median islet size of all non-diabetic patients vs. all type 2 diabetic patients assessed either by TMEM27/glucagon (G), BACE2/glucagon (H), or insulin/morphology (I). Median β-cell density of all non-diabetic patients vs. all type 2 diabetic patients assessed either by TMEM27 (J) or BACE2 positive cells (K). Values in Figure D-K were assessed by the automated pipeline.
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pone-0098932-g002: Assessment of β-cell area scores, islet size, and β-cell density.Correlation between the median of manually obtained area scores and the median of automated area scores per patient in the whole cohort (non-diabetic patients and type 2 diabetic patients) for TMEM27 (A), BACE2 (B), and insulin (C). Median area scores of all non-diabetic patients vs. all type 2 diabetic patients for TMEM27 (D), BACE2 (E), and insulin (F). Median islet size of all non-diabetic patients vs. all type 2 diabetic patients assessed either by TMEM27/glucagon (G), BACE2/glucagon (H), or insulin/morphology (I). Median β-cell density of all non-diabetic patients vs. all type 2 diabetic patients assessed either by TMEM27 (J) or BACE2 positive cells (K). Values in Figure D-K were assessed by the automated pipeline.

Mentions: In addition to the immunofluorescence stainings for TMEM27 and BACE2, insulin expression was assessed by immunohistochemistry (IHC) which is nowadays used as gold standard in pathology to determine the amount of β-cells in human pancreatic islets. The insulin positive area per islet of 905 islets in total was determined automatically using the commercial software analySIS from Olympus and manually using the scoring system described in Figure S1. The resulting median area scores of all islets per patient are plotted in Figure 2. We found a highly significant correlation between automatically and manually scored specimens with regard to TMEM27 (R2 = 0.48, p<0.001), BACE2 (R2 = 0.13, p<0.001), and insulin (R2 = 0.59, p<0.001), respectively (Figure 2A-C).


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)

Assessment of β-cell area scores, islet size, and β-cell density.Correlation between the median of manually obtained area scores and the median of automated area scores per patient in the whole cohort (non-diabetic patients and type 2 diabetic patients) for TMEM27 (A), BACE2 (B), and insulin (C). Median area scores of all non-diabetic patients vs. all type 2 diabetic patients for TMEM27 (D), BACE2 (E), and insulin (F). Median islet size of all non-diabetic patients vs. all type 2 diabetic patients assessed either by TMEM27/glucagon (G), BACE2/glucagon (H), or insulin/morphology (I). Median β-cell density of all non-diabetic patients vs. all type 2 diabetic patients assessed either by TMEM27 (J) or BACE2 positive cells (K). Values in Figure D-K were assessed by the automated pipeline.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098932-g002: Assessment of β-cell area scores, islet size, and β-cell density.Correlation between the median of manually obtained area scores and the median of automated area scores per patient in the whole cohort (non-diabetic patients and type 2 diabetic patients) for TMEM27 (A), BACE2 (B), and insulin (C). Median area scores of all non-diabetic patients vs. all type 2 diabetic patients for TMEM27 (D), BACE2 (E), and insulin (F). Median islet size of all non-diabetic patients vs. all type 2 diabetic patients assessed either by TMEM27/glucagon (G), BACE2/glucagon (H), or insulin/morphology (I). Median β-cell density of all non-diabetic patients vs. all type 2 diabetic patients assessed either by TMEM27 (J) or BACE2 positive cells (K). Values in Figure D-K were assessed by the automated pipeline.
Mentions: In addition to the immunofluorescence stainings for TMEM27 and BACE2, insulin expression was assessed by immunohistochemistry (IHC) which is nowadays used as gold standard in pathology to determine the amount of β-cells in human pancreatic islets. The insulin positive area per islet of 905 islets in total was determined automatically using the commercial software analySIS from Olympus and manually using the scoring system described in Figure S1. The resulting median area scores of all islets per patient are plotted in Figure 2. We found a highly significant correlation between automatically and manually scored specimens with regard to TMEM27 (R2 = 0.48, p<0.001), BACE2 (R2 = 0.13, p<0.001), and insulin (R2 = 0.59, p<0.001), respectively (Figure 2A-C).

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