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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

Correlation of TMEM27, BACE2, and insulin area scores to BMI.Automated area scoring of TMEM27 is presented as median of individual patients and correlated to BMI of non-diabetics (straight line; A) and patients with type 2 diabetes (dotted line; B). The slopes of the fitted lines were significantly different for the TMEM27 area scores (C). The same is plotted for BACE2 (D-F) and insulin (H-J).
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pone-0098932-g004: Correlation of TMEM27, BACE2, and insulin area scores to BMI.Automated area scoring of TMEM27 is presented as median of individual patients and correlated to BMI of non-diabetics (straight line; A) and patients with type 2 diabetes (dotted line; B). The slopes of the fitted lines were significantly different for the TMEM27 area scores (C). The same is plotted for BACE2 (D-F) and insulin (H-J).

Mentions: We found a significant positive correlation between TMEM27 area score and body mass index (BMI) in the ND (R2 = 0.223, p<0.05; Figure 4A) and a negative correlation in T2D (R2 = 0.04, p<0.05; Figure 4B). After correlating TMEM27 to BMI of ND and T2D separately, the resulting slopes were significantly different (p<0.001, Figure 4C). BACE2 and insulin expression did not correlate with BMI (Figure 4 D-J). By combining the three values of the area scores and taking the mean per patient did not result in a more stringent separation of the two slopes (ND vs. T2D) than considering TMEM27 area scores alone (data not shown).


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)

Correlation of TMEM27, BACE2, and insulin area scores to BMI.Automated area scoring of TMEM27 is presented as median of individual patients and correlated to BMI of non-diabetics (straight line; A) and patients with type 2 diabetes (dotted line; B). The slopes of the fitted lines were significantly different for the TMEM27 area scores (C). The same is plotted for BACE2 (D-F) and insulin (H-J).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098932-g004: Correlation of TMEM27, BACE2, and insulin area scores to BMI.Automated area scoring of TMEM27 is presented as median of individual patients and correlated to BMI of non-diabetics (straight line; A) and patients with type 2 diabetes (dotted line; B). The slopes of the fitted lines were significantly different for the TMEM27 area scores (C). The same is plotted for BACE2 (D-F) and insulin (H-J).
Mentions: We found a significant positive correlation between TMEM27 area score and body mass index (BMI) in the ND (R2 = 0.223, p<0.05; Figure 4A) and a negative correlation in T2D (R2 = 0.04, p<0.05; Figure 4B). After correlating TMEM27 to BMI of ND and T2D separately, the resulting slopes were significantly different (p<0.001, Figure 4C). BACE2 and insulin expression did not correlate with BMI (Figure 4 D-J). By combining the three values of the area scores and taking the mean per patient did not result in a more stringent separation of the two slopes (ND vs. T2D) than considering TMEM27 area scores alone (data not shown).

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