Scientific Papers

Circularity of islets is a distinct marker for the pathological diagnosis of adult non-neoplastic hyperinsulinemic hypoglycemia using surgical specimens | Diagnostic Pathology


We identified four cases of ANHH in the pathological database of Kyoto University Hospital between 1988 and 2022. The clinicopathological features of the patients are summarized in Table 2A. All four cases had PHH. Two of the four cases (Cases 1 and 3) were suspected of having ANHH owing to the lack of tumor-like nodules on computed tomography (CT), and the other two cases (Cases 2 and 4) were suspected of having an insulinoma or ANHH before distal pancreatectomy. Two cases of the ANHH specimens used for the evaluation were from the pancreatic tail (cases 1 and 2), and the other two were from the pancreatic body (cases 3 and 4).

Table 2 Clinicopathological features of the current cases

Controls were obtained from autopsied pathological specimens at the Department of Diagnostic Pathology, Kyoto University Hospital, and the Department of Pathology, Iwate Medical University. Suitable conditions for controls were as follows: (1) no pancreatic tumor (including neuroendocrine tumor); (2) no metastasis or invasion of the tumor into the pancreatic parenchyma or peripancreatic tissue (adipose tissue, blood, or lymphatic vessels); (3) no history of diabetes; (4) no chronic pancreatitis; (5) no intensive systemic treatment; and (6) good fixation and no advanced autolysis. One control specimen was obtained from the pancreatic tail (cases 1 and 2), two from the pancreatic head (case 3), and two from the pancreatic body (cases 4 and 5). Clinicopathological features of the control cases are summarized in Table 2B.

Pathological diagnoses

The pancreas in all four ANHH cases was entirely resected and examined (representative case shown in Fig. 1). All pancreatic specimens were cut perpendicular to the main pancreatic duct and preserved as formalin-fixed paraffin-embedded (FFPE) samples. Hematoxylin-eosin (H&E) staining and immunostaining for insulin (guinea pig polyclonal, Cat# A05664; DAKO, Glostrup, Denmark), glucagon (rabbit polyclonal, Cat# N1541; DAKO), pancreatic polypeptide (rabbit polyclonal, Cat# A0619; DAKO), somatostatin (rabbit polyclonal, Cat# N1551; DAKO), Ki-67 (mouse monoclonal, Cat# M7240; DAKO), and proliferating cell nuclear antigen (PCNA) (rabbit monoclonal, Cat# 13,110; Cell Signaling Technology, Boston, MA, USA) were performed using a Ventana Benchmark Ultra autoimmunostainer (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocols. H&E staining was also performed on all FFPE samples in the control cases, and immunostaining for insulin was performed in some cases. A microscopic examination was performed according to the criteria proposed in Ref. 8.

Fig. 1
figure 1

Pathological features of ANHH. (A) Macroscopic and (B) microscopic examinations (H&E) and (C) insulin immunostaining reveal no mass lesion in the sections of the resected pancreas. (D) Ductuloinsular complex. H&E. (E) Multiple β-cells with an enlarged and hyperchromatic nucleus and abundant clear cytoplasm. H&E. (F) Macronucleoli in β-cells. H&E. (G-J) Immunostaining. Islets with normal spatial distribution of the various cell types. (G) Insulin, (H) Glucagon, (I) Somatostatin, and (J) Pancreatic polypeptide staining. (K) Ki-67 immunostaining reveals no proliferative activity of the islet cells. Scale bars in B, C, G-K are 250 μm; the scale bar in D is 100 μm; the scale bar in E is 50 μm; and the scale bar in F is 25 μm

The measurement of the numbers, maximum diameters, total areas, and circularities of the islets

Representative sections of ANHH and control specimens were digitized using whole-slide imaging (WSI) (Nanozoomer RS or Nanozoomer S360; Hamamatsu Photonics KK, Hamamatsu, Japan). The number, maximum diameter, perimeter, and area of each islet in each specimen were measured using an image analysis system (NIS-Element D software program, version 5.30.00; Nikon, Tokyo, Japan). Islets of any size were evaluated. The measurement method is shown in the schematic illustration (Fig. 2A). The cross-sectional area of pancreatic parenchyma was measured in the same manner. The circularity ranged from 0 to 1.0, with a higher circularity meaning that the shape of the given islet was closer to a circle [9,10,11]. In this study, circularity was calculated using the following formula: 4π × (area of islet) / (perimeter of islet)2. We adopted 0.71 as a threshold value because it was the value that generated the largest statistically significant difference.

Fig. 2
figure 2

Measurement procedure of the islets (A), representative images with each circularity value (B) and representative images showing an irregular shape of the islets in ANHH cases compared to controls (insulin immunostaining) (C)

The measurement of cell size, the percentage of cells with enlarged nuclei, and the percentage of cells with recognizable nucleolus of the representative islets

For each of the three representative islets, we measured the average cell size (number of cells in the islet divided by the area of the islet), percentage of cells with enlarged nuclei (nucleus size: ≥9 μm), and percentage of cells in which the nucleoli could be recognized (nucleolus size: ≥1.5 μm) using the virtual slides above.

Interobserver analyses

We conducted a blind assessment of the virtual slides of four ANHH cases (cases 1–4) and four control cases (cases 1–4) to evaluate interobserver diagnostic concordance. The evaluations were performed by 5 experienced pathologists (≥ 10 years’ experience) and 7 less-experienced pathologists (< 10 years’ experience). After presenting the diagnostic criteria in Table 1, pathologists were asked to identify whether they were ANHH or controls. The number of ANHH and controls are not presented.

Statistical analyses

Data in Table 3; Fig. 3 are expressed as the mean ± standard error. Differences between the groups were examined for statistical significance using an unpaired t-test (Excel; Microsoft, Redmond, WA, USA). P was set at p < 0.05.

Table 3 The measured parameters and aspect ratio of the islets of ANHH and control cases
Fig. 3
figure 3

Number of islets with circularity < 0.71 per total area of pancreas parenchyma in patients with diffuse adult-onset ANHH compared with control specimens

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