PMC8579051_01
Flow chart of diagnostic work-up. Clinical presentation with headache, visual disturbances, and the intracranial location required differential diagnosis with a wide range of CNS conditions age-related. The patient underwent to clinical examination, instrumental and biochemical analyses. During the hydrocephalus decompression procedure, a sample of CSF fluid was collected. The CSF was clear and the cell count and protein content were normal. Disorders such as multiple sclerosis and Bickerstaff's encephalitis, conceivable for the clinical symptoms and young age, were excluded. The tissue biopsy needed to exclude high-grade glioma, pineocytoma, germinoma or infectious and parasitic diseases was not recommended. Clinicians opted for performing a protocol of short-time culture on chamber slide to obtain cytological preparations from the blood. Pathological evaluation on liquid biopsy cytological specimens suggested the final diagnosis of pilocytic astrocytoma further confirmed by post-shunting MRI and 68 months of clinical outcome.
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Flow chart adopted in the diagnostic phase is reported in  Figure 1 .
MRI post-shunting (20 days). (A) MRI (T1-weighted image) shows a hypointensity signal periaqueductal area (red circle) with irregular profile suggesting the presence of the lesion. (B) MRI scan (T2-weighted image) shows a hyperintensity signal in the same area (red circle). (C) Gadolinium T1-weighted image shows a low focal contrast enhancement of the lesion in the periaqueductal area. (D) Proton magnetic resonance spectroscopy (MRS) reveals elevated cholin peaks (cholin/creatinine ratio at 1,9) in addition to reduced NAA (N-acetylaspartate). (E) Perfusion-weighted imaging (PWI) shows a low cerebral blood volume (CBV) in the area of interest.
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MRI showed a significant decrease of the ventricular size. Moreover, the MRI revealed a nodular lesion within the mesencephalic tectum with hypo-intense areas on the T1-weighted images ( Figure 2A ) and hyper intense signals on the T2-weighted sequence ( Figure 2B ). Gadolinium- T1-weighted image showed a low focal enhancement of the lesion in the periaqueductal area which suggested a reduced damage of the blood-brain barrier ( Figure 2C ). Proton magnetic resonance spectroscopy (H-MRS) revealed elevated choline peaks (choline/creatinine ratio at 1,9) in addition to reduced NAA (N-acetylaspartate) ( Figure 2D ). The perfusion-weighted imaging (PWI) of the lesion showed a low value of relative cerebral blood volume (CBV) ( Figure 2E ).
Blood-derived glial cells in cytological preparations. (a) Peripheral blood sample collection. (b) Blood sample gradient. (c) From the gradient, a cellular suspension including non-haematological cells (N-HC) and residual of haematological cells (HC) was isolated. (d) The entire cell suspension was seeded in chamber slides and, after 14 days, adherent cells on slide were fixed for cytological analysis. (A, B) H&E staining of CTCs deriving from the presented case shows different cellular shapes. (C, D) Immunostaining shows CTCs S-100 and (E, F) GFAP-positive deriving from the presented case. (G, H) Cytological pattern of CTCs derived from one glioblastoma and one cerebral metastasis from melanoma used as positive controls. (I) Cytological pattern of cells from a frontal lobe scar used as negative control.
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The cellular suspension isolated in correspondence of the working density phase ( Figures 3a, b ) was seeded on chamber slides ( Figure 3d ) for a short-time expansion of 14 days. In this case, as shown in  Figure 3 , the Hematoxylin and Eosin (H&E) stained cytological preparations revealed the presence of bulky, polinucleated or spindled cell atypical elements ( Figures 3A, B ). In addition, these atypical cells found in the blood resulted, at the immunohistochemical analysis performed through methods previously described, positive for the expression of the astrocytes markers S-100 ( Figures 3C, D ) and glial fibrillary acidic protein (GFAP) ( Figures 3E, F ). Moreover, as positive control for the presence of CTCs from cerebral lesions in the peripheral blood we used cytological preparations obtained with the same method from peripheral blood collected from one case of glioblastoma and from one case of brain metastasis from melanoma (cerebral metastasis) ( Figures 3G, H ). As negative control was used the cytological pattern isolated by peripheral blood collected from a case of post-stroke frontal lobe scar ( Figure 3I ). The final cytological diagnosis was of pilocytic astrocytoma because the protean features of the proliferating glial elements ( Figures 3A-F ).