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2.
Figure 3

Figure 3. From: Consensus criteria for sensitive detection of minimal neuroblastoma cells in bone marrow, blood and stem cell preparations by immunocytology and QRT-PCR: recommendations by the International Neuroblastoma Risk Group Task Force.

Immunocytological analysis. Consensus guidelines for light microscopic evaluation, cell counting and reporting of results.

K Beiske, et al. Br J Cancer. 2009 May 19;100(10):1627-1637.
3.
Figure 2

Figure 2. From: Consensus criteria for sensitive detection of minimal neuroblastoma cells in bone marrow, blood and stem cell preparations by immunocytology and QRT-PCR: recommendations by the International Neuroblastoma Risk Group Task Force.

Immunocytological analysis. Consensus guidelines for cell preparation and immunocytochemical staining. Abbreviations: r.p.m., rounds per min; PBS, phosphate-buffered saline; BSA, bovine serum albumin; APAAP, alkaline phosphatase anti-alkaline phosphatase.

K Beiske, et al. Br J Cancer. 2009 May 19;100(10):1627-1637.
4.
Figure 1

Figure 1. From: Consensus criteria for sensitive detection of minimal neuroblastoma cells in bone marrow, blood and stem cell preparations by immunocytology and QRT-PCR: recommendations by the International Neuroblastoma Risk Group Task Force.

Consensus guidelines for sampling bone marrow (BM), peripheral blood (PB) and peripheral blood stem cell (PBSC) harvest for immunocytology (IC) and reverse transcriptase (RT)–PCR.

K Beiske, et al. Br J Cancer. 2009 May 19;100(10):1627-1637.
5.
Figure 4

Figure 4. From: Consensus criteria for sensitive detection of minimal neuroblastoma cells in bone marrow, blood and stem cell preparations by immunocytology and QRT-PCR: recommendations by the International Neuroblastoma Risk Group Task Force.

(AL) Immunostaining of cytospins containing mononuclear cells from BM aspirates pulled from children with NB stage M. The red label shows binding of monoclonal anti-GD2 antibody 14.G2a visualised by a commercial alkaline phosphatase anti-alkaline phosphatase kit and Fuchsin+ substrate (Swerts et al, 2005). All images are taken with a × 60 dry lens and reproduced with identical magnification to enable comparison of different cell types by size. A and B show the mandatory cytomorphological and immunological criteria of neuroblasts, that is high nuclear/cytoplasmic ratio and strong GD2 membrane expression. A granular appearance of the chromatin is frequently, but not always recognised. Neuroblasts are usually larger than small lymphocytes (arrows). If all criteria are fulfilled, even single neuroblasts can be identified (B). Reliable recognition of cytomorphological and immunological criteria depends on paraformaldehyde fixation. (C) Typical clump formation of neuroblasts with moulding nuclei. Some tumour cells may show increased amount of cytoplasm (arrow), but the nuclear/cytoplasmic ratio should not decline below 2. Note adjacent GD2-negative lymphocytes. Cells that fulfil the criteria as detailed in AC are classified as criteria-positive cells (CPC). (DF) Shedding of GD2 from a clump (D) or a single neuroblast (arrow in E) on adjacent haematopoietic cells creates the impression of an almost complete (arrow in D) or partial membrane staining. GD2 may even shed from fibrillary acellular material, for example fragment of neuropile (arrow in F) on to normal cells. Note: no neuroblasts are identified in F. (GI) Not convincingly interpretable cells (NCICs). Two of the cells in G (arrows) do not display a complete membrane staining. However, their nuclear size, morphology and clump formation strongly favour a neuroblastic origin. In H, form and size of the nucleus suggest a neuroblast, but the cell membrane is widely avoided of GD2. Also the cell in I is incompletely stained and in addition somewhat smaller than typical neuroblasts. The true nature of these cells can only be revealed by means of ancillary methods (eg FISH, single-cell PCR). Their potential clinical significance remains to be established. (JL) Macrophages with ingested GD2 are often found after chemotherapy and must not be interpreted as tumour cells. Hallmarks of macrophages are a very low nuclear/cytoplasmic ratio (usually <1), a granular compartmentalisation of the antigenic material in vesicles and a negative membrane stain (arrows in JL), although the label may even occupy the whole cytoplasm (L). The colour quality of ingested GD2 is usually not purple red as in neuroblasts (see AE), but rather brownish due to increased iron storage after blood transfusions.

K Beiske, et al. Br J Cancer. 2009 May 19;100(10):1627-1637.

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