Miniaturized FISH for Screening of Onco-Hematological Malignancies

Figures & data

Figure 1. Microfluidic device for miniaturized FISH.

(A) Sketch of the microfluidic PDMS pad. The pad has a size of 20 × 10 × 1 mm, and the microchannel is 1 cm long, 300 µm wide, and 50 µm deep. Two wells connect the microchannel with the top surface: an IN well (1.2-mm diameter) for reagent loading and an OUT well (0.7-mm diameter) for aspiration. (B) Complete structure of the FISH device (vertical section view). Glass slides are functionalized with ns-TiO₂, then treated with oxygen plasma to increase wettability (R means chemisorbed oxygen radicals); finally, the PDMS microfluidic pad is assembled on the slide, by spontaneous adhesion. An exemplary picture of a FISH device is presented; a 10-µL tip is used for cell loading.

Figure 2. Miniaturized FISH with CEP and LSI probes on tumor cells.

(A) Panel of tumor cells with CEP probes for sex chromosomes. Images from representative cells are acquired at 40× objective magnification. Red signal represents chromosome X; green signal represents chromosome Y; scale bar, 10 µm. (B) PB from normal donor (control cells) and U937 cells analyzed with LSI AML1/ETO probe. Images from representative cells are acquired at 100× objective magnification. Red signal represents chromosome 8, while green signal represents chromosome 21; U937 cells showed trisomy for both chromosomes; scale bar, 4 µm.

Figure 3. Validation of miniaturized FISH on hematopoietic cells from normal donor.

Miniaturized FISH analysis on hematopoietic cells from BM of normal donor with a panel of onco-hematology probes (Abbott Molecular, unless otherwise indicated): (A) Vysis CEP X SpectrumOrange/YSpectrumGreen Direct labeled fluorescent DNA probe kit; (B) Vysis LSIp53/ATM; (C) Vysis LSI D13S319/LSI 13q34/CEP 12 Multicolor probe; (D) Vysis LSI BCR/ABL Dual Color Dual Fusion Translocation probe; (E) Vysis LSI IGH Dual Color Break Apart Rearrangement probe; (F) Vysis LSI IGH/CCDN1 Dual Color Dual Fusion Translocation probe; (G) Poseidon Repeat Free 6q21 © SEM 6 Control probe (Kreatech); and (H) Vysis LSI AML1/ETO Dual Color, Dual Fusion Translocation probe. Representative images were acquired at 100× oil objective magnification. Scale bar, 5 µm.

Figure 4. Comparative FISH analysis: miniaturized FISH versus standard FISH.

(Upper panel) Miniaturized FISH (A–D) and standard FISH (E–H) for sex chromosome chimerism detection (A, B, E, and F) and in a CLL case (C, D, G, and H) with chromosome 12 trisomy, respectively. (A and E) Merged images at 20× objective magnification; scale bar, 20 µm. (B and F) Merged images at 100× oil objective magnification; scale bar, 5 µm. (C and G) Raw images at 100× oil objective magnification representing single channels of the different fluorescent probes (orange, chr.13q14.3; green, chr. 12p11.1-q11; aqua, chr.13q34). (D and H) Merge of different channels in panels C and G, respectively. Scale bar, 5 µm. (Lower panel) Miniaturized FISH (I–N) and standard FISH (O–R): genetic lesions detected are p53/ATM deletion in a CLL case (I and O), 13q14.3 deletion in a CLL case (L and P), chromosome 16 inversion in an AML case (M and Q), and BCR-ABL translocation in a CML case (N and R). Images are acquired at 100× oil objective magnification. White arrows on each image indicate nuclei with the relative specific genetic lesion detected.