Table 1.

Comparison of two major, validated variations of the Flow-FISH method

Step	Immunophenotyping → FISH (Flow-FISH) Hultdin et al. (Hultdin, Gronlund et al. 1998) and modifications (Schmid, Dagarag et al. 2002, Kapoor, Hakim et al. 2009)	FISH → Immunophenotyping (FISH-Flow) Rufer et al. 1998 (Rufer, Dragowska et al. 1998) and modifications (Baerlocher, Vulto et al. 2006)
Calibration of instrument	DNA calibration beads	Calibration (MESF) beads
Cells	PBMC and 1301 control cells	PBMC and bovine thymocytes
Immunophenotyping	Few fluorochromes retain their fluorescence after the harsh conditions required for DNA denaturation in Flow-FISH (heat affects the fluorochrome) Stable fluorescent probes: Alexa Fluor 488 (Schmid, Dagarag et al. 2002), Alexa Fluor 546 (Schmid, Dagarag et al. 2002), Alexa Fluor 647 (Kapoor, Hakim et al. 2009), Qdot 605 (Kapoor, Hakim et al. 2009), Qdot 655(Kapoor, Hakim et al. 2009), Qdot 705 (Kapoor, Hakim et al. 2009) for cell surface staining Antibodies: anti-CD4, anti-CD3, anti-CD8, anti-CD14, anti-CD28 BS3 cross-linking to stabilize fluorochrome-antigen-antibody complexes	Very few antibodies recognize epitopes that survive the harsh conditions required for DNA denaturation in Flow-FISH (heat affects the epitope) Antibodies: CD45RA–Cy5 (clone 8d2) and CD20– (PE) (clone L26), Biotinylated anti-CD57 (clone HNK-1) No use of cross-linking fixatives (Baerlocher and Lansdorp 2003)
Flow-FISH	Covalent cross-linking DNA denaturation: 10 min at 80°C in a shaking water bath Hybridization: overnight in the dark.	No fixation; permeabilization only with formamide and heat DNA denaturation: 15 min at 87°C in a shaking water bath Hybridization: 2 h at RT in the dark.
DNA staining	Propidium iodide (PI), Hoechst 33342, LDS751	LDS751
Data analysis	Relative telomere length determination (TLR method)	Absolute telomere length determination (MESF/Kb)