During acquisition/analysis of flow data: what is the best practice? Can one use Mean Fluorecent Intensity (MnI) even when data has a nice dual population showing distinct negative and positive population in a sample?
Oskar, you meant both distinct NEGATIVE and POSITIVE subsets (?). Using both % and MFI you may indicate that your population is made of x% of negative cells (MFI = w a.u.) + z% of positive cells with a relative level MFI = y a.u. (arbitrary units of fluorescence). For that, you need to measure MFI on each separated subset,not on the whole population.
Let's now the case where you have two distinct subsets of dim and bright positive subsets. Again, you may describe your population as being made of e.g. 30% dim cells (MFI= xxx a.u.) + 70% bright cells (MFI= zzz a.u.). This is the case (as an example) for T4 lymphocytes with Fas (CD95) low and Fas (CD95) high, corresponding grossly to naive T4 Ly and memory T4 Ly respectively. Current % are variable in aging: 90-10% in newborns, 30-70% to 70-30% during life and 10-90% in centenaries. If you would opt for QFCM (I mean absolute quantitation of cell-surface Ags*), then a.u. could be changed to real expression levels, in that case ~500 Fas molecules/Ly in the naive subset and ~5000 Fas molecules/Ly on memory T cells.
If you would opt for MFI (geometric mean or Mn rather than arithmetic mean or MnI), then global MFI is somehow representative of immune age of a person, i.e. close to 500-1000 molec/cell in newborns up-to almost 5000 molec/cell in centenarians.
Interestingly enough, patients with viral infections show both enhanced % of Fas high and elevated density of Fas on Fas high (up-to 20000 fas molec/cell in acute infection like HIV or HepB). Same, after BM transplantation, transplanted patients may wait for over one year before their global Fas level (so-called "immune age") starts to decline, corresponding to the eappearance of new naive T cells.
Thus, you can measure MFI on clearly delineated subsets (also characterized by their relative %) and/or MFI (geometric mean preferably, although that point may be a matter of indefinite debate) on the whole, bi-modal (or multi-modal) population. Both types of MFI bear different significance.
Hope I am clear.
PP
*) Poncelet et al, Clinical applications of quantitative immuno-phenotyping, in:
CC Stewart & JKA Nicholson (eds), Immunophenotyping, Wiley-Liss, 2000.
We use both, depending on what you wan to measure, % of positive cells ans mean fluorescence intensity of positive cells to compare level of expression of a given receptor
We also use both depending on the questions to ask. if you want to determine how a given receptor is expressed on your whole cell population I would recommend MFI, but if you see that there are two distinct populations it would be better to use propotions.
Oskar, you meant both distinct NEGATIVE and POSITIVE subsets (?). Using both % and MFI you may indicate that your population is made of x% of negative cells (MFI = w a.u.) + z% of positive cells with a relative level MFI = y a.u. (arbitrary units of fluorescence). For that, you need to measure MFI on each separated subset,not on the whole population.
Let's now the case where you have two distinct subsets of dim and bright positive subsets. Again, you may describe your population as being made of e.g. 30% dim cells (MFI= xxx a.u.) + 70% bright cells (MFI= zzz a.u.). This is the case (as an example) for T4 lymphocytes with Fas (CD95) low and Fas (CD95) high, corresponding grossly to naive T4 Ly and memory T4 Ly respectively. Current % are variable in aging: 90-10% in newborns, 30-70% to 70-30% during life and 10-90% in centenaries. If you would opt for QFCM (I mean absolute quantitation of cell-surface Ags*), then a.u. could be changed to real expression levels, in that case ~500 Fas molecules/Ly in the naive subset and ~5000 Fas molecules/Ly on memory T cells.
If you would opt for MFI (geometric mean or Mn rather than arithmetic mean or MnI), then global MFI is somehow representative of immune age of a person, i.e. close to 500-1000 molec/cell in newborns up-to almost 5000 molec/cell in centenarians.
Interestingly enough, patients with viral infections show both enhanced % of Fas high and elevated density of Fas on Fas high (up-to 20000 fas molec/cell in acute infection like HIV or HepB). Same, after BM transplantation, transplanted patients may wait for over one year before their global Fas level (so-called "immune age") starts to decline, corresponding to the eappearance of new naive T cells.
Thus, you can measure MFI on clearly delineated subsets (also characterized by their relative %) and/or MFI (geometric mean preferably, although that point may be a matter of indefinite debate) on the whole, bi-modal (or multi-modal) population. Both types of MFI bear different significance.
Hope I am clear.
PP
*) Poncelet et al, Clinical applications of quantitative immuno-phenotyping, in:
CC Stewart & JKA Nicholson (eds), Immunophenotyping, Wiley-Liss, 2000.
- Badges
- Science method