Thanks for your strong defense of what is clearly a methodology that is
headed in the right direction, viz., the employing of fluorescent standard
beads to convert the measured average cellular fluorescence intensity to
some equivalent number of target receptor molecules per average cell.
I have no argument with the use of beads; indeed, I was among the first
persons in flow to attempt to elucidate the relationship between average
cellular fluorescence intensity and the underlying number of target
receptor/marker/antigen molecules which bound the fluorescent ligand to
produce that fluorescence. It was clear to me even then, as Bohm and Sklar
had both indicated before me, that a bead standard was the necessary
vehicle to enable the conversion from numbers of fluorescent photons
emitted by labelled cells to the numbers of bound fluorochromes underlying
those fluorescences.
In my original letter to this List, I stated the two main technical
assumptions in employing beads then went on to elaborate some other factors
which lead to what is the inhererent inaccuracy (not imprecision, thankyou
Mario Roederer for your gentle distinction) in the numbers which are
finally held aloft by Neptune [or was it Laertes or Medusa (like this
issue)] emerging from the primeval soup: Spake Neptune, "Eureka, the number
of CD71 receptors on a proliferating lymphocyte is 745,250 +/- 25,000!!"
My concern is based on the observation that numbers have a way of becoming
the gospel truth. I want to caution all players to the reality that
although they may get very reproducible data (high precision), the numbers
are still quite rubbery, because of the many uncertainties that occur
through the chain of assumptions that is followed.
A couple of examples:
Consider an antigen whose expression can be up- and down- regulated. At low
density, the Mab-FITC binding will be entirely monovalent. At high density,
the Mab-FITC binding will be entirely bivalent once the targets are dense
enough. This geometry needs to be addressed, as the numbers of bound FITC
can be the same while the antigen target numbers changes dramatically.
The second is pertinent to clinical states, where the number of marker
molecules may change, but so also may the affinity be altered due to
molecular defects in the marker itself. The fact is that the number of
Mab-FITC ligands that bind is a function of both variables: the number of
receptors expressed and their affinity for the binding ligand. Antibodies
can vary considerably in their affinities for a particular receptor,
indeed, affinity maturation is a feature of the immune system, so why could
it not work the other way around?
In summary, our task is not just to get a number, but to get the affinity
as well. In this way the number data can be made unequivocal.
Cheers, Bob