I'm crossposting this to bionet.immunology, where I think you will
find a rather greater concentration of flow cytometry expertise.
In article <33717308.32EA at usa.net>, Jumbuck <jumbuck at usa.net> wrote:
>Martin Offterdinger wrote:
>>>> On Fri, 2 May 1997 13:21:21 +0100 (BST), BMBRNL at leeds.ac.uk (R.N.
>> Leach) wrote:
>>>> >Could anyone tell me the limit of sensitivity of fluorescence activated
>> >cell sorting? Is it possible to detect, for example, one labelled cell
>> >out of a population of 1 million cells?
>> >Thank you in advance.
>> >Rob Leach,Ph.D.,
>> >Dept. of Biochemistry and Molecular Biology,
>> >University of Leeds
>>>> Theoretically it is, but
>> 1) You will need enourmous amounts of cells, to achieve statistical
>> significance-in your case at least 1000 million cells.
>> 2)the experiment will take quite a lot of time, because you have to
>> pass these 1000million cells through the FACScan!
>>>> hope this helps!
>> Martin
>>>what if all your intereseted in is the positive/negative aspect of the
>marking, rather than the level of positivity (flourescence) per cell?
>>Are any other antibody related assays more sensitive?
>>Robert
>University of Melbourne
Three reasons that people have been trying to pursue rare-event flow
cytometry are 1) the detection of fetal cells in maternal blood, thus by-
passing the need for an invasive procedure like amniocentesis: 2) the de-
tection of residual metastatic tumor cells in the circulation, post-chemo-
therapy; 3) identification and sorting of hematopoietic stem cells for bone
marrow transplants. In at least the first two cases the cells are likely
well-endowed with some discriminating marker; there just aren't many cells
to find. The issue of statistical significance is a relative one. If you
want to accurately assess the *frequency* of your roughly 1:1,000,000 rare
event against the total cell count, then Martin is correct -- you need a
lot. On the other hand, if you just want to look at a secondary character-
istic of the rare cells -- e.g., looking for chromosomal trisomies in
fetal cells -- then you might not need very many.
Hans-Joachim Gross and Diether Recktenwald presented at least one
abstract at an International Society for Analytical Cytology (ISAC) meeting
that detailed their efforts to detect one cell in a million by flow cytom-
etry. They accomplished this in a model system in which PBMC were spiked
with decreasing numbers of epithelial cells. (And yes, they collected
data files with 100 million events.) There were basically two findings.
First, an ordinary flow cytometer is "dirty". The sample injection port,
the flow cell, and even the sheath line have small quantities of fluores-
cent particles in them. They may be floating free or they may be tempo-
rarily attached to the tube walls. So the first thing they did was to
clean the entire system very well. Second, false positive events can occur
when antibody aggregates pass through the flow cell at the same time as an
unlabeled cell. Therefore, in order to unambiguously identify a rare event,
they used multi-color flow cytometry. One channel was used for a rare-cell
positive marker, but the second channel was a negative marker for the rare
cell which also stained every other cell type in the preparation. In prac-
tice, this can mean mixing several antibodies together for the exclusion of
all the unwanted cells.
--
Unique ID : Ladasky, John Joseph Jr.
Title : BA Biochemistry, U.C. Berkeley, 1989 (Ph.D. perhaps 1998???)
Location : Stanford University, Dept. of Structural Biology
Keywords : immunology, music, running, Green
