At 11/15/2001, OSCAR K. KAI wrote:
>Has anyone out there tried doing agglutination assays with flow machine e.g
>autoagglutination assay in coombs work, or any work on autoagglutination?
>
>how does one control clogging of the machine/ incase big agglutinates form?
>apart from sieving the test mixture through a mesh with small pores?(wild
thinking ,
>not done it before)
One method to avoid clogging is to let the large aggregates settle out
(don't stir the sample while acquiring), and measure only the scatter-gated
single cells. Remember that the shear during sample intake (also mixing
before acquisition) is likely to disaggregate. You can't tell how many
cells are in each aggregate anyway. So one method is called the 'single
cell loss' assay for aggregation.
The MFI free program (re-announced here yesterday,
http://www.umass.edu/microbio/mfi/) has sample data files and a description
of this assay in its tutorial, section 4,
http://www.umass.edu/microbio/mfi/tutorhlp/mfitutor.htm This illustrates
using a ratio of events in two separate scatter gates, one for (single)
beads, and one for single cells.
Because flow cytometers generally don't guarantee to acquire a fixed sample
volume per minute, you can't tell the single cell density without having a
volume reference, such as minimally-aggregating 2-micrometer beads added at
a known concentration just before acquisition. The single cell to bead
ratio then tells you single cells/ml. To quote from the above URL:
'Reading Howard Shapiro's 3rd edition of Practical Flow Cytometry, I
learned that this method was patented by Becton-Dickinson in the 1970's,
and also reported by C. C. Stewart and J. A. Steinkamp ("Quantitation of
cell concentration using the flow cytometer", Cytometry 2:238-43, 1982).
The ratio of single cells per bead is proportional to the single cells per
milliliter. Thus, the percentage of cells in aggregates can be calculated
from the reduction in single cells per bead, relative to a reference sample
known to have no cell aggregation. This type of assay is termed a "single
cell loss assay".'
-Eric Martz
----
Eric Martz, Professor (Immunology), Dept Microbiology
Morrill IVN 203, U Mass, Amherst MA US 01003-5720
413-545-2325/FAX 413-545-2532 emartz@microbio.umass.edu
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