Biomedical Engineering Reference
In-Depth Information
viability, and so on can be obtained simultaneously. High content analysis such as this
can provide increased efficiency and cost reduction since a study may require fewer
reagents, animals, and time to completion. Presently available instrumentation and
fluorescent reporters make multiparameter (10
colors) immunophenotyping rou-
tine. It is often underappreciated that a single 10-color experiment provides directly
correlative data that simply cannot be obtained with two separate five-color panels. In
addition, the two-panel approach is inherently inefficient since some antigens are
undoubtedly repeated to identify the main populations of interest, hence the savings in
resources. There is also the real potential for increased analytical precision. In the case
of manual bonemarrowanalysis, the current “gold standard,” it is easy to see howflow
cytometry provides increased analytical precision. Basic statistics dictates that the
absolute error is larger when counting fewer objects. Manual analysis allows only
hundreds of cells to be counted whereas flow cytometry allows from thousands to
millions. Flow cytometric cell lineage classification using monoclonal antibodies is
also less subjective than manual cytologic methods. A comparison of flow cytometry
and manual bone marrow assessment in mice is included in this chapter.
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6.4 CHALLENGES
Along with the many opportunities come some challenges. Three major challenges
are readily identified. The first is antibody availability. Antibodies raised against
murine and human antigens, especially hematopoietic cells, are readily available
directly conjugated to a plethora of fluorescent reporters. It is more difficult to find
directly conjugated antibodies raised against certain rat, dog, and nonhuman primate
antigens. In some cases, antihuman antibodies will cross-react; but this is not always
true, not even for nonhuman primate (e.g., some anti-CD3 clones). Antibodies for
certain antigens/cell types are not available at all. This makes it difficult, if not
impossible, to develop identical panels across species. This can be overcome if you
have the time and budget to have antibodies developed, else you must wait until the
antibodies become commercially available. The second challenge is related speci-
fically to multiparameter, high-content flow cytometry. Assuming antibodies are
available for all antigens of interest, it is fairly easy to develop a three- or four-color
panel as most antibody vendors provide three or four conjugation choices. FITC, PE,
PECy5, PECy7, APC, and APC-Cy7 as well as AlexaFluor variants with similar
fluorescence emissions are common choices. And this could be accomplished with
one (488 nm) or two laser (488 nm and
640 nm) excitation. But building a 10-color
panel can prove a little more difficult. It can be more challenging to identify suitable
antibody-fluor combinations and may also require additional lasers for excitation.
Furthermore, considerable thought should be given to pairing fluors and antigens.
“Brighter” flours should be used to label less prevalent antigens. Consideration should
also be given to how spectral overlap (a.k.a. spillover) will affect results. The third
major challenge is data analysis. A 10-color analysis will likely provide many unique
populations, some of them very rare. The wealth of data provided is extremely
valuable but does pose a challenge. Manual analysis usingmultiple two-parameter dot
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