Immunological Response to Experimental Intracerebral Hemorrhage: Morphological Assessments - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 601

using FC ( 4 ). In a similar animal model of ICH, but using IP, the
number of CD8-alpha positive cells was increased at 2-3 days and
remained detectable for 28 days after ICH ( 14 ) (Table 1 ). These
differences could be related to time of assessment or immunophe-
notyping. Also, other methods such as volumetric cytometry or
magnetic microspheres (beads) cell sorting can be more effi cient.
In regards to method sensitivity, the new multi-analyte profi ling
beads (xMAP) technology (i.e. Luminex assays) looks promising.
This technology combines proven platforms and assays—fl ow
cytometry, fl uorescent-dyed, microbeads, lasers, digital signal pro-
cessing, and traditional chemistry. Luminex assays enable the detec-
tion and quantifi cation of multiple ribonucleic acid (RNA) or
protein targets simultaneously and provide data with concordance
in ELISA and mass spectrometry.
Each method of immune system assessment has its advantages
and disadvantages. ELISA quantifi cation is rapid and highly sensi-
tive, but is an expensive method as it recognizes only one specifi c
binding site (epitope) and requires specifi c monoclonal antibodies.
Western Blotting is technically demanding and also expensive. It is
subject to interpretation (presence or absence of bands; intensity of
those bands) but remains the gold standard quantitative technique
to validate immunohistochemistry. Immunohistochemistry has the
tremendous advantage of being able to localize given proteins
within the tissue examined at relatively low cost. Its major disad-
vantage is that, unlike immunoblotting techniques where staining
is checked against a molecular weight ladder, it is impossible to
show in immunohistochemistry that the staining corresponds with
the protein of interest. Flow cytometry allows high-speed identifi -
cation and sorting of individual cells by their size, structure, or
specifi c identifi ers such as cell receptors based on optical signal
(fl uorescence-activated and volumetric methods) ( 15 ). The clear
advantage of FC over other technologies is that a large number of
parameters can be analyzed on each and every cell. However, the
disadvantage is that fl ow cytometry, even with high-speed systems,
is very much slower than automated image-processing systems ( 16 ).
While FC is a valuable technique for blood cell sorting, it is a
subject of interpretation for testing cells within parenchymatous
tissue; depending on the method of cell collection the cell mem-
branes may be damaged and results can be misinterpreted. In addi-
tion, there are some common problems which fl uorescent
immunophenotyping share between all fl uorescent-based assess-
ment techniques. When the primary antibody used to detect an
antigen on the blot is labeled with an enzyme or fl uorescent dye
(i.e. direct detection method), its immunoreactivity can be altered
which results in reduced signal amplifi cation. When a primary anti-
body is fi rst bound to the antigen and then followed by a labeled
secondary antibody that is directed against the primary antibody
(indirect detection method), the secondary antibody does not