FLOW CYTOMETRY IN PRECLINICAL TOXICOLOGY/SAFETY ASSESSMENT - Flow Cytometry in Drug Discovery and Development - page 137

Ovation Biotin System. Size and integrity of amplified cDNA (SPIA) products and

fragmented and biotin-labeled SPIA product were assessed using the Agilent 2100
BioAnalyzer RNA 6000 Nano LabChip system. Fragmented and biotin-labeled
SPIA yields and purities were determined by measuring O.D.260, O.D.280, and
O.D.320 usingNanoDrop. Targets containing 2.2 g of fragmented and biotin-labeled

SPIA were prepared for hybridization onto the Affymetrix GeneChip Rat
Genome 230

m

2.0 arrays. Samples were hybridized for approximately 18 h at 45 C
in a hybridization oven at 60 rpm rotation. Following hybridization, arrays were
washed and stained with R-phycoerythrin streptavidin using the Affymetrix EukGE-
WS2v4_450_OneFill protocol. Arrays were scanned using a GeneChip Scanner
3000. Array quality was analyzed by principal component analysis; results indicated
zero outliers due to overall array quality.

þ

6.6.6 Results and Discussion

Statistical evaluation of linear dose response was used to identify toxicologically
relevant genes, that is, genes whose expression changed linearly with increasing
dose and hence increasing severity of vascular injury. For each gene, dose response
was evaluated by application of a statistical trend test to expression values to obtain
a P-value for linear response to increasing dose. Specifically, a one-way ANOVA
analysis across dose groupings was followed by calculation of a t-statistic using
a post hoc contrast for linear trend. A gene-specific reference distribution for the
observed t-statistic was obtained using 100,000 permutations of the dose labels and
recalculating the t-statistic for each permutation. A P-value for dose response was
then obtained by comparing the observed t-statistic against the reference distribu-
tion. Over 1602 statistically significant dose-responsive genes were identified
(P 0.01). This is above the number expected by chance based on a chip of this

size (31,099 genes). Representative data from four genes of interest is presented in
Figure 6.4. These results demonstrated that gene expression analysis of sorted CECs
in preclinical vascular toxicity studies is feasible. Many other tool compounds were
investigated using a similar study design providing a wealth of data that is still being
explored.

<

6.7 MOUSE BONE MARROW ASSESSMENT

Bone marrow differentials in preclinical toxicology studies are routinely assessed by
manual microscopic evaluation. This method is tedious and labor intensive, with
several hundred cells being counted per slide. Evidence suggests that flow cytometric
analysis of rodent bone marrow can identify major hematopoietic subsets and reveal
hematologic abnormalities [31-34]. Therefore, flow cytometric bone marrow differ-
ential analysis was investigated as a replacement to the manual cytologic technique.
To this end, a multiparameter flow cytometric panel consisting of antibodies against
erythroid (ter119), leukocyte (CD45), and myeloid (CD11b) antigens and the DNA
binding dyes Hoechst 33342 (HO) and 7-aminoactinomycin D (7AAD) was devised