autologous blood and marrow transplantation - Blog Science ...

238 Chapter 5: Myeloma
of CD34 +
cells by fluorescence-activated cell sorter (FACS) analysis. Mononuclear
cells (MNC) from BM and LP samples were obtained by Ficoll-Hypaque density
centrifugation (Biochrom, Berlin, Germany).
Immunofluorescence analysis
1X10 6
MNC of the LP or 20-50 pL of whole blood were incubated for 30
minutes at 4°C with fluorescein isothiocyanate-conjugated anti-CD34 antibody
(HPCA-2) obtained from Becton Dickinson (Heidelberg, Germany). Isotypeidentical
antibodies served as controls. The immunofluorescence analysis was
performed using a five-parameter FACScan (Becton Dickinson) as previously
described. 13
Quantitative assessment of tumor cells and statistical analysis
RNA and DNA extraction, CDR3 consensus PCR, cloning, and sequencing of
the patient-specific CDR3 regions with the subsequent generation of allele-specific
oligonucleotides complementary to the specific CDR3 regions were performed as
described previously. 14
Quantitative ASO-PCR was carried out essentially as reported. 15
In brief, per
aliquot of LP, 2 pg of DNA (equivalent to 3.3X10 5
cells) was serially diluted in
10-fold steps with placental DNA (Clontech, Palo Alto, CA) and amplified using
the patient-specific ASO together with the antisense JH-consensus primer. 16
At
each dilution level, five or 10 identical PCR reactions were performed simultaneously
in a volume of 50 pL with 2 pg genomic DNA, 2.5 U AmpliTaq Gold
DNA-polymerase (Perkin Elmer, Weiterstadt, Germany), 2 mM MgCl2, 5 pL
GeneAmp 10X PCR buffer II, 0.2 mM of dNTPs, and 0.6 pM of each primer.
The percentages of malignant cells in LP were determined by using a maximum
likelihood function based on the Poisson distribution of positive and negative
reactions at each sample dilution. The calculation was done by x 2
minimization.
The absolute number of malignant cells in LP was deduced from the MNC obtained
from the differential white cell count less the number of neutrophils.
Tests whether two quantitative variables are uncorrelated were performed using
Spearman's rank correlation coefficient. Linear regression analysis was used to
examine the influence of the pretreatment cycles on tumor cell load and stem cell
yield of LP using Box-Cox power transformed data for tumor cell load and stem
cell yield (y A
lambda) with parameter lambda = 0.25. Graphical displays of the
model fit also include 95% confidence intervals. An effect was considered statistically
significant if the P value of the corresponding test value was < 0.05.
Statistical calculations were done using Statistica for Windows (StatSoft, Tulsa,
OK), and S-Plus, Version 3.4 for Sun SPARC (Math Soft, Seattle, WA).