autologous blood and marrow transplantation - Blog Science ...

Moss et al.
tests, and bilateral posterior iliac crest marrow aspirates and biopsies.
Clinical follow-up was calculated using the day of PBPC infusion, and the
last follow-up was obtained in November 1997. The information obtained
included diagnosis date, mobilization regimen, clinical status at the time of
harvesting, date of relapse, site of relapse, date of death, and last clinical
follow-up.
Marrow and PBPC specimens
Approximately 3-5 mL of bone marrow and 1-3 mL of PBPC product were
obtained from each patient. Samples from referring hospitals arrived within 24
hours, and all specimens were processed within 48 hours of collection. On
arrival in the laboratory, the marrow/PBPC product was diluted with Liebowitz
L-15 medium (L-15) supplemented with 10% fetal bovine serum (FBS) (Gibco,
Grand Island, NY). Diluted samples were layered over Ficoll-Hypaque
(Pharmacia, Uppsala, Sweden) and then subjected to density gradient centrifu-
gation. The mononuclear cell fraction was tested for viability (Trypan
exclusion), cytocentrifuged onto slides, and stored at 4°C for future immuno-
staining.
Immunostaining
Patient specimens were removed from storage the next day and tested for
immunoreactivity. Immunostaining was accomplished using the avidin biotinalkaline
phosphatase technique. Slides were fixed in a paraformaldehyde solution
at 4°C for 30 minutes and washed thoroughly in phosphate-buffered saline (PBS)
(Gibco). Before March 1995, immunostaining was performed using the Zymed
immunoperoxidase kit. After fixation, slides were incubated with anti-breast
carcinoma antibodies for 45 minutes at room temperature. This was followed by
incubation in normal goat serum, followed by Zymed reagents as per protocol.
After March 1995, following fixation, slides were placed on the Biotek
automated immunostainer (TechMate; Ventana, Tucson, AZ) and incubated with
anti-breast carcinoma antibodies for 30 minutes at 37°C. Biotek immunoreagents
for alkaline phosphatase staining were performed as per protocol. The specimen
was then incubated with Ehrlich's hematoxylin counterstain and coverslipped
with cytoseal.
Positive control slides consisted of cultured breast cancer cells (e.g., CAMA-1)
seeded into normal leukapheresis product or bone marrow and immunostained as
above. Negative control slides were the patient's specimen immunostained with normal
mouse serum at the same concentration as used for the anti-breast carcinoma
antibodies.
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