Tissue samples received in transport media (RPMI 1640 with 16% fetal calf serum [FCS], 99 U/ml penicillin, 99 μg/ml streptomycin, and 41 U/ml heparin) were manually disaggreated by scraping with glass slides and washing two times in buffer (phosphate buffered saline [PBS], pH 7.4, with 2% FCS and 0.1% sodium azide). The cells were enumerated with a Sysmex K-1000 (TOA Medical Electronics, Kobe, Japan). The cell number and viability were determined manually by a trypan blue dye exclusion test in a hemocytometer chamber. Cytospins were prepared for morphologic review by the pathologists. Bone marrow aspiration samples received in transport media were centrifuged, with all except 2 ml of the supernatant discarded, and lysed in 50 ml of ammonium chloride lysing solution (pH 7.4, 0.15 M ammonium chloride, 0.009 M sodium bicarbonate, and 0.001 M EDTA). The marrow samples were washed twice and enumerated. Cell viability was determined and cytospins were prepared as described above for tissue samples. The initial CD45− CD56very bright+ dual-color and CD45− CD56very bright+ NSEcytoplasmic+ three-color methods evolved into the three-color GD2 cell membrane method. A total of 1 × 106 isolated tissue or marrow aspirate cells were labeled with 15 μl of GD2FITC (G2005-15, clone 8.S.077, IgM kappa [US Biological, Swampscott, MA] or 870633, clone GMR7, IgM kappa, fluorescein isothiocyanate [FITC; Seikagaku America, Falmouth, MA]), 5 μl of 1:5 diluted CD56PE (R7127, clone MOC-1, phycoerythrin [PE], Dako, Carpinteria, CA), and 20 μl of CD45PerCP (340665, clone 2D1, peridinin chlorophyll protein [PerCP], Becton Dickinson, San Jose, CA). Depending on the lot, the optimum titer ranged from 2 to 15 μl for GD2 and from 5 μl of a 1:5 dilution to 3 μl of “neat” CD56 based on testing against the SK-N-DZ cell line. Some of the results successfully substituted optimally titered CD56 from Becton Dickinson or Beckman Coulter (Hialeah, FL). A similar cocktail of IgMFITC IgG1PE IgG1PerCP class/subclass controls was used to label another tube of patient cells. CD45FITC, CD45PE, and CD45PerCP compensator controls were used separately to label daily control peripheral blood cells from a normal donor to help fine-tune the flow cytometer compensation. Cells were labeled for 15 min at 4°C, washed in buffer, lysed a second time for 3–5 min at room temperature in 1 ml of lysing solution (349202 FACS lysing solution, Becton Dickinson), and fixed in 1% formaldehyde solution. Samples were analyzed on a FACScan, FACSort, or FACSCalibur flow cytometer (Becton Dickinson) using either Lysis II or CellQuest software. The analysis strategy was to maximize the count of suspected neuroblastoma cells, minimize data storage, minimize computer time for analysis of the acquired data, but still report the percent suspected neuroblastoma cells in terms of all tissue or marrow cells to help monitor the patient. Approximately 10,000 events were collected and stored on a forward scatter (FSC) versus side scatter (SSC) plot, an R1 gate was established around a lymphocytic/blast/neuroblastoma cell area, and 50,000 or more events were collected and stored only within the R1 gate. If positive events were seen, the cell acquisition count was increased and/or more cells were labeled to achieve the customary goal for rare event analysis of 100 positive events for statistical relevance. The percentage of cells of interest within the R1 gate multiplied by the percentage of the R1 gate (the 10,000 events described above) estimated the percentage of cells of interest in the entire sample. The International Society of Hematology and Graft Engineering (ISHAGE) method was used for stem cell assays. It was helpful at the end of the assay to “back-gate” any CD45− CD56very bright+ GD2+ events on a FSC/SCC light scatter plot to confirm a cluster of cells centrally/evenly located in the lymphocytic/neuroblastoma/blast area that did not nonspecifically cluster toward the perimeter of the light scatter gate (perhaps implying nonspecific events). A cluster of cells was considered positive whereas one to three cells arbitrarily was considered to be suspicious. A successful positive control for the assay could either be cryogenically stored/aliquoted neuroblastoma tissue (example not shown) or a routinely used SK-N-DZ neuroblastoma cell line from the American Type Culture Collection (Manassas, VA, CRL-2149) that successfully demonstrated CD45− CD56very bright+ GD2+ CD81+ expression (Fig. 1).
Tissue and marrow samples included in this study were either sent to us for evaluation for suspected leukemia/non-Hodgkin's lymphoma or to confirm neuroblastoma. From 1992 to the end of 2001, 36 patients were diagnosed at our institution with neuroblastoma. Counting bilateral marrow samples as one sample unless separately analyzed by flow cytometry, we received 300 samples from these 36 patients. In addition to DNA ploidy analysis, stem cell enumeration, and “cell holds,” 123 samples from 25 of these 36 patients (Table 1) were analyzed by either the dual-color assay (N = 48), the three-color NSE assay (N = 16), or the three-color GD2 assay (N = 59). The INSS stages of the 25 patients were Stage I (N = 2), Stage III (N = 3), Stage IV (N = 18), or Stage 4S (N = 2). Of the 25 patients, 15 are currently alive (15 received transplants).