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MB Sample ID: SA043609
| Local Sample ID: | ms6274-17 |
| Subject ID: | SU000816 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Species Group: | Human |
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Collection:
| Collection ID: | CO000810 |
| Collection Summary: | In order to analyze the metabolites of clonal PCs (intracellular) and BM plasma (extracellular) separately, they have to be separated out from the BM samples. Thus, upon acquiring the BM samples from patients they will be placed in centrifuged at 2500 rpm for 10 minutes to separate out the plasma from the cellular fraction. The separated BM plasma is then stored in separate vials and snap frozen under liquid nitrogen for 20 seconds before storing at -80οC for further analysis. The leftover cellular component present as a pellet will be washed and reconstituted with an equal volume of RPMI 1640 medium. Erythrocytes are lysed using ammonium chloride lysing solution. After incubation on ice for 5 min, the cell suspension is diluted with RPMI medium. The cells are again pelleted by centrifugation and then suspended in RoboSep buffer (250ml PBS, 2% BSA, 1mM EDTA). The clonal CD138 positive PCs are purified using positive selection by mixing the cells with a CD138 positive selection cocktail and anti-CD138 magnetic-activated cell separation microbeads (ROBOSEP™ cell separation system, StemCell Technologies Inc) in the automated RoboSep cell separation system. The purified samples containing only CD138 positive cells are re‐suspended and then centrifuged to form a cell pellet. The goal will be to obtain at least 1-2 x 107 clonal PCs per sample. The cell pellet will be snap frozen under liquid nitrogen for 20 seconds before storing at -80οC for further analysis. Both the BM plasma samples as well as the clonal PCs pellet will be provided to the metabolomics core for sample preparation for LC-MS analysis. For Aim 2, we will be using stored BM samples from SMM patients that have already had their BM plasma and clonal PCs separated from each other and stored at -80οC. It is important to note that all these samples were collected and frozen in a timely manner. Furthermore consistency in sample collection, storage and processing is imperative for the optimal conduct of metabolomics-based experiments. This ensures that all samples being analyzed and compared with each other would have been manipulated similarly, limiting any handling biases. All patient samples in the Predolin Foundation Biobank were collected and stored by following a standardized operating procedure. BM samples were processed for BM plasma separation and clonal PCs enrichment but were then immediately snap frozen for storage at -80οC within approximately 3 hours of collection from the patient. All samples were collected in patients who have been fasting for at least 6 to 8 hours prior. To further ensure consistency in our analysis of this study, we will only use samples that have never been thawed since initial storage to preserve the stability of the metabolites originally present in the samples. |
| Sample Type: | Bone marrow |
