Apoptosis Rates in Myeloma Cells Can Be Significantly Affected by CD138 Sorting and by the Specific Method Used To Detect Apoptotic Cells.

Background: The clonal plasma cells in multiple myeloma have relatively low rates of apoptosis. We have previously shown that the apoptotic rates of the plasma cells have prognostic value in these patients. However, several methods are used both for targeting the myeloma cells for study as well as for detection of apoptosis in these cells. It is not clear which method is optimal for studying apoptotic rates of plasma cells from primary patient samples. We examined two different ways of studying the plasma cells, namely CD138 magnetic bead based positive selection of myeloma cells (sorted) and gating on the plasma cells in whole bone marrow (unsorted) using CD38/CD45 expression patterns. Additionally, we compared two different methods of identifying apoptotic plasma cells.

Methods: Paired samples processed by two different methods were studied. The bone marrow aspirate samples were initially processed using ACK lysis to remove the red cells. The samples were then studied using flow cytometry (unsorted sample) or subjected to a second step of magnetic bead based separation of myeloma cells based on CD138 expression (sorted sample). Each of the samples was studied using Annexin/PI and Apo 2.7 expression to identify the plasma cells undergoing apoptosis. Plasma cells were identified in the unsorted samples by virtue of their CD38/CD45 expression. Samples were studied at collection and after 48 hours of culture.

Results: At collection, the median percentages of cells considered apoptotic by annexin/PI (Annexin pos, PI neg) were higher than that detected by the Apo 2.7 method (Apo 2.7 + cells), for both sorted (6.5% vs. 1.5%) and unsorted cells (26% vs. 10%). However, after 48 hours of culture the percentage of apoptotic cells detected using the Apo 2.7 method were higher than that by Annexin/PI for both sorted (22.5% vs. 14.5%) and unsorted cells (43.5% vs. 10%). When the 48 hr study was compared with the initial study, percent apoptotic cells detected by Apo 2.7 had increased at 48 hrs for both sorted and unsorted cells. However, the percentage of apoptotic cells by annexin/PI had decreased in the unsorted cells at 48 hrs compared to initial study. The sorted samples had an overall increase in apoptosis at 48 hours as measured by annexin/PI. We then looked at the percentage of apoptotic cells detected by either method on initial study and compared the sorted cells and unsorted cells. Using either method, the percentage of apoptotic cells was significantly less (P=0.01; paired t-test) among the sorted cells compared to the unsorted cells.

Conclusion: Previous studies have shown that CD138 expression is lost from PCs as they enter apoptosis. Here we show in paired samples that the percentage of apoptotic myeloma cells significantly decreases following CD138 sorting. CD138 sorting should not be used when studying apoptosis of myeloma cells in patient samples. Use of unsorted cells also allows for examination of the effects of interventions on other cell populations as well. When serial studies of apoptosis are done with unsorted samples, we recommend using Apo 2.7 rather than Ann/PI, since the percentage of apoptotic cells by Ann/PI expression actually appear to decrease with time in culture, thus making it difficult to assess effects of intervention such as in drug treatment studies. We hypothesize that this is due to an artificially high level of Annexin positivity at the initial study that might be brought on by non-specific damage of the membranes secondary to cell processing.