Analysis of Variables Affecting the Flow Cytometric Recovery of Plasma Cell Myeloma Cells in Bone Marrow Aspirates.
Christopher H Cogbill, Michael D Spears, Alexandra M Harrington, Horatiu Olteanu, Steven H Kroft. Medical College of Wisconsin, Milwaukee
Background: Flow cytometry (FC) is playing an increasing role in the evaluation of plasma cell myeloma (PCM). However, it is widely recognized that plasma cells (PCs) are generally underrepresented in FC analyses, with losses averaging 70% and sometimes exceeding an order of magnitude compared with aspirate counts. Hemodilution due to "second pull" aspirates is often implicated, but concentration of PCs in lipid-rich bone marrow particles has also been hypothesized to play a role. We further hypothesized that biologic and physical characteristics of PCs may also affect FC recovery, and that selective loss of PCs may occur during processing of FC samples.
Design: We retrospectively analyzed diagnostic and post-treatment PCM bone marrows with ≥2% aspirate smear PCs. 500 non-erythroid cells were counted on both routine aspirate preparations and post-erythrocyte lysis cytospins. The predominant PC morphology was classified as lymphoplasmacytic, mature, intermediate maturity, or plasmablastic. FC PC enumeration was performed in the following tubes with surface CD14/CD56/CD45/CD38 and intracytoplasmic kappa/lambda/CD45/CD38 after exclusion of erythroid events/debris. PCs were defined as CD38(bright+), CD45(dim to -) events. PC recovery was calculated as: cytospin/aspirate%, FC/aspirate%, and FC/cytospin%. Cytogenetic and clinical data were obtained by chart review.
Results: There were 54 FC analyses from 32 pts. The mean aspirate PC% was 30.3±3.0%. The PC% was decreased in both the post-lysis cytospin (mean 9.1±2.2%) and by FC (mean 7.2±2.0%) (p<.001 for both). FC PC% was also significantly lower than cytospin% (p<0.01). Poorer PC recovery was associated with del(13q)(cytospin/aspirate%, p=0.021; FC/aspirate%, p= 0.031; FC/cytospin%, p=0.021). Cases with del p53 showed a trend towards poorer recovery by FC/cytospin% (p=0.063). There were trends towards poorer recovery from aspirates with lower PC%. Immunophenotypic features (CD19, CD20, CD45, CD56), heavy chain type, prior treatment, morphologic category, and β-2 microglobulin were not significantly associated with PC recovery.
Conclusions: Similar to prior reports, we found an average decrement in FC PC recovery versus aspirate differential count of ≈75%. The most dramatic decrease occurs between the morphologic aspirate and post-erythrocyte lysis specimens, but a small, albeit significant, loss is incurred on further processing. Interestingly, we also found that the presence of 13q deletions affected PC recovery, suggesting that biologic as well as technical variables may play a role in PC loss.
Tuesday, March 1, 2011 1:00 PM
Poster Session IV # 164, Tuesday Afternoon