Comparison of CD45+ Depletion Methods for Enrichment of Disseminated Tumor Cells in Bone Marrow Samples
Poster Dec 07, 2017
Birte Möhlendick, Imke Hoffmann, Swetlana Seidschner, Sarah Schumacher, Alexander Smyczek, Carina Vaerst, Wolfram Trudo Knoefel, Nikolas Hendrik Stoecklein
Disseminated tumor cells (DTC) in bone marrow (BM) samples are, with a frequency of only one DTC per 1x106 cells, quite rare. DTCs are usually detected utilizing immunofluorescence staining of 1-2x106 mononuclear cells (MNC) and consecutive microscopic analysis. To increase the detection rate of DTCs a higher number of cells should be analyzed. Since microscopic detection of these cells is very laborious DTC enrichment strategies are needed. Here, we investigated the efficiency of three different depletion strategies of CD45+ cells for DTC enrichment in BM samples.
CD45+ cell depletion was performed on BM samples from cancer patients with different tumor entities. Immunomagnetic depletion (DYN) of CD45+ cells was performed after Ficoll density gradient centrifugation on 1x107 MNCs utilizing CD45 Dynabeads® (Invitrogen) with i) 4x107 beads/ml cell suspension (DYNLO) and ii) 1x108 beads/ml cells suspension (DYNHI). Antibody-based depletion (ROS) of CD45+/CD66b+ cells was performed on 1x108 BM cells with the RosetteSep® Human CD45 Depletion Cocktail (Stemcell Technologies). For the different strategies at least 60 samples were analyzed, the depletion efficiency was calculated and DTC numbers were extrapolated to compare the respective methods.
We were able to detect DTCs in 32.7% (DYNLO), 12.5% (DYNHI) and 66% (ROS) of the patient samples, respectively. CD45+ cells could be depleted most efficiently up to 440-fold using ROS, followed by 44-fold with DYNHI and 20-fold using DYNLO. DTC numbers were significantly higher (p<0.0001) in the ROS samples (mean=3.69) with up to 21 DTCs per 1x106 cells. With the immunomagnetic procedures DYNLO samples (mean=0.48) had slightly higher DTC counts (8.33 vs 6.35 DTCs per 1x106 cells) compared to DYNHI (mean=0.39).
In our hand the ROS procedure showed the best results for DTC enrichment from BM samples. This method enables the analysis of a high number of BM cells since it depletes unwanted CD45+ cells most efficiently and thus increases DTC detection rates in the patient samples.
Cerebral Malaria Insights: Pathogenesis, Host Parasite Interactions including Host ResistancePoster
Cerebral malaria is a dreadful disease transmitted by mosquito. The major preventive approach is focused more in vector control than development of anti-malarial drug. The purpose of this presentation is to analyze different aspects of disease manifestations including clinical symptoms and pathogenesis in the context of mosquito borne infections in different geographical regions of the world.READ MORE
Amoebic Meningoencephalitis: Etiology, Infection and PreventionPoster
The presentation covers the different types of organisms that can cause amoebic meningioencephalitis. The two main types focused on are primary amoebic meningioencephalitis and granulomatous amoebic meningioencephalitis. The method of transmission and target hosts are vastly different. Prevention of these diseases is imperfect at best, as complete avoidance is the only way to not contract the disease.READ MORE
Depressive Symptoms Related to Biological Markers of Immune Functioning among Young People with HIVPoster
Youth living with HIV (YLWH) are at risk for depression. This study aimed to identify trends in depressive symptoms for YLWH in a specialty-care clinic and follow up clinical treatment procedures.READ MORE