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Megakaryocyte colony-forming unit growth is enhanced by alemtuzumab: in vitro experiments and a case report of acquired amegakaryocytic thrombocytopenic purpura

Leukemia volume 20, pages 1618–1619 (2006)Cite this article

Alemtuzumab induces the destruction of CD52 antigen-bearing cells. As a potent T-cell-depleting agent, alemtuzumab (or previously Campath-1H) is used for graft-versus-host disease (GvHD) prophylaxis, being given to the cell suspension containing hematopoietic stem cells and lymphocytes, either intravenously or directly ‘in the bag’.1, 2 Unlike bone marrow-derived grafts, experience is limited concerning alemtuzumab being added to the peripheral blood progenitor cell (PBPC) suspension,3 and major questions concerning GvHD prophylaxis with alemtuzumab still remain.2, 4 Therefore, it was decided to further study the effect of short-time incubation of PBPC samples with alemtuzumab using flow-cytometry and cultivation assays.

After gaining informed consent, a sample of PBPC product (15 ml) was obtained from the collection bag immediately after a leukapheresis (COBE Spectra Apheresis System, Gambro BCT, Denver, CO, USA) in 22 healthy donors mobilized by granulocyte colony-stimulating factor (Neupogen, Amgen Inc., Thousand Oaks, CA, USA). Cells were washed using 4% human albumin and then incubated at 37°C with: (1) alemtuzumab (MabCampath, Schering AG, Germany) at a dose of 5 μg/106 cells and (2) water for injection as a control. The reason for using water for injection as a control is owing to the manufacturer's recommendation, according to which alemtuzumab is diluted with water for injection. Autologous plasma as a source of complement was added after 15 min of incubation and cell suspensions were further incubated for 15 min.3 Each sample of 2 ml contained 150 × 106 cells/ml. Cell suspensions were then washed, re-suspended in 4% human albumin and samples were collected for blood count, triple-color flow-cytometric analysis (CD34, CD4, CD8, CD19, CD16, CD56, CD11c, CD83, CD123, HLA DR; Cytomics FC 500, Beckman Coulter Inc., Miami, FL, USA) and cultivation assays. The number of colony-forming unit granulocyte–macrophage (CFU-GM), and erythroid burst-forming units (BFU-E) were determined using a standard colony assay system with a complete methylcellulose-based medium MethoCult HCC-4434 (StemCell Technologies Inc., Vancouver, BC, Canada). Colonies were scored after 14–16 days of cultivation (37°C, 5% CO2, humidified atmosphere) according to their shape, color and other characteristics. Numbers of megakaryocyte colony-forming units (CFU-Meg) were also evaluated using a standard colony assay MegaCult-C (StemCell Technologies Inc., Canada).

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Acknowledgements

We thank Monika Muzikářová, Tissue Bank, University Hospital Brno, for her kind assistance.

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  1. M Doubek and Z Kořístek: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Internal Medicine – Hematooncology, Masaryk University Hospital Brno, Brno, Czech Republic

    M Doubek, Z Kořístek, L Šmardová & J Mayer

  2. Tissue Bank, University Hospital Brno, Brno, Czech Republic

    D Havranová

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  1. M Doubek
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  2. Z Kořístek
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  3. D Havranová
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  5. J Mayer
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Correspondence to J Mayer.

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Doubek, M., Kořístek, Z., Havranová, D. et al. Megakaryocyte colony-forming unit growth is enhanced by alemtuzumab: in vitro experiments and a case report of acquired amegakaryocytic thrombocytopenic purpura. Leukemia 20, 1618–1619 (2006). https://doi.org/10.1038/sj.leu.2404337

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