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no conflict of interest

Immunomagnetic CD34 positive (CD34+) selection is a graft manipulation technique to lower the risk of graft versus-host-disease (GvHD) in patients receiving stem cell boosts for graft failure after allogeneic hematopoietic cell transplantation (HCT). We report about the successful administration of stem cell boosts with CD34+ selected cells from a portion of previously cryopreserved stem cell products in three children who initially underwent allogeneic HCT for malignant diseases.

Cryopreserved stem cells were thawed carefully, supplemented with 10 Vol% citrate, 2500 IE DNase and 0.3 ml MgCl2 0.5 molar, and centrifuged to reduce DMSO and platelets to a minimum. The cells were incubated with immunomagnetic beads against CD34 for 30 minutes. Subsequently, two washing steps using PBS/EDTA buffer with 0.5% human albumin and a soft centrifugation program (300 g, 15 minutes) were performed. Before selection, the cells were adjusted to a volume of 150 ml and filtered through a 200 µm filter system to reduce cell aggregates and clots. CD34+ selection was performed using the CliniMacs plus device (Miltenyi Biotec). CD34+/CD45+ cells were analyzed by flow cytometry and sterility was tested by bacterial culture tests.

Two cryopreserved haploidentical allogeneic peripheral stem cell grafts and one cryopreserved erythrocyte depleted bone marrow graft from an unrelated HLA-identical donor were thawed and CD34+ selected. Indications were secondary graft failure, cytopenia due to meta-iodobenzylguanidine (miBG) therapy and prolonged cytopenia after CAR-T-cell infusion, respectively. Basic values were 88, 129, and 39 x106 and after selection 44, 63, and 16 x106 viable CD34+ cells which results in a CD34 recovery rate of 51, 49, and 42%. Sterility testing was negative. The patients were transfused on day of selection with 1.2, 4.5, and 0.9 x106 CD34+ cells /kg body weight. The transfusions were well tolerated. All three patients engrafted and no GvHD occurred.

CD34+ selection of cryopreserved stem cell grafts was safe and efficient in three cases of allogeneic stem cell boost. After thawing, the CD34 recovery rates were lower than in fresh grafts. Despite of extra-addition of anticoagulant one has to be aware of the risk, that cell aggregates and clots may occure and may hamper the processing. Thus, grafts should be filtered through a transfusion filter system before selection.