Widely applicable, extended flow cytometric stem cell enumeration panel for quality control of advanced cellular products.

The most widely used quality control assay for CD34 + hematopoietic stem cell product characterization is the protocol established by the International Society of Hematotherapy and Graft Engineering (ISHAGE). While this protocol is still the gold standard for stem cell enumeration and viability assessment, it does not include T cell enumeration, which is nowadays mandatory for assaying standard allogeneic grafts and various advanced therapy medicinal products (ATMPs). In accordance, we have developed and extensively validated a new approach for a more comprehensive characterization of hematopoietic cellular products using a pre-formulated dried antibody format panel. In addition to the counting beads, the typical markers CD45 fluorescein isothiocyanate (FITC) and CD34 phycoerythrin (PE), as well as the viability dye 7-amino actinomycin D (7-AAD), our novel pre-formulated panel also contains CD3 Pacific Blue (PB) and CD19 allophycocyanin (APC) in the same tube, thereby allowing a combined calculation of leucocytes, stem cells, T and B cells. Showing high linearity, sensitivity and accuracy, our approach is easy to implement and enables a more in-depth characterization of the cellular product under release testing conditions. In addition, the dried pre-formulated antibody approach increases assay reliability compared to the standard antibody panel.

Paired Donor and Recipient Immunophenotyping in Allogeneic Hematopoietic Stem Cell Transplantation: A Cellular Network Approach.

Success and complications of allogeneic hematopoietic stem cell transplantation (alloHSCT) are closely connected to the transferred graft and immune reconstitution post alloHSCT. Due to the variety of immune cells and their distinct roles, a broad evaluation of the immune cellular network is warranted in mobilization and reconstitution studies in alloHSCT. Here, we propose a comprehensive phenotypic analysis of 26 immune cell subsets with multicolor flow cytometry from only 100µl whole blood per time point. Using this approach, we provide an extensive longitudinal analysis of almost 200 time points from 21 donor-recipient pairs. We observe a broad mobilization of innate and adaptive immune cell subsets after granulocyte-colony stimulating factor (G-CSF) treatment of healthy donors. Our data suggest that the relative quantitative immune cell subset composition in recipients approaches that of healthy donors from day +180 post alloHSCT onwards. Correlation of donor and recipient cell counts reveals distinct association patterns for different immune cell subsets and hierarchical clustering of recipient cell counts identifies distinct reconstitution groups in the first month after transplantation. We suggest our comprehensive immune subset analysis as a feasible and time efficient approach for a broad immune assessment for future clinical studies in the context of alloHSCT. This comprehensive cell composition assessment can be a critical step towards personalized graft composition strategies and individualized therapy management in areas such as GvHD prophylaxis in the highly complex immunological setting of alloHSCT.

Molecular monitoring of minimal residual disease in two patients with MLL-rearranged acute myeloid leukemia and haploidentical transplantation after relapse.

This report describes the clinical courses of two acute myeloid leukemia patients. Both had MLL translocations, the first a t(10;11)(p11.2;q23) with MLL-AF10 and the second a t(11;19)(q23;p13.1) with MLL-ELL fusion. They achieved a clinical remission under conventional chemotherapy but relapsed shortly after end of therapy. Both had a history of invasive mycoses (one had possible pulmonary mycosis, one systemic candidiasis). Because no HLA-identical donor was available, a haploidentical transplantation was performed in both cases. Using a specially designed PCR method for the assessment of minimal residual disease (MRD), based on the quantitative detection of the individual chromosomal breakpoint in the MLL gene, both patients achieved complete and persistent molecular remission after transplantation. The immune reconstitution after transplantation is described in terms of total CD3+/CD4+, CD3+/CD8+, CD19+, and CD16+/CD56+ cell numbers over time. The KIR and HLA genotypes of donors and recipients are reported and the possibility of a KIR-mediated alloreactivity is discussed. This report illustrates that haploidentical transplantation may offer a chance of cure without chronic graft-versus-host disease in situations where no suitable HLA-identical donor is available even in a high-risk setting and shows the value of MRD monitoring in the pre- and posttransplant setting.