Automated Cell Separation Method Enhances Leukemia Patient Care: Part 1

05 Jun 2014
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Allogeneic stem cell transplantation (allo-SCT) is used to treat a wide range of haematological malignant diseases and bone marrow disorders1. One of the major complications to occur following such a procedure in patients with malignant disease is relapse of the original disease2-4. The re-emergence of patient cells may cause mixed chimerism in the patient’s bone marrow and blood, which is the presence of blood cells originating from both the donor and the recipient stem cells. Chimerism analysis is usually performed by analysing polymorphic DNA sequences, most commonly by polymerase chain reaction (PCR)5, to determine the origin of cells.

Investigations to assess chimerism following allo-SCT may be useful for monitoring minimal residual disease and thereby early detection of imminent relapse in the treatment of malignant diseases, such as acute myeloid leukemia (AML), acute lymphoblastic leukaemia (ALL) and chronic myeloid leukemia (CML)1-6. Such investigations, if timely, can help to guide interventions for the prevention of relapse, such as a reduction in immunosuppressive therapy or donor lymphocyte infusion (DLI)1-4,6,7.

Chimerism analysis has now become a routine part of patient management following allo-SCT5. The cell-type of interest for chimerism analysis will depend on the type of cancer being treated and may be relatively few in number within the total cell population of the patient’s blood or bone marrow. As a result, chimerism analysis of whole blood or whole bone marrow lacks sensitivity3,5. This can be addressed by separating out the cell type(s) of particular interest, such as B cells or T cells, which can be achieved by flow analysis or by using antibody coated magnetic beads5. Such lineage-specific chimerism analysis increases sensitivity significantly 2,5,6, allowing even earlier detection of mixed chimerism.
 

Faster, more efficient cell separation

In the Transplantation and Immunology Unit of the Department of Immunology and Transfusion Medicine at the Karolinska University Hospital, Huddinge, Stockholm, one of the leading transplant centres in Sweden, lineage-specific chimerism investigations are performed on a daily basis.

“We found that there is at least a 10-fold increase in sensitivity when DNA is extracted from specific cell populations rather than whole blood,” comments Medical Director for Transplant Immunology, Associate Professor Dan Hauzenberger. “For example, if we are just interested in B cells, then separating out the B cells from all the other cells in the patient’s sample results in higher sensitivity. We find that specific analysis of B cell, T cell and myeloid cell populations is of prognostic value for allo-SCT patients .”

In the past, cell separation was achieved manually, which was labour intensive and time consuming. Now, however, a new automated method for cell separation is available. The CellSep Advanced assay, in combination with the Liaison® Ixt/Arrow Instrument, uses magnetic bead technology to isolate up to 3 cell types in every sample. Processing from one to 12 whole blood or buffy coat samples per run in 32 minutes per cell type, it offers significant time savings when compared to manual cell separation methods.

CellSep Advanced requires minimal hands-on intervention and produces good yields of purified cell preparations ready for use in a wide range of downstream applications, including lineage-specific chimerism analysis8. The automated platform minimises human error and ensures reproducibility between runs for consistent and reliable downstream analysis.

“Previously, the separation of specific cell types of interest from blood or bone marrow was very laborious, involving sequential separations of one cell type at a time,” explains Dr Mehmet Uzunel, PhD, Technical Manager at the Transplantation and Immunology Unit. “We used a manual immunomagnetic bead method to separate B cells, T cells and myeloid cells. The number of samples that can be processed simultaneously with the manual work is limited to 3-4 samples. In the past we had fewer samples, but now we process around 12-15 samples per day. For this volume of work, the manual method would require 2 dedicated technicians every day just to perform sequential cell separations and nothing else.”

“As a development site for CellSep Advanced, we have now been using this automated method routinely for about a year. It has reduced our technician requirement for isolating cell types considerably, so that we now need just one person to load and unload samples. Once the samples are loaded, the technician can walk away and is free to perform other tasks, which has resulted in greater efficiency in the laboratory.”

Read part 2 of the article here.


Figure 1. The Cell Isolation Rack within the Arrow Instrument


References:


1. El-Cheikh, J., Vazquez, A., Crocchiolo, R. et al (2012) Acute GVHD is a strong predictor of full donor CD3+ T cell chimerism after reduced intensity conditioning allogeneic stem cell transplantation. Am J Hematol 87: 1074-1078.
2. Mattsson, J., Uzunel, M., Tammik, L. et al (2001) Leukemia lineage-specific chimerism analysis is a sensitive predictor of relapse in patients with acute myeloid leukemia and myelodysplastic syndrome after allogeneic stem cell transplantation. Leukemia 15: 1976-1985.
3. Rosenow, F., Berkemeier, A., Krug, U. et al (2013) CD34+ lineage-specific donor cell chimerism for the diagnosis and treatment of impending relaps of AML or myelodysplastic syndrome after allo-SCT. Bone Marrow Transplantation 48, 1070-1076.
4. Zetterquist, H., Mattsson, J., Uzunel, M. et al (2000) Mixed chimerism in the B cell lineage is a rapd and sensitive indicator of minimal residual disease in bone marrow transplant recipients with pre-B cell acute lymphoblastic leukemia. Bone Marrow Transplantation 25: 843-851.
5. Lion, T. Detection of Impending Graft Rejection and Relapse by Lineage-Specific Chimerism Analysis. Chapter 14 in Methods in Molecular Medicine, volume 134: Bone Marrow and Stem Cell transplantation.
6. Thiede, C. (2004) Diagnostic Chimerism Analysis After Allogeneic Stem Cell Transplantation: New Methods and Markers. Am J Pharmacogenomics 4(3):177-187.
7. Bader, P., Miethammer, D., Willasch, A. et al (2005) Review – How and when should we monitor chimerism after allogeneic stem cell transplantation? Bone Marrow Transplantation 35: 107-119.
8. DiaSorin Arrow and LIAISON® Ixt CellSep Advanced Instructions for Use (2012/06 version 5) 6.12.02-607-01,EN 09/13.


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Sonia Nicholas
Clinical Diagnostics Editor