Christopher A Lazarski1, Keri Toner1,2, Michael D Keller1,3, Naomi Luban4,2, Pampee P Young5,6, Catherine M Bollard1,7,2, Stephen J Wagner5, Patrick J Hanley1,7,2. 1. Center for Cancer and Immunology Research, Children's National Hospital, Washington, DC, United States of America. 2. School of Medicine and Health Sciences, George Washington University, Washington, DC, United States of America. 3. Division of Allergy and Immunology, Children's National Hospital, Washington, DC, United States of America. 4. Division of Haematology, George Washington University, Washington, DC, United States of America. 5. Holland Lab for the Biomedical Sciences, American Red Cross, Rockville, MD, United States of America. 6. Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN, United States of America. 7. Division of Blood and Marrow Transplantation, Children's National Hospital, Washington, DC, United States of America.
Abstract
BACKGROUND: Leukoreduction to eliminate mononuclear cells within blood products is necessary to prevent graft-versus-host disease after transfusion. Published reports document low concentrations of mononuclear cells leftover in fresh-frozen plasma products, however the phenotype and the proliferative potential of these cells has not been tested. MATERIALS AND METHODS: We investigated residual cellular components contained within fresh and fresh-frozen plasma products and characterised their proliferative potential in co-cultures with unrelated allogeneic cells. We designed a flow-based assay to phenotype cells and quantify cell division by measuring the dilution of fluorescently labeled protein as cells divide. Leukocytes from consenting donors were purified from fresh liquid or fresh-frozen plasma units and cultured for three to seven days with unrelated irradiated allogeneic targets. RESULTS: We discovered a median of 1.6×107 viable lymphocytes were detectable in fresh plasma units after collection (n=8), comprised of a mixture of CD3+ CD8+ and CD3+ CD4+ cells. Furthermore, we identified a median of 8.4% of live CD3+ plasma lymphocytes divided as early as Day 4 when co-cultured with unrelated allogeneic cells, expanding to a median 88.8% by Day 7 (n=3). Although freezing the plasma product reduced the total number of viable leukocyte cells down to 2.3×105 (n=10), residual naive CD3+ cells were viable and demonstrated division through Day 7 of co-culture. DISCUSSION: The evidence of viable proliferative lymphocytes in fresh and fresh-frozen plasma products derived from centrifugation suggests that additional leukoreduction measures should be investigated to fully eradicate reactive lymphocytes from centrifuged plasma products.
BACKGROUND: Leukoreduction to eliminate mononuclear cells within blood products is necessary to prevent graft-versus-host disease after transfusion. Published reports document low concentrations of mononuclear cells leftover in fresh-frozen plasma products, however the phenotype and the proliferative potential of these cells has not been tested. MATERIALS AND METHODS: We investigated residual cellular components contained within fresh and fresh-frozen plasma products and characterised their proliferative potential in co-cultures with unrelated allogeneic cells. We designed a flow-based assay to phenotype cells and quantify cell division by measuring the dilution of fluorescently labeled protein as cells divide. Leukocytes from consenting donors were purified from fresh liquid or fresh-frozen plasma units and cultured for three to seven days with unrelated irradiated allogeneic targets. RESULTS: We discovered a median of 1.6×107 viable lymphocytes were detectable in fresh plasma units after collection (n=8), comprised of a mixture of CD3+ CD8+ and CD3+ CD4+ cells. Furthermore, we identified a median of 8.4% of live CD3+ plasma lymphocytes divided as early as Day 4 when co-cultured with unrelated allogeneic cells, expanding to a median 88.8% by Day 7 (n=3). Although freezing the plasma product reduced the total number of viable leukocyte cells down to 2.3×105 (n=10), residual naive CD3+ cells were viable and demonstrated division through Day 7 of co-culture. DISCUSSION: The evidence of viable proliferative lymphocytes in fresh and fresh-frozen plasma products derived from centrifugation suggests that additional leukoreduction measures should be investigated to fully eradicate reactive lymphocytes from centrifuged plasma products.