Contributions
Abstract: PB2423
Type: Publication Only
Background
Mesenchymal stem cells (MSCs) possess unique properties of self-renewal, wide differentiation potential and an immunosuppressive effect, are enriched in the bone marrow and their isolation relies on culture in special media.
Aims
Present study aimed to demonstrate MSCs in the peripheral blood (PB) of individuals undergoing hematopoietic stem cell transplant (HSCT) using flow cytometry and correlate their counts with the post-transplant outcomes.
Methods
A total of 33 subjects which included autologous arm (n=25) [patients of MM (n=20), lymphoma (n=5)] and allogeneic arm (n=8) [healthy donors of patients undergoing allogeneic HSCT] during the period extending from 1-1-2016 to 30-06-2017 were prospectively included in the study. Baseline PB sample of the eligible subjects was analyzed for MSCs by flow cytometry. Stem cell mobilization of the subjects was done using granulocyte-colony stimulating growth factor (G-CSF, given for 4 days @ 10mcg/kg/day). Two milliliters of PB sample was analyzed for CD34 and MSC counts using a panel of monoclonal antibodies (CD34-APC, CD45-APC H7, CD73-PE, CD90-FITC and CD105-PE-Cy7) immediately prior to the apheresis. MSCs were defined by an absence of CD34/CD45 and any dual positivity for CD90/73/105. Mononuclear cells were harvested using cell separator and 3 ml of sample from the apheresis product (AP) was analyzed for CD34 and MSC by flow cytometry. Subjects with inadequate CD34 count after the first apheresis (< 2 x 10⁶/kg) underwent a second procedure next day after receiving G-CSF (10 mcg/kg/day) and a single dose of plerixafor (0.24 mg/kg, given 8 hours prior to the procedure). CD34 and MSC were analyzed by flow cytometry at day 6 (both PB and AP). AP with a minimum of 2.0 x 106/kg CD34 dose was infused to the patient following conditioning. Patients were followed up for a minimum of 100 days post-HSCT and complications in the post-transplant period were recorded till the last follow up. Analyses were conducted using IBM SPSS statistics (version 22.0).
Results
Baseline MSCs were seen in 14 (42%) subjects. The mean baseline MSC (PB) (x 10 ⁻⁴) % in the autologous and allogeneic group was 19.7 + 41.83 and 7.9 + 9.95 respectively (p-0.852). Patients with baseline MSC (PB) had a higher mean MSC (PB) at D5 and D6 (p-<0.001 and 0.030 respectively). Overall 19 (58%) subjects had MSCs at D5 (PB). Administration of more chemotherapy cycles was associated with an absence of D5 MSCs (PB) in patients of MM (p-0.045). A total of 17 (52%) subjects had D5 MSC (AP). Mean D5 MSC (AP) (x 10⁻⁴ %) in the autologous and allogeneic groups was 5.4 + 8.7 and 25 + 39 respectively (p-0.019). Patients with D5 MSC (AP) had a trend towards requiring anti-fungal drugs for a greater duration (p-0.051). Only 6 (18%) patients had detectable D6 MSC (PB). Patients with D6 MSC (PB) required anti-fungal drugs for a greater duration (p-0.041) and had a lesser duration of GI mucositis (p-0.015). A total of 6 (18%) subjects had D6 MSC (AP). Post-HSCT events were not associated with the D6 MSC (AP) except for a greater duration of anti-fungal drug requirement (p-0.041). Day-100 outcome of MM patients was significantly associated with D5 MSC (AP) and D6 MSC (PB) (p-0.043 and 0.046 respectively). No significant difference was found between the type of chemotherapy received by MM and lymphoma patients and baseline, D5 (PB/AP) and D6 (PB/AP) MSC.
Conclusion
MSCs can be demonstrated by flow cytometry in the PB and AP of individuals undergoing HSCT. MSC count in the PB and AP correlates with post-HSCT events.
Session topic: 23. Stem cell transplantation - Clinical
Keyword(s): Acute graft-versus-host disease, Allogeneic hematopoietic stem cell transplant, flow cytometry, Mesenchymal stem cell
Abstract: PB2423
Type: Publication Only
Background
Mesenchymal stem cells (MSCs) possess unique properties of self-renewal, wide differentiation potential and an immunosuppressive effect, are enriched in the bone marrow and their isolation relies on culture in special media.
Aims
Present study aimed to demonstrate MSCs in the peripheral blood (PB) of individuals undergoing hematopoietic stem cell transplant (HSCT) using flow cytometry and correlate their counts with the post-transplant outcomes.
Methods
A total of 33 subjects which included autologous arm (n=25) [patients of MM (n=20), lymphoma (n=5)] and allogeneic arm (n=8) [healthy donors of patients undergoing allogeneic HSCT] during the period extending from 1-1-2016 to 30-06-2017 were prospectively included in the study. Baseline PB sample of the eligible subjects was analyzed for MSCs by flow cytometry. Stem cell mobilization of the subjects was done using granulocyte-colony stimulating growth factor (G-CSF, given for 4 days @ 10mcg/kg/day). Two milliliters of PB sample was analyzed for CD34 and MSC counts using a panel of monoclonal antibodies (CD34-APC, CD45-APC H7, CD73-PE, CD90-FITC and CD105-PE-Cy7) immediately prior to the apheresis. MSCs were defined by an absence of CD34/CD45 and any dual positivity for CD90/73/105. Mononuclear cells were harvested using cell separator and 3 ml of sample from the apheresis product (AP) was analyzed for CD34 and MSC by flow cytometry. Subjects with inadequate CD34 count after the first apheresis (< 2 x 10⁶/kg) underwent a second procedure next day after receiving G-CSF (10 mcg/kg/day) and a single dose of plerixafor (0.24 mg/kg, given 8 hours prior to the procedure). CD34 and MSC were analyzed by flow cytometry at day 6 (both PB and AP). AP with a minimum of 2.0 x 106/kg CD34 dose was infused to the patient following conditioning. Patients were followed up for a minimum of 100 days post-HSCT and complications in the post-transplant period were recorded till the last follow up. Analyses were conducted using IBM SPSS statistics (version 22.0).
Results
Baseline MSCs were seen in 14 (42%) subjects. The mean baseline MSC (PB) (x 10 ⁻⁴) % in the autologous and allogeneic group was 19.7 + 41.83 and 7.9 + 9.95 respectively (p-0.852). Patients with baseline MSC (PB) had a higher mean MSC (PB) at D5 and D6 (p-<0.001 and 0.030 respectively). Overall 19 (58%) subjects had MSCs at D5 (PB). Administration of more chemotherapy cycles was associated with an absence of D5 MSCs (PB) in patients of MM (p-0.045). A total of 17 (52%) subjects had D5 MSC (AP). Mean D5 MSC (AP) (x 10⁻⁴ %) in the autologous and allogeneic groups was 5.4 + 8.7 and 25 + 39 respectively (p-0.019). Patients with D5 MSC (AP) had a trend towards requiring anti-fungal drugs for a greater duration (p-0.051). Only 6 (18%) patients had detectable D6 MSC (PB). Patients with D6 MSC (PB) required anti-fungal drugs for a greater duration (p-0.041) and had a lesser duration of GI mucositis (p-0.015). A total of 6 (18%) subjects had D6 MSC (AP). Post-HSCT events were not associated with the D6 MSC (AP) except for a greater duration of anti-fungal drug requirement (p-0.041). Day-100 outcome of MM patients was significantly associated with D5 MSC (AP) and D6 MSC (PB) (p-0.043 and 0.046 respectively). No significant difference was found between the type of chemotherapy received by MM and lymphoma patients and baseline, D5 (PB/AP) and D6 (PB/AP) MSC.
Conclusion
MSCs can be demonstrated by flow cytometry in the PB and AP of individuals undergoing HSCT. MSC count in the PB and AP correlates with post-HSCT events.
Session topic: 23. Stem cell transplantation - Clinical
Keyword(s): Acute graft-versus-host disease, Allogeneic hematopoietic stem cell transplant, flow cytometry, Mesenchymal stem cell