Oਉਇਉਁ Aਔਉਃਅ Fully automated, clinical-grade bone marrow processing: a single-centre experience Benedetta Mazzanti1, Serena Urbani1, Simone Dal Pozzo1, Paola Bufano1, Lara Ballerini1, Alessia Gelli1, Irene Sodi1, Irene Donnini1, Massimo Di Gioia1, Stefano Guidi1, Julien Camisani2, Riccardo Saccardi1 Cell Therapy and Transfusion Medicine Unit, Cord Blood Bank, Careggi University Hospital, Florence, Italy; Biosafe, Eysin, Switzerland
1 2
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Background. Clinical grade processing of harvested bone marrow is required in various clinical situations, particularly in the management of ABO mismatching in allogeneic haematopoietic stem cell transplantation (HSCT) and in regenerative medicine. Material and methods. We report a single-centre experience using a fully automated, clinical grade, closed system (Sepax, Biosafe, Switzerland). From 2003 to 2015, 125 procedures were performed in our laboratory, including buffy-coat production for HSCT (n=58), regenerative medicine in an orthopaedic setting (n=54) and density-gradient separation in a trial for treatment of critical limb ischaemia (n=13). Results. Buffy coat separation resulted in a median volume reduction of 85% (range, 75-87%), providing satisfactory red blood cell depletion (69%, range 30-88%) and a median recovery of CD34 cells of 96% (range, 81-134%) in the setting of allogeneic HSCT. Significantly greater volume reduction (90%; range, 90-92%) and red blood cell depletion (88%; range, 80-93%) were achieved by the new SmartRedux software released for Sepax2, validated in the last eight allogeneic HSCT. The density gradient separation programme resulted in complete red blood cell depletion associated with high CD34 recovery (69%; range, 36-124%). No reactions related to the quality of the product were reported. Time to engraftment following allogeneic HSCT was in the normal range. No cases of microbiological contamination related to the manipulation were reported. Discussion. Clinical grade, automated bone marrow manipulation with Sepax was shown to be effective, giving operator-independent results and could be used for a broad range of clinical applications. Keywords: bone marrow, automated processing, HSCT, regenerative medicine.
Introduction
©
Haematopoietic stem cell transplantation (HSCT) is a medical procedure commonly used for the treatment of a wide variety of haematological malignancies, metabolic disorders and immunodeficiency states1. Different sources of stem cells can be used: bone marrow (BM), mobilised peripheral blood stem cells and umbilical cord blood. Peripheral blood stem cells have replaced BM in the autologous setting, while about 30% of allogeneic transplants still use BM as the source of stem cells, probably because of the higher incidence of chronic Graft-versus-Host disease following peripheral blood stem cell transplantation2,3. In the setting of allogeneic HSCT, 30% of transplants from HLA-identical siblings and 50% from unrelated donors are red blood cell (RBC) incompatible4,5. The immuno-haematological consequences of major and minor ABO incompatibilities have been
summarised by Rowley et al. and include acute and delayed haemolysis, delayed RBC recovery and pure red cell aplasia6. The risk of reactions, which can have an abrupt onset and may be fatal, is lowered by graft processing and proper blood component support. Standard procedures for ABO incompatible transplants consist of RBC and/or plasma depletion7 carried out by apheresis devices, densitygradient separation and simple centrifugation, possibly associated with the use of sedimentation agents, such as hydroxyethyl starch8,9. Centrifugation of the graft is performed in order to enable collection of the total nucleated cell layer at the interphase between the plasma and RBC pellet (buffy coat), or the overall cellular pellet after plasma removal (plasma depletion). An algorithm for the management of RBC incompatibility was proposed6 with the aim of standardising the graft manipulation.
Blood Transfus 2017; 15: 577-84 DOI 10.2450/2016.0057-16 © SIMTI Servizi Srl
All rights reserved - For personal use only No other use without premission
577
Mazzanti B et al
SI M
TI
Se rv
Cell processing: the Sepax system BM was processed in either a Sepax S-100 or Sepax 2 device (Biosafe), managed through software specifically developed for different clinical targets, in closed, disposable kits. The Sepax cell processing system uses a rotating syringe technology that allows separation of blood components through rotation of the syringe chamber. Blood components (plasma, buffy coat and red cells) are detected by an optical sensor and transferred into different output bags by diverting the output flow of the syringe piston.
Sr l
Materials and methods
one kit. The time for SmartRedux processing of 1 L of BM ranges between 20 and 210 minutes, according to the final product (plasma depletion or buffy coat). Once the operator has set the desired parameters, the whole procedure is carried out in a fully automated manner. Either saline or autologous plasma can be added to the product in order to decrease the product density before infusion into the patient. The density-gradient based separation software (NeatCell, Biosafe) is designed to manage automated separation of BM on a density gradient of 1.077 g/mL, in order to obtain a purified MNC fraction. It was used either in regenerative medicine protocols, when a purified MNC fraction was required, or in the case of major incompatibility with a high titre of natural anti-ABO antibodies. The separation process is carried out in a specific closed, disposable kit (CS-900). After the separation, the product is washed two or three times, according to the operator's settings, in saline with added human serum albumin. The input volume ranges between 30 and 120 mL while the final output volume can be set up by the operator in a range between 8 and 45 mL. The processing time with three washing cycles is about 90 minutes.
iz i
Processing of intermediate/small volumes of BM harvests is also requested for graft preparation in regenerative medicine protocols. Autologous BM mononuclear cells (MNC) have been safely used in different clinical experimental trials for the treatment of critical limb ischaemia10 and myocardial infarction11 and buffy coat has been used in orthopaedic conditions12. According to the clinical target, the volume of the harvested BM and the final characteristics of the product may vary largely and flexible processing methodologies are, therefore, required. Manipulation of BM is a key factor in the transplantation process and may influence both engraftment and overall survival7,9. Standardisation of cell processing would decrease both variability of the final product quality and the need for specialised staff training. Here we report a single-centre experience of the use of a fully automated, clinical-grade, closed system (Sepax, Biosafe, Eysins, Switzerland) for BM processing in different clinical settings.
©
Operating software Non-density gradient separations in ABOincompatible transplant procedures and in the orthopaedic setting were performed with either generic volume reduction (GVR) or SmartRedux software (Biosafe). The GVR protocol enables the collection of BM buffy coat or plasma-free BM. CS-490 disposable kits were used for all the procedures. As the volume of the syringe chamber is 220 mL, multiple cycles of centrifugation must be performed for larger BM volumes. The GVR protocol allows initial product volumes from 50 mL to 880 mL, so multiple procedures are required for larger BM volumes. The protocol is user-adaptable and various parameters can be adjusted in order to maximise cell recovery or decrease RBC contamination of the final product. The processing time for 880 mL of BM is about 60 minutes. A further evolution of GVR software is SmartRedux, available only with the Sepax2 device: this software can deal with an input volume ranging between 30 and 3,300 mL and a large volume product can, therefore, be processed in a single procedure with
Patients
Haematopoietic stem cell transplantation Between 2003 and 2013, 24 patients with haematological malignancies received a BM transplant in our facility in Florence (Italy): 19 had allogeneic ABO mismatching (11 major, 4 minor and 4 double) and five required volume reduction of autologous BM before cryopreservation of the product following failure of peripheral blood stem cell mobilisation. Between December 2014 and January 2016, eight more patients with haematological malignancies underwent allogeneic transplantation with ABO mismatching (5 major and 3 minor). The patients' characteristics are reported in Table I. Engraftment was assessed as time (days) to reach a neutrophil count of 0.5×109/L.
Regenerative medicine In the same period, autologous BM processing was carried out within approved trials of regenerative medicine: volume reduction was achieved by either simple buffy-coat separation (GVR programme) in a protocol of bone regeneration in the orthopaedic setting (n=54) or density-gradient mononuclear cell separation (NeatCell) in a trial on the treatment of critical limb ischaemia (n=13). Bone marrow harvesting BM units were collected in our centre or in other accredited centres for mismatched unrelated donor marrow collection, according to the standard procedure
Blood Transfus 2017; 15: 577-84 DOI 10.2450/2016.0057-16 578
All rights reserved - For personal use only No other use without premission
Clinical grade bone marrow processing Table I - Characteristics of the allogeneic and autologous HSCT recipients. Patients' characteristics Allogeneic HSCT
Autologous HSCT
Diagnoses
n.
Diagnoses
Acute myeloid leukaemia
14
Acute myeloid leukaemia
n. 3
Acute lymphoblastic leukaemia
9
Multiple myeloma
1
Non-Hodgkin's lymphoma
2
Non-Hodgkin's lymphoma
1
Myeloproliferative neoplasm
1
Myelodysplastic syndrome
1
Matched sibling donor
4
Matched family donor
7
Matched unrelated donor
Sr l
Type of donor
Statistical analysis Data were extracted retrospectively from our leucapheresis/BM processing database. Data are expressed as mean ±SD or median (range) and comparisons were carried out using the Student's t-test or the non-parametric Mann-Whitney test.
16
3
A−/O−
1
B+/O+
4
B+/A+
3
AB+/O+
1
O+/AB+
1
O+/A+
2
O−/A−
1
O+/A−
1
O+/B+
Se rv
A+/O−
Results
Volume reduction Initial and final BM volumes as well as percentage volume reduction (median and range) are reported in Table II. Data are divided for allogeneic HSCT, autologous HSCT, buffy coat and density-gradient separation for regenerative medicine. In all cases the BM volume reduction was greater than 72% (Table II).
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A+/O+
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ABO-mismatch (donor/recipient) A+/B+
Haematopoietic progenitor cell assays were performed in duplicate, with the methylcellulose-based medium containing recombinant cytokines (MethoCult GF H4434; Stem Cell Technologies, Vancouver, Canada) as previously described16. Total colony-forming cells, defined as the total number of colonies, regardless of their lineage, were used to calculate clonogenic potential. The number of fibroblast colony-forming units (CFU-F) was determined as previously described16, and was used as a surrogate marker for mesenchymal stem cells progenitors. Before and after separation, 1×106 total nucleated cells were plated in duplicate in 100 mm Ø Petri dishes. After 14 days, the dishes were fixed with methanol and stained with Giemsa; visible colonies formed by 50 or more cells were counted and reported as the number of CFU-F/106 seeded total nucleated cells.
2
Patients are grouped based on diagnosis, type of donor and ABO mismatch. HSCT: haematopoietic stem cell transplantation.
©
of each centre. BM was harvested by multiple aspirations from the iliac crest and cells were collected into transfer bags containing ACD and heparin. The volume collected ranged between 150 and 1,727 mL, depending on the clinical application. Assessment of bone marrow content White blood cell (WBC) and RBC counts and haematocrit were determined before and after processing using an automated haematology analyser (XS-1000i, Sysmex Corp., Kobe, Japan). CD34+ cell count, MNC quantification and viability as well as WBC viability were determined by immunofluorescence analysis (FACScanto, BD Pharmingen, San Diego, CA, USA) using the ISHAGE protocol with the single platform technique13-15. 7-aminoactinomycin D (BD Pharmingen) was used to exclude dead cells.
Bone marrow processing for ABO mismatch When the GVR protocol for RBC and plasma removal (buffy coat extraction) was used, multiple procedures were needed if the initial volume exceeded 880 mL; overall 50 procedures were carried out. For allogeneic HSCT the median CD34+ cell recovery was 95.9% (range, 81.2-134.3%); CD34+ recovery was not available for autologous HSCT. The median percentage WBC recovery was 86.0% (range, 76.0-104.2%) in allogeneic HSCT and 74.0% (range, 57.1-76.3%) in autologous HSCT (Figure 1). Reductions in packed RBC volume with the GVR protocol in the HSCT setting are reported in Table III. The median reduction in RBC volume was 69% (range, 30-88%) for allogeneic transplantation and 73% (range, 59-85%) for autologous transplantation. The SmartRedux protocol was used with the Sepax 2 machine, allowing an input volume up to 3,300 mL and enabling the final volume to be set. This software was validated in five allogeneic HSCT with major and three with minor ABO mismatching; the data are summarised in Table IV, and compared with data obtained when the
Blood Transfus 2017; 15: 577-84 DOI 10.2450/2016.0057-16 All rights reserved - For personal use only No other use without premission
579
Mazzanti B et al Table II - BM volumes before and after processing and percentage volume reduction for each protocol. Patients
N
Input volume (mL)
Output volume (mL)
Volume reduction (%)
Allogeneic HSCT
19
1,221 (773-1,727)
200 (120-300)
85 (75-87)
Autologous HSCT
5
1,047 (1,021-1,129)
150 (119-153)
86 (85-88)
BC/RM
54
210 (150-300)
30 (22-45)
86 (79-90)
DGS/RM
13
300 (200-360)
42 (27-51)
86 (73-90)
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Data are expressed as median (range). BM: bone marrow; HSCT: haematopoietic stem cell transplantation; BC: buffy coat; DGS: density-gradient separation; RM: regenerative medicine.
©
Figure 1 - Percentage recovery of CD34 cells and WBC for the GVR protocols for BM buffy coat extraction for infusion in allogeneic HSCT, autologous HSCT and in regenerative medicine for patients with orthopaedic disorders (BC/RM). Each bar represents the median, quartiles, minimum and maximum values. WBC: white blood cells; GVR: generic volume reduction; BM: bone marrow; HSCT: haematopoietic stem cell transplantation; BC: buffy coat; RM: regenerative medicine.
Table III - RBC volumes before and after processing and percentage RBC reduction for HSCT and densitygradient separation for regenerative medicine protocols. RBC volume before processing (mL)
RBC volume after processing (mL)
RBC volume reduction (%)
Allogeneic HSCT
349 (193-525)
116 (36-217)
69 (30-88)
Autologous HSCT
344 (225-355)
93 (49-99)
73 (59-85)
67 (32-107)
0.4 (0-0.6)
100 (99-100)
DGS/RM
Data are expressed as median (range). RBC: red blood cell; HSCT: haematopoietic stem cell transplantation; DGS: density-gradient separation; RM: regenerative medicine.
Blood Transfus 2017; 15: 577-84 DOI 10.2450/2016.0057-16 580
All rights reserved - For personal use only No other use without premission
Clinical grade bone marrow processing
Table IV - BM processing for allogeneic HSCT: GVR vs Smartredux protocol. SmartRedux (n=8)
Pre-processing volume (mL)
1,221 (773-1,727)
1,128 (87-1,440)
Post-processing volume (mL)
200 (120-300)
110 (75-150)
Volume reduction (%)
85 (75-87)
90 (90-92)
CD34 recovery %
96 (81-134)
105 (82-114)
WBC recovery %
86 (76-104)
92 (87-111)
Pre-processing RBC volume (mL)
349 (193-525)
380 (234-589)
Post-processing RBC volume (mL)
116 (36-217)
44 (24-50)
RBC volume reduction (%)
69 (30-88)
88 (80-93)
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p-value
TI
GVR (n=19)
Neutrophil engraftment was achieved at a median of 23 days (range, 13-31 days) for allogeneic HSCT and 19 days (range, 9-31 days) for autologous HSCT. One patient did not engraft after a haploidentical HSCT as rescue for failed engraftment of a cord blood transplant; the patient died of sepsis 17 days after the rescue transplant. No infusion-related side effects were reported; in particular, no transfusion reactions occurred in the first group (ABO-mismatched allogeneic HSCT) except in one patient who developed a reaction after the infusion of 20 mL of the product, possibly due to the presence of irregular antibodies. The graft was then rescued by MNC separation with the NeatCell protocol and the infusion was carried out without any side effects. No microbiological contamination due to the BM manipulation process was reported.
Se rv
Mononuclear cell separation for regenerative medicine The density-gradient separation procedure (NeatCell protocol) resulted in a satisfactory recovery of CD34+ cells
Clinical outcome
Sr l
Bone marrow buffy coat extraction for regenerative medicine The GVR programme was applied for a bone regeneration protocol in the orthopaedic setting in 54 patients. The median volume of autologous BM processed was 210 mL (range, 150-300 mL). The median CD34+ and WBC recovery was 79.4% (range, 41.1109.2%) and 50.8% (range, 23.0-84.4%), respectively (Figure 1). The final, median reduced volume was 30 mL (range, 22-45 mL) (Table II).
(69%; range 36-124%), MNC (67%; range, 17-119%) and colony-forming cells (49%; range, 9-124%), but a quite low CFU-F recovery (30%; range, 8-50%) according to our previous data16. The procedure resulted in a median polymorphonuclear cell reduction of 99.8% (range, 99.7-100%); complete RBC depletion (100%) was also achieved (Table III), therefore making this procedure suitable for BM processing in the case of allogeneic HSCT with major ABO mismatching and a high titre of natural antibodies or irregular anti-ABO antibodies.
iz i
GVR programme was used. The SmartRedux protocol was associated with greater volume reduction (90%; range, 90-92% vs 85%; range, 75-87%: p
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TI
Se rv
iz i
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Background. Clinical grade processing of harvested bone marrow is required in various clinical situations, particularly in the management of ABO mismatching in allogeneic haematopoietic stem cell transplantation (HSCT) and in regenerative medicine. Material and methods. We report a single-centre experience using a fully automated, clinical grade, closed system (Sepax, Biosafe, Switzerland). From 2003 to 2015, 125 procedures were performed in our laboratory, including buffy-coat production for HSCT (n=58), regenerative medicine in an orthopaedic setting (n=54) and density-gradient separation in a trial for treatment of critical limb ischaemia (n=13). Results. Buffy coat separation resulted in a median volume reduction of 85% (range, 75-87%), providing satisfactory red blood cell depletion (69%, range 30-88%) and a median recovery of CD34 cells of 96% (range, 81-134%) in the setting of allogeneic HSCT. Significantly greater volume reduction (90%; range, 90-92%) and red blood cell depletion (88%; range, 80-93%) were achieved by the new SmartRedux software released for Sepax2, validated in the last eight allogeneic HSCT. The density gradient separation programme resulted in complete red blood cell depletion associated with high CD34 recovery (69%; range, 36-124%). No reactions related to the quality of the product were reported. Time to engraftment following allogeneic HSCT was in the normal range. No cases of microbiological contamination related to the manipulation were reported. Discussion. Clinical grade, automated bone marrow manipulation with Sepax was shown to be effective, giving operator-independent results and could be used for a broad range of clinical applications. Keywords: bone marrow, automated processing, HSCT, regenerative medicine.
Introduction
©
Haematopoietic stem cell transplantation (HSCT) is a medical procedure commonly used for the treatment of a wide variety of haematological malignancies, metabolic disorders and immunodeficiency states1. Different sources of stem cells can be used: bone marrow (BM), mobilised peripheral blood stem cells and umbilical cord blood. Peripheral blood stem cells have replaced BM in the autologous setting, while about 30% of allogeneic transplants still use BM as the source of stem cells, probably because of the higher incidence of chronic Graft-versus-Host disease following peripheral blood stem cell transplantation2,3. In the setting of allogeneic HSCT, 30% of transplants from HLA-identical siblings and 50% from unrelated donors are red blood cell (RBC) incompatible4,5. The immuno-haematological consequences of major and minor ABO incompatibilities have been
summarised by Rowley et al. and include acute and delayed haemolysis, delayed RBC recovery and pure red cell aplasia6. The risk of reactions, which can have an abrupt onset and may be fatal, is lowered by graft processing and proper blood component support. Standard procedures for ABO incompatible transplants consist of RBC and/or plasma depletion7 carried out by apheresis devices, densitygradient separation and simple centrifugation, possibly associated with the use of sedimentation agents, such as hydroxyethyl starch8,9. Centrifugation of the graft is performed in order to enable collection of the total nucleated cell layer at the interphase between the plasma and RBC pellet (buffy coat), or the overall cellular pellet after plasma removal (plasma depletion). An algorithm for the management of RBC incompatibility was proposed6 with the aim of standardising the graft manipulation.
Blood Transfus 2017; 15: 577-84 DOI 10.2450/2016.0057-16 © SIMTI Servizi Srl
All rights reserved - For personal use only No other use without premission
577
Mazzanti B et al
SI M
TI
Se rv
Cell processing: the Sepax system BM was processed in either a Sepax S-100 or Sepax 2 device (Biosafe), managed through software specifically developed for different clinical targets, in closed, disposable kits. The Sepax cell processing system uses a rotating syringe technology that allows separation of blood components through rotation of the syringe chamber. Blood components (plasma, buffy coat and red cells) are detected by an optical sensor and transferred into different output bags by diverting the output flow of the syringe piston.
Sr l
Materials and methods
one kit. The time for SmartRedux processing of 1 L of BM ranges between 20 and 210 minutes, according to the final product (plasma depletion or buffy coat). Once the operator has set the desired parameters, the whole procedure is carried out in a fully automated manner. Either saline or autologous plasma can be added to the product in order to decrease the product density before infusion into the patient. The density-gradient based separation software (NeatCell, Biosafe) is designed to manage automated separation of BM on a density gradient of 1.077 g/mL, in order to obtain a purified MNC fraction. It was used either in regenerative medicine protocols, when a purified MNC fraction was required, or in the case of major incompatibility with a high titre of natural anti-ABO antibodies. The separation process is carried out in a specific closed, disposable kit (CS-900). After the separation, the product is washed two or three times, according to the operator's settings, in saline with added human serum albumin. The input volume ranges between 30 and 120 mL while the final output volume can be set up by the operator in a range between 8 and 45 mL. The processing time with three washing cycles is about 90 minutes.
iz i
Processing of intermediate/small volumes of BM harvests is also requested for graft preparation in regenerative medicine protocols. Autologous BM mononuclear cells (MNC) have been safely used in different clinical experimental trials for the treatment of critical limb ischaemia10 and myocardial infarction11 and buffy coat has been used in orthopaedic conditions12. According to the clinical target, the volume of the harvested BM and the final characteristics of the product may vary largely and flexible processing methodologies are, therefore, required. Manipulation of BM is a key factor in the transplantation process and may influence both engraftment and overall survival7,9. Standardisation of cell processing would decrease both variability of the final product quality and the need for specialised staff training. Here we report a single-centre experience of the use of a fully automated, clinical-grade, closed system (Sepax, Biosafe, Eysins, Switzerland) for BM processing in different clinical settings.
©
Operating software Non-density gradient separations in ABOincompatible transplant procedures and in the orthopaedic setting were performed with either generic volume reduction (GVR) or SmartRedux software (Biosafe). The GVR protocol enables the collection of BM buffy coat or plasma-free BM. CS-490 disposable kits were used for all the procedures. As the volume of the syringe chamber is 220 mL, multiple cycles of centrifugation must be performed for larger BM volumes. The GVR protocol allows initial product volumes from 50 mL to 880 mL, so multiple procedures are required for larger BM volumes. The protocol is user-adaptable and various parameters can be adjusted in order to maximise cell recovery or decrease RBC contamination of the final product. The processing time for 880 mL of BM is about 60 minutes. A further evolution of GVR software is SmartRedux, available only with the Sepax2 device: this software can deal with an input volume ranging between 30 and 3,300 mL and a large volume product can, therefore, be processed in a single procedure with
Patients
Haematopoietic stem cell transplantation Between 2003 and 2013, 24 patients with haematological malignancies received a BM transplant in our facility in Florence (Italy): 19 had allogeneic ABO mismatching (11 major, 4 minor and 4 double) and five required volume reduction of autologous BM before cryopreservation of the product following failure of peripheral blood stem cell mobilisation. Between December 2014 and January 2016, eight more patients with haematological malignancies underwent allogeneic transplantation with ABO mismatching (5 major and 3 minor). The patients' characteristics are reported in Table I. Engraftment was assessed as time (days) to reach a neutrophil count of 0.5×109/L.
Regenerative medicine In the same period, autologous BM processing was carried out within approved trials of regenerative medicine: volume reduction was achieved by either simple buffy-coat separation (GVR programme) in a protocol of bone regeneration in the orthopaedic setting (n=54) or density-gradient mononuclear cell separation (NeatCell) in a trial on the treatment of critical limb ischaemia (n=13). Bone marrow harvesting BM units were collected in our centre or in other accredited centres for mismatched unrelated donor marrow collection, according to the standard procedure
Blood Transfus 2017; 15: 577-84 DOI 10.2450/2016.0057-16 578
All rights reserved - For personal use only No other use without premission
Clinical grade bone marrow processing Table I - Characteristics of the allogeneic and autologous HSCT recipients. Patients' characteristics Allogeneic HSCT
Autologous HSCT
Diagnoses
n.
Diagnoses
Acute myeloid leukaemia
14
Acute myeloid leukaemia
n. 3
Acute lymphoblastic leukaemia
9
Multiple myeloma
1
Non-Hodgkin's lymphoma
2
Non-Hodgkin's lymphoma
1
Myeloproliferative neoplasm
1
Myelodysplastic syndrome
1
Matched sibling donor
4
Matched family donor
7
Matched unrelated donor
Sr l
Type of donor
Statistical analysis Data were extracted retrospectively from our leucapheresis/BM processing database. Data are expressed as mean ±SD or median (range) and comparisons were carried out using the Student's t-test or the non-parametric Mann-Whitney test.
16
3
A−/O−
1
B+/O+
4
B+/A+
3
AB+/O+
1
O+/AB+
1
O+/A+
2
O−/A−
1
O+/A−
1
O+/B+
Se rv
A+/O−
Results
Volume reduction Initial and final BM volumes as well as percentage volume reduction (median and range) are reported in Table II. Data are divided for allogeneic HSCT, autologous HSCT, buffy coat and density-gradient separation for regenerative medicine. In all cases the BM volume reduction was greater than 72% (Table II).
TI
7
SI M
1
A+/O+
iz i
ABO-mismatch (donor/recipient) A+/B+
Haematopoietic progenitor cell assays were performed in duplicate, with the methylcellulose-based medium containing recombinant cytokines (MethoCult GF H4434; Stem Cell Technologies, Vancouver, Canada) as previously described16. Total colony-forming cells, defined as the total number of colonies, regardless of their lineage, were used to calculate clonogenic potential. The number of fibroblast colony-forming units (CFU-F) was determined as previously described16, and was used as a surrogate marker for mesenchymal stem cells progenitors. Before and after separation, 1×106 total nucleated cells were plated in duplicate in 100 mm Ø Petri dishes. After 14 days, the dishes were fixed with methanol and stained with Giemsa; visible colonies formed by 50 or more cells were counted and reported as the number of CFU-F/106 seeded total nucleated cells.
2
Patients are grouped based on diagnosis, type of donor and ABO mismatch. HSCT: haematopoietic stem cell transplantation.
©
of each centre. BM was harvested by multiple aspirations from the iliac crest and cells were collected into transfer bags containing ACD and heparin. The volume collected ranged between 150 and 1,727 mL, depending on the clinical application. Assessment of bone marrow content White blood cell (WBC) and RBC counts and haematocrit were determined before and after processing using an automated haematology analyser (XS-1000i, Sysmex Corp., Kobe, Japan). CD34+ cell count, MNC quantification and viability as well as WBC viability were determined by immunofluorescence analysis (FACScanto, BD Pharmingen, San Diego, CA, USA) using the ISHAGE protocol with the single platform technique13-15. 7-aminoactinomycin D (BD Pharmingen) was used to exclude dead cells.
Bone marrow processing for ABO mismatch When the GVR protocol for RBC and plasma removal (buffy coat extraction) was used, multiple procedures were needed if the initial volume exceeded 880 mL; overall 50 procedures were carried out. For allogeneic HSCT the median CD34+ cell recovery was 95.9% (range, 81.2-134.3%); CD34+ recovery was not available for autologous HSCT. The median percentage WBC recovery was 86.0% (range, 76.0-104.2%) in allogeneic HSCT and 74.0% (range, 57.1-76.3%) in autologous HSCT (Figure 1). Reductions in packed RBC volume with the GVR protocol in the HSCT setting are reported in Table III. The median reduction in RBC volume was 69% (range, 30-88%) for allogeneic transplantation and 73% (range, 59-85%) for autologous transplantation. The SmartRedux protocol was used with the Sepax 2 machine, allowing an input volume up to 3,300 mL and enabling the final volume to be set. This software was validated in five allogeneic HSCT with major and three with minor ABO mismatching; the data are summarised in Table IV, and compared with data obtained when the
Blood Transfus 2017; 15: 577-84 DOI 10.2450/2016.0057-16 All rights reserved - For personal use only No other use without premission
579
Mazzanti B et al Table II - BM volumes before and after processing and percentage volume reduction for each protocol. Patients
N
Input volume (mL)
Output volume (mL)
Volume reduction (%)
Allogeneic HSCT
19
1,221 (773-1,727)
200 (120-300)
85 (75-87)
Autologous HSCT
5
1,047 (1,021-1,129)
150 (119-153)
86 (85-88)
BC/RM
54
210 (150-300)
30 (22-45)
86 (79-90)
DGS/RM
13
300 (200-360)
42 (27-51)
86 (73-90)
SI M
TI
Se rv
iz i
Sr l
Data are expressed as median (range). BM: bone marrow; HSCT: haematopoietic stem cell transplantation; BC: buffy coat; DGS: density-gradient separation; RM: regenerative medicine.
©
Figure 1 - Percentage recovery of CD34 cells and WBC for the GVR protocols for BM buffy coat extraction for infusion in allogeneic HSCT, autologous HSCT and in regenerative medicine for patients with orthopaedic disorders (BC/RM). Each bar represents the median, quartiles, minimum and maximum values. WBC: white blood cells; GVR: generic volume reduction; BM: bone marrow; HSCT: haematopoietic stem cell transplantation; BC: buffy coat; RM: regenerative medicine.
Table III - RBC volumes before and after processing and percentage RBC reduction for HSCT and densitygradient separation for regenerative medicine protocols. RBC volume before processing (mL)
RBC volume after processing (mL)
RBC volume reduction (%)
Allogeneic HSCT
349 (193-525)
116 (36-217)
69 (30-88)
Autologous HSCT
344 (225-355)
93 (49-99)
73 (59-85)
67 (32-107)
0.4 (0-0.6)
100 (99-100)
DGS/RM
Data are expressed as median (range). RBC: red blood cell; HSCT: haematopoietic stem cell transplantation; DGS: density-gradient separation; RM: regenerative medicine.
Blood Transfus 2017; 15: 577-84 DOI 10.2450/2016.0057-16 580
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Clinical grade bone marrow processing
Table IV - BM processing for allogeneic HSCT: GVR vs Smartredux protocol. SmartRedux (n=8)
Pre-processing volume (mL)
1,221 (773-1,727)
1,128 (87-1,440)
Post-processing volume (mL)
200 (120-300)
110 (75-150)
Volume reduction (%)
85 (75-87)
90 (90-92)
CD34 recovery %
96 (81-134)
105 (82-114)
WBC recovery %
86 (76-104)
92 (87-111)
Pre-processing RBC volume (mL)
349 (193-525)
380 (234-589)
Post-processing RBC volume (mL)
116 (36-217)
44 (24-50)
RBC volume reduction (%)
69 (30-88)
88 (80-93)
SI M
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p-value
TI
GVR (n=19)
Neutrophil engraftment was achieved at a median of 23 days (range, 13-31 days) for allogeneic HSCT and 19 days (range, 9-31 days) for autologous HSCT. One patient did not engraft after a haploidentical HSCT as rescue for failed engraftment of a cord blood transplant; the patient died of sepsis 17 days after the rescue transplant. No infusion-related side effects were reported; in particular, no transfusion reactions occurred in the first group (ABO-mismatched allogeneic HSCT) except in one patient who developed a reaction after the infusion of 20 mL of the product, possibly due to the presence of irregular antibodies. The graft was then rescued by MNC separation with the NeatCell protocol and the infusion was carried out without any side effects. No microbiological contamination due to the BM manipulation process was reported.
Se rv
Mononuclear cell separation for regenerative medicine The density-gradient separation procedure (NeatCell protocol) resulted in a satisfactory recovery of CD34+ cells
Clinical outcome
Sr l
Bone marrow buffy coat extraction for regenerative medicine The GVR programme was applied for a bone regeneration protocol in the orthopaedic setting in 54 patients. The median volume of autologous BM processed was 210 mL (range, 150-300 mL). The median CD34+ and WBC recovery was 79.4% (range, 41.1109.2%) and 50.8% (range, 23.0-84.4%), respectively (Figure 1). The final, median reduced volume was 30 mL (range, 22-45 mL) (Table II).
(69%; range 36-124%), MNC (67%; range, 17-119%) and colony-forming cells (49%; range, 9-124%), but a quite low CFU-F recovery (30%; range, 8-50%) according to our previous data16. The procedure resulted in a median polymorphonuclear cell reduction of 99.8% (range, 99.7-100%); complete RBC depletion (100%) was also achieved (Table III), therefore making this procedure suitable for BM processing in the case of allogeneic HSCT with major ABO mismatching and a high titre of natural antibodies or irregular anti-ABO antibodies.
iz i
GVR programme was used. The SmartRedux protocol was associated with greater volume reduction (90%; range, 90-92% vs 85%; range, 75-87%: p
