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Correspondence: Steven L. Highfill, PhD, Department of Transfusion Medicine, Center for Cellular Engineering, National Institutes of Health Clinical Center, 10 Center Drive, MSC-1184, Building 10, Room 3C720, Bethesda, Maryland 20892-1184, USA.
Hematopoietic stem cell transplantation using bone marrow as the graft source is a common treatment for hematopoietic malignancies and disorders. For allogeneic transplants, processing of bone marrow requires the depletion of ABO-mismatched red blood cells (RBCs) to avoid transfusion reactions. Here the authors tested the use of an automated closed system for depleting RBCs from bone marrow and compared the results to a semi-automated platform that is more commonly used in transplant centers today. The authors found that fully automated processing using the Sepax instrument (Cytiva, Marlborough, MA, USA) resulted in depletion of RBCs and total mononuclear cell recovery that were comparable to that achieved with the COBE 2991 (Terumo BCT, Lakewood, CO, USA) semi-automated process.
Methods
The authors optimized the fully automated and closed Sepax SmartRedux (Cytiva) protocol. Three reduction folds (10×, 12× and 15×) were tested on the Sepax. Each run was compared with the standard processing performed in the authors’ center on the COBE 2991. Given that bone marrow is difficult to acquire for these purposes, the authors opted to create a surrogate that is more easily obtainable, which consisted of cryopreserved peripheral blood stem cells that were thawed and mixed with RBCs and supplemented with Plasma-Lyte A (Baxter, Deerfield, IL, USA) and 4% human serum albumin (Baxalta, Westlake Village, CA, USA). This “bone marrow-like” product was split into two starting products of approximately 600 mL, and these were loaded onto the COBE and Sepax for direct comparison testing. Samples were taken from the final products for cell counts and flow cytometry. The authors also tested a 10× Sepax reduction using human bone marrow supplemented with human liquid plasma and RBCs.
Results
RBC reduction increased as the Sepax reduction rate increased, with an average of 86.06% (range of 70.85–96.39%) in the 10×, 98.80% (range of 98.1–99.5%) in the 12× and 98.89% (range of 98.80–98.89%) in the 15×. The reduction rate on the COBE ranged an average of 69.0–93.15%. However, white blood cell (WBC) recovery decreased as the Sepax reduction rate increased, with an average of 47.65% (range of 38.9–62.35%) in the 10×, 14.56% (range of 14.34–14.78%) in the 12× and 27.97% (range of 24.7–31.23%) in the 15×. COBE WBC recovery ranged an average of 53.17–76.12%. Testing a supplemented human bone marrow sample using a 10× Sepax reduction resulted in an average RBC reduction of 84.22% (range of 84.0–84.36%) and WBC recovery of 43.37% (range of 37.48–49.26%). Flow cytometry analysis also showed that 10× Sepax reduction resulted in higher purity and better recovery of CD34+, CD3+ and CD19+ cells compared with 12× and 15× reduction. Therefore, a 10× reduction rate was selected for the Sepax process.
Conclusions
The fully automated and closed SmartRedux program on the Sepax was shown to be effective at reducing RBCs from “bone marrow-like” products and a supplemented bone marrow product using a 10× reduction rate.
Hematopoietic stem cells from bone marrow or mobilized peripheral blood stem cells (PBSCs) are commonly used in transplantation for hematological malignancies and immunodeficiencies. The goal of bone marrow transplant is to replace a patient's diseased hematopoietic compartment with a healthy donor marrow graft [
]. Although transplantation using mobilized PBSCs has all but replaced bone marrow as the source of healthy progenitor cells, bone marrow still accounts for approximately 25% of all allogeneic transplants in the US [
]. Bone marrow is still preferred in some scenarios because of a decreased risk of graft-versus-host disease compared with allogeneic PBSC transplants, especially in children [
Allogeneic Bone Marrow Transplantation versus Peripheral Blood Stem Cell Transplantation for Hematologic Malignancies in Children: A Systematic Review and Meta-Analysis.
Major complications associated with infusion of bone marrow grafts, such as acute hemolysis of donor erythrocytes, can be observed as a result of ABO blood group incompatibility between the marrow donor and recipient and the presence of pre-existing recipient antibodies [
]. In addition, incompatible ABO antibodies in donor plasma present in the marrow graft can cause hemolysis of recipient red blood cells (RBCs). Therefore, it is important that RBCs and plasma are removed from ABO-incompatible grafts prior to infusion [
]. This manipulation of bone marrow is an important factor, as it can directly affect the quality of the graft and overall engraftment and patient survival. It is important that the maximum amount of CD34+ hematopoietic progenitor cells is recovered during the RBC depletion process [
Peripheral blood stem cells (PBSCs) collected after recombinant granulocyte colony stimulating factor (rhG-CSF): an analysis of factors correlating with the tempo of engraftment after transplantation.
Factors affecting volume reduction and red blood cell depletion of bone marrow on the COBE Spectra cell separator before haematopoietic stem cell transplantation.
Allogeneic blood stem cell transplantation: peripheralization and yield of donor-derived primitive hematopoietic progenitor cells (CD34+ Thy-1dim) and lymphoid subsets, and possible predictors of engraftment and graft-versus-host disease.
]. There are no official guidelines on what constitutes the “safe criterion” for what the maximum volume of RBCs may be in the graft, but most institutions have agreed to aim for 20–30 mL [
Since the quantity of CD34+ cells in bone marrow samples is generally very small, it is crucial to ensure that processing events do not lead to excessive loss and that CD34 cell recovery is high enough to reach the transplant dose. The COBE 2991 (Terumo BCT, Lakewood, CO, USA) and Sepax C-Pro (Cytiva, Marlborough, MA, USA) both use centrifugation of the starting product to produce a buffy coat with the desired cells and remove the unwanted ABO-mismatched RBCs. The Sepax instrument utilizes a specific processing kit (CT-49.1), which includes a tubing set and a 220-mL separation chamber that will perform multiple centrifugation cycles for bone marrow products, which may be larger in volume than the chamber volume [
Multicenter cell processing for cardiovascular regenerative medicine applications: the Cardiovascular Cell Therapy Research Network (CCTRN) experience.
]. The Sepax is a fully automated and closed system, whereas the COBE is semi-automated and requires significant user interaction to complete a process. The Sepax instrument uses SmartRedux software (Cytiva), which is listed as class I medical device software with the Food and Drug Administration. The instrument and software applications are used for research and in clinical and commercial manufacturing environments where the process is validated by the end user. Since large-volume human bone marrow products are extremely difficult to obtain for research purposes, the authors optimized the Sepax SmartRedux software with a “bone marrow-like” product to demonstrate reproducibility [
]. This product consisted of RBCs and cryopreserved/thawed PBSCs from healthy donors, and RBC content was fixed to 30% hematocrit to have similar characteristics to what is observed in clinical bone marrow products [
]. The authors then supplemented this “bone marrow-like” product with Plasma-Lyte A (Baxter, Deerfield, IL, USA) and 4% human serum albumin (Shire, Lexington, MA, USA) to increase the volume.
The Sepax cell processing system is a fully automated and closed system that uses controlled-speed centrifugation to separate blood components. A pneumatic force and optical sensor are then employed to separate each fraction through movement of the piston in the chamber. The SmartRedux protocol is flexible, allowing the user to manipulate the starting volume, fixed volume, proportional volume, reduction rate, reprocessed buffy coat and reprocessed volume.
The authors tested the performance of the Sepax with regard to RBC reduction of a “bone marrow-like” product and compared it with the standard COBE processing procedure. Reduction rates on the Sepax were evaluated on the basis of RBC reduction, white blood cell (WBC) and CD34 cell recovery and changes in cell populations during the process, and an optimal reduction rate was determined. A 10× reduction on the Sepax was tested using an actual human bone marrow product (StemExpress, Folsom, CA, USA) supplemented with human plasma and RBCs to ensure the process was consistent and efficient. The Sepax SmartRedux software was qualified at our center to be an alternative process to the standard COBE processing for reducing the volume of RBCs in bone marrow while not affecting the recovery of the CD34+ stem cells.
Methods
“Bone marrow-like” cell preparation
“Bone marrow-like” products were prepared using patient-derived cryopreserved PBSCs and unmatched healthy donor RBCs from the National Institutes of Health Clinical Center Blood Bank. Cryopreserved PBSCs were thawed and transferred to a 3-L transfer pack (Terumo BCT). The PBSC bag was rinsed with a wash solution of Plasma-Lyte A and 4% human serum albumin and then transferred to the 3-L transfer pack. The healthy donor RBCs were transferred to the 3-L pack containing PBSCs, and each RBC bag was rinsed with the wash solution. The product was brought up to approximately 1200 mL and split into two 600-mL starting products for each instrument. Before splitting the product, a sample was collected from the original bag to process a complete blood count (CBC) to ensure the hematocrit was approximately 30%.
Semi-automated cell processing on the COBE
“Bone marrow-like” products were processed on the COBE 2991 using a blood processing set with a single bag. The COBE 2991 cell processor is a semi-automated instrument that concentrates/separates cells based on centrifugation. It consists mainly of a centrifuge and a flexible diaphragm. When the centrifuge is spinning, the flexible diaphragm located inside the centrifuge bowl is inflated with hydraulic fluid. This presses against the bottom of the cell processing bag to expel fluids and/or cells for removal or collection during centrifugation. “Bone marrow-like” products were processed on the COBE 2991 for RBC reduction. A single donut bag with a centrifugation speed of 3000 rpm is used for the process. The process duration per run varies depending on the starting bone marrow volume. In addition, there is a packed RBC loading limit of 125–200 mL per run. If a marrow product contains >200 mL of packed RBCs, it needs to be divided and processed on multiple COBE 2991 instruments. For the authors’ purposes, the starting product contained at least 125 mL of RBCs and a total nucleated cell (TNC) count of at least 5 × 109.
Fully automated cell processing on the Sepax
“Bone marrow-like” products were processed on the Sepax C-Pro using SmartRedux software. The Sepax uses a rotating syringe in the chamber of the closed CT-49.1 kit to separate the blood components. An optical sensor detects the plasma, buffy coat and RBC layer and transfers each into separate bags connected to the kit. The SmartRedux protocol can process 30–3300 mL using one kit to get to either a user-selected fixed final volume or a proportional final volume. The proportional volume is calculated as a percentage of the initial volume, whereas the fixed volume is selected by the technician [
]. In this study, the authors tested 10×, 12× and 15× fixed reductions using a reprocessed volume of 20 mL and reprocessed buffy coat of either 20 mL or 25 mL. Testing parameters were set per suggestions from the instrument manufacturer. For a volume of 600 mL, the fully automated process takes approximately 120 min or four cycles. The final buffy coat product was collected in a 150-mL transfer pack.
Testing a supplemented bone marrow product on the Sepax
A human bone marrow product was processed on the Sepax C-Pro using a 10× reduction. The initial bone marrow volume of 100 mL was transferred to a 2-L transfer pack, and the container was rinsed with human plasma from the National Institutes of Health Clinical Center Blood Bank. Two bags of RBCs and a bag of human plasma were added to the 2-L transfer pack for a final volume of 1340.9 mL. A sample from this “large bag pre-sample” was collected before it was split into two bags. Samples were collected from each smaller pre-bag before they were run on the Sepax for CBC and flow cytometry. Pre-bag 1 had an initial volume of 679.3 mL and pre-bag 2 had an initial volume of 663.8 mL. The final buffy coat product was collected in a 150-mL transfer pack, and post-samples were collected from each bag for cell counts and flow cytometry analysis.
Evaluation of samples
Values of pre- and post-samples were compared to determine the efficiency of the Sepax C-Pro SmartRedux software. Samples were collected before the large bag was split into two (large bag pre-) for the two smaller pre-samples (Sepax pre-, COBE pre-) and all final samples (Sepax buffy coat, Sepax RBCs, Sepax plasma, COBE buffy coat, COBE packed RBCs). CBCs were performed before and after processing using an ADVIA 2120i Hematology System automated blood cell analyzer (Siemens Medical Solutions USA, Inc, Malvern, PA, USA). The proportion of cell populations was determined using a two-tube multi-color antibody staining strategy, which consisted of the following: tube 1, which contained CD4 (fluorescein isothiocyanate), CD34 (phycoerythrin [PE]), CD3 (PECy7), CD8 (allophycocyanin [APC]) and CD45 (APCCy7), and tube 2, which contained CD14 (fluorescein isothiocyanate), CD16 (PE), CD56 (PECy7), CD19 (APC), CD45 (APCCy7), CD3 (BV421) and CD15 (BV510). Flow cytometry was performed on a 10-color BD FACSCanto X (BD Biosciences, San Jose, CA, USA) after labeling with fluorescent-labeled antibodies (BD Biosciences and BioLegend, San Diego, CA, USA). The authors used 7-aminoactinomycin D (BD Biosciences) to gate out dead cells. Measurements of CD34 cell counts were performed using a dual platform method consisting of cell counts obtained from an ADVIA 2120i Hematology System analyzer and a BD FACSCanto X flow cytometer. Cells were gated on singlets, CD45+ cells, live cells (7-aminoactinomycin D-negative) and CD34+ cells. Calculations were made to determine RBC depletion, RBC volume in buffy coats and recovery of WBCs and each immune cell population.
Statistical analysis
Prism 7 software (GraphPad Software, San Diego, CA, USA) was used to generate graphs. Unpaired t-tests were used to compare groups.
Results
“Bone marrow-like” products
RBC volume reductions
Although the COBE is capable of depleting RBCs from bone marrow products for the purposes of transplantation, multiple technicians are required to process one donor. Since bone marrow starting products can be up to 1200 mL, two COBE machines are required, and multiple people are needed to operate each instrument. To provide a safer, faster and more consistent and efficient process and to potentially reduce the number of technicians needed, the authors decided to test the SmartRedux protocol on the Sepax. Since the Sepax allows for the input of a fixed final volume of the buffy coat, the authors were able to test different reduction rates. The authors compared 10×, 12× and 15× reductions on the Sepax with the standard COBE process using split paired “bone marrow-like” products. The total volume of starting samples used in this study was 600 mL, and the final processing volume on the Sepax was a fixed volume of 120 mL, whereas the COBE final processing volume ranged between 150 mL and 200 mL. Initial and final RBC volumes as well as the percentage of volume reduction for RBCs were measured for each product run on the different instruments.
Although the 10× and 12× reductions on the Sepax were comparable to the COBE in depleting RBCs in the buffy coat, there was a larger difference between the two processes for the 15× reduction (average 98.89% for the Sepax versus 74.65% for the COBE) (Table 1). With respect to the Sepax, there was an increase in RBC depletion for the 12× and 15× reductions compared with the 10× reduction (Figure 1A). The Sepax had an average 98.8% RBC reduction at 12× and 98.89% RBC reduction at 15× and a slightly lower (86.06%) average RBC reduction at 10×. Even though the 10× RBC reduction was not as efficient as the 12× or 15× reduction, it was comparable to the standard COBE reduction process, which demonstrated an average 88.43% RBC reduction. It is important to point out that the COBE process was unchanged for these paired samples; only the Sepax process was modified. The variance in recovery of the COBE between runs was likely due to donor variability and served as a good control with regard to what would be expected from the authors’ standard process. In an effort to determine the significance between different methods, the authors normalized RBC depletion by calculating the percent difference in RBC depletion. The authors showed that the largest difference between the Sepax and COBE in terms of RBC depletion percentage occurred when comparing the 15× Sepax with the COBE, and the smallest difference occurred when comparing the 10× Sepax with the COBE (Figure 1B).
Table 1Sepax and COBE RBC volume reductions.
Reduction
N
Sepax RBC pre-volume, mL (Avg)
Sepax RBC post-volume, mL (Avg)
COBE RBC pre-volume, mL (Avg)
COBE RBC post-volume, mL (Avg)
Sepax RBC volume reduction, % (Avg)
COBE RBC volume reduction, % (Avg)
10× comparison
3
76.82 166.40 173.04 (138.75)
22.39 6.00 15.66 (14.68)
84.60 175.10 172.54 (144.08)
19.37 10.70 9.83 (13.3)
70.85 96.39 90.95 (86.06)
77.10 93.89 94.30 (88.43)
12× comparison
2
176.00 164.85 (170.42)
0.83 3.20 (2.02)
182.00 168.44 (175.22)
22.56 2.194 (12.38)
99.53 98.06 (98.80)
87.60 98.70 (93.15)
15× comparison
2
178.84 187.78 (183.31)
2.06 1.92 (1.99)
180.24 189.55 (184.89)
55.92 37.26 (46.59)
98.80 98.98 (98.89)
69.00 80.30 (74.65)
RBC volumes before and after processing for both the Sepax and COBE. Values from each run are shown along with Avg of each individual comparison. Three runs were carried out for the 10× comparison, and two runs were carried out for the 12× and 15× comparisons.
Figure 1Comparison of RBC depletion of and WBC recovery from “bone-marrow-like” products using Sepax and COBE. For each run, a single donor product was split into two pre-samples that were processed on the Sepax and COBE. The 10×, 12× and 15× Sepax settings were evaluated for optimization. (A) RBC depletion percentages in the buffy coat compared with the starting pre-product. (B) To account for donor-to-donor variability among samples, the percent difference in RBC depletion between the Sepax and COBE devices was calculated. Unpaired t-test was used to determine significance between groups. (C) WBC recovery percentage in the buffy coat. (D) To account for donor-to-donor variability among samples, the percent difference in WBC recovery between the Sepax and COBE devices was calculated. Unpaired t-test was used to determine significance between groups. (E) Total volume of RBCs in the starting and buffy coat samples. (F) WBC viability of pre-processing and buffy coat samples from the Sepax and COBE. (Color version of figure is available online.)
WBC recovery was highest on the Sepax at the 10× reduction (average 47.65%). There was no significant difference between the 10× Sepax and the COBE with regard to WBC recovery. The 12× and 15× reductions on the Sepax suffered from significantly decreased WBC recovery (14% and 27.97%, respectively) (Figure 1C). There was a significant difference in WBC recovery for the 12× and 15× reductions between the Sepax and COBE (P < 0.05). To account for donor-to-donor variability between runs, the authors plotted the percent difference between the Sepax and COBE. Upon doing this, the data showed that the 10× process was most similar to the COBE (showing the least amount of difference), whereas the 12× and 15× processes were the most different from the COBE and not significantly different from one another (Figure 1D). The absolute TNC numbers of the Sepax and COBE are shown in Table 2. The difference in viable TNCs pre- and post-processing between the Sepax and COBE at the 10× reduction rate was minimal (3.53 × 109 for the Sepax versus 3.46 × 109 for the COBE). As the reduction rate on the Sepax was increased, however, these differences increased as a result of much higher loss in recovery (12× reduction, 7.66 × 109 for the Sepax versus 4.10 × 109 for the COBE, 15× reduction, 7.85 × 109 for the Sepax versus 3.07 × 109 for the COBE).
Table 2TNC differences in each reduction condition.
Reduction
N
Sepax TNC pre ×109 (Avg)
Sepax TNC buffy coat post ×109 (Avg)
COBE TNC pre ×109 (Avg)
COBE TNC buffy coat post ×109 (Avg)
10× comparison
3
2.52 10.01 6.05 (6.19)
1.58 3.89 2.52 (2.66)
4.24 10.91 6.10 (7.08)
2.71 5.12 3.01 (3.61)
12× comparison
2
11.30 7.63 (9.46)
1.70 1.90 (1.80)
11.10 7.29 (9.20)
7.00 3.18 (5.09)
15× comparison
2
9.66 12.65 (11.16)
2.70 3.95 (3.32)
9.51 11.90 (10.71)
7.28 7.98 (7.63)
Viable total cell counts before and after processing for the Sepax and COBE. Values from each run are shown along with Avg of each individual comparison. Three runs were carried out for the 10× comparison, and two runs were carried out for the 12× and 15× comparisons.
RBC volumes in the Sepax buffy coat samples were comparable for all reduction rates and below 25 mL. RBC volumes in the COBE buffy coat samples were comparable in the 10× and 12× reductions. However, a total of 55 mL and 37 mL were noted in the 15× comparison samples (Figure 1E), indicating that the Sepax may be more consistent among donors than the COBE. With the exception of one run, the viability of cells post-processing was consistently high for each Sepax reduction tested and comparable to the viability of the COBE samples. Samples were taken immediately after the Sepax and COBE runs were complete and processed within 1 h. The decrease in viability on the 12× Sepax reduction for the COBE post-sample may be explained by the fact that this particular sample was not analyzed within this time frame but was instead analyzed several hours later (Figure 1F).
Frequency and recovery of different cell populations
Flow cytometry was performed to examine the frequencies of various cell populations (CD34, CD3, CD19, CD14/CD16, CD15, CD56) between the two processing methods. There was little difference in the frequency of CD34 cells between the groups (Figure 2A); however, differences in CD34 recovery were more apparent (Figure 2B). The inadvertent exclusion of anti-CD34 in the 15× 01 sample resulted in a reduction in sample size; however, the other two samples analyzed (15× 02, 15× 03) showed a high level of consistency. The recovery of 10× Sepax groups was very similar among the donors, with a slight increase compared with the COBE. At the 12× and 15× reduction rates, however, the authors observed a dramatic loss in the recovery of CD34+ cells compared with the COBE. Frequencies of CD3+ T cells and CD19+ B cells generally increased from pre- to post-processing on the Sepax, whereas the frequencies of these populations in the COBE-processed samples remained more similar (Figure 2C,E). Recovery of CD3+ T cells and CD19+ B cells was higher in the 10× Sepax-processed samples but dropped significantly in the 12×- and 15×-processed samples (Figure 2D,F). Frequencies of monocytes (CD14/CD16+) were mostly decreased in post-processing Sepax samples, whereas they remained more similar in the COBE samples (Figure 2G). Frequencies of granulocytes (CD15+) remained more constant, with the exception of a decrease in the 12×-1 Sepax post-sample (Figure 2I). The recovery of monocytes and granulocytes was higher among COBE-processed samples in all patients at all comparisons (Figure 2H,J). Overall, these data showed that 10× processing on the Sepax resulted in higher purity and better recovery of CD34+, CD3+ and CD19+ cells than 12× and 15× processing, and this process was used for further experiments. Because of the poor recovery of cells at the end of the process, 12× and 15× processing on the Sepax was not continued.
Figure 2Flow cytometry analysis of buffy coats from “bone-marrow-like” products using Sepax and COBE. Buffy coats were evaluated by flow cytometry. (A,C,E,G,I) Frequencies of CD34, CD32, CD19, CD14/CD16 and CD15 cell populations in pre- and post-processing samples were plotted for each run. (B,D,F,H,J) Cell recovery percentages were calculated for each run using pre-processing and buffy coat samples for both Sepax and COBE. (Color version of figure is available online.)
To confirm that the optimized 10× Sepax reduction is suitable for a bone marrow product, the authors tested supplemented human bone marrow on the Sepax using the 10× reduction. After supplementing 100 mL of donor bone marrow with human plasma and RBCs, the large bag was split into two bags for separate runs, with a starting hematocrit of 28.3% and 28.5% for run 1 and run 2, respectively. The total volume of RBCs in the buffy coat was reduced significantly from an average of 190 mL to an average of 30 mL (Figure 3A). This reduction in volume resulted in an RBC depletion percentage of 84.04% and 84.36% for bag 1 and bag 2, respectively (Figure 3B). The average WBC recovery from both bags was approximately 43.4%, with the recovery from bag 2 being slightly higher (37.48% versus 49.26%) (Figure 3C). The recovery observed here was comparable to the “bone marrow-like” samples in the 10× reduction Sepax runs. The viability of the two products was between 97% and 99% for post-processing samples (Figure 3D).
Figure 3Results of 10× Sepax reduction of human bone marrow are comparable to those observed for “bone marrow-like” products. One bone marrow donor sample was split into two bags for separate Sepax runs. (A) Total volume of RBCs in starting and buffy coat samples. (B) RBC depletion percentages in the buffy coat from the starting product for each bag. (C) WBC recovery percentage in the buffy coat. (D) Viability of pre-processing and buffy coat samples from each bag. (E) Percentages of different cell populations from pre- and post-samples were plotted for each run. (F) Cell recovery percentages were calculated for both runs using pre-processing and buffy coat samples for the Sepax.
Flow cytometry was performed to examine the frequency of various cell populations pre- and post-processing (CD34, CD14, CD19, CD56, CD3, CD15). With the exception of CD15 granulocytes, where the authors observed a decrease in frequency, the frequencies of these cell populations all demonstrated a slight increase (Figure 3E). Importantly, there was a respective 75% and 100% recovery of CD34+ cells for bag 1 and bag 2, and the recovery of CD14, CD19, CD56, CD3 and CD15 cells ranged from 40% to 80% and was in line with that observed for the “bone marrow-like” product (Figure 3F).
Discussion
ABO-incompatible bone marrow transplant risks, such as intravascular immune hemolysis and delay of RBC engraftment, can be minimized by depletion of RBCs in the product prior to transplantation [
]. The study described herein highlights the use of the automated closed system RBC depletion protocol for clinical-scale reductions of bone marrow for stem cell transplantation. Current methods used at the authors’ center and elsewhere utilize the COBE 2991 to deplete RBCs from bone marrow products prior to transplantation [
Preparation of red-blood-cell-depleted marrow for ABO-incompatible marrow transplantation by density-gradient separation using the IBM 2991 blood cell processor.
]. The authors tested and optimized a fully automated closed system method to reduce the number of laboratory staff required for processing and to enhance the consistency of producing a reduced volume of RBCs from the bone marrow product.
The authors report here a detailed analysis comparing the Sepax and COBE 2991 processing of split paired products. Such a detailed analysis has been very difficult to perform because of the lack of volunteers willing to contribute the large volume of bone marrow required for this research [
]. One of the novelties of this study is that the authors devised a method whereby we were able to mimic a large-volume bone marrow product by mixing RBCs with mobilized PBSCs. This large 1200-mL product was split into two identical 600-mL starting products to test the Sepax and COBE RBC volume reductions head-to-head. The clinical manufacturing process that was used for the COBE 2991 instrument was developed in the authors’ center and has been in use for these purposes since 2003. The Sepax is more customizable and has many different options. Per the manufacturer's suggestion, the authors tested 10×, 12× and 15× volume reductions on the Sepax using the SmartRedux protocol. This software allows for a wide range of initial volumes to be used to initiate the reduction process and provides a final buffy coat volume based on the initial volume. Once each run was complete, samples were collected for CBC and flow cytometry analysis.
The authors report here that the 10× volume reduction on the Sepax is comparable to the reduction observed using the COBE 2991. A 10× reduction had an average RBC volume reduction of 86.06% compared with the COBE, which demonstrated an average reduction of 88.43%. There was an increase in RBC volume reduction on the Sepax as the reduction rate increased to 12× and 15×. However, the increase in RBC reduction was at the cost of lower WBC recovery. The 10× reduction rate on the Sepax had similar WBC recovery as the COBE. However, compared with the COBE, the Sepax showed a drastic decrease in WBC recovery when performing a 12× or 15× reduction. The viability remained consistent among all samples for each reduction comparison. Each Sepax reduction was able to reduce the RBC volume in the buffy coat to under 25 mL.
The authors then tested healthy donor bone marrow on the Sepax to verify that the “bone marrow-like” product behaved similarly to an actual bone marrow product. The bone marrow product (100 mL) was supplemented with RBCs and human liquid plasma and split in half to increase the authors’ number of replicates using the same donor. Both runs had nearly identical RBC reduction, demonstrating 84.2 ± 0.1% depletion and a respective 29 mL and 30 mL of RBCs in the buffy coat for run 1 and run 2. Both runs also had a WBC recovery that was comparable to the “bone marrow-like” 10× reduction runs. Flow cytometry results showed that pre- and post-buffy coat viability was between 97% and 99%. Cell recovery for CD34 cells was also very high, at 87.5 ± 12%.
Currently, there are only two studies on the topic of RBC reduction in bone marrow that utilize the Sepax SmartRedux software. Fantin et al. [
] compared bone marrow RBC reduction using two different software programs on the Sepax: NeatCell (Cytiva), which is a Ficoll-Paque (GE Healthcare, Uppsala, Sweden) based process the authors opted not to test, and SmartRedux, which was utilized here. The researchers’ results for the latter are similar to what the authors show here for the human bone marrow product in terms of erythrocyte depletion (95.3 ± 2% versus the authors’ 84.2 ± 0.1%) and CD34 recovery (86.4 ± 14.6% versus the authors’ 87.5 ± 12%). Similarly, in a study by Mazzanti et al. [
], the Sepax SmartRedux system was used in the manufacture of eight separate products. Here, again, the authors fall within the range reported by this group for erythrocyte depletion and CD34 cell recovery (30–80% and 81–134%, respectively). These studies give the authors more confidence in the present results given that we did not have a large number of samples to test.
In terms of impact on staff, the authors found that the Sepax instrument is much more conducive to smaller processing centers that may not operate with a large number of staff. Even though bone marrow processing on the COBE has been utilized for many years, there are limitations to the instrument. For example, at the authors’ center, because of the large volume (2 L) usually associated with this type of product, three instruments are required to be run simultaneously, with multiple technicians operating each instrument. Specifically, for each instrument used, two technicians are tasked with performing the hands-on work while one technician acts to verify and record data in the batch record. The amount of time spent on one product can be up to 3 hours, multiplied by nine technicians (if using three instruments, as in most circumstances).
Other limitations include potential breaches in sterility of the product. Connection of the sample/buffer bag to the COBE kit by spiking is performed under non-sterile conditions, which increases the chances of microbial contamination. In the same vein, on at least six separate occasions spanning approximately 15 years, the authors have observed leaks or breakages in the bags—some of which were reportedly due to contact with the internal mechanisms of the instrument—that have resulted in loss of the product. In comparison, because of its fully automated and closed nature, the Sepax instrument does not suffer from these issues. The Sepax tubing set (CT-49.1) associated with this process can be easily connected sterilely to the product and buffer bag using a sterile tubing welder, eliminating non-sterile spiking of bags. The SmartRedux software is Title 21 Code of Federal Regulations Part 11 compliant, and the electronic record keeping on the instrument serves in place of handwritten batch records. Finally, the manufacturer reports that the instrument is capable of processing a large-volume product (up to 3300 mL) on a single instrument, although the authors were unable to verify this because of the lack of availability of such a large product. Taken together, all of this results in the ability to process a large bone marrow product with just one technician per instrument.
Conclusions
The authors demonstrate the utility of using a “bone marrow-like” product for testing equipment such as the Sepax in instances where bone marrow may not be available. The present data support the hypothesis that the Sepax is comparable to the authors’ standard process using the COBE 2991 for RBC reduction of bone marrow products. Because of its closed nature and the lack of necessity to spike bags under non-sterile conditions, the product is less prone to contamination. Finally, since the Sepax is fully automated, it is better suited for clinical manufacturing when performing RBC reductions and the overall cost of processing a single product is significantly reduced.
Funding
Open access funding was provided by the National Institutes of Health (NIH). Human bone marrow was purchased by the Center for Cellular Engineering. This study was supported by the NIH with awards from the Intramural Research Program of the NIH Clinical Center (Z99CL999999) and the National Center for Advancing Translational Sciences (UL1TR000445). This study was completed in part by AC as her project work in the Specialist in Blood Banking program at the NIH (https://clinicalcenter.nih.gov/dtm/research/sbb.html). The views expressed by the authors do not necessarily represent the views of the NIH, Department of Health and Human Services or US federal government.
Author Contributions
Conception and design of the study: SLH, VAR, AC, LM, JJ and KMB. Acquisition of data: YC and MP. Analysis and interpretation of data: YC, MP, SLH, VAR, AC, LM and JJ. Drafting or revising the manuscript: VAR and AC. All authors have approved the final article.
Declaration of Competing Interest
The authors have no commercial, proprietary or financial interest in the products or companies described in this article.
Acknowledgments
The authors thank the staff of the Department of Transfusion Medicine, Center for Cellular Engineering, NIH for their help in preparing samples and operating the COBE. The authors also thank the staff of the Clinical Center Blood Bank for providing RBCs and human liquid plasma as well as Dr Eu Han Lee, Sonia Bulsara and Zachary Nelson for their helpful discussion and insight into the Sepax instrument.
References
Georges G.E.
Doney K.
Storb R.
Severe aplastic anemia: allogeneic bone marrow transplantation as first-line treatment.
Allogeneic Bone Marrow Transplantation versus Peripheral Blood Stem Cell Transplantation for Hematologic Malignancies in Children: A Systematic Review and Meta-Analysis.
Peripheral blood stem cells (PBSCs) collected after recombinant granulocyte colony stimulating factor (rhG-CSF): an analysis of factors correlating with the tempo of engraftment after transplantation.
Factors affecting volume reduction and red blood cell depletion of bone marrow on the COBE Spectra cell separator before haematopoietic stem cell transplantation.
Allogeneic blood stem cell transplantation: peripheralization and yield of donor-derived primitive hematopoietic progenitor cells (CD34+ Thy-1dim) and lymphoid subsets, and possible predictors of engraftment and graft-versus-host disease.
Multicenter cell processing for cardiovascular regenerative medicine applications: the Cardiovascular Cell Therapy Research Network (CCTRN) experience.
Preparation of red-blood-cell-depleted marrow for ABO-incompatible marrow transplantation by density-gradient separation using the IBM 2991 blood cell processor.
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