Automated Cell Processor Research Articles

Impact of input volume on red cell quality in deglycerolized RBCs using a modified ACP-215 protocol.

The ACP 215 automated cell processor is used to glycerolize and deglycerolize red cell concentrates (RCCs). Its primary advantage over the COBE 2991, previously used to cryopreserve RCCs, is that it maintains a closed system enabling extended post-thaw expiry. However, it was observed that post-deglycerolization hematocrits (Hct) of units processed with the LN236 kit are markedly lower than those processed using the COBE 2991. Therefore, we intended to determine whether a modified process using a smaller volume deglycerolization kit (LN235) could increase the final Hct with limited deleterious effects on product characteristics. Two proof-of-concept (POC) studies, conducted to determine the feasibility of using the LN235 processing kit for deglycerolization, identified the necessary modifications to the pre- and post-deglycerolization process, after which a two-part study characterized the modified protocol. The impact of pre-cryopreservation storage duration (7-21 days), input red cell mass, and the type of CPD/SAGM RCC production method (red cell filtration and whole blood filtration) were investigated. Using the LN235 kit in conjunction with a volume reduction step for RCCs with a red cell mass exceeding 180 mL allowed for an ~8% increase in Hct. As expected, slightly lower recoveries were seen for large RCCs due to volume reduction; however, there were no other detrimental outcomes on product quality. Leveraging the LN235 kit, recommended by Haemonetics for units with a red cell mass of ≤180 mL, can be used to increase the post-deglycerolization Hct of RCCs that exceed this volume.

Modification of deglycerolization procedure improves processing and post-thaw quality of cryopreserved sickle trait red cell concentrates

Red blood cell (RBC) transfusion is a critical therapy for those with sickle cell disease (SCD). Alloimmunization is frequent for those with SCD and may limit the availability of matched RBC. Cryopreserved RBCs, from family members or donors with a similar RBC antigen profile could provide a viable alternative to avoid further alloimmunization and prevent hemolytic transfusion-related events. However, cryopreserved SCD and Sickle Cell trait (S-trait) donor RBC units suffer from reduced recovery following deglycerolization. This study proposes and tests a modified deglycerolization protocol using an automated cell processor to mitigate RBC loss. Six red cell concentrates (RCC) from donors with S-trait and six control RCCs were glycerolized, frozen (<−65 °C) and deglycerolized on the ACP 215 using modified parameters (decreased hypertonic solution flow rate (100 mL/min) and hypertonic equilibration delay (120 s), and increased NaCl dilution volumes (500 mL). Quality testing included: hematocrit (HCT), hemolysis, indices, extracellular potassium, morphology, osmotic fragility, osmotic gradient ektacytometry, hemoglobin (HGB), and recovery. Canadian standards (CS) indicate that acceptable deglycerolized units for transfusion require a HCT ≤0.80 L/L, HGB ≥35 g/unit, and hemolysis <0.8 % in 90 % of units tested. No significant differences in HGB or RBC recovery were observed between study groups. Significant differences between study groups were identified in osmotic fragility and osmotic gradient ektacytometry parameters. Of the 6 S-trait RCCs, 3/6 units were within the HCT, HGB and hemolysis thresholds set by the CS. The modified deglycerolization protocol provides a path for the routine cryopreservation of S-trait RBCs.

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor.

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant diseases. CAR-T cells are genetically modified T cells that express a chimeric protein that recognizes and binds to a cell surface target, resulting in the killing of the target cell. Traditional CAR-T cell manufacturing methods are labor-intensive, expensive, and may carry the risk of contamination. The CliniMACS Prodigy, an automated cell processor, allows for manufacturing cell therapy products at a clinical scale in a closed system, minimizing the risk of contamination. Processing occurs semi-automatically under the control of a computer and thusminimizes human involvement in the process, which saves time and reduces variability and errors. This manuscript and video describes the T cell transduction (TCT) process for manufacturing CAR-T cells using this processor. The TCT process involves CD4+/CD8+ T cell enrichment, activation, transduction with a viral vector, expansion, and harvest. Using the Activity Matrix, a functionality that allows ordering and timing of these steps, the TCT process can be customized extensively. We provide a walk-through of CAR-T cell manufacturing in compliance with current Good Manufacturing Practice(cGMP) and discuss required release testing and preclinical experiments that will support an Investigational New Drug (IND) application. We demonstratethe feasibility and discuss the advantages and disadvantages of using asemi-automatic process for clinical CAR-T cell manufacturing. Finally, we describe an ongoing investigator-initiated clinical trial that targets pediatric B-cell malignancies [NCT05480449] as an example of how this manufacturing process can be applied in a clinical setting.

Platelet vesicles are potent inflammatory mediators and washing red blood cell products with an automated cell processor reduces the inflammatory phenotype

Platelet vesicles are potent inflammatory mediators and washing red blood cell products with an automated cell processor reduces the inflammatory phenotype

A novel protocol for cryopreservation of paediatric red blood cell units allows increased availability of rare blood types.

There is a growing concern for shortage in future blood supply, caused by a predicted decrease in eligible blood donors and simultaneous increase in recipients. Cryopreservation of split red blood cell units could increase stock supply by reducing waste of rare blood. This would be particularly useful in paediatric care where very small volumes often are transfused. The aim of this study was to develop a cryopreservation protocol for split units using the closed-system automated cell processor ACP215, as such protocols are currently missing. Using a pool-and-split design, red blood cell units (N=8) were glycerolized and frozen, either as standard volume reference units, or further divided into three smaller split units each. After thawing/deglycerolization, the supernatant of the smaller splits was reduced by additional centrifugation, and new SAGM was added to 60% haematocrit. The units were analysed for storage lesion effects up to ten days post-thawing. Haemolysis and extracellular potassium ion levels were lower in the split units than in the whole units from day three onwards. The metabolic parameters pH, ATP, glucose and lactate were also lower, though likely caused by lower additive solution pH rather than storage. Split units of red blood cells can be successfully cryopreserved using the ACP215. In addition to favourably low haemolysis and potassium, they also have higher haematocrit than corresponding whole units and enable involvement of fewer donors at repeated transfusions. These characteristics are all desirable features in paediatric care.

Characterization of clinical grade CD19 chimeric antigen receptor T cells produced using automated CliniMACS Prodigy system.

BackgroundChimeric antigen receptor (CAR) T-cell therapy is highly effective for treating acute lymphoblastic leukemia and non-Hodgkin’s lymphoma with high rate complete responses. However, the broad clinical application of CAR T-cell therapy has been challenging, largely due to the lack of widespread ability to produce and high cost of CAR T-cell products using traditional methods of production. Automated cell processing in a closed system has emerged as a potential method to increase the feasibility of producing CAR T cells locally at academic centers due to its minimal reliance on experienced labor, thereby making the process less expensive and more consistent than traditional methods of production.MethodIn this study, we describe the successful production of clinical grade CD19 CAR T cells using the Miltenyi CliniMACS Prodigy Automated Cell Processor at University of Colorado Anschutz Medical Campus in a rapid manner with a high frequent CD19 CAR expression.ResultsThe final CAR T-cell product is highly active, low in immune suppression, and absent in exhaustion. Full panel cytokine assays also showed elevated production of Th1 cytokines upon IL-2 stimulation when specifically killing CD19+ target cells.ConclusionThese results demonstrate the feasibility of producing CAR T cells locally in a university hospital setting using automated cell processor for future clinical applications.

Comparison between in vitro properties of washed platelet concentrates suspended in M-sol and those in BRS-A, both of which were prepared with an automated cell processor

BackgroundWashed platelet concentrate (WPC) is prepared manually in general, but automated preparation is desirable to minimize variation in the WPC quality and enhance WPC production. Recently, the software was improved for an automated cell processor (ACP) to control all processes of WPC preparation. M-sol and BRS-A, which are mixtures of medical solutions, are widely used for WPC preparation with a manual method in Japan. In this study, we prepared WPC suspended in M-sol (WPC-M) or BRS-A (WPC-B) with the ACP, and compared their in vitro properties during 7-day storage. Study design and methodsPC was divided into two equal aliquots for WPC-M and WPC-B. A divided PC, medical solutions and disposable materials were set in the ACP, and it was started to prepare WPC-M or WPC-B on Day 0. Prepared WPC was stored on a flatbed shaker until Day 7. ResultsThe pH of WPC-M and WPC-B was maintained above 6.8 during the 7-day storage. The differences in aggregation (%), HSR (%), P-selectin expression, GPIbα expression, and phosphatidylserine expression between WPC-M and WPC-B were minimal until Day 3. ConclusionThe in vitro properties of WPC-B are not markedly different from those of WPC-M until Day 3.

Quality of red blood cells washed using an automated cell processor with and without irradiation

Sterile washing of red blood cells (RBCs) and use of an additive solution permits longer postwash storage. The effect of irradiation during this extended storage time is unclear. RBCs were washed 14 days after collection using an automated cell processor and stored in saline-adenine-glucose-mannitol. To determine how long washed and irradiated RBCs could be stored, units were irradiated 1, 4, 5, and 7 days after washing and in vitro quality was assessed. Determined limits of postwash storage time for washed and washed and irradiated RBCs were validated. Quality assessment included percent recovery, hemoglobin (Hb), hemolysis, extracellular K(+) , and adenosine triphosphate. Immunoglobulin (Ig)A levels were measured in the nonirradiated arm. RBCs irradiated 1 and 4 days after washing had unacceptably high hemolysis by Day 7 postwash, not meeting the acceptance criterion (<0.8% hemolysis in 98% of units with 95% confidence). Therefore, a 48-hour maximum storage time after irradiation was chosen. Storage limits tested in the validation phase were as follows: washing on Day 14 and subsequent storage for 7 days (washed RBCs) and washing on Day 14, irradiation on Day 19, and subsequent storage for 48 hours (washed and irradiated RBCs). All units met criteria for Hb, hematocrit, hemolysis, and sterility for washed RBCs. However, RBCs were washed with less than 2 L of saline, and IgA levels in 27 of 40 units were too high to be suitable for transfusion to IgA-deficient recipients. The extended expiry for washed and washed and irradiated RBCs met requirements for all indications except transfusion to IgA-deficient recipients.

Comparative study of automated cryopreservation of red blood cells

BackgroundStrategic blood reserves are an important component in meeting blood needs and this can be accomplished through the establishment of a frozen blood program. MethodsOne hundred units of packed RBC were glycerolized using the Haemonetics ACP 215 automated cell processor and placed in a −86 °C deep freezer for freezing and storage. Product weight, hematocrit, RBC count, WBC count and hemoglobin were recorded prior to freezing. Twenty five bags were thawed and deglycerolized after every three months starting at one year from the date of first glycerolization In addition to the earlier parameters the bags were assessed for supernatant osmolality, pH, supernatant hemoglobin, ATP levels and supernatant potassium and from these red cell recovery, percentage hemolysis, supernatant glycerol and red cell viability were estimated. All tests were repeated at the end of 7 and 14 days. ResultsThe mean red cell recovery was found to be 86.12% on Day 0 and 84% on Day 14. All the bags showed residual glycerol and pH within the acceptable limits upto Day 14. Percentage hemolysis, Mean ATP levels and mean supernatant potassium levels were within acceptable limits upto Day 14. All the units were sterile upto Day 14. ConclusionThe data in this study showed that the red cells which were glycerolized using the automated platform ACP 215, frozen at −80 °C for more than a year and deglycerolized again using the ACP 215 had excellent viability while being stored at 4 °C during the 14 days of post-thaw storage.

Quality of red blood cells washed using the ACP 215 cell processor: assessment of optimal pre‐ and postwash storage times and conditions

Washing of red blood cell concentrates (RCCs) is required for potassium-sensitive transfusion recipients, including neonates in need of large-volume transfusions. When open, nonsterile washing systems are used, postwash outdate time is limited to 24 hours, often leading to problems providing the component to the patient before expiry. A closed, automated cell processor, the ACP 215 from Haemonetics Corporation, was used to wash RCCs and determine optimal pre- and postwash storage times. Two postwash storage solutions, additive solution (AS)-3 and saline-adenine-glucose-mannitol (SAGM), were compared. The in vitro quality of leukoreduced RCCs, prepared from citrate-phosphate-dextrose-anticoagulated whole blood, was determined postwash and compared to existing guidelines for RCC quality (hemoglobin content, hematocrit, and hemolysis) and predetermined criteria for ATP and supernatant potassium levels. A criterion for visual hemolysis was also applied. The prewash storage time, postwash storage time, and the postwash resuspension solution all contributed to RCC quality postwash. Levels of hemolysis were greater when washed RCCs were resuspended in SAGM (p = 0.01), while AS-3 proved worse at maintaining ATP levels postwash (p < 0.01). Immediately postwash, all units had supernatant K+ levels below the detection limit of the instrument (<1 mmol/L), but these increased to above acceptable levels within 14 days. Based on all acceptance criteria, a maximum 14-day prewash storage period and 7-day postwash storage period in SAGM preservative was found to be optimal. The longer outdate time postwashing should help lessen challenges in providing components to patients before expiry.

The in vitro quality of red blood cells frozen with 40 percent (wt/vol) glycerol at -80 degrees C for 14 years, deglycerolized with the Haemonetics ACP 215, and stored at 4 degrees C in additive solution-1 or additive solution-3 for up to 3 weeks.

Red blood cells (RBCs) frozen with 40 percent (wt/vol) glycerol, stored at -80 degrees C (mean temperature; range, -65 to -90 degrees C) for 14 years, deglycerolized in the Haemonetics automated cell processor (ACP) 215 with the 325-mL disposable bowl, and stored at 4 degrees C in additive solution (AS)-1 or AS-3 for 21 days were evaluated. A total of 106 units of citrate phosphate dextrose adenine-1 RBCs were frozen with 40 percent (wt/vol) glycerol in the original 800-mL polyvinylchloride plastic bag and stored in corrugated cardboard boxes at -80 degrees C for 14 years. The thawed units were deglycerolized with the ACP 215 with a 325-mL disposable bowl and stored in AS-1 or AS-3 at 4 degrees C for 21 days. The freeze-thaw recovery value was 94 +/- 4 percent (mean +/- SD), the freeze-thaw-wash recovery value was 80 +/- 7 percent, and there was no breakage. Thirty-eight units were processed as 19 pairs. Two units of ABO-matched units were thawed, pooled, divided equally into two units, and deglycerolized. One unit was stored in AS-1 and the other in AS-3 at 4 degrees C for 21 days. Units stored in AS-1 exhibited significantly greater hemolysis than those stored in AS-3. Acceptable results were achieved when RBCs frozen at -80 degrees C for 14 years were deglycerolized in the ACP 215. Deglycerolized RBCs in AS-1 exhibited significantly higher hemolysis than those in AS-3 after storage at 4 degrees C for 7 to 21 days.