Impact of Cryopreservation on Extracorporeal Photopheresis (ECP)-Treated Leukocyte Subsets.

BACKGROUND: Extracorporeal photopheresis (ECP) is frequently utilized in the treatment of steroid-refractory acute and chronic graft-versus-host disease (GVHD). Although the mechanism of action is not fully understood, it has been postulated that its therapeutic effect is immunologic tolerance linked to the associated apoptosis of the treated cells. Despite significant advances in allogeneic hematopoietic stem cell transplantation (HSCT), prophylaxis and treatment of GVHD remain a challenge and major limitation associated with this therapy. Use of ECP is a valuable strategy; however, it is time, cost, resource intensive, and not readily accessible. OBJECTIVE: In an effort to expand access to this therapy, we are investigating the use of cryopreserved ECP-treated cells. This will provide the ability to administer a significant proportion of the treatment at a facility closer to the patient's residence, thereby decreasing the number of visits to the primary treatment center with the goal of improving and expanding access to this therapy. Here we report the effects of cryopreservation on ECP-treated leukocytes. STUDY DESIGN: Mononuclear cells were pheresed from human patients, ECP-treated, and collected for viability and apoptotic analysis. Cells were then cryopreserved at -80°C or -150°C for 1 week, 1 month, and 3 months. Following thaw, repeat viability and apoptosis studies were performed on the leukocytes. RESULTS: WBC viability for freshly ECP-treated leukocytes was 84.5% ± 3.5 at 1 week, 87.3% ± 5.2 at 1 month, and 79.1% ± 1.1 at 3 months post thaw. Similar results were seen for cells frozen in cryovials. Leukocytes frozen the day after ECP treatment had 1 week and 1 month WBC viabilities of 84.0 ± 4.1 and 83.1 ± 2.1, respectively. Apoptotic potential was well preserved at 3 months, with cryopreserved ECP-treated lymphocytes being 19.2%, 44.5%, 75.5%, and 94.0% apoptotic after thaw on days 0, 1, 2, and 3 in culture, respectively. CONCLUSIONS: ECP-treated leukocytes cryopreserved at -80°C or -150°C for 3 months remain viable and as capable of apoptosis as freshly treated cells. Cryopreservation of an ECP-product warrants further in vivo investigation as a strategy to facilitate access to this needed therapy.

Pilot study of a new online extracorporeal photopheresis system in patients with steroid refractory or dependent chronic graft vs host disease.

BACKGROUND: A new protocol has been developed on the Amicus Separator that enables the device to perform online extracorporeal photopheresis (ECP) procedures when used in conjunction with the Phelix photoactivation device and associated disposable kit. The objective of this study was to evaluate the safety and performance of the Amicus ECP System in adult subjects with steroid-refractory or dependent chronic graft vs host disease (cGVHD). STUDY DESIGN AND METHODS: Eight subjects with mild to severe cGVHD underwent 31 procedures. Subject safety evaluations were performed pre and post procedure and adverse events (AEs) were recorded during treatment and 24 hours after the last procedure. In vitro evaluations of the treated cells included hematology counts and lymphocyte apoptosis, viability and proliferation as measures for ECP procedure validation. RESULTS: For n = 23 evaluable procedures, median (range) procedure time was 88 (78-110) minutes, during which 2.9 (0.6-4.7) × 109 TNCs (approximately 90% MNCs) were treated and reinfused to the subjects. All subject safety evaluations (vitals, cell counts, plasma hemoglobin and bacterial and endotoxin testing) were within expected ranges. All device or procedure related AEs were mild in nature. After 24 hours in culture, 86 (52-98)% of treated lymphocytes were apoptotic compared to 27 (15-51)% in controls. Inhibition of lymphocyte proliferation was >91% in all procedures. CONCLUSION: ECP procedures were safely completed in adult subjects with SR-cGVHD treated using the new online Amicus ECP system.

Cryopreserved ECP-treated lymphocytes maintain apoptotic response and anti-proliferative effect.

BACKGROUND: The ability to cryopreserve a portion of the cells treated during extracorporeal photopheresis (ECP) would improve therapy logistics, particularly for pediatric patients, by allowing multiple therapeutic doses to be collected from a single apheresis session. However, the effect of cryopreservation on ECP-treated cells is unknown (e.g., ECP-induced lymphocyte apoptosis and inhibition of proliferation). STUDY DESIGN AND METHODS: Mononuclear cell (MNC) apheresis products collected from healthy subjects were ECP-treated using offline methods. Fresh samples of ECP-treated and control cells were placed immediately in culture. The remainder of the cells were frozen in cryovials (n = 8) or cryobags (n = 8) at -80°C. After 1 week of -80°C storage, ECP-treated and control cells were thawed rapidly and samples were placed in culture. Lymphocyte apoptosis was assessed by phosphatidylserine exposure using Annexin V/7-AAD labeling. Lymphocyte proliferation after 3 days culture was measured using the carboxyfluorescein succinimidyl ester labeling technique. RESULTS: On Day 0, apoptosis levels were <5% in fresh ECP-treated and control cells and approximately 20% on thawing of cryopreserved ECP-treated and control cells. Apoptosis levels were comparable between the two cryopreserved groups immediately on thawing, indicating that ECP-treated cells were no more sensitive to the cryopreservation process than control cells. During 72-h culture, apoptosis levels increased to >80% in fresh and cryopreserved ECP-treated cells but remained near constant in both control groups. Inhibition of lymphocyte proliferation was >95% in all ECP-treated cells with no significant difference between fresh and cryopreserved cells (P = 0.12). CONCLUSION: Cryopreservation did not impair the apoptotic response or anti-proliferative effect of ECP-treated lymphocytes, thereby demonstrating early feasibility of this approach.

Separation of in-vitro-derived megakaryocytes and platelets using spinning-membrane filtration.

In-vitro-derived platelets (PLTs) could potentially overcome problems associated with donated PLTs, including contamination and alloimmunization. Although several groups have produced functional PLTs from stem cells in vitro, the challenge of developing this technology to yield transfusable PLT units has yet to be addressed. The asynchronous nature of in vitro PLT generation makes a single harvest point infeasible for collecting PLTs as soon as they are formed. The current standard of performing manual centrifugations to separate PLTs from nucleated cells at multiple points during culture is labor-intensive, imprecise, and difficult to standardize in accordance with current Good Manufacturing Practices (cGMP). In an effort to develop a more effective method, we adapted a commercially-available, spinning-membrane filtration device to separate in-vitro-derived PLTs from nucleated cells and recover immature megakaryocytes (MKs), the precursor cells to PLTs, for continued culture. Processing a mixture of in-vitro-derived MKs and PLTs on the adapted device yielded a pure PLT population and did not induce PLT pre-activation. MKs recovered from the separation process were unaffected with respect to viability and ploidy, and were able to generate PLTs after reseeding in culture. Being able to efficiently harvest in-vitro-derived PLTs brings this technology one step closer to clinical relevance.

The effects of red blood cell preparation method on in vitro markers of red blood cell aging and inflammatory response.

BACKGROUND: Studies are currently under way examining whether the age of stored red blood cells (RBCs) affects clinical outcome in transfusion recipients. The effects of storage duration on the RBC storage lesion are well documented, while fewer studies are available regarding the effect of RBC production method. In this study, we compared in vitro RBC quality variables and markers of inflammatory response in apheresis and whole blood (WB)-derived RBCs, specifically those prepared after an overnight room temperature hold (RTH) of WB. STUDY DESIGN AND METHODS: SAGM RBCs, prepared from WB after overnight RTH (n = 10), were compared to SAGM RBCs prepared using an apheresis device (Alyx, n = 10). As a control, SAGM RBCs were also prepared within 2 hours of WB collection (2-hr WB, n = 10). All RBCs were stored at 4°C for 42 days with weekly assay of in vitro variables, cytokines and/or chemokines, and neutrophil activation after incubation with RBC supernatant. RESULTS: RTH WB RBCs exhibited decreased levels of 2,3-diphosphoglycerate acid (2.3 µmol/g hemoglobin [Hb] ± 2.1 vs. 13.7 ± 1.3 µmol/g Hb) and morphology (160 ± 10 vs. 192 ± 5) on Day 1 and increased hemolysis (0.45 ± 0.21% vs. 0.31 ± 0.09%) and microparticles (6.1 ± 2.8/10(3) RBCs vs. 3.9 ± 1.1/10(3) RBCs) on Day 42 compared to apheresis RBCs. Gro-α and ENA-78 cytokine levels were significantly higher in RTH WB than Alyx RBCs during storage. CD11b expression was highest in neutrophils exposed to supernatant from RTH WB RBCs (p < 0.05). CONCLUSION: RBC preparation method has a meaningful effect on the RBC storage lesion, which should be taken into account in addition to length of storage.

The role of bicarbonate in platelet additive solution for apheresis platelet concentrates stored with low residual plasma.

BACKGROUND: Complex platelet additive solutions (PASs) are required to store platelet (PLT) concentrates with plasma levels below 30%. Previously, apheresis PLTs stored with 5% plasma in acetate- and bicarbonate-containing PAS maintained stable pH and bicarbonate levels during 7-day storage. Due to this observation, the necessity of added bicarbonate in PAS was investigated and whether the concurrent increase in PAS pH after bicarbonate addition had any effect on PLT storage. STUDY DESIGN AND METHODS: Apheresis PLTs were stored in 5% plasma-95% high- or low-pH PAS, with or without bicarbonate (n=10 per arm). Bicarbonate PAS PLTs were paired and nonbicarbonate PAS PLTs were paired (split from same double-dose collection). PLTs were evaluated for in vitro variables on Days 1 and 7 and up to Day 14 if the Day 7 pH was higher than 6.2. RESULTS: PLT pH was maintained above 7.3 to Day 14 in bicarbonate PAS PLTs while pH failures below 6.2 were observed in 4 of 10 and 2 of 10 units on Day 7 in low- and high-pH nonbicarbonate PAS arms, respectively. Day 7 in vitro variables in nonbicarbonate PAS PLTs with pH values of higher than 6.2 were comparable to Day 7 variables in bicarbonate PAS PLTs. The pH of bicarbonate PAS did have a small effect on pH and bicarbonate levels in PLT units, but did not have an effect on functional variables and metabolism. CONCLUSION: Bicarbonate was not required to maintain in vitro PLT function in 5% plasma-95% PAS, but was required as a pH buffer and increased PAS pH did not significantly contribute to this effect.

Exploratory in vitro study of red blood cell storage containers formulated with an alternative plasticizer.

BACKGROUND: The plasticizer di-2-ethylhexyl phthalate (DEHP) is a common component in medical plastics. There is motivation to replace this component; however, DEHP is necessary to prevent excessive hemolysis in stored red blood cells (RBCs). Our objective is to evaluate a candidate replacement plasticizer (Hexamoll, di-isononyl cyclohexane-1,2-dicarboxylic acid [DINCH], BASF Corp.) compared to DEHP in an in vitro feasibility study. We hypothesize that the candidate will provide at least equivalent protection against hemolysis for RBCs stored for 42 days and periodic mixing of RBCs will add additional protection against hemolysis. STUDY DESIGN AND METHODS: Whole blood was collected into citrate-phosphate-dextrose; combined into pools of 2 ABO identical whole blood units; and divided, leukoreduced, centrifuged, and separated into plasma and RBCs. Additive solution was added, and the RBCs were stored for 42 days at 1 to 6°C. In three parts of this study, split pools were paired as DINCH-polyvinyl chloride (PVC) with weekly mixing versus DINCH-PVC with no mixing, DINCH-PVC mixed versus DEHP-PVC no mix, and DINCH-PVC versus DEHP-PVC with neither mixed. A standard panel of in vitro RBC characteristics was determined on Days 0 and 42. RESULTS: Mixing DINCH-PVC weekly improved Day 42 hemolysis (0.36 ± 0.07% vs.0.56 ± 0.15%, p = 0.002), and mixed DINCH-PVC bags were noninferior to unmixed DEHP-PVC bags (p ≤ 0.05). DINCH-PVC bags stored without weekly mixing were inferior to unmixed DEHP-PVC bags for hemolysis on Day 42, although no individual bag exceeded 0.8% hemolysis. CONCLUSION: Periodic mixing of RBCs stored in DINCH-PVC provides additional protection against hemolysis. Unmixed DINCH-PVC bags were inferior to DEHP-PVC bags for prevention of hemolysis, but remain a candidate for replacement DEHP in RBC storage bags.

In vitro variables of apheresis platelets are stably maintained for 7 days with 5% residual plasma in a glucose and bicarbonate salt solution, PAS-5.

BACKGROUND: Platelet additive solutions (PASs) facilitate improved recovery of plasma and may reduce the severity and/or frequency of plasma-associated transfusion reactions. Current apheresis platelet (PLT) PAS products contain approximately 30 to 40% residual plasma. In an effort to further decrease the residual plasma, two in vitro studies were conducted with PLTs suspended in 5% plasma and a reformulated PAS-3, named PAS-5, that contains additional salts, glucose, and bicarbonate. STUDY DESIGN AND METHODS: In Study 1, PLTs suspended in 5% plasma/95% PAS-5 were prepared directly on a separator (Amicus, Fenwal, Inc.) without additional centrifugation or washing. In Study 2, a double unit of hyperconcentrated Amicus PLTs in plasma was collected, divided, and centrifuged to prepare a control unit in 100% plasma and a paired test unit in 5% plasma/95% PAS-5. The in vitro properties of PLTs were assessed in both studies during 7-day storage at 20 to 24°C with continuous agitation. RESULTS: In Study 1, PLT concentration, pH, mean PLT volume (MPV), HCO(3)(-), pCO(2), pO(2), lactate dehydrogenase, and hypotonic shock response (HSR) did not significantly change during storage. By Day 7, glucose levels and morphology scores modestly decreased (17.6 and 14.4%, respectively) and lactate levels modestly increased (to 7.2 mmol/L). In Study 2, MPV, pH, glucose, pO(2), HSR, and morphology were comparable in control and test PLTs during 7-day storage. Glucose consumption and lactate production were significantly less in test versus control PLTs (p≤0.0015). Extent of shape change and %CD62P-positive test PLTs were less than those of controls (p<0.001). CONCLUSION: Apheresis PLTs suspended in 5% plasma/95% PAS-5 maintained in vitro properties during 7-day storage.