Optimization of the Spectra Optia for Leukapheresis of Very Small Subjects

Leukapheresis procedures in very small volume subjects require procedure modifications related to anticoagulant, venous access, and volume. Historically, we accomplished this procedure in 4-5 kg rhesus macaques using a modified CS3000Plus cell separator, but this instrument is no longer available. The Spectra Optia apheresis system and its mononuclear cell (MNC) collection set were evaluated to develop leukapheresis procedures for such subjects.

Five rhesus macaques, weight 7-9 kg, blood volume 60 mL/kg, were studied. They pre-donated 100-120 mL of autologous blood (25-30 mL/wk x 4 wks). Aspirin 81 mg was given daily for 3 days prior to 5 of 8 procedures. AMD3100 1 mg/kg SQ 2-3 hours prior to leukapheresis was given as a mobilizing agent. Autologous blood was pooled, diluted 1:1 with saline, and washed to remove citrate and plasma. The Optia was primed first with saline, and then with a customized albumin option in which 30-35 mL of the 40-50 mL of packed RBCs were pumped into the disposable apheresis kit at 10 mL/min. The albumin option shortens procedure ramp-up time. A closed circuit was created between the return line and the packed RBC bag and the flow rate increased to 40 mL/min until 400 mL was processed. To minimize citrate toxicity and avoid volume overload, animals received heparin 50 U/kg bolus IV followed by an ACDA solution containing heparin 5U/mL at an inlet:AC ratio of 25:1. A Collection Preference (CP) setting of 20 was selected using the automatic interface management mode. In the last procedure a semi-automatic mode was utilized so that collection deeper than CP of 20 was possible. Procedures were terminated at 120 min. Animals were anesthetized and catheters placed in the saphenous and cephalic as draw and return lines. Blood counts, chemistries, and ionized calcium (iCa) levels were monitored every 30 min. The elutriation chamber, which concentrates MNCs and allows platelets to return to the donor, was filled 1-2 times per procedure resulting in a product volume of 17-34 mL.

A total of eight leukapheresis procedures were performed. Inlet flow rates were typically 10.5 mL/min. In one animal, an inlet rate of 12.0 mL/min was achieved; however, at 120 minutes severe citrate toxicity developed with bradycardia and an iCa level of 0.62 mmol/L, requiring IV calcium gluconate. Excluding this case, a mean(SD) baseline iCa value of 1.17(0.2) dropped to 0.84(0.03) mmol/L at120 min. Mean hematocrit (HCT) decreased from 40.4(1.7)% to a nadir of 36.8(2.6)% at 90 min. Mean platelets declined to a greater degree, from 342(42) x10³/mcL to 109(33) x10³/mcL at 60 min and 121(43) x10³/mcL at 120 min (p<0.001). This drop was reflected in the large number of platelets in the product, 2.49(1.09)x10¹⁰ total. Occasional platelet aggregate formation was observed in animals both receiving and not receiving aspirin. Product WBC differential was 65.5(11.1)% MNC and 34.5(10)% granulocytes. Total product MNC content was 1.95(0.54)x10⁹ . MNC collection efficiency (CE) was 26.2(8.5)% using CP=20 and improved to 38.1% using the semi-automatic mode. Product MNC composition consisted of 5.1(2.0)x10⁸ CD4⁺ , 5.6(2.1)x10⁸ CD8⁺, and 3.0(2.0)x10⁸ CD20⁺ lymphocytes, and 9.8(7.0)x10⁸ CD14⁺ monocytes. As a measure of product contamination, the CE for granulocytes was 5.8(3.0)% and for platelets 11.2(4.4)%. In 3 procedures, two-chamber collections were obtained, the remaining 5 procedures had one-chamber collections (all within 120 min). The HCT of the product was low, 3.7(1.2)%. The product also was enriched for reticulocytes (10.57(2.44)%) and CD4⁺CD8⁺ cells(3.4(2.2)x10⁷). Relatively low numbers of CD34+ cells were collected, 5.2(1.9)x10⁶; however, the mobilization protocol was not optimized for CD34+ collection in that AMD3100 alone and older monkeys were used. We are currently optimizing the procedure for even smaller rhesus and combination cytokine mobilization.

MNC collections through the process of leukapheresis have proven to be effective in isolating cells for transplantation and cellular therapeutics. The use of leukapheresis in a pediatric setting is difficult due to small blood volumes and a high risk of adverse events. Here we have identified a novel means to perform leukapheresis procedures safely and effectively in small volume subjects. This methodology will have numerous therapeutic applications in both human and veterinary medicine.