Optimizing the Apheresis Product

Definition and Early Beginnings of Apheresis

Apheresis, named after the ancient Greek word meaning “taking away,” is a medical procedure in which blood is drawn from a donor or patient and separated into its component parts outside of the body, allowing selective retention of a particular component and return of the remainder by reinfusion. The most common modern application of apheresis is the collection of specific blood components for transfusion from allogeneic donors, such as platelets, plasma, red cells, and granulocytes. Apheresis may also be employed in a therapeutic context to treat disease by removing pathologic cells or plasma-bound substances. Leukapheresis is the general term for white blood cell (WBC) collection by apheresis.

The history of modern leukapheresis had its beginnings in the National Cancer Institute at the National Institutes of Health. In the 1960s, George Judson, an IBM engineer whose son had chronic myelogenous leukemia, worked with Dr. Emil Freireich to develop an instrument comprising a reusable centrifugal bowl connected to the patient, to separate and deplete excess leukocytes from venous whole blood (WB) in a continuous manner, reinfusing the remaining red cells, platelets, and plasma. Subsequently, leukapheresis was optimized for granulocyte collections, hematopoietic progenitor cell (HPC) and lymphocyte collection from healthy donors. Over 50 years later, collection of autologous lymphocytes from patients has been increasing with the development of novel cellular therapies such as CAR T-cells. Compared with healthy donors, patients undergoing CAR T-cell treatment have a number of characteristics that may impact the collection procedure, the quality of the apheresis material, and downstream processing in the cell manufacturing facility. Managing each unique apheresis procedure ultimately affects the successful manufacture of the CAR T-cell product and is therefore of the utmost importance.

Principles and Technical Considerations in Apheresis

Modern apheresis devices for cellular collections work on the principle of centrifugal separation of blood components based on their density or specific gravity (see Fig. 2.1 ). Mononuclear cell (MNC) apheresis targets the layer comprising mostly lymphocytes (60%) and monocytes (20%), as well as granulocytes (15%), red blood cells (RBCs; 3%–5%) and platelets; however, absolute demarcation of specific cell populations (e.g., T-lymphocytes) is not possible. Further processing or purification of the apheresis product by elutriation, or antibody-bound magnetic beads, is required to select for the targeted cell type.

The apheresis procedure begins with the removal of WB from the patient via an IV catheter or central line. The apheresis machine is generally prepared before the patient arrives and contains a sterile, single-use, functionally closed disposable tubing set or kit that has been primed with normal saline. The extracorporeal circuit must be anticoagulated to prevent blood clotting in the tubing and kit during the procedure. Citrate-based anticoagulant (AC) is routinely used, generally AC citrate dextrose solution A (ACD-A), which is added to tubing sets during apheresis procedures and returns to the patient with the reinfusion of blood from the circuit. It exerts its AC effect by chelating ionized calcium (iCa), a necessary cofactor in the clotting cascade, as well as other divalent cations such as magnesium (iMg). Citrate AC may cause symptoms due to hypocalcemia and/or hypomagnesemia during the apheresis procedure; however, these are generally mild, and due to its rapid metabolism, with a short half-life of 36 minutes, residual effects are not prolonged. Because citrate is quickly metabolized, it is not a systemic AC and therefore will not cause sustained bleeding risk in patients postprocedure. Supplemental heparin anticoagulation may be used routinely in some centers according to institutional practice or limited to select cases at the discretion of the Apheresis Medical Director to minimize the risk of bleeding or heparin-induced thrombocytopenia (HIT).

Citrate delivery rates generally range from 1.0 to 1.8 mg/kg/minute. The ratio of WB to AC in the circuit (WB:AC) is typically 12:1 but can be changed depending on the instrumentation and procedure involved. The rate of citrate infusion is coupled to the WB flow rate and therefore to the length of the procedure. The faster the AC infusion rate, the greater the decline in iCa and iMg, with proportionally increased risk of symptoms and complications. When citrate accumulation outpaces metabolism, toxicity occurs. Citrate is metabolized by the Krebs (citric acid) cycle, mainly in the liver, but also in muscles and kidneys. Thus, patients predisposed to citrate toxicity include those with renal and hepatic insufficiency, low body mass, female gender, small children, and those undergoing longer procedures. An example worksheet used to calculate the ACD-A infusion rates, inlet flow rate, and prophylactic calcium and magnesium replacement in a pediatric patient is shown in Table 2.1 .

During the procedure, a portion of the patient's blood volume, termed the extracorporeal volume (ECV), will be outside the body within the apheresis apparatus. The ECV is dependent on the specific device and type of kit used. For a given patient, the ECV should not exceed 15% of the patient's total blood volume (TBV), as this will result in unsafe volume and RBC loss. In patients for whom the ECV slightly exceeds 15% of their TBV, the apheresis device may be primed with colloid (albumin) to limit the effects of the volume shift. Small children under 25 kg generally require a prime with an RBC unit to prevent severe intraprocedural anemia. Due to infusion of crystalloids and AC solution, the patient is often fluid positive after leukapheresis and may even have peripheral edema, with adequate renal function, this excess fluid is eliminated shortly after the procedure.

The patient's blood passes through the extracorporeal circuit in a continuous fashion; hence, apheresis procedures are described in terms of liters processed or blood volumes processed. To collect sufficient numbers of lymphocytes (≈60–600 × 10 ⁶ /kg body weight) for CAR T-cell manufacture, processing at least two to four times the patient's TBV, termed large-volume leukapheresis (LVL), is required. Occasionally, up to six times the patient's TBV may be processed in pediatric cases to collect sufficient cells, provided that patient is tolerating the procedure well. Typical flow rates range from 50 to 90 mL/minute in adult patients and 30–50 mL/minute in children; thus, the procedure takes several hours to complete. The number of targeted cells to be collected and the peripheral blood counts determine the duration of the procedure. Therefore, patients with reduced absolute lymphocyte count (ALC) require a longer procedure to collect sufficient lymphocytes for CAR T-cell manufacture.

Investigators at the National Institutes of Health demonstrated that the CD3 ⁺ count preapheresis correlates to the yield of CD3 ⁺ cells in the collected product. The use of preapheresis CD3 ⁺ count as a prediction tool builds on the long established experience in apheresis clinics using preapheresis peripheral blood CD34 ⁺ count to extrapolate collection yield during HPC harvest. To utilize this approach, the apheresis clinic must have access to a flow cytometry laboratory during the collection procedure.