BLOOD COMPONENT MANUFACTURE.docx

**BLOOD COMPONENT MANUFACTURE** Whole-blood donations are commonly manufactured into components. This facilitates the treatment of different patients with requirements for RBCs, plasma, or platelets. The goals of component manufacture are to maintain viability and function, and to prevent detriment...

**BLOOD COMPONENT MANUFACTURE** Whole-blood donations are commonly manufactured into components. This facilitates the treatment of different patients with requirements for RBCs, plasma, or platelets. The goals of component manufacture are to maintain viability and function, and to prevent detrimental changes or contamination of desired constituents. **RED BLOOD CELLS** RBCs are prepared from whole blood by centrifugation and removal of plasma and platelets. RBCs prepared in the preservative solution CPDA-1, supplemented with dextrose and adenine to preserve red cell adenosine triphosphate (ATP) levels, may be stored for up to 35 days at 1°C to 6°C. RBCs may also have an additive solution containing glucose and other substrates added during manufacture. Such additive solutions permit a longer storage period (42 days) and have a lower hematocrit (Hct). During storage, red cells undergo senescence changes similar to aging in vivo such that a portion of transfused red cells are rapidly cleared by the spleen. The maximum allowable storage time for RBCs is defined by the requirement for recovery of 75% of transfused cells 24 hours after transfusion. Leakage of intracellular potassium occurs during red cell storage due to lack of ATP to fuel the potassium pump. The potassium concentration in the supernatant of RBCs can reach levels of 76 mmol/L, which may appear alarmingly high. However, the total amount of potassium in a unit of RBCs at outdate is small compared with daily physiologic requirements, and hyperkalemia following transfusion is rare except in special circumstances (see later discussion). Hypokalemia may also occur secondary to RBC transfusion due to influx of potassium into transfused RBCs. After transfusion, the intracellular ATP levels are restored and the ATPase potassium pump resumes activity. Red cells lose intracellular 2,3-diphosphoglycerate (2,3-DPG) during storage, resulting in a shift of the Hb-oxygen dissociation curve to the left. Thus, shortly after transfusion, stored red cells have relatively high oxygen affinity. Normal levels of 2,3-DPG are restored within 24 hours of transfusion. The shift in oxygen association with storage is rarely clinically significant.  **PLASMA** Plasma is separated from whole blood and frozen for extended preservation. In the liquid state at refrigerator temperatures, there is loss of labile clotting factors, particularly factor VIII and factor V. Fresh frozen plasma (FFP) is separated from the RBCs and is frozen at -18°C within 8 hours of collection. Plasma frozen within 24 hours after phlebotomy is manufactured similarly to FFP but may be frozen at any time up to 24 hours after collection. If this plasma is held at 4°C prior to freezing, it is labeled as PF24. If it is held at room temperature prior to freezing, it is labeled as PF24RT24. The coagulation factor content of FP24 or PF24RT24 is essentially equivalent to FFP (Scott et al., 2009). Frozen plasma may be stored for up to 1 year at -18°C or lower. Before transfusion, both FFP and FP24 are thawed at 37°C and must be transfused within 24 hours. Thawed plasma not used within 24 hours may be relabeled as "thawed plasma." Thawed plasma can be kept at refrigerator temperatures for up to 5 days with maintenance of adequate levels of factors V and VIII (Downes et al., 2001; Scott et al., 2009).  **CRYOPRECIPITATED ANTIHEMOPHILIC FACTOR** Cryoprecipitated antihemophilic factor (cryoprecipitate or cryo) is the cold insoluble portion of plasma remaining after FFP has been thawed at refrigerator temperatures. It contains approximately 50% of factor VIII and 20% to 40% of the fibrinogen present in the original plasma unit. Cryo also contains von Willebrand factor (vWF) and factor XIII. FDA regulations require that a unit of cryoprecipitate contain at least 80 IU of factor VIII (21CFR640.56), although most blood centers achieve higher levels routinely. A unit of cryoprecipitate contains approximately 250 mg of fibrinogen; however, testing for the fibrinogen content is not required. Cryoprecipitated antihemophilic factor was a major advance in the treatment of hemophilia A before the development of safe purified clotting factor concentrates. Currently, cryo is used mainly as a source of fibrinogen.  **PLATELET CONCENTRATES** Platelet concentrates (PCs) are prepared from whole blood by centrifugation of platelet-rich plasma and removal of platelet-poor plasma. PCs must contain at least 5.5 × 1010 platelets per unit. They are stored at room temperature (20°C--24°C) because platelets stored at refrigerator temperature (1°C--6°C) have greatly diminished posttransfusion survival. Current FDA regulations allow PCs to be stored for up to 5 days with continuous gentle agitation to encourage gas exchange. At the end of storage, the pH of the PCs must be 6.0 or higher. PCs typically contain a small number of red cells, which are visibly apparent and can cause alloimmunization to red cell antigens. PCs contain 30 to 50 mL of plasma. It is typically necessary to pool five or more PCs to obtain a therapeutic dose for a typical adult patient (3.0 × 1011 platelets). PCs that are pooled using an open system must be transfused within 4 hours. PCs can be pooled and leukocyte reduced at the time of manufacture using a system that maintains sterility, often referred to as *prepooled platelets*. Because the integrity of the container is not compromised in this process, prepooled platelets can be stored for up to 5 days. PCs prepared by apheresis (platelets, pheresis, or single-donor platelets) are stored and handled in the same manner as platelet concentrates prepared from whole blood. Each apheresis platelet unit should contain a minimum of 3.0 × 1011 platelets. It is possible to collect two or more platelet units in a single apheresis session from some donors. One apheresis platelet unit will typically provide a therapeutic dose for an adult patient. Apheresis platelet units are considered leukoreduced in the process of collection due to the apheresis collection selectivity for the platelets. Apheresis platelets can be further processed by removal of plasma and addition of a platelet additive solution (PAS). PAS platelets may pose a lower risk of allergic reactions. On September 30, 2019, the FDA issued a Final Guidance, "Bacterial Risk Control Strategies for Blood Collection Establishments and Transfusion Services to Enhance the Safety and Availability of Platelets for Transfusion" with recommendations to enable blood banks and transfusion services to comply with 21 CFR 606.145(a): "Blood collection establishments and transfusion services must assure that the risk of bacterial contamination of platelets is adequately controlled using FDA approved or cleared devices or other adequate and appropriate methods found acceptable for this purpose by FDA." The Guidance may be accessed at https://www.fda.gov/regulatory-information/search-fdaguidance-documents/bacterial-risk-control-strategies-blood-collectionestablishments-and-transfusion-services-enhance. Several protocols for compliance for both apheresis platelets and whole blood--derived platelets are described. These include options for bacterial culture, pathogen reduction, and rapid testing for bacteria.  **LEUKOCYTE COMPONENTS** Granulocytes can be prepared by apheresis. Granulocytes may be stored at room temperature for up to 24 hours. However, after even brief in vitro storage, granulocytes may have reduced ability to circulate and migrate to areas of inflammation (McCullough et al., 1983). It is desirable that they be transfused as soon as possible after collection. Donor stimulation with granulocyte colony-stimulating factor (G-CSF) is usually necessary to obtain a sufficient number of granulocytes for a therapeutic dose for an adult patient (Heuft et al., 2002). Granulocyte units contain a substantial number of RBCs and must be ABO compatible with the recipient. Mononuclear cells collected by apheresis can be a source of hematopoietic progenitor cells (HPCs) for autologous or allogeneic transplantation. The number of circulating HPCs can be increased by growth factor (G-CSF or granulocyte-macrophage colony-stimulating factor \[GMCSF\]) stimulation, recovery from chemotherapy, or the chemokine receptor blocker plerixafor (Cashen, 2009; Nakasone et al., 2009). Autologous HPCs for transplantation in patients with lymphoma or other malignancies may be collected when the bone marrow is recovering from chemotherapy because there are relatively high numbers of circulating stem cells at that time. HPCs may be stored frozen after addition of a cryoprotective agent, such as dimethylsulfoxide (DMSO), for an extended period of time. After thawing at 37°C, HPCs should be transfused as soon as possible. Mononuclear cells can also be used as a source of lymphocytes for induction of graft-versus-tumor effect, known as *donor lymphocyte infusion* (Castagna et al., 2016) or for future manufacturing to produce cellular therapy products.  **LEUKOCYTE-REDUCED BLOOD COMPONENTS** Leukocytes present in blood components, particularly RBCs and PCs, may cause adverse effects. Such untoward effects include febrile nonhemolytic transfusion reactions, immunization to leukocyte (particularly HLA) antigens with subsequent refractoriness to platelet transfusions, transmission of leukocyte-associated viruses, and graft-versus-host disease. To minimize most of these adverse impacts, many blood centers and transfusion services have instituted the use of leukocyte-reduced components for all transfusions (universal leukocyte reduction). It must be noted that leukocyte reduction has not been conclusively shown to prevent posttransfusion graft-versus-host disease and is not used for this purpose (Hayashi et al., 1993). To be considered leukocyte reduced, blood components must be prepared by a method known to reduce the total number of residual leukocytes to fewer than 5 × 106 per unit for RBCs and fewer than 8.3 × 105 for whole blood--derived PCs (Levitt, 2014). Leukocyte reduction is typically accomplished by filtration at the time of component manufacture (prestorage leukocyte reduction) or at the time of transfusion (poststorage leukocyte reduction). Both methods are effective for removing leukocytes. However, prestorage leukocyte reduction has the advantage of preventing accumulation of leukocyte-derived biological response modifiers, particularly cytokines, which may cause adverse reactions (Silliman et al., 2017). In addition, filtration at the time of manufacture allows for better process control. Certain apheresis devices can reliably produce platelet concentrates containing fewer than 1 × 106 leukocytes. Subtle differences have been noted in the distribution of leukocyte subsets between such components and those produced by filtration (Pennington et al., 2001). These differences, however, are unlikely to be clinically significant. Leukocyte reduction failures may occur during filtration of blood from donors with sickle trait. Such filtration failures may be due to Hb S polymerization in an environment of low oxygen tension and high osmolality (Beard et al., 2004). Clearly, granulocytes and hematopoietic progenitor cells cannot be leukocyte reduced.  **SPECIAL COMPONENTS** Cryoprecipitate-reduced plasma (cryopoor plasma) is the supernatant remaining from the production of cryoprecipitate. It is relatively deficient in high molecular weight forms of vWF but retains close to normal levels of the vWF-cleaving metalloprotease ADAMTS 13 (a disintegrin and metalloprotease with eight thrombospondin) (Raife et al., 2006). For these reasons, cryoprecipitate-reduced plasma has been an alternative to FFP for the treatment of patients with thrombotic thrombocytopenic purpura **PART 4** **775** (TTP). However, at present, no evidence indicates that plasma exchange with cryoprecipitate-reduced plasma results in improved patient outcome. RBCs can be stored in the frozen state after addition of a cryoprotective agent, such as glycerol. Frozen RBCs can be stored in mechanical freezers or liquid nitrogen for up to 10 years. Frozen units are thawed rapidly at 37°C. The cryoprotective agent must be removed by progressive addition of washing solutions with decreasing osmolality. Failure to properly deglycerolize frozen RBCs can result in hemolysis. Cryopreservation and deglycerolization of sickle trait red cells requires a modified process (Meryman & Hornblower, 1976). After deglycerolization, red cells can be stored for up to 1 day at 1°C to 6°C if processed by an open method or up to 14 days if processed by a closed method. The main use of frozen RBCs is to maintain an inventory of rare antigen-negative units. RBCs and PCs can be washed to remove plasma proteins and electrolytes. Washing can be accomplished by manual or automated methods. Loss of cells during the washing process can be substantial. In addition, washing of platelets can result in clumping and activation with reduced viability (Pineda et al., 1989). If this is performed using an open process, washed red cells may be stored for 24 hours at refrigerator temperatures, and washed platelets must be transfused within 4 hours of initiation of washing, when prepared using an open system. The main use of washed components is the prevention of severe allergic reactions to plasma components. Washing is not an effective means of leukocyte reduction. Transfusion-associated graft-versus-host disease (TA-GVHD) can be prevented by irradiation of components containing viable lymphocytes (RBCs, PCs, granulocytes, and nonfrozen plasma). This can be accomplished by exposure to γ-rays or x-rays. The minimum dose should be 25 Gy delivered to the center of the blood container and no less than 15 Gy to the periphery. Irradiation causes chromosomal damage, which prevents replication of transfused lymphocytes in the recipient. However, irradiated cells remain immunogenic. Thus, irradiation is not equivalent to leukocyte reduction. Irradiation also causes damage to red cell membranes, with increased potassium leakage and decreased posttransfusion survival (Hauck-Dlimi et al., 2015). Irradiated red cell units must have their outdate shortened to no more than 28 days from the date of irradiation. Platelets appear to sustain minimal damage from irradiation. Thus, their expiration date need not be altered, although the increment in platelet count may be reduced (Sweeney et al., 1994; Slichter et al., 2005). Clearly, hematopoietic progenitor cells must not be irradiated. Irradiation is not sufficient to prevent transmission of viral infections, including cytomegalovirus (CMV), or bacterial contamination.