postthaw results have been found after storage at −80°C for 37 years [32–35]. The cryoprotectant commonly used is glycerol, which must be removed before transfusion to avoid osmotic hemolysis when the cells are transfused. The method of freezing and storage must preserve at least 80% of the original red cells, and at least 70% of those cells must survive 24 hours after transfusion.
Freezing of red cells is based on work from more than 50 years ago showing that glycerol protected human red cells from freezing injury [36], and that red cells preserved with glycerol were clinically effective [37–39]. From this work, “high‐” and “low‐concentration” glycerol methods were developed [40, 41]. These methods actually relate to the concentration of glycerol, which determines the nature of the freezing injury to the cells. When freezing is slow, extracellular ice forms, which increases the extracellular osmolarity, causing intracellular water to diffuse out of the cell and resulting in intracellular dehydration and damage [40]. This type of injury is prevented by solutes such as glycerol that penetrate the cell and minimize the dehydration [40]. Because the freezing process is slow, high concentrations of cryoprotectant, usually 40% glycerol, are required. Red cells preserved with this high concentration of glycerol can be stored at about −85°C, a temperature that is achievable by mechanical freezers.
Rapid freezing causes intracellular ice crystals and resulting cell damage [40]. However, because the freezing is faster, lower concentrations of cryoprotectant, usually about 20% glycerol, are effective [42]. This lower concentration of glycerol necessitates storage of red cells at a temperature of about −196°C, achievable only by using liquid nitrogen.
These two methods—with different freezing rates, concentrations of glycerol, storage conditions, and processes for removing the glycerol— involve different technologies [40, 42]. Technology development played a major role in making red cell freezing clinically available. During the 1970s, a disposable plastic bowl and semiautomated washing system were developed that greatly facilitated glycerol removal from high glycerol concentration red cells [43–45]. In the rapid‐freeze method, the concentration of glycerol is low enough that glycerol removal can be done by washing in ordinary blood bags; complex instruments are not required. To summarize, the high‐concentration glycerol method involves more simple freezing and storage but complex deglycerolizing procedures. The low‐concentration glycerol method involves complex freezing and storage but simple deglycerolizing procedures. Frozen deglycerolized red cells are composed of essentially red cells suspended in an electrolyte solution. Most of the plasma, platelets, and leukocytes have been removed either by the freezing, thawing, or washing step necessary to remove the glycerol cryoprotectant. Thus, the deglycerolized red cells have a 24‐hour storage period, which is a major factor in the logistics of their use. Frozen RBCs are used primarily for storage of rare RBC types [46].
Red cells must be frozen within 6 days of collection to provide acceptable posttransfusion survival. Red cells that have been stored longer than 6 days can be frozen if they are “rejuvenated” [47]. Rejuvenation restores metabolic functions after the red cells are incubated with solutions containing pyruvate, inosine, glucose, phosphate, and adenine followed by freezing [47]. This is a helpful strategy to freeze red cells in situations such as: (a) red cells found after 6 days of storage have a rare phenotype, (b) red cells donated for autologous transfusion but the surgery is postponed, and (c) rare phenotype red cells thawed but not used. The rejuvenation and subsequent freezing process is complex, expensive, and not widely used.
Washed red cells
Washing is indicated for severe allergic reactions and for removal of potassium in large‐volume transfusion in pediatrics. The definition of washed red cells is rather vague. These are the red cells remaining after washing with a solution that will remove almost all of the plasma [24]. Thus, the requirements for this component do not specify the nature of the washing solution or the exact composition of the final component. Red cells can be washed by adding saline to the red cells in an ordinary bag, centrifuging them and removing the supernatant, or by using semiautomated washing devices, such as those used for deglycerolization [48–50]. Depending on the solution and technique used, the washed red cells may have a variable content of leukocytes and platelets. There is usually some red cell loss during the washing step, and the resulting red cell unit may contain a smaller dose of red cells than a standard unit. In general, the characteristics of washed red cells are the removal of approximately 85% of the leukocytes, loss of about 15% of the red cells, and loss of more than 99% of the original plasma [48–50]. Because the washing usually involves entering the storage container, the washed red cells have a storage period of 24 hours at 1–6°C.
Leukocyte‐reduced red blood cells
Definition of component
Leukocyte‐reduced red cells are cells prepared by a method known to retain at least 80% of the original red cells and reduce the total leukocyte content to less than 5 × 106 [24].
History of leukodepletion
The blood filters used for routine transfusions have a pore size of 170–260 mm. They filter out clots and fibrin strands but do not effectively remove leukocytes. During the 1960s and 1970s, it was recognized that leukocytes were important in the pathogenesis of febrile nonhemolytic transfusion reactions and could cause alloimmunization, which would later interfere with organ transplantation or platelet transfusion (see Chapter 16). Thus, considerable interest developed in removing leukocytes before transfusion. Early methods involved centrifuging the red cells (either upright or inverted); sedimenting red cells with dextran or hydroxyethyl starch; filtration with nylon or cotton wool, which removed only granulocytes [51]; or washing, freezing, and deglycerolizing [50]. These methods removed from 65% to 99% of the original leukocytes and from 5% to 20% of the original red cells. A huge body of literature developed describing the advantages and disadvantages of the different methods and some of the clinical effects of their use. Although they are of historical interest, these studies are not described extensively in this chapter because the methods are not used today. Leukodepletion continues to be a major issue in transfusion medicine because even more adverse effects of leukocytes contained in blood components have been identified (Table 5.8). The consequences of these effects are described in more detail in Chapter 16. As these adverse effects of leukocytes have been more extensively described, the technology for producing leukodepleted red cells has evolved to provide more extensive leukocyte depletion than was possible using earlier methods [52].
Clinical and animal studies suggested that red cells intended to prevent febrile nonhemolytic transfusion reactions must contain fewer than 5 × 108 leukocytes, and those intended to prevent alloimmunization contain fewer than 5 × 106 leukocytes [24]. The latter requires removal of about 99.9% of the leukocytes. Sophisticated filters have been developed to accomplish this.
Leukocyte depletion filters
The filter material may be modified to alter the surface charge and improve the effectiveness. The mechanism of leukocyte removal by the filters currently in use is probably a combination of physical or barrier retention and also biological processes involving cell adhesion to the filter material.
Because leukocytes are contained in red cell and platelet components, filters have been developed for both of these components. Filters are available as part of multiple‐bag systems, including additive solutions, so that leukocytes can be removed soon after collection and the unit of WB converted into the usual components. Filtration removes 99.9% of the leukocytes, along with a loss of 15–23% of the red cells [53]. Thus, bedside leukodepletion is not used. Filters fail to achieve the desired leukodepletion from 0.3 to 2.7% of units. Red cell components from donors with sickle cell trait often occlude white cell reduction filters.
It appears that febrile nonhemolytic transfusion reactions are caused not only by leukocyte antigen–antibody reactions but also by the