has also been given to normal donors to increase the peripheral granulocyte count to improve the yield of granulocytes for transfusion. Depending on the dose schedule, the granulocyte count increases to between 20,000 and 40,000 per microliter after several days of G‐CSF treatment [70–74]. Using G‐CSF‐stimulated normal donors, it is possible to obtain granulocyte concentrates containing about 4 × 1010 granulocytes or more [72–75]. More recently, use of dexamethasone has been combined with G‐CSF to provide even higher granulocyte levels in the donor, resulting in granulocyte concentrates containing up to 6 × 1010 granulocytes [72]. A large multicenter randomized trial to evaluate these high‐dose granulocyte concentrates has been completed.
Filtration leukapheresis
This method of granulocyte collection is described because of historical interest, but it is not used today. A nylon fiber filter system was developed to collect granulocytes [76]. Although this system yielded a larger number of cells than the centrifuge procedures, granulocytes obtained by filtration leukapheresis had a mild‐to‐moderate functional impairment and decreased intravascular recovery and survival [77, 78]. Also, a severe transient neutropenia occurred a few minutes after the donor’s blood came into contact with the nylon fibers [78–81] due to activation of the complement system [81, 82]. Reports of donor complications [83] led to the discontinuation of filtration leukapheresis.
Function of granulocytes obtained by leukapheresis
Granulocytes collected by centrifuge leukapheresis techniques demonstrate normal bacterial killing, phagocytosis, granulocyte metabolism), chemiluminescence, superoxide production, and chemotaxis [77, 84–86]. In vivo studies using isotope‐labeled cells showed that granulocytes have normal intravascular recovery and survival, and migrated to sites of inflammation [77, 87–89]. The use of corticosteroids or G‐CSF in donors to improve the granulocyte yield does not adversely affect their function in vitro or in vivo [75, 84, 87].
Storage of granulocytes for transfusion
Granulocytes have a life span in circulation of only a few hours, so storage of granulocytes as part of a routine blood bank operation is difficult. Granulocytes retain bactericidal capacity and metabolic activity related to phagocytosis and bacterial killing for 1–3 days with storage at refrigerator temperatures, although chemotactic response declines by 30–50% after 24 hours [88–91]. Studies using 111In‐labeled granulocytes showed that storage of granulocytes between 1 and 6°C for 24 hours was associated with a reduction in the percentage of transfused cells that circulated and about a 75% reduction in migration into a skin window [88], but storage at room temperature for 8 hours did not reduce the intravascular recovery, survival, or migration into a skin chamber [88]. In vivo recovery, survival, or migration was reduced further when granulocytes were stored longer than 8 hours at room temperature or for even 8 hours between 1 and 6°C. Thus, it appears that granulocytes can be stored for up to 8 hours at room temperature before transfusion.
Granulocyte concentrates from G‐CSF‐stimulated donors contain large numbers of granulocytes with increases in IL‐1B and IL‐8, and decreases in pH during storage [75]. Thus, storage of granulocyte concentrates obtained from G‐CSF‐stimulated donors is probably even less effective than these data indicated. It is recommended that granulocytes be transfused within a very few hours. AABB standards allow storage for up to 24 hours at 20–24°C [47].
Donor–recipient matching for granulocyte transfusion
ABO antigens are probably not present on granulocytes (see Chapter 8), but granulocyte concentrates must be ABO‐compatible with the recipient because of the substantial volume of red cells in the concentrates. The clinical impact of ABO incompatibility on granulocyte transfusion was evaluated in one study [92]. A small number of 111In‐labeled granulocytes free of RBCs were injected into ABO‐incompatible recipients. The intravascular recovery, survival, and tissue localization of the cells were not different from those seen when similar injections were given to ABO‐compatible subjects [92]. This study was not intended to encourage the use of ABO‐incompatible granulocyte transfusions, but this could be considered if granulocyte concentrates that are depleted of RBCs could be prepared.
Incompatibility by leukoagglutination or lymphocytotoxicity is associated with the failure of transfused CML cells to circulate or localize at sites of inflammation [93, 94]. Studies using 111In‐labeled granulocytes in humans established that granulocyte‐agglutinating antibodies were associated with decreased intravascular recovery and survival, failure of the cells to localize at known sites of inflammation [95], and excess sequestration of transfused granulocytes in the pulmonary vasculature [95, 96]. However, applying these research data to the practical operation of a blood bank and granulocyte transfusion service is difficult because granulocytes can be stored for only a few hours, and cells are not usually available for crossmatching to allow advance selection of compatible donors. The only practical approach has been to monitor the recipient plasma for the presence of granulocyte‐agglutinating antibodies, traditionally done by screening the patients’ serum against a panel of cells periodically. More recently, in vitro techniques such as flow cytometry have been developed for detecting anti–human leukocyte antigen antibodies.
If a patient becomes alloimmunized, trials of human leukocyte antigen–matched unrelated donors or family members can be selected for leukapheresis, if available. However, the problem of donor–recipient matching and compatibility testing for granulocyte transfusion has not been solved.
6.5 Leukapheresis for the collection of mononuclear cells
Lymphocytes or monocytes are being used increasingly as starting material for the production of cells for adoptive immunotherapy, genetically engineered immunotherapy like chimeric antigen receptor (CAR) T‐cell therapy, or as a concentrate enriched in PBSCs (see Chapter 19). With the exception of donor lymphocyte infusion in the setting of transplant, lymphocytapheresis is almost always performed on patients. Thus, there are no established criteria for normal donor selection and management; however, yields of 1–3 × 1010 MNCs are typical.
6.6 Leukapheresis for the collection of peripheral blood stem cells
For malignancies in which there was suspected marrow involvement, bone marrow aspiration is unsuitable for autologous transplant because of the presence of tumor cells.
However, hematopoietic stem cells are present not only in the marrow but also in the peripheral circulation and can be collected by cytapheresis. Normally the number of circulating PBSCs is much less than in the marrow, but after chemotherapy‐induced marrow suppression, there is a rebound and the number of PBSCs increases substantially. The PBSCs—expected to contain few, if any, malignant cells—can be used for marrow rescue after high‐dose chemotherapy. These autologous transplants of PBSCs made new chemotherapy regimens possible and established that PBSCs could be used successfully for autologous marrow transplantation [97–102].
For several years, the use of PBSCs was limited to autologous transplants. It was feared that the large number of T‐lymphocytes contained in the PBSC concentrates would cause severe graft versus host disease, and that T‐depletion would result in an unacceptably large loss of PBSCs. However, this did not occur [102–107]. PBSCs result in more rapid engraftment [108], give results equivalent to marrow [107, 109], and may provide faster lymphocyte return, resulting in fewer infections [110]. Thus, there has been considerable interest in the methods to obtain PBSCs from both patients and normal donors.
PBSCs can be obtained from the peripheral blood by apheresis, but because of the small number of circulating PBSCs, multiple procedures would be necessary to obtain enough cells for transplantation from unstimulated donors. To further increase the level of circulating PBSCs, donors are given the growth factor G‐CSF. In