Jeffrey McCullough - Transfusion Medicine

b Source : Smith JW, Gilcher RO. Red blood cells, plasma, and other new apheresis‐derived blood products: improving product quality and donor utilization. Transfus Med Rev 1999; 13:118–123.

Leukapheresis from an unstimulated donor produced only a marginally adequate dose of granulocytes for therapeutic benefit and never gained widespread use. The resulting blood component is a suspension of granulocytes in plasma prepared by cytapheresis. Early on, patients with chronic myelogenous leukemia (CML) were used as granulocyte donors. However, there were the obvious problems of the use of abnormal or malignant cells, as well as the limited number of patients with CML available to donate. The two additional strategies used to increase the granulocyte yield are the addition of the blood sedimentation agent hydroxyethyl starch (HES) to improve granulocyte separation within the centrifuge and the treatment of donors with corticosteroids, and more recently with G‐CSF, to increase the level of circulating granulocytes.

Leukapheresis procedures in general are usually more complex and lengthier than plateletpheresis. The leukapheresis procedure takes 2–3 hours, compared with about 1 1/2 hours for plateletpheresis, to improve the granulocyte yield. Usually 6,500–8,000 mL of the donor’s blood is processed through the instrument, with removal of about 50% of the granulocytes, resulting in a granulocyte concentrate with a volume of about 200 mL. Because granulocytes do not completely separate from the red cells, granulocyte concentrates usually contain a substantial number of red cells (hematocrit 10% or about 20 mL of red cells); therefore, red cell crossmatching is necessary.

A granulocyte concentrate must contain at least 1 × 10 10granulocytes in at least 75% of the units tested [47]. Neither the American Association of Blood Banks (AABB) Standards nor US Food and Drug Administration regulations specify the number of units that must be tested for quality‐control purposes, but because only a few granulocyte concentrates are prepared by most blood banks, it is customary to test all concentrates.

The separation between granulocytes from the upper layer of red cells is poor because the density of granulocytes is similar to that of some red cells. Although several agents can be used to sediment red cells in vitro, HES is used because it is licensed in the United States for in vivo use and is not associated with unacceptable reactions or alteration of coagulation tests. The granulocyte yield is doubled when HES is added to the leukapheresis system by constant infusion [63–65]. Several studies of the effects of HES established that the nature and incidence of reactions are acceptable for use on normal donors, the potential for blood volume overload when administered to normal donors can be easily managed during the procedure, there is no adverse effect on laboratory values or platelet or granulocyte function, and there are no adverse long‐term effects. Pentastarch has a shorter in vivo half‐life than HES and can also be used in leukapheresis [66, 67].

Another approach to increase the granulocyte yield is to increase the donor’s circulating granulocyte count. Corticosteroids have been the drug of choice, and dexamethasone was selected because it could be given either orally several hours before leukapheresis or parenterally at the beginning of the procedure. Dexamethasone 60 mg can be given orally the evening before, or hydrocortisone 4 mg/m 2can be given intravenously 6–12 hours before leukapheresis. This is a very effective method to increase the granulocyte yield, even more than is accomplished by adding HES to the separation system [64]. It has been suggested that corticosteroids may cause cataracts in granulocyte donors [68], although this was not substantiated in a larger study [69].

G‐CSF 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 × 10 10granulocytes 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 × 10 10granulocytes [72]. A large multicenter randomized trial to evaluate these high‐dose granulocyte concentrates has been completed.

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.

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].

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].