Extended storage of granulocyte concentrates mobilized by G-CSF with/without dexamethasone and collected by bag separation method

The aim of this study was to examine the extended storage of granulocyte concentrates mobilized by granulocyte-colony-stimulating factor (G-CSF) with/without dexamethasone (DEX) and collected by a bag separation method. Ten healthy adult volunteers donated blood three times: twice after granulocyte mobilization by (1) injecting G-CSF at 3 microg kg(-1) subcutaneously (s.c.) and (2) injecting G-CSF at 3 microg kg(-1) s.c. + DEX at 8 mg per oral and once (3) for a baseline control without any forms of mobilization. Granulocytes were collected by a bag separation method. The functions (phagocytosis and oxidative killing levels), viability and levels of interleukin (IL)-1beta, IL-8, IL-6 and tumour necrosis factor-alpha of granulocytes were measured. The average numbers of granulocytes collected from 200-mL samples of whole blood from the G-CSF and G-CSF + DEX groups were 35.1 x 10(8) and 49.4 x 10(8), respectively. Phagocytosis level, oxidative killing level and the viability of the granulocytes mobilized by G-CSF with/without DEX were well maintained for up to 72 h of storage after collection. The levels of the cytokines increased in a time-dependent manner. The in vitro phagocytosis level, oxidative killing level and the viability of granulocytes mobilized by G-CSF with/without DEX and collected by bag separation method can be maintained for as long as 72 h after collection.     

The determinants of granulocyte yield in 1198 granulocyte concentrates collected from unrelated volunteer donors mobilized with dexamethasone and granulocyte–colony-stimulating factor: a 13-year experience

BACKGROUND: The combination of granulocyte–colony-stimulating factor (G-CSF [filgrastim]) and dexamethasone (G-CSF/dex) is an effective granulocyte mobilization regimen, but the variables that affect donor neutrophil response and granulocyte collection yield are not well characterized.
STUDY DESIGN AND METHODS: A computerized database containing records of 1198 granulocyte collections from 137 unrelated volunteer apheresis donors during a 13-year period was retrospectively analyzed. Donors were categorized by age, sex, and cumulative number of granulocyte donations. Complete blood counts at baseline and after G-CSF/dex stimulation were recorded. The outcome variables include the preprocedure absolute neutrophil count (preANC), which reflects G-CSF/dex stimulation, and the granulocyte product yield per liter processed (BagGranYield/L).
RESULTS: Higher baseline ANC and platelet (PLT) counts were significantly associated with higher preANC while a larger number of prior granulocytapheresis procedures was associated with lower preANC. Total filgrastim dose (used in weight-based dosing) did not significantly impact preANC or the granulocyte yield; weight-based dosing at 5 µg per kg and a uniform 480-µg dose produced equivalent preANC. PreANC and weight were the key determinants of granulocyte yield (BagGranYield/L).
CONCLUSION: Apheresis donors with higher baseline PLT counts and ANCs have higher ANCs after G-CSF/dex stimulation; donor age, weight, and sex do not have a significant impact. A uniform G-CSF dose of 480 µg is as effective as weight-based dosing at 5 µg per kg. Donor ANC monitoring should be considered after serial granulocytapheresis procedures.     

The sterility of platelet and granulocyte concentrates collected by discontinuous flow centrifugation

Platelet concentrates prepared by a discontinuous flow centrifugal technique (Haemonetics) were examined for evidence of bacterial contamination and subsequent growth from 3 to 11 days after collection. Of the 126 platelet concentrates examined, 4 revealed bacterial growth. However, the growth patterns indicated contamination during the microbiologic manipulations rather than contamination of the units during preparation. These studies indicate that platelet concentrates prepared using the Haemonetics Blood Processor can be safely transfused for up to three days after collection if stored at 4 to 6 C.     

Collection and transfusion of granulocyte concentrates from donors primed with granulocyte stimulating factor and response of myelosuppressed patients with established infection

Fifteen patients with prolonged neutropenia (a median of 23 days with granulocyte [PMN] ≤ 500/μl) and established fungal infections that had not responded to adequate antifungal therapy were transfused with PMN concentrates collected from 35 cytokine-primed granulocyte colony-stimulating factor (GCSF) donors. Patients received a median of six transfusions. Leukocytosis and granulocytosis were observed within 24 hours of the first GCSF injection, which yielded concentrates averaging 55 × 10⁹ white blood cells and 41 × 10⁹ PMN. Data analysis suggested that response might be related to the duration of neutropenia and known infection, as patients given PMN tx earlier in the infectious course tended to have a better response. No significant toxicity was observed in donors.     

Functional characteristics of neutrophils collected and stored after administration of G-CSF

BACKGROUND: Granulocyte transfusion may be used in neutropenic patients with severe bacterial or fungal infections that are unresponsive to antibiotic therapy. However, the inability to store granulocyte concentrates limits their clinical usefulness. STUDY DESIGN AND METHODS: Neutrophil chemotaxis and NADPH oxidase activity and the integrity of the neutrophil NADPH oxidase system were examined after apheresis collection and during storage to 48 hours. Neutrophils were mobilized in vivo by G-CSF, collected by apheresis techniques, and stored in apheresis bags in the presence and absence of additional G-CSF. For all experiments, cells were further purified by standard techniques of dextran sedimentation and hypotonic RBC lysis. RESULTS: Neutrophil chemotaxis was preserved to 24 hours of storage but was not affected by the G-CSF added to storage units. The NADPH oxidase system was also preserved as a functioning complex, and both cytosolic proteins and membrane-associated proteins were normal to 48 hours. However, there were divergent responses by intact cells to activating stimuli and reduced oxidase activity in the cell-free system. G-CSF did not appear to significantly affect NADPH oxidase activity or NADPH oxidase system integrity during storage. CONCLUSION: Neutrophils collected after the administration of G-CSF retained functional and biochemical characteristics for at least 24 hours of storage, which suggests additional effects of G-CSF mobilization beyond enhancing PMN yields and the possibility of storage of these components after collection.     

Characteristics of stored granulocytes collected from donors stimulated with dexamethasone

Two hours after normal donors were given intravenous dexamethasone, their leukocytes were collected by intermittent flow centrifugation. Neutrophils were tested immediately after collection and following storage at 4 to 6 C for 24, 48, 72 and 96 hours. Tests included total leukocyte and absolute neutrophil counts, plasma glucose concentrations, the percentage of phagocytic neutrophils, the ability of phagocytes to accumulate particles, candidacidal activity, bactericidal capacity and chemotaxis. Total leukocyte and absolute neutrophil counts in the stored suspensions were decreased after 48 hours (p = .005). Plasma glucose levels in the suspensions declined at first, then stabilized at 48 hours of storage probably because of loss of cellular integrity. Chemotaxis, candidacidal activity, phagocytosis and dye exclusion showed statistically significant decreases at 24 hours. Chemotaxis deteriorated rapidly, with a mean 63 per cent functional loss at 48 hours. We conclude that treatment of donors with dexamethasone does not extend the storage limits of granulocyte concentrates used for clinical transfusions. Based on these and our previous observations, unless the storage changes should be shown to be reversible, granulocyte concentrates should probably not be stored more than 24 hours before transfusion.     

Collection of two peripheral blood stem cell concentrates from healthy donors

When peripheral blood stem cell (PBSC) concentrates are used for allogeneic transplants, two or more apheresis procedures must often be performed. To determine how many cells could be collected from healthy people by two back-to-back apheresis procedures and what effect these collections would have on donors, we gave 19 healthy people 5 micrograms kg-1 day-1 and 21 people 10 micrograms kg-1 day-1 of granulocyte colony stimulating factor, filgrastim, for 5 days. We then collected two PBSC concentrates, one on day 5 and one on day 6. A third group of six people was given filgrastim 10 micrograms kg-1 day-1 for 5 days but had no PBSC concentrates collected. PBSC concentrate cell counts and donor cell counts, symptoms, and blood chemistries were assessed for up to 1 year. On day 5, three times more CD34+ cells were collected from donors given 10 micrograms kg-1 day-1 than those given 5 micrograms kg-1 day-1 (P = 0.009) but on day 6 the quantity of cells collected was the same (P = 0.23). The total number of CD34+ cells collected was two times greater in donors given the higher dose of filgrastim (median = 579 x 10(6); range = 174-1639 x 10(6) compared to 237 x 10(6); 103-1670 x 10(6); P = 0.061). Platelet counts fell after each PBSC concentrate collection, but there were no differences between the two groups of donors in platelet counts measured immediately after each collection. The platelet counts also fell in people who did not donate PBSC concentrates. The lowest counts in all three groups of people also occurred on day 10. In PBSC donors given 10 micrograms kg-1 day-1 of filgrastim the absolute neutrophil count (ANC) fell below premobilization counts on day 14. In donors given 5 micrograms kg-1 day-1 the ANC fell below premobilization counts on days 21, 28 and 49, CD34+ cell counts were significantly lower than premobilization counts on days 14 and 28 in donors given 10 micrograms kg-1 day-1 of filgrastim and on day 14 in those given 5 micrograms kg-1 day-1. No decrease in neutrophil or CD34+ cell counts occurred after filgrastim was given in the people who did not donate PBSC concentrates. The incidence of symptoms was similar in both groups of PBSC concentrate donors, except that those given 10 micrograms kg-1 day-1 were more than twice as likely to experience myalgias as those receiving the lower dose (P = 0.029). Several blood chemistries changed. Levels of alkaline phosphatase, LDH, SGPT, SGOT, uric acid and sodium increased. Levels of bilirubin, total protein, potassium, calcium and chloride decreased. In conclusion, twice as many CD34+ cells were collected from donors given 10 micrograms kg-1 day-1 of filgrastim. Platelet, neutrophil and CD34+ cell counts fell after the PBSC concentrate collections. The fall in platelet counts was due to both the collection and the administration of filgrastim. The falls in neutrophil and CD34+ cell counts were due to the loss of haematopoietic progenitor cells in the PBSC concentrates. Allogeneic PBSC concentrate donors should be given 10 micrograms kg-1 day-1 of filgrastim, and if possible only one component should be collected in order to avoid thrombocytopenia.     

Maintenance of surface antigens and the absence of an apoptotic marker are observed during storage of granulocyte concentrates collected by bag separation method

Granulocytes were collected by the bag separation method and stored in whole blood for up to 72h. We evaluated the expressions of various surface antigens: CD62L, CD11b, CD18, CD64, CD16b, and CD95. Apoptosis was assessed both by flow cytometry and by light microscopy. Expression levels of all the surface antigens were shown to be maintained during storage for up to 72h. Approximately 80% of granulocytes were annexin V negative until 72h after collection. The storage of granulocyte concentrates collected by the bag separation method may maintain granulocyte surface antigens and lack an apoptotic marker.     

Composition of peripheral blood progenitor cell components collected from healthy donors

BACKGROUND: Peripheral blood progenitor cell (PBPC) components are being collected from healthy donors for allogeneic transplantation, but the quantity, quality, composition, and variability of PBPCs collected from healthy people given granulocyte-colony-stimulating factor (G-CSF) have not been evaluated. STUDY DESIGN AND METHODS: PBPC components were collected from 150 healthy people who were given G-CSF (5, 7.5, or 10 microg/kg/day) for 5 days. The components were evaluated for white cell (WBC), mononuclear cell, CD34+ cell, neutrophil, platelet, and red cell (RBC) composition. RESULTS: The quantities collected were: WBCs, 35.0 +/? 16.4 × 10(9) (range, 11.9–163.3 × 10(9)); mononuclear cells, 33.3 +/? 14.4 × 10(9) (range, 11.9–139.6 × 10(9)); CD34+ cells, 412 +/? 287 × 10(6) (range, 70–1658 × 10(6)); neutrophils, 1.71 +/? 3.59 × 10(9) (range, 0–27.6 × 10(9)); RBCs, 7.2 +/? 4.0 mL (range, 0–22.1 mL); and platelets, 480 +/? 110 × 10(9) (range, 250–920 × 10(9)). PBPC components collected from people given G-CSF at 7.5 or 10 microg per kg per day contained significantly more CD34+ cells (respectively, 428 +/? 300 × 10(6); range, 70–1658 × 10(6) and 452 +/? 294 × 10(6); range, 78- 1380 × 10(6)) than those from people given G-CSF at 5 microg per kg per day (276 +/? 186 × 10(6); range, 91–767 × 10(6)) (p = 0.007 and p = 0.002). When 10 microg per kg per day of G-CSF was given, 50 percent of the components contained enough CD34+ cells for transplantation to a 75- kg recipient (375 × 10(6) CD34+ cells), but 10.6 percent of the components contained less than 150 × 10(6) CD34+ cells and thus would provide a transplantable dose only for a 30-kg patient. CONCLUSION: One PBPC component collected from a healthy donor given 7.5 or 10 microg per kg per day of G-CSF should contain 70 to 1660 × 10(6) CD34+ cells, with 0 to 22 mL of RBCs. Because of the great variability in the number of CD34+ cells collected, the quantity of CD34+ cells in each component should be measured after each procedure to ensure that sufficient quantities of cells are present for a successful transplant.     

Storage of platelet concentrates harvested from blood collected into dextrose-free preservative without agitation

Summary. This study addresses the possibility of platelet quality being maintained during storage by an endogenous metabolic fuel, while avoiding dextrose-induced lactate accumulation. This was achieved by harvesting platelet concentrates from blood donations collected into a dextrose-free anticoagulant. Adequate maintenance of all metabolic and functional parameters was observed in platelets from blood collected into 4% citrate. The requirement for platelets stored in CPD plasma to be agitated during storage was confirmed, but agitation could be omitted for dextrose-free platelets without increased lactate generation and a drop in pH. These results indicate that platelet concentrates from dextrose-free blood may be stored without some of the constraints accompanying platelet storage in conventional media, and may thus result in improved delivery of this product.     

Effects of intensive granulocyte donation on donors and yields

Thirteen HLA-identical bone marrow donors served as the sole source of daily granulocyte transfusions for respective marrow recipients during the period of severe neutropenia between transplantation and engraftment. They experienced 12 to 29 (median, 17) daily, continuous flow centrifugation leukapheresis procedures using hydroxyethyl starch, but no corticosteroids, with little serious difficulty. No immediate clinical reactions occurred in 90 percent of 228 procedures. Mild citrate reactions were noted in 9 percent, and only two procedures (0.8%) were discontinued due to severe reactions. Ten donors (77%) answered a questionnaire mailed weeks later, and six reported transient, late clinical adverse effects. Five had moderate dermatologic problems; one had minimal hypertension requiring no therapy. Donors were monitored daily for laboratory abnormalities while donating granulocytes. Hemoglobin concentration and platelet counts remained stable (autologous red cell transfusions had been given). Blood leukocyte counts gradually fell (p less than 0.05), particularly after 10 or more daily granulocyte donations, and this fall was associated with a decrease of about 33 percent in leukocyte yields. No attempts were made to improve yields by giving higher doses of hydroxyethyl starch or by corticosteroid stimulation. With primary emphasis on donor safety, it seems feasible for a few compatible donors to provide prolonged granulocyte transfusion support for designated patients. However, diminishing leukocyte yields may result from intensive, repeated leukapheresis.     

Improved granulocyte collection from normal donors by combination of continuous-flow centrifugation and filtration leukapheresis

A total of 30 leukaphereses were performed with the Aminco Cellseparator on 27 healthy donots. Pretreatment by steroids and addition of fluid gelatin to the input line was performed in all runs. The combination of continuous flow centrifugation and filtration leukapheresis (CFC-FL) in nine of the 30 runs resulted in a highly significant increase of the yield to a mean of 59.6 X 1o(9) granulocytes as compared with a mean of 30.8 X 10(9) granulocytes obtained with conventional CFC. This significance is valid for the total yield of granulocytes, the yield per hour of run, or per liter of blood processed. No adverse reaction occurred.     

Predictive factors that affect the mobilization of CD34(+) cells in healthy donors treated with recombinant granulocyte colony-stimulating factor (G-CSF)

No specific characteristics have been identified as predictors of peripheral blood stem cells (PBSC) mobilization in healthy donors. In this study, clinical characteristics and laboratory data for 122 healthy donors who underwent apheresis on day 5 of treatment with recombinant granulocyte colony-stimulating factor (G-CSF) were retrospectively analyzed for correlations with CD34(+) cell mobilization. The variables that were analyzed included age, sex, body weight, basal complete blood count, and maximum white blood count (WBC) before apheresis, G-CSF type, and dosage. Median age and body weight were 42.5 years (range 16-65) and 72.5 kg (range 47-121), respectively. By univariate analysis, male sex (P = 0.007), body weight (< or = 70 vs. >70 kg, P = 0.04), and donor's age (< or = 50 vs. > 50 years; P = 0.015) were correlated with the number of CD34(+) cells mobilized. By multivariate analysis, donor's age and male sex were the only two variables that significantly predicted a high CD34(+) cell level. In conclusion, our data suggest that male sex and younger age are the only factors that significantly affect CD34(+) mobilization in healthy donors.     

Survey on the current use of leukapheresis and the collection of granulocyte concentrates

This study was designed to define leukapheresis practice. A voluntary questionnaire on leukapheresis was sent to 280 FDA-registered blood-collecting establishments performing leukapheresis. Of the facilities questioned, 67.9 percent responded. The survey results indicate that most facilities use intermittent-flow blood cell separators, while 22.6 percent use more than one separation method. Establishments routinely use 6% hydroxyethyl starch (HES 450/0.70) and the majority using trisodium citrate as the anticoagulant. Forty-eight percent use corticosteroids, primarily dexamethasone, to pretreat the donor. The frequency of donation was not specified by 25.3 percent of the report. Forty-two percent chose an individual donation frequency of two times per week. A limit on the total number of donations allowed per donor was not specified by 78.4 percent of the facilities. Community blood banks (including regional centers) performed 55.6 percent of all leukocyte concentrate collections. The donor reaction incidence was of 3.64 percent. Hospitals, of all types, performed 37.8 percent of the collections. The adverse reaction rate ranged from 2.84 to 9.72 percent. Adverse reactions occurred in donors 54.9 times per 1000 procedures. Ninety-four percent of reported reactions were mild, whereas moderate and severe reactions accounted for 5.9 and 0.4 percent, respectively. Granulocyte yields varied by the type procedure and the use of corticosteroids as well as among facilities. The majority (56.3%) held leukocytes at 22 to 25 degrees C prior to transfusion, while most of the remainder stored at 4 degrees C.     

Effective storage of granulocytes collected by centrifugation leukapheresis from donors stimulated with granulocyte-colony-stimulating factor

BACKGROUND: Donor stimulation with granulocyte-colony-stimulating factor (G-CSF) has increased the number of neutrophils (PMNs) that can be collected for granulocyte transfusion therapy. Clinical utility, however, has been limited by the inability to store functional PMNs ex vivo. This study was conducted to determine whether granulocyte products from G-CSF-stimulated donors could be effectively stored at reduced temperature (22 degrees C vs. 10 degrees C) with maintenance of functional properties in vitro and in vivo. STUDY DESIGN AND METHODS: Nine normal subjects received G-CSF (600 microg subcutaneously) 12 hours before centrifugation leukapheresis. Granulocyte products were divided and stored for 24 and 48 hours under four conditions: 1) 22 degrees C; 2) 22 degrees C, with supplemental G-CSF (100 ng/mL); 3) 10 degrees C; and 4) 10 degrees C, with supplemental G-CSF. Functional PMN activity during ex vivo storage was assessed in vitro and in vivo by the skin-window technique for granulocytes stored at 10 degrees C for 24 hours. RESULTS: Surface expression of CD11b/CD18, CD14, CD16, CD32, and CD64 was maintained during 48-hour storage at reduced temperature. Inducible respiratory burst activity, bactericidal activity, and fungicidal activity were preserved during storage for 48-hour storage at 10 degrees C. Proinflammatory cytokine production was decreased in product stored at 10 degrees C. Supplemental G-CSF ex vivo did not substantially improve functional activity during storage. After storage at 10 degrees C for 24 hours, in vitro chemotactic potential was maintained, and transfused granulocytes retained capacity to circulate and migrate appropriately in vivo. CONCLUSIONS: Granulocyte product collected by centrifugation leukapheresis from G-CSF-stimulated donors can be effectively stored at subphysiologic temperature for 24 hours with preservation of functional activity. Storage at 10 degrees C appears to be slightly superior to storage at 22 degrees C.     

Platelet concentrates derived from buffy coat and apheresis: biochemical and functional differences

Today, platelet concentrates are generally produced from whole blood by differential centrifugation (buffy coat-derived platelet concentrates--PCs) or by plateletpheresis (apheresis-derived platelet concentrates--APCs). As PCs are characterized by a lower number of platelets than APCs, four to six PCs are customarily combined in order to obtain an equivalent dose. In the 1970s and 1980s, the use of PCs exceeded that of APCs by far; in contrast, since the beginning of the 1990s, APCs comprise more than half of all transfused platelets. However, the selection of PCs or APCs for transfusion to thrombocytopenic patients is still a matter of debate. The present paper compares biochemical and functional properties of both platelet preparations in vitro. Besides plasma parameters (e.g. platelet factor 4 (PF4), P-selectin, C3a-desarginin, plasma coagulation factors), platelet function was analysed by aggregometry and the PFA 100 system. APCs are characterized by a better preservation of ADP and collagen-induced platelet aggregation, and shorter closure times of the PFA 100 test system during storage. The improved primary in vitro haemostatic capacity of APCs is presumed to be owing to a lower cellular activation rate in these preparations. This hypothesis is supported by the higher plasma concentrations of PF4, beta-thromboglobulin and P-selectin found in PCs compared with APCs. The concentrations of C3a-desarginin in PCs exceed those in APCs by far. Additionally, thrombin generation is higher in PCs than in APCs. These data suggest that APCs are characterized by a superior haemostatic capacity over PCs in vitro. However, in vivo studies should be performed to confirm these findings in the patients' circulation also.