Effect of storage conditions on function of granulocytes

The effect of collection technique, anticoagulant, pH, glucose, and temperature on in vitro granulocyte function were studied after 24 hr of storage in the liquid state. Collection by CL did not adversely affect granulocyte function, however, cells collected by FL had accelerated loss of bactericidal activity and chemotactic response. Citrate anticoagulants provided better maintenance of bacteridical activity, NBT reduction, and chemotactic response than heparin, EDTA, and ion-exchange anticoagulants. Chemiluminescence was well maintained when the initial pH of the preservative solution (CPD plasma) was between 6.5 and 8.0 but maintenance of chemotaxis required pH of 7.0–7.5. Glucose concentrations of 80–1000 mg/dl provided adequate maintenance of chemiluminescence and chemotaxis. Bacterial killing was well maintained by storage at either 1–6 or 20–24 °C. Storage at 1–6 °C caused decreased chemotaxis, decreased ability of granulocytes to adhere and spread on a foreign surface, and a decreased intravascular recovery and shortened half-life after transfusion. Although short-term liquid storage may be practical, at present, granulocytes should be transfused as soon as possible after collection.     

Kinetics of neutrophil-releasing activity of filtration leukapheresis and postleukapheresis plasma

A rat model was used to study the effect of filtration leukapheresis on the kinetics of granulocyte mobilization in normal rat donors and in recipients of plasma obtained from these donors. Our earlier observations showed that infusion of postpheresis plasma (PPP) into normal homologous recipients is consistently capable of inducing a marked granulocytosis. The effects of such a transfusion is dose related and duration dependent. This study shows that the granulocytosis occurring subsequent to administration of PPP was directly related to the duration of pheresis in the donor. Its maximum effect occurred 2–3 hr after transfusion. Animals with a low granulocyte count were less capable of mobilizing granulocytes during filtration leukapheresis than animals with a higher prepheresis count, possibly due to low endogenous stores of granulopoietin. Additionally, rats < 300 g were capable of mobilizing fewer granulocytes per volume of circulating blood than were larger (older?) animals. The maximum ability of PPP to increase circulating granulocyte numbers was found when postpheresis plasma was injected along with concurrent removal of circulating granulocytes by filtration leukapheresis.     

Immunomagnetic bead separation of mononuclear cells from contaminating granulocytes in cryopreserved blood samples

Density gradient centrifugation usually allows efficient separation of mononuclear cells from granulocytes using fresh human blood samples. However, we have found that with cryopreserved blood samples, density gradient centrifugation fails to separate granulocytes from mononuclear cells and have explored using immunomagnetic anti-CD15 microbeads as an alternate method to separate these cell populations. Using cryopreserved blood samples from 10 healthy donors we have shown that granulocytes express a significantly higher level of CD15 antigen than monocytes and lymphocytes, which allows for their efficient separation from mononuclear cells using anti-CD15 microbeads. This procedure is critical for purification of individual cell populations from cryopreserved leukocyte samples and could also potentially be applied to avoid granulocyte contamination of mononuclear cells isolated from stored blood and from patients with sepsis or thermal injury.     

16. Environmental specimen cryobanking

Density gradient centrifugation usually allows efficient separation of mononuclear cells from granulocytes using fresh human blood samples. However, we have found that with cryopreserved blood samples, density gradient centrifugation fails to separate granulocytes from mononuclear cells and have explored using immunomagnetic anti-CD15 microbeads as an alternate method to separate these cell populations. Using cryopreserved blood samples from 10 healthy donors we have shown that granulocytes express a significantly higher level of CD15 antigen than monocytes and lymphocytes, which allows for their efficient separation from mononuclear cells using anti-CD15 microbeads. This procedure is critical for purification of individual cell populations from cryopreserved leukocyte samples and could also potentially be applied to avoid granulocyte contamination of mononuclear cells isolated from stored blood and from patients with sepsis or thermal injury.     

Segregation of mouse hemopoietic progenitor cells using the monoclonal antibody,YBM/42

A rat monoclonal antibody, YBM/42, directed against mouse leukocyte common antigen, was used for the analysis and separation of hemopoietic progenitor cells from mouse bone marrow and fetal liver. Cells were fractionated on a FACS-II cell sorter and the resulting subpopulations examined for their morphology and ability to form colonies in agar (for day 7 colonies) and methylcellulose (for day 2 erythroid clones). The antibody bound to all leukocytes, including blast cells and day 7 hemopoietic progenitor cells (day 7 colony forming cells, CFC), but not to erythrocytes or nucleated erythroid cells. This antibody can be used to advantage to enrich for early progenitor cells from mouse fetal liver, in which the majority of cells (70%) are nucleated erythroid cells. In day 12 fetal liver, approximately 10% of all cells bind this antibody strongly and, of these approximately 70% are blast cells. Contained within this positive population are 95% of all day 7 CFC. In the most enriched fraction about 20% of the cells formed day 7 colonies. This represents a 25-fold enrichment over unsorted fetal liver. The negative fractions contain 94% of all cells forming erythroid clones (≥8 cells) on day 2 of culture (day 2 CFU-E). In the most enriched fraction, 20% of the cells are day 2 CFU-E. Day 7 CFC can therefore be well separated from day 2 CFU-E, with good recovery of both cell types, by use of a single label. Day 7 colony forming cells were classified as granulocyte (G-CFC), macrophage (M-CFC), mixed granulocyte/macrophage (GM-CFC), pure erythroid (E), or mixed erythroid (E_mix). A high enrichment for multipotential cells is achieved and constitues 3–5% of cells in the most enriched fraction. Most types of day 7 CFC could not be separated with YMB/42, but GM-CFC and M-CFC exhibit a broader distribution than the other CFC with regard to fluorescence intensity. This implicit heterogeneity in GM-CFC and M-CFC is further substantiated by the finding that myeloid progenitors in the different FACS fractions also share a differential reactivity to different sources of growth factors.     

Separation of leucocytes in the dogfish (Scyliorhinus canicula) using density gradient centrifugation and differential adhesion to glass coverslips

Summary The blood of the dogfish, S. canicula, contains several types of leucocytes, namely thrombocytes, monocytes, lymphocytes and four populations of granulocytes. Three of these granulocyte types, G1, G3 and G4, are eosinophilic while G2 is heterophilic/neutrophilic. All of the leucocyte types, with the exception of G2 granulocytes and monocytes, can be separated by means of their differential adherent properties to glass and by density gradient centrifugation. Thrombocytes, G3 and G4 granulocytes can be separated in good purity by single-step methods while G1 granulocytes and lymphocytes require a combination of density gradient centrifugation followed by adherence to glass to remove contaminating thrombocytes. Depending on the cell type, between 11–45% of cells with consistently high viability can be recovered after separation. Separated populations of the thrombocytes and granulocytes will be especially useful for studies on the role of such cell types in inflammation.     

小鼠骨髓内皮细胞条件培养液复合FL及TPO对HPP—CFC及CFU—GM增殖的影响

通过传代培养小鼠骨髓内皮细胞,收集无血清条件培养液（ECM）,并经超滤得到大于10kD的浓缩液,分别观察ECM和大于10kD的浓缩液复合flt3ligand(FL)及thrombopoietin(TPO)对体外培养HPP-CFC、CFU-GM的影响,结果表明：ECM或大于10kD的浓缩液对HPP-CFC、CFU-GM的生长均有支持作用;FL或/和TPO与ECM或大于10kD的浓缩液合用能加强对HPP-CFC、CFU-GM生长的刺激作用;FL加TPO与ECM或大于10kD的浓缩液合用对HPP-CFC、CFU-GM生长的刺激作用更加明显;选择FL和TPO特异性的引物,用RT-PCR技术未能检测到小鼠骨髓内皮细胞有FL和TPOmRNA的表达。     

Phagocytosis and digestion of pH-sensitive fluorescent dye (Eos-FP) transfected E. coli in whole blood assays from patients with severe sepsis and septic shock

The function of phagocytic and antigen presenting cells is of crucial importance to sustain immune competence against infectious agents as well as malignancies. We here describe a reproducible procedure for the quantification of phagocytosis by leukocytes in whole blood. For this, a pH-sensitive green-fluorescent protein- (GFP) like dye (Eos-FP) is transfected into infectious microroganisms. After UV-irradiation, the transfected bacteria emit green (≈5160 nm) and red (≈581 nm) fluorescent light at 490 nm excitation. Since the red fluorescent light is sensitive to acidic pH, the phagocytosed bacteria stop emitting red fluorescent light as soon as the phagosomes fuse with lysosomes. The green fluorescence is maintained in the phagolysosome until pathogen degradation is completed. Fluorescence emission can be followed by flow cytometry with filter settings documenting fluorescence 1 (FL 1, FITC) and fluorescence 2 (FL 2, phycoerythrin, PE). Eos-FP transfected bacteria can also be traced within phagocytes using microscopical techniques. A standardized assay has been developed which is suitable for clinical studies by providing clinicians with syringes pre-filled with fixed and appropriately UV-irradiated Eos-FP E. coli (TruCulture™). After adding blood or body fluids to these containers and starting the incubation at 37°C, phagocytosis by granulocytes proceeds over time. Cultures can be terminated at a given time by lysing red blood cells followed by flow cytometry. A pilot study demonstrated that Eos-FP E. coli phagocytosis and digestion was up-regulated in the majority of patients with either severe sepsis or septic shock as compared to healthy donors (p < 0.0001 after o/n incubation). Following treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF) in selected patients with sepsis, phagolysosome fusion appeared to be accelerated.     

Adhesion of human T lymphocytes and granulocytes to endothelial cells stimulated by cell-surface antigens of Bacteroides thetaiotaomicron

The aim of this study was to assay the degree of human T lymphocyte and granulocyte adhesion to the vascular endothelial cells stimulated by Bacteroides thetaiotaomicron lipopolysaccharides, components of LPS and capsular polysaccharide. HMEC-1 cells were activated with bacterial preparations in concentration 10 micrograms/ml for 4 and 24 hours. T lymphocytes and granulocytes were isolated from peripheral blood of healthy blood donors. Thereafter, the adhesion tests of granulocytes and adhesion tests of non-activated and activated with PMA (in concentration 10 ng/ml) T lymphocytes to the resting and stimulated vascular endothelium were performed. The number of viable cells, which adhered to the endothelium, was determined using inverted microscope (magnification 200x). The results were presented as the number of viable cells adhering to 1 mm2 of the endothelial cell culture. The obtained results indicate that granulocytes and T lymphocytes (resting and activated with PMA) adhere to the endothelial cells stimulated by B. thetaiotaomicron cell-surface antigens. B. thetaiotaomicron lipopolysaccharides and capsular polysaccharide are weaker stimulants of human leukocyte adhesion to the HMEC-1 cells than E. coli O55:B5 LPS.     

Purification and characterisation of the in vitro colony forming cell in monkey hemopoietic tissue

Buoyant density gradient separation of Rhesus monkey bone marrow, spleen and blood leukocytes has demonstrated a reproducible and homogeneous light density distribution profile of cells capable of forming hemopoietic colonies in agar culture (in vitro colony forming cells — CFC). High resolution density gradient separation performed on a light density fraction of bone marrow produced on average a 100-fold enrichment of in vitro CFC with the most enriched fractions containing the majority of the in vitro CFC population present in the original marrow. Fractions were routinely obtained in which up to 23% of cells formed colonies and 33% were capable of proliferating to some degree upon stimulation. Tritiated thymidine suiciding showed the active proliferative status of the in vitro CFC and application of autoradiography and morphological characterisation to highly enriched density fractions has shown that the in vitro CFC in normal marrow is a transitional lymphocyte. Single cell transfer experiments have shown that in vitro CFC's formed colonies containing both granulocytes and macrophages, formally demonstrating the clonal origin of in vitro colonies and the common origin of granulocytes and macrophages.     

Blood and inflammatory cells of the lungfish Lepidosiren paradoxa

A special interest exists concerning lungfish because they may possess characteristics of the common ancestor of land vertebrates. However, little is known about their blood and inflammatory cells; thus the fine structure, cytochemistry and differential cell counts of coelomic exudate and blood leucocytes were studied in Lepidosiren paradoxa. Blood smear analyses revealed erythrocytes, lymphocytes, monocytes, polymorphonuclear agranulocytes, thrombocytes and three different granulocytes. Blood monocytes and lymphocytes had typical vertebrate morphology. Thrombocytes had large vacuoles filled with a myelin rich structure. The polymorphonuclear agranulocyte had a nucleus morphologically similar to the human neutrophil with no apparent granules. Types I and II granulocytes had eosinophilic granules. Type I granulocytes had round or elongated granules heterogeneous in size, while type II had granules with an electron dense core. Type III granulocyte had many basophilic granules. The order of frequency was: type I granulocyte, followed by lymphocyte, type II granulocyte, monocyte, polymorphonuclear agranulocyte and type III granulocyte. Peroxidase localized mainly at the periphery of the granules from type II granulocytes, while no peroxidase expression was detected in type I granulocytes. Alkaline phosphatase was localized in the granules of type II granulocyte and acid phosphatase cytochemistry also labelled a few vacuoles of polymorphonuclear agranulocyte. About 85% of the coelomic inflammatory exudate cell population was type II granulocyte, 10% polymorphonuclear agranulocyte and 5% macrophages as judged by the nucleus and granule morphology. These results indicate that this lungfish utilises type II granulocytes as its main inflammatory granulocytes and that the polymorphonuclear agranulocyte may also be involved in the inflammatory response. The other two granulocytes appear similar to the mammalian eosinophil and basophil. In summary, this lungfish appears to possess the typical inflammatory granulocytes of teleosts, however, further functional studies are necessary to better understand the polymorphonuclear agranulocyte.     

The generation of colony-forming cells (CFC) and the expansion of hematopoiesis in cultures of human cord blood cells is dependent on the presence of stem cell factor (SCF)

We have analyzed the effect of stem cell factor (SCF), alone or in combination with other growth factors, on the generation of colony-forming cells (CFC) and on the expansion of hematopoiesisin vitro from light density, soybean agglutinin^–, CD34⁺ cord blood cells under serum-deprived conditions. The growth factors were either added only once at the onset of the culture or added every few days when the cultures were demidepopulated and refed with fresh medium. No growth factor, alone, generated CFC or expanded hematopoiesis under these conditions. However, SCF, in combination with interleukin 3 (IL-3) or with late-acting factors (granulocyte colony-stimulating factor (G-CSF) or erythropoietin (Epo)), generated large numbers of mature cells as well as CFC. The number of CFC generated depended on the refeeding procedure adopted. In cultures never refed, the CFC numbers increased from > 160 CFC/culture at day 0 to > 3000 CFC at day 10. The CFC numbers stayed above the input levels for 25 days before declining. Almost no CFC were detectable after one month. In contrast, in cultures regularly refed, CFC were detectable for at least 40 days. The lineages of the mature cells and the types of CFC generated varied with the different growth factors. In the presence of SCF plus IL-3, erythroid burst-forming cells (BFU-E) and granulocyte/macrophage colony-forming cells (GM-CFC) were generated and erythroid as well as myelomonocytic precursors were present among the differentiated cells. In contrast, in the presence of SCF and G-CSF or Epo, the progenitor cells as well as the differentiated cells were dictated by the late-acting growth factor (i.e. mostly G-CFC and myeloid cells in the presence of SCF and G-CSF vs. BFU-E, erythroid colony-forming cells (CFU-E) and erythroblasts in the presence of SCF and Epo). Thus, marked expansion of erythropoiesis and granulopoiesis can be achievedin vitro by as few as two factors — SCF acting as the early factor along with the appropriate late-acting factor.Paper presented in part at the World Congress on Cell Cultures, Washington D.C., 21–24 June 1992.     

Interactions of basophilic granulocytes and fat metabolism as indicators in thrombophilia

In different groups of patients and blood donors with increased thrombophilic or arteriosclerotic risk we found some correlations between basophilic granulocyte values and lipid parameters. Basophilic granulocytes are one of the bodies own origin of sulfated mucopolysaccharides which protects endothelial cells and saves high density lipoprotein cholesterol, as we were able to find in blood donors.     

Cryopreservation of granulocytes for transfusion: Studies on human granulocyte isolation,the effect of glycerol on lysosomes,kinetics of glycerol uptake and cryopreservation with dimethyl sulfoxide and glycerol

Existing methods for the cryopreservation of granulocytes employ primarily dimethyl sulfoxide (Me₂SO) rather than glycerol as the cryoprotective additive of choice. Although Me₂SO has been demonstrated to be an effective cryoprotective additive for granulocyte preservation to yield viable cells (dye exclusion, phagocytosis, etc.), the inherent toxicity and clinical objections of Me₂SO as a cryoprotective additive for granulocyte preservation preclude its extensive and routine use in patients. Therefore, glycerol, with its important advantage of nontoxicity, has been investigated for its potential usefulness as a cryoprotective additive for preserving human granulocytes for transfusion.Granulocyte preparations were isolated from impure leukocyte concentrates obtained from the buffy coats of human whole blood. Studies on the isolation and purification of the granulocytes involved separation by sedimentation with dextran, removal of red cells by hypotonic shock with water, resuspension with Plasmatein and further purification by centrifugation. Intact viable granulocytes were obtained with a purity in excess of 90%.Lysosomes were studied as indicators of cryoinjury in granulocytes using β-glucuronidase as the key marker enzyme. This enzyme has been characterized as a sensitive indicator of damage to lysosomes and a direct linear relationship has been established between damage to granulocytes by freezing and amount of lysosomal enzyme released. Addition or presence of the cryoprotectant, glycerol, did not appear to have any adverse effect on lysosomes of intact granulocytes.Studies on the permeation kinetics of glycerol in granulocytes indicated that the additive was freely permeable and did not cause any potentially damaging osmotic changes in cell volume. Granulocytes in various concentrations of glycerol were then frozen at slow, moderate, and rapid cooling rates. Based on the small amount of β-glucuronidase released, good preservation of granulocyte lysosomes has been obtained with a slow cooling rate of 5 °C/min and a concentration of 15% glycerol. Further studies now are necessary to define those conditions of cooling rate and glycerol concentration required to develop a simple method for optimal preservation of granulocytes based on additional functional criteria of viability.     

Granulocyte interactions with GM-CSF and G-CSF secretion by endothelial cells and monocytes

We have, in previous studies, characterized the cytokine and cellular regulation of GM-CSF and G-CSF production by monocytes and endothelial cells. In this study, we investigated the regulatory role of granulocytes. The addition of granulocytes to endotoxin-stimulated monocytes dose-dependently decreased both GM-CSF and G-CSF concentrations, presumably by absorbing the cytokines. A similar dose-dependent decrease in GM-CSF concentration was found when granulocytes were added to IL-1-stimulated endothelial cells. In contrast, G-CSF secretion by endothelial cells responded to granulocytes in a biphasic fashion. At low granulocyte concentrations, endothelial cells responded with an increased G-CSF secretion, but at high concentrations of granulocytes G-CSF secretion was down modulated. Our results suggest that there exist two loops between granulocytes and endothelial cells for regulating G-CSF activity. Granulocytes can stimulate G-CSF secretion by activated endothelial cells but can also decrease the biological activity by absorbing the cytokine. These mechanisms might be involved in the regulation of the local and systemic levels of granulocytes.     

Expansion of CD34+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34+ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34+ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34+ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3±52.1)-fold, total progenitor cells (CFC) by (74.5±5.2)-fold and CD34+ cells by 15.7-fold.Maximal expansions of CFC and CD34+ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34+ cells within 28 days was found only in two combinations, I.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34+ cells can be extensively expanded ex vivo by using gene transfected stromal cells along with cytokines.     

Inhibition of human cytotoxic T lymphocyte activity in vitro by autologous peripheral blood granulocytes

Peripheral blood granulocytes from normal healthy donors were found to reproducibly inhibit the cytolytic effector function of specifically sensitized cytotoxic T lymphocytes in vitro when co-incubated with these effector cells and target cells in 8 hr 51Cr release assays. Inhibition required intact granulocytes, was proportional to the number of granulocytes present, and was independent of granulocyte adherence, phagocytic function, and viability. Equivalent numbers of enriched normal or leukemic peripheral T lymphocytes did not cause inhibition of 51Cr release, and preincubation of granulocytes with effectors did not significantly alter viability or cytotoxic function. Because granulocytes can inhibit natural killer cell function in vitro, these data indicate that granulocytes can regulate diverse antigen-specific and spontaneous cytotoxic functions in vitro, suggesting that circulating granulocytes may have the potential for in vivo regulation of these cytotoxic effectors.     

Perspectives of leukocyte activation in the microcirculation

Recent evidence for a role of granulocytes in ischemic organ injury and in hemorrhagic shock is provided. Compared to red cell, granulocytes are large cells and have a stiff cytoplasm, making them prone to entrapment in the microcirculation. After activation, granulocytes become adhesive, they can elaborate superoxide radicals and release proteolytic enzymes. In the circulation a subgroup of granulocytes are in a spontaneously activated state. If during shock such cells become trapped in the microcirculation they impose a risk for organ injury. In a short term shock protocol, the group of surviving and non-surviving animals can be sharply distinguished by the number of activated granulocytes before shock. Experimental forms of granulocyte activation in the coronary circulation cause temporary trapping of cells, an increase in vascular resistance, and a transient reduction of muscle contraction even in the presence of a normal perfusion pressure. Detection of spontaneous granulocyte activation requires the development of new tests which can be carried out on fresh unseparated blood samples. We provide here also a critical evaluation of experimental neutropenia as a test for granulocyte related hypotheses.     

Identification of polymorphonuclear leukocytes in cytologic samples for flow cytometry.

Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.     

Neutrophil migration in canines: In vivo comparison of granulocyte function following 24-hour storage at 6 or 20 °C of leukocyte concentrates or of granulocytes purified by counterflow centrifugation-elutriation

The use of granulocyte-rich concentrates from leukapheresis purified by counterflow centrifugation—elutriation to obtain pure granulocytes for transfusion studies in cyclo-phosphamide-induced neutropenic animal models is reported. Our data for granulocyterich leukapheresis concentrates indicate that room temperature (20 °C) appears to be preferred to 6 °C for short-term granulocyte storage. The data also indicate that although the granulocytes isolated by counterflow centrifugation—elutration may retain in vitro functions of chemotaxis, phagocytosis, and bactericidal activity, the in vivo function of migration into skin chambers for isolated granulocytes is seriously impaired after storage for 18 to 24 hr at both 6 and 20 °C. This loss of in vivo function of stored granulocytes occurs in isolated granulocytes obtained by both counterflow centrifugation-elutriation and dextran sedimentation, and it is not observed in the leukocyte concentrates held at 20 °C. The results of these studies are fourfold. First, freshly isolated granulocytes display no apparent loss of either in vivo or in vitro function. Second, granulocytes isolated by counterflow centrifugation-elutriation or dextran sedimentation and stored at 6 or 20 °C are severely impaired in terms of their in vivo chemotactic function but display no loss of in vitro efficacy. Third, 20 °C storage of granulocyte-rich leukapheresis concentrates for 18 to 24 hr is superior to 6 °C storage. Fourth, in vitro analysis may be limited in its ability to indicate in vivo function as a measure of success in granulocyte preservation studies.