Liquid and cryopreservation effects on in vitro function of dog granulocyte concentrates prepared for transfusion

The effects of liquid and Cryopreservation on in vitro function of dog granulocyte concentrates prepared by continuous flow centrifugation leukapheresis and counterflow centrifugation elutriation are presented. These homogeneous granulocyte concentrates (97 ± 2% granulocytes, 99.4 ± 0.3% viable) were cryopreserved in 5% DMSO and 5% HES dissolved in 2 g% BSA, 20% autologous citrated plasma in a modified a minimal essential medium. The granulocyte recovery was 87.6 ± 2.4% relative to the number of intact and viable granulocytes in the washed suspension of cells. In vitro functions of chemotaxis, phagocytosis, bactericidal activity, and selected enzymes were not affected by 12–24 hr storage at 4–6 °C. Frozen, thawed, and washed granulocytes showed a significant decrease (P < 0.01) in chemotactic recognition and response but not chemokinetic response, although it was depressed. Phagocytosis of latex beads and associated burst of O₂ consumption also decreased significantly (P < 0.05) to approximately 50% of the original prefreezing value. However, the killing of live Escherichia coli was not depressed to the extent expected and suggested by loss of O₂ consumption and selected enzyme activity. The cryopreservation of viable homogeneous granulocyte concentrates in sufficient quantity for transfusion in the neutropenic and/or septicemic dog model is demonstrated in these results.     

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.     

Liquid preservation of highly purified human granulocytes

Highly purified human granulocytes isolated from continuous flow centrifugation leukapheresis concentrates by counterflow centrifugation-elutriation were stored at 4 °C in concentrations of 6 × 10⁶ to 1 × 10⁷ granulocytes per milliliter for up to 14 days. The in vitro physiological function assays of phagocytosis, oxygen consumption associated with phagocytosis, bacterial growth inhibition, chemotaxis, and five enzyme analyses indicated good storage survival for up to 4 days. Stored granulocytes separated from other blood cells have greater storage stability than granulocytes stored as leukapheresis concentrates. After 14 days of storage a small percentage of granulocytes still maintained all physiological functions, with the exception of chemotaxis. Of the five enzymes assayed, only the enzyme activity of leucine aminopeptidase decreased significantly by the 14th day of storage. The storage stability of each physiological function assayed decreased as follows: bacterial growth inhibition (most stable), phagocytosis, oxygen consumption, and chemotaxis (least stable).     

Factors affecting the stability of cryogenically preserved granulocytes

Human granulocytes free of other cell types were obtained by counterflow centrifugation, cryogenically preserved, and studied for stability and function after thawing.Isolation of granulocytes by counterflow centrifugation was optimal at reduced temperatures (4–10 °C) in phosphate-buffered saline (or Ca²⁺-free buffers) at pH 7.1. A stabilizing protein, or HES was required. Routinely, 1.2% human or bovine serum albumin was used. Hyperosmolar (310 m0sm) buffers and post isolation handling in ice water baths was optimal for cryogenic preservation. Addition of DMSO at 22 °C produced transient shrinkage initially which depended on the rate of addition, concentration, and temperature. Within 10–15 min granulocytes returned to volume, but continued to swell, equilibrating for 1 hr at 20% larger volume. Ethidium uptake gradually increased. After 24 hr, extreme swelling, lysis, and ethidium uptake was observed at the highest concentration (10%) of DMSO. DMSO-induced swelling was prevented with HES.Granulocytes (30 × 10⁶ ? 50 × 10⁶) were frozen in 2.0-ml volumes in plastic tubes. The combination of 5% DMSO, 6% HES, 4% albumin, 0.056 M glucose in NormosolR at pH 7.1 produced the best yields. Granulocytes were first cooled to 4 °C, then to ?80 °C (approx rate 4 °C per min) in a mechanical freezer and finally stored in liquid nitrogen. Storage varied from days to months. Granulocytes were thawed at 42 °C by manually twirling the freezing tubes and they were subsequently maintained in ice water. They were diluted 3:1 dropwise with a room temperature solution of 7% HES, 1.2% albumin, and 0.026 M glucose in Normosol. Particle ingestion tests were conducted by incubation at room temperature for forty minutes with yeast or zymosan opsonized with autologous serum. Particles ingested were counted by microfluorimetry after two washings at 150g.Granulocytes could not be cryogenically preserved in plasma or serum. Heating or prefreezing of serum was ineffective, but dialysis or addition of EDTA overcame the destructive effect of serum. Neither treatment was an improvement over the standard freeze procedure using buffered albumin and cryoprotective components. β-mercaptoethanol added to the freezing medium caused the production of a single homogeneous population of osmotically inert, nonviable, ethidium-reactive granulocytes. This suggests that osmoregulation by granulocyte membranes is a critical requirement for cryopreservation.Preservation efficiency is species dependent, increasing in the order of human, baboon, guinea pig, and dog. Dog granulocytes can be stored for at least 8 months in liquid nitrogen with small loss of cells and functionality.The present efficiency of preservation of human granulocytes for 3–4 weeks of liquid nitrogen storage is 90–100% morphological and 40% functional recovery. Attempts to increase stability of thawed granulocytes with other additions to our current procedure have so far proved fruitless. These have consisted of inosine, adenine, pyruvate, gluconate, vitamin C, β-mercaptoethanol, para-phenylmethyl-sulfonylfluoride, and mannitol.     

Nicotine-induced membrane perturbation of intact human granulocytes spin-labeled with 5-doxylstearic acid Correlation with chemotaxis

The effects of nicotine on intact human granulocytes were examined, using 5-doxylstearic acid as a spin probe. At micromolar concentrations, (−)-nitocine produces a membrane perturbation in granulocytes not observable with oriented lipid bilayers. The effect, which is stereoselective for the (−)-isomer, occurs at concentrations of nicotine that bind to noncholinergic nicotine receptors on granulocytes and which are present in the blood after smoking. At comparable concentrations, (−)-nicotine modulates granulocyte chemotaxis towards a chemotactic peptide in a stereospecific and dose-dependent manner. Cotinine, the major metabolite of nicotine, does not bind to the receptor, does not produce the membrane perturbation observed with nicotine, and has no effect on chemotaxis. These results suggest that (−)-nicotine present in the blood after smoking binds to a receptor on granulocytes, perturbs granulocyte membranes and modulates chemotaxis.     

Recovery,structure, and function of dog granulocytes after freeze-preservation with dimethylsulfoxide

The recovery, structure and function of dog granulocytes were determined before and after freeze-preservation. Leucocytes were isolated from defibrinated or anti-coagulated whole blood and subsequent erythrocyte sedimentation on a column of 2:1 dextran (6%)-isopaque (33.9%). Granulocytes isolated by these procedures were examined for changes in O₂ consumption associated with phagocytosis, in vitro directed migration (chemotaxis), bactericidal activity, and ultrastructure before and after freezing. Granulocytes were frozen in DMSO (7.5%) and autologous serum or HBSS-minus and 20% autologous serum at the rate of ?1 °C/min to ?80 °C and stored in liquid N₂ vapor.After freeze-preservation, O₂ consumption associated with phagocytosis was decreased by 54 and 64% for granulocytes isolated from defibrinated or from ACD-anticoagulated blood, respectively. Bactericidal activity is only slightly depressed in samples from either isolation method after freeze-preservation when compared to the prefreeze controls, but granulocytes isolated from defibrinated blood are significantly less effective in killing bacteria than those from ACD-anticoagulated blood. Chemotactic response after freeze-preservation was completely inhibited in granulocytes isolated from defibrinated blood. Exposure of granulocytes to ACD inhibited chemotaxis prior to freezing, but the granulocytes responded chemotactically after freeze-thaw and additional washing. The ultrastructure of granulocytes observed before and after freeze-thaw was similar for cells isolated by both methods. However, nuclear, cytoplasmic, and granular changes observed were slightly greater in granulocytes isolated from defibrinated blood. Dog granulocytes isolated by either method withstood freeze-preservation in DMSO to a degree not previously reported.It is concluded that dog granulocytes freeze-preserved by these methods are functional in vitro, but that phagocytic, directed migration, and bactericidal functions and ultrastructure are impaired to different degrees, according to the method of isolation and preparation for storage. These results indicate the need for continued investigation on the effects of storage variables on the preservation of granulocytes.     

Human endothelial cells modulate granulocyte adherence and chemotaxis

Although human endothelial cells (EC) and granulocytes interact in several ways, the factors that regulate such interactions are not well defined. In this study we found that EC and their products directly altered granulocyte adherence (GA) and chemotactic activity. The spontaneous adherence of granulocytes to human umbilical vein EC monolayers was significantly reduced at 5, 15 and 30 min if the granulocytes were preincubated with EC that were stimulated by rocking. At 30 min the spontaneous adherence of EC-preincubated granulocytes was 36% of that of control granulocytes (P = 0.004). The augmented adherence stimulated by FMLP was also decreased (54% of control) by preincubation of the granulocytes with rocked EC. The ability of stimulated EC to inhibit GA was attenuated when the EC monolayers that were used for preincubation with the granulocytes were pretreated with the cyclooxygenase inhibitor indomethacin. GA to unstimulated EC was not significantly altered by indomethacin or aspirin, suggesting that cyclooxygenase products do not influence GA under resting conditions. Preincubation of granulocytes with rocked EC monolayers or supernatant media from rocked EC monolayers diminished their chemotactic response to FMLP by 45 to 65. This inhibition was also attenuated by pretreatment of the EC with indomethacin. EC supernatant medium caused a rapid increase in granulocyte intracellular cyclic AMP, with a maximum increase to 200% of control at 1 min. These data indicate that stimulated EC release one or more arachidonic acid products that alter spontaneous and inflammatory mediator-stimulated granulocyte activity. Prostacyclin, a major cyclooxygenase product of EC arachidonate, depressed inflammatory mediator-augmented GA to EC monolayers and chemotaxis when present in nanomolar concentrations. We conclude that EC-derived prostacyclin, alone or in combination with other EC products, alters GA and chemotaxis stimulated by inflammatory mediators. This provides a mechanism by which EC may modulate granulocyte distribution as well as granulocyte responses that are influenced by adherence, such as the release of toxic oxygen metabolites and granular enzymes.     

Nicotine-induced membrane perturbation of intact human granulocytes spin-labeled with 5-doxylstearic acid. Correlation with chemotaxis

The effects of nicotine on intact human granulocytes were examined, using 5-doxylstearic acid as a spin probe. At micromolar concentrations, (-)-nitocine produces a membrane perturbation in granulocytes not observable with oriented lipid bilayers. The effect, which is stereoselective for the (-)-isomer, occurs at concentrations of nicotine that bind to noncholinergic nicotine receptors on granulocytes and which are present in the blood after smoking. At comparable concentrations, (-)-nicotine modulates granulocyte chemotaxis towards a chemotactic peptide in a stereospecific and dose-dependent manner. Cotinine, the major metabolite of nicotine, does not bind to the receptor, does not produce the membrane perturbation observed with nicotine, and has no effect on chemotaxis. These results suggest that (-)-nicotine present in the blood after smoking binds to a receptor on granulocytes, perturbs granulocyte membranes and modulates chemotaxis.     

Chemotactic Responses of Marine Vibrio sp. Strain S14 (CCUG 15956) to Low-Molecular-Weight Substances under Starvation and Recovery Conditions

The chemotactic responses by starved cells of marine Vibrio sp. strain S14 differed from those elicited by cells that were not nutrient limited. The rate of chemotaxis at different concentrations of several attractants varied for starved and growing cells. Vibrio sp. strain S14 showed positive chemotaxis to leucine, valine, arginine, and glucose at the onset of energy and nutrient deprivation. A continued, though decreased, positive response was demonstrated fro leucine, arginine, and glucose at 10 h of starvation. Cells starved for 3 h displayed a stronger response to glucose than those starved for shorter or longer times. However, cells starved for 5 and 10 h responded more strongly to a lower concentration of glucose than did cells starved for 0 and 3 h. Starvation for 24 h elicited no measurable chemotaxis to leucine, arginine, or glucose. The motility decreased by over 95% in the cell population after 24 h of starvation, which resulted in a low sensitivity in the chemotaxis assay. A switch in the response to valine was observed by 3 h of starvation. The addition of nutrients of 22-h-starved cells elicited a temporary positive chemotactic response to leucine by 2 and 4 h of nutrient recovery, while cells at 1 and 6 h of recovery showed no response. At 2 h of recovery, the greatest response was recorded to 10⁻⁴ M leucine, whereas at 4 h it was to 10⁻² M leucine. Ten to fifty percent of the 22-h-starved cell population regained their motility after 4 h of nutrient-aided recovery. It is possible that two types of chemosensory systems exist in marine bacteria. Starved and growing cells responded to different concentrations of the attractant, and growing cells displayed a saturated chemotactic system with leucine as the attractant, unlike the response during starvation.     

Stimulation of Locomotion of Peripheral Blood Monocytes by Human Plasma Fibronectin

The motility of human peripheral blood granulocytes and monocytes in response to human plasma fibronectin was quantified by an in vitro assay using blind-well chemotaxis chambers. Purified fibronectin under nondenaturing conditions produced increased migration of granulocytes only at concentrations higher than 100 nm, and induced increased chemotactic and random locomotion of monocytes at concentrations higher than 0.1 nm. The monocyte migration-inducing activity of fibronectin was concentration dependent, and was strongly inhibited by low concentrations of colchicine (100 nm–100 μm). These findings suggest the possibility that plasma fibronectin serves as a chemotactic stimulus for monocytes in vivo and attracts these cells to sites of microscopic tissue injury where plasma fibronectin is deposited.     

Use of a modified rotor and enlarged separation chamber for isolation of human granulocytes by counterflow centrifugation-elutriation

Human granulocytes were isolated from leukapheresis blood by counterflow centrifugation-elutriation using the Beckman J21B centrifuge and JE6 rotor. Attempts to increase the absolute number of human granulocytes recovered by a single- or double-chamber procedure revealed the physical limitations of the chamber capacity in granulocyte isolation. To improve the absolute number of granulocytes from leukapheresis blood, an enlarged separation chamber and accompanying rotor were fabricated. The enlarged chamger has three times the capacity of the standard 4.5-ml Beckman chamber. Aside from increased yield of granulocytes, the 13.2-ml chamber affords a 25% saving in isolation time compared to the Beckman chamber for isolation of a comparable number of cells. In vitro analysis of the isolated granulocytes for viability, latex bead ingestion, and chemotactic response indicate no apparent loss of granulocyte function as a result of the isolation procedure.     

THE REGULATORY ROLE OF DIVALENT CATIONS IN HUMAN GRANULOCYTE CHEMOTAXIS : Evidence for an Association between Calcium Exchanges and Microtubule Assembly

Optimal human granulocyte chemotaxis has been shown to require both calcium and magnesium. Exposure of granulocytes to three different chemotactic factors (C5a, kallikrein, and dialyzable transfer factor) yielded a rapid calcium release, depressed calcium uptake, and was associated with a shift of calcium out of the cytoplasm and into a granule fraction. Colchicine, sodium azide, and cytochalasin B, in concentrations that inhibited chemotaxis, also inhibited calcium release while low concentrations of cytochalasin B, which enhanced chemotaxis, also enhanced calcium release. Microtubule assembly was visualized both in cells suspended in C5a without a chemotactic gradient and in cells actively migrating through a Micropore filter. The data suggest microtubule assembly is regulated, at least, in part, by the level of cytoplasmic calcium. It is proposed that asymmetric assembly of microtubules may be instrumental in imparting the net vector of motion during chemotaxis.     

Evaluation as a function of granulocytes in hyperimmunoglobulinemia E syndrome with recurrent infections

The hyper-IgE syndrome with recurrent infections (HIESRI) is characterized by skin and respiratory infections due to Staphylococcus aureus and several fungi infections which are frequently associated with tissue damage. A deficiency in the chemotaxis of phagocytic cells has been documented to explain these findings; however, the expression of adhesion molecules, the secretion of cytokines that activate granulocytes and the production of oxygen reactive molecules have not been evaluated in HIESRI. Six HIESRI patients were evaluated for the following parameters: (1) secretion of GM-CSF and IL-5 by mitogen and antigen-activated mononuclear cells, (2) the chemotactic response of FMLP-activated granulocytes, (3) the respiratory burst of PMA-activated granulocytes, and (4) the expression of L-selectin and CD11b in PMA-activated granulocytes. Human recombinant GM-CSF and culture supernatants were evaluated for capacity to modulate granulocytic function. Compared to controls, HIESRI patients showed a normal production of GM-CSF and an increase in the basal secretion of IL-5. No significant differences were observed for chemotaxis, respiratory burst or L-selectin and CD11b expression. The GM-CSF did not modulate these functions in granulocytes from HIESRI patients, but culture supernatants applied to granulocytes inhibited chemotaxis, increased respiratory burst and caused the shedding of L-selectin from the granulocyte surface. The 6 HIESRI patients were nonsymptomatic during the time of this research due to a program of continued treatment; findings suggest that granulocytes are activated more easily in response to proinflammatory factors and that production of these factors is higher in HIESRI.     

Frozen fungi: cryogenic storage is an effective method to store Fusarium cultures for the long‐term

Microbial culture collections provide a vast amount of genotypic and phenotypic information which are invaluable resources for future advancements in research. For most microbial strains, cryopreservation in the vapour phase above liquid nitrogen provides the most stable and long‐term storage method. However, in the case of fungal microbes, not all are suited for cryogenic storage and few studies have addressed the effectiveness of storage in the vapour phase above liquid nitrogen on a diverse collection of Fusarium species. In this work, a collection of 374 Fusarium strains from the Fungal Genetics Stock Center, including 24 unique species, were duplicated and sent to the National Laboratory for Genetic Resource Preservation for storage in the vapour phase above liquid nitrogen. After 5 years of storage the entire collection was tested for viability and phenotypic stability by using plating, cellular staining assays, assessing the number of viable cells and measuring the rate of growth of each isolate. Additionally, the rate of growth for ～10% of the isolates were compared with the same isolates which had been stored at ?80°C at the Fungal Genetics Stock Center over the same timeframe to determine if cryopreservation in liquid nitrogen vapour provided a comparable method of storage. All National Laboratory for Genetic Resources Preservation isolates grew after being stored at ?165°C for 5 years. In general, the isolates that were stored at ?165°C grew at a faster rate than the isolates stored at ?80°C for the same period. Of the isolates stored at ?165°C, most had greater than 80% cell viability, however, those isolates that had less than 50% cell viability generally also had fewer conidia germinate. These isolates may be at a greater risk for storage over longer times. In conclusion, storage at ?165°C liquid nitrogen provided reliable preservation of a diverse collection of Fusarium spp. over 5 years, and culture viability data indicates that they will remain viable during additional storage for longer periods.     

Cryopreservation of bovine hemopoietic progenitor cells in liquid nitrogen

Bovine marrow granulocyte/macrophage and erythroid progenitor cells maintained viability after storage in liquid nitrogen for 2 to 4 weeks. The granulocyte/macrophage progenitor cells maintained 100% viability for 4 weeks, while the erythroid progenitor cells maintained 100% viability for at least 2 weeks. The optimum concentration of either DMSO or glycerol was found to be 5–10%. DMSO was superior to glycerol as a cryopreservative of bovine granulocyte/ macrophage progenitor cells. Glycerol was found to be unable to cryopreserve bovine erythroid progenitor cells.     

Chemotaxis toward amino acids inBacillus subtilis

Quantitative measurement of positive chemotaxis inBacillus subtilis was performed by means of adaption of the procedure used in studies withEscherichia coli. The motility ofB. subtilis was optimal in the presence of an exogeneous energy source and a nonionic detergent,e. g. Tween 80 or Brij-36. B. subtilis is chemotactic toward the commonly occurringL-amino acids except arginine, lysine, aspartate and glutamate. No chemotactic response was observed towardD-amino acids. Threshold, optimal response and peak concentration were determined. Chemotaxis toward glutamine was optimal at pH 6-7 and a temperature of 32°C. The maximum response toward a particular attractant was presumably influenced by the aerotactic behavior ofB. subtilis.     

Safety of Bacillus thuringiensis for earthworms.

In vitro chemotaxis by invertebrate hemocytes is demonstrated by the attraction of granulocytes from the operculate snail Viviparus malleatus to heat-killed Staphylococcus aureus and to N-acetyl-d-glucosamine. A soluble constituent of the hemolymph, a bacterial agglutinin, was necessary for this positive response to both of these chemotactic agents. Agglutination studies revealed the presence of two nonhomologous agglutinins in the hemolymph of V. malleatus.     

Cellular changes in bone marrow of malaria-infected mice. III. Chemotaxis of granulocytes

The number of bone marrow cells and their chemotactic activity was studied during malaria infection. Two days after infection of Balb/c mice with Plasmodium berghei, an increase in granulocyte number was observed in the blood. A modified Boyden chamber chemotaxis assay was employed to investigate the mechanism of granulocyte accumulation in the blood. Bone marrow cells from normal mice, from mice during a primary lethal infection and from immune mice after challenge were compared. The complement factor C5a showed chemotactic activity for bone marrow cells; a significant decrease of chemotaxis was only observed after 6 days of primary infection. Extracts of spleen, liver and infected erythrocytes lacked chemotactic activity, or caused inhibition of cell migration. Serum from mice with a 2-day primary infection contained chemotactic activity. The active component was heat labile, protease sensitive and had an estimated molecular weight of 250,000.     

A subpopulation analysis of f-MLP stimulated granulocytes migrating in filters

Leukocyte migration in vitro has been studied extensively during many years without providing satisfactory theoretical models for the different migratory behaviors (chemotaxis and chemokinesis) of leukocyte populations. The present study utilized the fluid gradient chamber, which is a new method to study leukocyte migration in filters. Human neutrophils were applied between two stacked filters and migrated in all directions under the influence of constant concentrations or chemotactic gradients of f-MLP, maintained in fluid phase density gradients. The distributions of the granulocytes over filter depth were fitted to theoretical functions composed by 1–3 Gaussian distributions, representing subpopulations. The results showed that the neutrophils migrated as two discrete subpopulations during chemokinetic stimulation (a constant concentration of f-MLP). One of the subpopulations showed less active and passive (slow sedimentation under the influence of gravity) translocation. The most mobile subpopulation was divided into two new subpopulations when exposed to chemotactic stimulation (concentration gradient of f-MLP), one of which responded chemotactically and one of which migrated in random directions. The properties of the different subpopulations where characterized in terms of diffusion coefficient (random migration), convection velocity (chemotactic migration) and sedimentation coefficient (passive translocation).     

Cryopreservation of human granulocytes.

Granulocyte preservation was undertaken using hydroxyethylstarch for both sedimentation of red cells and cryopreservation of buffy coat white cells from CPD whole blood. Buffy coats were mixed with HES to a final concentration of 4% (w/v) and hematocrit of 30%, and sedimented in inverted plastic syringes. The leukocyte enriched (100–500×) supernatant was frozen at 2.0 °C/min to ?80 °C (and stored frozen up to 3 months). Alternatively, sedimented leukocytes were frozen after a slow addition of 10% DMSO to 5%. Tubes were thawed at 37 °C, and DMSO was removed by dilution with Hank's solution containing CPD and centrifugation. The pellets of granulocytes were resuspended in Normosol.Buffy coat from 10 units yielded 60 ± 9.7% of the available whole blood leukocytes, of which 43 ± 14% were recovered after sedimentation in HES. Freezing in DMSO yielded all, 101% of the prefrozen leukocytes. Postthawed viability of granulocytes was estimated morphologically and by their ability to inhibit the rate of growth of E. coli. Complete inhibition was observed at a ratio of one E. coli to one granulocyte. Postthawed granulocytes were characterized by high myeloperoxidase activity and exclusion of trypan blue. Approximately 25% of the total available granulocytes in CPD whole blood were recovered.