Human blood cells at microgravity: the NASA Initial Blood Storage Experiment.

The Initial Blood Storage Experiment (IBSE) probed the behavior of human red cells, white cells, and platelets during exposure to microgravity for 6 days and 2 hours on a National Aeronautics and Space Administration (NASA) shuttle mission, named STS 61-C, which was launched on January 12, 1986. IBSE involved carefully controlled comparisons between two identical sets of blood cells, one exposed to microgravity and the other held on the ground. Specially designed and fabricated, electrically powered environmental chambers provided appropriate environmental temperatures and air flow to support cell metabolism throughout the experiment. To circumvent the need for constant agitation of platelets during storage, a new thin-layer compression method for platelet preservation was developed. Blood cell samples were allocated to the two arms of the experiment, microgravity and earth gravity, by blind assignment. Moreover, to ensure unbiased assessment of the experiment's findings, postexperiment samples for measurement were identified by code. To optimize the chances of detecting possible gravitational effects, a wide array of measurements of cellular function, morphology, metabolism, and immunology were made. Analysis of variance was used in analyzing the data. The most striking finding was that platelets displayed markedly superior structural and functional integrity at microgravity. Granulocytes held on the ground were preserved slightly better than those that orbited in the shuttle, whereas red cells displayed few effects that were attributable to the gravitational variable. Polyvinylchloride-di-(2-ethylhexyl)phthalate (PVC-DEHP) was the plastic of choice for storage of red cells, while PVC-trioctyltrimellitate (TOTM) was superior to PVC-DEHP and polyolefin (PO) for platelets.

The role of transmembrane cationic gradients in immune complex stimulation of human polymorphonuclear leukocytes.

The role of monovalent cationic gradients in human polymorphonuclear leukocyte (PMNL) stimulation was investigated by monitoring immune complex-stimulated transmembrane depolarization and superoxide production, events which accompany--and have been used as indicators of --PMNL activation. Abolishing only the Na+ gradient by substitution of choline for extracellular Na+ did not affect the resting membrane potential but reduced the rate of stimulus-induced transmembrane depolarization to 50% of control. In contrast, collapsing both Na+ and K+ gradients by suspension in K+ buffer (high K-PRK) depolarized the cells and reduced the stimulus-induced rate of depolarization to 11% of control. Pretreatment of cells suspended in Na+ buffers with 5-(N,N-dimethyl)amiloride hydrochloride (DMA) or with valinomycin reduced by one-half the rate of immune complex induced membrane depolarization. Conversely, in the absence of either or of both Na+ or K+ gradients, or in the presence of valinomycin, immune complex elicited an enhanced rate of superoxide production. However, PMNL prepared via NH4Cl (NH4Cl-PMNL) instead of H2O (H2O-PMNL) lysis of residual red blood cells exhibited an absolute requirement for an intact Na+ gradient in cell stimulation. The present results thus demonstrate that: 1) both Na+ and K+ gradients participate equally in the membrane depolarization elicited by immune complex; 2) neither a Na+ or a K+ gradient is required for immune complex activation, or for activity of the respiratory burst; and 3) an artifactual requirement for an intact Na+ gradient occurs in neutrophils prepared by the NH4Cl lysis technique.

Isolation and cryopreservation of monocytes from plateletapheresis cellular residues.

Human monocytes were isolated from the cellular residues remaining after plateletapheresis of donors using an automated blood cell processor. Mononuclear cells were obtained with density gradients and separated from lymphocytes by stepwise elutriation. The isolated cells were frozen using extracellular hydroxyethyl starch and intracellular dimethylsulfoxide. In three procedures, approximately 1 X 10(9) monocytes were obtained. Ninety-nine percent of isolated monocytes were viable in a fluorescein diacetate (FDA)-ethidium bromide (EB) test. Myeloperoxidase-positive cells were 95 percent and 90 percent in the two chambers. Ninety-four percent of monocytes ingested five or more opsonized polycyclic hydrocarbon particles and 95 percent ingested one or more ethidium-treated zymosan particles. After storage in liquid nitrogen for up to 9 weeks, 99 percent of the cells were recovered after thawing. Of these, 95 percent were myeloperoxidase-positive, 94 percent showed intact membranes in the FDA-EB test, 95 percent ingested five or more opsonized polycyclic hydrocarbon particles, and 96 percent ingested one or more ethidium-treated zymosan particles. These results demonstrate the utility of elutriation as a means to isolate large numbers of monocytes. The isolated cells can be cryogenically preserved for at least 2 months with small loss of phagocytic function.

Alternative complement pathway-dependent ingestion of fluolite particles by human granulocytes.

Fluorescent particles (Fluolite) with an average size of 0.1 micrometers were ingested by human granulocytes after incubation in fresh normal human serum (NHS). Ingestion was assessed by visual counting in a fluorescent microscope of cells containing particles. Ingestion required fresh normal serum and did not occur when serum was heated for 30 min at 50 degrees C or in the presence of ethylenediaminetetraacetic acid (EDTA). It did not occur in serum genetically deficient in C3b inactivator or in C3. Phagocytic activity was restored to C3-deficient serum by purified human C3 and to heat inactivated serum by purified factor B. Opsonic activity was present in NHS containing 5 mM Mg++ and 10 mM ethyleneglycoltetraacetic acid (EGTA) and in human serum genetically deficient in human C components C2 and C5. Agammaglobulinemic sera had normal opsonic activity. Opsonization of particles in this system is mediated through the alternative pathway of C activation, and its measurement serves as a simple quantitative functional assay for this system.

Temperature effects on shape and function of human granulocytes.

Scanning microscopic and functional studies were made of granulocytes isolated from CPD anticoagulated whole blood by counterflow centrifugation in a Beckman JE-6 rotor. The collection buffer was phosphate (20 mM) buffered saline (280 mOsM) with glucose (29 mM) and human serum albumin (1.2% w/v). The final suspension contained less than 2% mononuclear cells and 5% red cells. Incubation and fixation at various temperatures revealed two distinct temperature dependent shape transformations. At 22, 37, 40 and 45 degrees C granulocytes were ameboid with extensive highly textured veils. These smoothed progressively, bullae and blebs formed, and membranes peeled finally leaving nonfunctional spheres with smooth surfaces. At 4 degrees C, granulocytes were irregular spheres, less rugose but with numerous microvilli and nodules. Veiling was absent. Phagocytosis, initially low, progressively declined over 48 h while cell surfaces become smooth. Some formed blebs, but all terminated as nonfunctional spheres with untextured surfaces containing occasional large single holes. Cellular stability estimated from changes in volume distributions, and phagocytosis by microfluorescence measurements of yeast and latex particle ingestion were also temperature dependent and paralleled the shape progressions. It is concluded that at body (37 degrees C) or fever (40 degrees C) temperatures, granulocytes have dynamic membrane surfaces characterized by extensive veiling and high function. At 4 degrees C they are relatively inactive spheres devoid of pseudopodia or veils, yet functional at slow rates.

Preservation of human granulocytes. III. Liquid preservation studied by electronic sizing.

Human granulocytes were isolated from blood either by counterflow centrifugation in a Beckman JE-6 rotor or by sedimentation of the red blood cells with dextran and centrifugation of the granulocyte-rich plasma. The stability of the granulocytes was assessed during storage in the liquid state by measurements of granulocyte loss, volume distribution characteristics, and ability to produce fluorescein in their cytoplasm and to exclude ethidium bromide from their nuclei. After storage at 4 C for 2 days in phosphate-buffered saline with a pH of 7.1 containing 5 g/dl human albumin + 0.46 g/dl dextrose or 1 g/dl Physiogel + 0.46 g/dl dextrose, the granulocytes were adequately preserved from the in vitro measurements.