Humoral Na+-K+ pump inhibitory activity in essential hypertension and in normotensive subjects after acute volume expansion.

Plasma from black male patients with essential hypertension was bioassayed for vascular Na+-K+ pump inhibitory activity. Halves of the same rat tail artery were incubated for two hours in boiled plasma supernates from a hypertensive patient and a paired age-, sex-, and race-matched normotensive subject and then ouabain-sensitive 86Rb uptake was measured. Ouabain-sensitive 86Rb uptake by their leukocytes was also measured. Eighteen pairs of subjects were studied. The uptakes were not significantly different in the hypertensive patients and control subjects. However, when we selected from the eighteen hypertensive patients, nine with low plasma renin activity on the day of the study, uptakes were reduced in the hypertensive patients relative to the paired control subjects. We also assayed plasma supernates from normotensive black and white male subjects before and after acute volume expansion (2.5 L saline IV + 1.5 L distilled water orally over a three-hour period) and from paired normotensive subjects before and after sham volume expansion and obtained a positive bioassay in the expanded subjects both on intraindividual and interindividual comparisons. These studies demonstrate increased vascular Na+-K+ pump inhibitory activity in the plasma of black male patients with low renin essential hypertension and in the plasma of normotensive subjects after acute volume expansion. The findings suggest that the inhibitory activity in the hypertensive subjects' plasma is related to volume expansion, relative or absolute.

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

The use of granulocyte-rich concentrates from leukaphresis purified by counterflow centrifugation--elutriation to obtain pure granulocytes for transfusion studies in cyclophosphamide-induced neutropenic animal models is reported. Our data for granulocyte-rich leukapheresis concentrates indicate that room temperature (20 degrees C) appears to be preferred to 6 degrees 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 degrees 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 degrees C. The results of these studies are four-fold. 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 degrees C are severely impaired in terms of their in vivo chemotactic function but display no loss of in vitro efficacy. Third, 20 degrees C storage of granulocyte-rich leukapheresis concentrates for 18 to 24 hr is superior to 6 degrees 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.

Bone marrow reconstitution of lethally irradiated canines using autologous bone marrow fractions obtained by counterflow centrifugation-elutriation.

Canine bone marrow fractionated by counterflow centrifugation-elutriation results in three areas of nucleated cell recovery. Fraction 1 accounts for 50% of the total nucleated cells and 25-40% of the total recovered CFU-GM activity. Fraction 2 contains less than 2% of the total nucleated cells and less than 0.2% of the CFU-GM activity. Fraction 3 accounts for approximately 50% of the total nucleated cell recovery and 60-75% of the total recovered CFU-GM activity. Animal survival was not directly correlated with the levels of CFU-GM activity in the transfused fractions. Autologous infusion of these fractions into irradiated canines (9 Gy, 0.1 Gy/min) resulted in distinct survival profiles. Canines receiving autologous fraction-2 cells showed no haematological reconstitution, with death occurring on days 10-11 post-irradiation. Canines receiving autologous fraction-3 cells showed limited myeloid repopulation of both the bone marrow and peripheral blood with a mean survival time for 24 d. Canines receiving autologous fraction-1 cells showed complete haematological reconstitution after 48 d and long-term survival. The data may indicate a separation or enrichment of pluripotential stem cells (fraction 1) from committed myeloid progenitor cells (fraction 3).

Fractionation of mammalian bone marrow by counterflow centrifugation-elutriation using a continuous albumin gradient: analysis of granulocyte-macrophage colony-forming units.

Bone marrow cells from murine, canine, primate and human donors were fractionated by counterflow centrifugation-elutriation (CCE) using a continuous albumin gradient. Fractionation of 9 x 10(8) nucleated bone marrow cells (without prior removal of the erythrocytes) can be done in less than 1.5 h with 92% nucleated cell recovery, depending on donor species. Murine bone marrow cell recovery after fractionation averaged 52% whereas cell recoveries for canine, primate and human donors averaged 85%, 92% and 89%, respectively. The fractions were evaluated for total nucleated cell counts and granulocyte-macrophage colony-forming units (GM-CFC). Each species studied presented a unique profile for nucleated cell recovery and associated GM-CFC activity. These profile variations may suggest significant differences in the modes of GM-CFC expression in the individual species that may depend on cell-to-cell interactions. Although CCE is unable to isolate a single population responsible for GM-CFC activity, CCE does permit a rapid, reproducible fractionation of a large numbers of cells with minimal manipulation of sample. This makes the isolated sample ideal for further purification of progenitor cell populations

Human blood monocytes: characterization of negatively selected human monocytes and their suspension cell culture derivatives.

Normal human monocytes were negatively selected from leucapheresis cell suspensions by countercurrent centrifugation-elutriation in high yield with a mean purity of 93.5%. The combination of the novel methods of negative cell selection and suspension cell culture has provided the opportunity to study serially over several days the morphologic and functional changes of monocytes from a single donor as they matured in culture to typical macrophages. Human monocytes nearly double in size during the first week of culture, experiencing near daily increases in cell volume. This was associated with changes in the ultrastructure of these cells, including the development of numerous small knob-like projections on the cell membrane and the proliferation of microtubules and filamentous structures within the cell cytoplasm during the first 6 days of culture. Peroxidase activity declined during the first 4 days of culture, whereas 5'-nucleotidase activity was acquired during the first 48 h of culture. Lysozyme activity in the cultures increased form day 2 to day 6 of culture. The phagocytic capacity of monocytes for igG-coated erythrocytes increased dramatically during the first week of culture, but the cytotoxic capability of monocytes against similar targets in an antibody-dependent cytotoxicity assay declined to nearly half of base-line levels by day 2 of culture and remained at this diminished level during subsequent days of culture.

Experimental Hematology Department, Armed Forces Radiobiology Research Institute, Bethesda, Maryland.

Granulocytes from dogs obtained by continuous-flow centrifugation leukapheresis were purified by counterflow centrifugation-elutriation using a modified rotor and enlarged separation chamber. The separation chamber has a threefold increase in volume capacity, as compared to the commercial Beckman Instruments' separation chamber, and the quantity and purity of the granulocytes recovered are suitable for purified granulocyte transfusion studies in a 10-kg canine animal model. Transfusion of these cells into cyclophosphamide-induced neutropenic animals permits an analysis of granulocyte chemotactic ability in terms of migration into skin chambers filled with endotoxin-activated serum. The purified granulocyte function in vivo was compared to the activity of granulocytes present in leukapheresis concentrates. The data show that transfused granulocytes isolated by counterflow centrifugation-elutriation from leukapheresis concentrates retain identical in vivo chemotactic activity, as compared to granulocytes present in transfused leukapheresis concentrates.

Effect of plastic containers on liquid preservation of human granulocytes. I. With and without deoxyribonuclease and hydrocortisone.

We have characterized the effects of four types of plastic polymer containers--polyvinyl chloride (PL-146), bioriented-biaxial polyolefin, ethylene-vinyl acetate, and polypropylene--on the in vitro recovery, viability, membrane integrity, and phagocytosis of human granulocytes (PMNL). Cells were collected by continuous-flow centrifugation leukapheresis (CFCL), then further isolated by counterflow centrifugation-elutriation (CCE), and stored at 4 to 6 C for up to 14 days at concentration of 6 X 10(6) PMNL/ml in medium with or without hydrocortisone (HC) or deoxyribonuclease (DNAase). Regardless of the containers, there were no significant differences observed in the responses of PMNL within the first 48 hours of storage. However, PMNL stored in the polyvinyl chloride (PL-146) containers either with or without HC and DNAase maintained a significant higher viability and slower mean cell volume (MCV) expansion rate after 48 to 96 hours compared with that stored in other containers. The phagocytic response was comparable in surviving PMNL from all containers during the first seven days. Addition of HC and DNAase to the storage medium reduced the rate of PMNL volume expansion in all containers, improved the viability in all but the polypropylene containers, but had no effect on the phagocytic response. Since the results show a strong correlation between the rate of PMNL volume expansion during storage and subsequent loss of PMNL viability and function, the containers made with PL-146 polyvinyl chloride are superior to those made of bioriented-biaxial polyolefin, ethylene-vinyl acetate, or polypropylene.

Human granulocyte isolation by continuous flow centrifugation leukapheresis and counterflow centrifugation elutriation (CFCL/CCE).

Human granulocytes (PMNL) isolated by continuous flow centrifugation leukapheresis (CFCL) were further purified by counterflow centrifugation elutriation (CCE). Studies were conducted to determine the maximal granulocyte capacity of the 4.5-ml elutriation chamber and to determine the efficiency of granulocyte recovery from CFCL concentrates. The maximal capacity of the elutriation chamber was determined to be 1.30 x 10(9) granulocytes with a 98.8 percent granulocyte purity and a 51:1 PMNL/RBC ratio. The efficiency of recovery was 82.2 percent with a 95.1 percent granulocyte purity and a 68:1 PMNL/RBC ratio. The CCE flow system was designed to allow continuous elutriation runs without need of breakdown time between runs. In four to five hours, approximately 4 to 5 x 10(9) granulocytes can be isolated with purity in excess of 95 percent. In vitro analysis of the viability and function of CFCL/CCE-isolated granulocytes indicates that the morphologic integrity and physiologic function were not comprised as a result of the combined isolation procedure relative to granulocytes isolated by CCE from whole blood samples. Granulocytes were held at 4 to 6 C for 16 to 24 hours in granulocyte storage medium (GSM) plus 20 percent autologous plasma at concentrations of about 6 x 10(6) PMNL/ml prior to in vitro analysis.

Technique for increased granulocyte recovery from human whole blood by counterflow centrifugation elutriation I. In vitro analyses.

Human granulocytes were isolated from 120 ml of whole blood by a modified counterflow centrifugation-elutriation (CCE) technique. Overall recovery of isolated granulocytes averaged 2.82 +/- 0.25 x 10(8) cells or 77 per cent yield from whole blood with 96 per cent granulocyte purity and 4 per cent mononuclear leukocytes. The granulocyte fraction was assayed in vitro to determine chemotactic response, stimulated oxygen consumption in the presence of latex beads, bactericidal capacity, and enzyme activities. Cellular integrity was determined by scanning and transmission electron microscopy as well as by cell volume analysis. The data suggest that granulocytes isolated by CCE suffered no discernible loss of function or morphologic damage. The granulocytes are free of platelets and most mononuclear leukocytes and erythrocytes, and have not been exposed to sedimenting agents or surface adhesive agents such as dextran, nylon or glass wool.

Liquid preservation of canine granulocytes obtained by counterflow centrifugation-elutriation.

Granulocytes (PMN) were isolated from 120 ml of canine whole blood by a modification of the counterflow centrifugation-elutriation technique. Isolated cell suspensions of 96% granulocytes and 4% mononuclear leukocytes with a 21:1 PMN/RBC ratio were stored at 4 degrees C in 4:4:2 medium consisting of four parts HBSS minus Ca++ and Mg++, four parts MEM, twp parts autologous plasma, and 20 microgram/ml gentamicin for 15 days. Granulocytes were stored at concentrations of approximately 4 x 10(6) PMN/ml in polypropylene centrifuge tubes. The stored granulocyte suspensions were assayed in vitro 0, 1, 4, 8, and 15 days to monitor chemotaxis, bacterial growth inhibition, O2 consumption associated with phagocytosis, and enzyme activities. Cell volume analysis was used to evaluate cellular integrity of the liquid-stored granulocytes. Canine granulocytes isolated by the modified dilution technique of counterflow centrifugation-elutriation can be preserved for up to 15 days with 77 +/- 6% granulocyte survival with maintenance of morphological and organelle integrity, as well as retention of in vitro functions of recognition, migration, phagocytosis, and killing of gram-negative bacteria.

Granulocyte isolation by counterflow centrifugation-elutriation of canine blood obtained by continuous-flow centrifugation leukapheresis.

Granulocytes have been isolated by counterflow centrifugation-elutriation (CCE) from canine leukocyte-rich blood obtained by continuous-flow centrifugation leukapheresis (CFCL). We have attempted to define both the maximal granulocyte recovery and the efficiency of granulocyte purification for the Beckman JE6 elutriation rotor when large volumes of leukocyte rich blood are utilized. The efficiency of granulocyte purification by CCE is 81% (1.31 +/- 0.1 x 10(9) PMNL) if the number of granulocytes entered into the Beckman JE-6 rotor as leukocyte rich blood is limited to 1.60 +/- 0.13 x 10(9) PMNL. Approximately 96 +/- 2% of the leukocytes in the purified fraction were of the granulocytic series with mononuclear leukocytes comprising the residual 4% of the cell population. In vitro analysis of the isolated granulocytes indicated that the cells did not lose their morphological integrity or physiological function as a result of the dual CFCL/CCE procedure relative to granulocytes isolated by CCE from freshly drawn peripheral blood.

Improved technique for increased granulocyte recovery from canine whole blood samples by counterflow centrifugation-elutriation. I. In vitro analysis.

Granulocytes were isolated from 120 ml of canine whole blood by a modification in counterflow centrifugation-elutriation technique. The leukocytes were concentrated in a buffy coat fraction and diluted to a fixed RBC density prior to entry into the rotor. Overall recovery of granulocytes from canine whole blood averaged 82% with 96% purity from contaminating mononuclear leukocytes. The purified cellular fraction was assayed in vitro to monitor O2 consumption, chemotaxis, bacterial growth inhibition, and enzyme activities. Cellular integrity was monitored by scanning and transmission electron microscopy as well as by cell volume analysis. The data suggest that the granulocytes isolated by counterflow centrifugation-elutriation suffered no noticeable morphological damage or loss of function in terms of recognition of a toxic agent, migration, phagocytosis, or bactericidal capacity. Granulocytes isolated by counterflow centrifugation-elutriation may be physiologically more active than cells obtained by filtration leukapheresis, continuous-flow centrifugal leukapheresis or discontinuous flow centrifugation both in vitro and in vivo.