Polymorphonuclear leukocyte accumulation in brain regions with low blood flow during the early postischemic period.

In an anesthetized canine model in which ischemia was induced by incremental air embolism, 16 animals were exposed to 1 hr of ischemia and monitored for 10 min (n = 4), 60 min (n = 6), or 240 min (n = 6). Fourteen animals were observed for corresponding periods without being subjected to ischemia 70 min (n = 4), 120 min (n = 4), or 300 min (n = 6). Autologous granulocytes were labeled with 111In and reinfused just before ischemia. At the conclusion of each experiment, a 14C-iodoantipyrine autoradiographic blood flow study was performed. Granulocyte accumulation measured by gamma scintigraphy (cpm/gm) occurred in the injured hemisphere of ischemic animals at 60 min in anterior brain segments and at 240 min in anterior, middle, and posterior segments. By means of a double-label autoradiography technique, clustering of punctate granulocyte images was detected in regions of low flow or heterogeneous flow in half of the animals at both 60 min and 240 min postischemia. Granulocyte clustering did not occur in the autoradiograms of nonischemic animals. The results implicate granulocyte participation in the acute phase of ischemic brain injury and signal a convergence of hemostatic and inflammatory processes during the immediate postischemic period.

Horse red blood cells frozen with 20% (w/v) glycerol and stored at -150 C for five years.

When equine RBC were frozen with 20% (w/v) glycerol and stored at -150 C for as long as 5 years, there were no adverse effects on freeze-thaw or freeze-thaw-wash recovery or oxygen transport function. The manner in which the glycerol was added to, and removed from, the equine RBC was shown to be an important consideration in ensuring optimal freeze-thaw-wash recovery values.

Rhesus macaque red blood cells frozen with 40% glycerol and stored at -80 C.

Red blood cells of rhesus macaques cryopreserved with 40% (w/v) glycerol and stored at -80 C had freeze-thaw-wash recovery values of 87%, 24-hour posttransfusion survival values of 85%, and life-span values of 13 days. Liquid and freezing methods of preserving RBC were studied in the macaques.

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).

Freeze-preserved baboon red blood cells: effects of biochemical modification and perfusion in vitro.

Nonrejuvenated and rejuvenated baboon RBC were freeze-preserved with 40% (w/v) glycerol at -80 C. To prepare rejuvenated RBC, a 50-ml solution containing pyruvate, inosine, glucose, phosphate, and adenine was used and RBC were incubated with this solution before glycerolization and freezing. Appropriate steps were taken to minimize osmotic damage to the RBC during glycerolization and deglycerolization. Nonrejuvenated and rejuvenated cryopreserved RBC had freeze-thaw recovery values of 98%, freeze-thaw-wash recovery values of 92%, and 24-hour post-transfusion survival values of 85%. Some units of cryopreserved RBC were autotransfused after thawing, washing, and storage at 4 C for 24 hours. Other units were perfused in vitro before autotransfusion. After 24 hours of postwash storage, the RBC were concentrated by centrifugation and suspended in a plasma protein fraction and/or an electrolyte solution, and then were exposed to extracorporeal perfusion. Serious adverse effects were not observed on posttransfusion survival, function, or hemolysis in nonrejuvenated or rejuvenated baboon RBC as a result of perfusion in vitro.

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.

Liquid preservation of baboon red blood cells in acid-citrate-dextrose or citrate-phosphate-dextrose anticoagulant: effects of washing liquid-stored red blood cells.

Autologous baboon RBC stored at 4 C in acid-citrate-dextrose (ACD) or in citrate-phosphate-dextrose (CPD) for 3 weeks after collection had 24-hour 51Cr posttransfusion survival values of about 77%. When 20-day-old ACD and CPD baboon RBC were washed and then stored at 4 C for 24 hours before autotransfusion, the 24-hour 51Cr posttransfusion survival values were about 81%. These values were similar to those seen in studies of human RBC preserved in an identical manner. Our results indicated that the baboon can be used to evaluate RBC preservation techniques before human volunteers are studied.

Measurement of red blood cell volume, plasma volume, and total blood volume in baboons.

Red blood cell volume was measured directly in baboons by infusion of 51Cr-labeled autologous RBC, and was indirectly estimated from the plasma volume measured with 125I-labeled albumin and the total body hematocrit. The total body hematocrit was calculated from the peripheral venous hematocrit multiplied by a correction factor; for nonanemic baboons the correction factor was 0.87, and for anemic baboons, 0.75. Within 2 weeks after the phlebotomy (150 ml of blood), the baboon's RBC volume was restored to normal. Posttransfusion survival of baboon RBC can be measured accurately in nonanemic baboons; the preserved RBC can be labeled with 51Cr and the RBC volume of the baboon can be measured indirectly from the plasma volume measured with 125I-labeled albumin and the total body hematocrit.

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.

Liquid and freeze-preservation of dog red blood cells.

After storage in the liquid state at 4 C for up to three weeks, washing with sodium chloride solutions, and storage in a sodium chloride-glucose-phosphate solution for 24 hours at 4 C, dog red blood cells had excellent post-transfusion survival. After freeze-preservation with 40% W/V glycerol at -80 C or with 20% W/V glycerol at -150 C, thawing, washing with sodium chloride solutions, and storage in a sodium chloride-glucose-phosphate solution for 24 hours at 4 C, dog red blood cells had satisfactory recovery values in vitro, acceptable 24-hour post-transfusion survival and long-term survival values, and normal oxygen transport function. Controlled addition and removal of the cryoprotectant, glycerol, helped reduce the amount of osmotic damage to the red blood cells and enhanced freeze-preservation. Osmotic damage can also be prevented by warming the dog blood to a temperature of 22 +/- 2 C prior to centrifugation to concentrate the red blood cells and remove the plasma. This step enhances removal of the cold agglutinins. Another processing step used by the authors was to add a sodium chloride solution to the dog red blood cells before adding the glycerol solution in order to eliminate rouleaux formation.

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.

Occurrence of HBsAg, anti-HBs, the anti-CMV following the transfusion of blood products.

A retrospective study of the serum of 104 patients treated at Chelsea Naval Hospital between 1969 and 1972 was done. The donor blood products were not tested for the hepatitis B antigen before transfusion. The incidence of hepatitis B antigen following transfusion was about 2.8 per cent. The incidence of antibody to HBsAg prior to transfusion was 16 per cent, and about 27 per cent of the patients developed antibody to HBsAg following transfusion. The incidence of antibody to cytomegalovirus was about 22 per cent before transfusion, and 22 per cent of the patients developed complement fixing antibody against cytomegalovirus after transfusion. Since the patients received a variety of blood products it was not possible to determine retrsopectively which product, if any, produced the lowest incidence of hepatitis B antigen and transmission of cytomegalovirus.

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.