The effect of temperature on cellular reactions of Crassostrea virginica to the injection of avian erythrocytes

Intracardial injection of chicken red blood cells (RBC) into the oyster, Crassostrea virginica, at 15–19°C elicited ingestion, digestion, and removal of the cells by the leukocytes. A rapid response of fibroblastlike cells and characteristic cell aggregates were also detected as early as 1 hr post-injection. While the former was engaged in an unsuccessful attempt to encapsulate red blood cell laden leukocytes intravascularly, the function of the latter cell type was undetermined. The process of disposing the red blood cells in the oysters under running seawater conditions was not noticeably different from that observed in the oysters under standing water conditions.In oysters previously sensitized with red blood cells, response to a challenge injection was similar to that observed following the first injection, and there was no indication that the activities of the leukocytes were enhanced. However, it was demonstrated that oyster leukocytes remained functionally viable for at least 22 days. There is evidence indicating that the number of the characteristic cell aggregates increases considerably after a secondary injection of red blood cells, although the specificity of this response is yet to be determined.At 6°C, although phagocytosis of the blood cells occurred quite extensively, leukocytes exhibited considerably low digestive and migratory activity which resulted in far less effective disposal of the blood cells. Preliminary observations also revealed possible adhesion of oyster leukocytes to the wall of blood vessels following the inoculation at this temperature.     

Peripheral blood admixture in bone marrow aspirates

A simple method is described by which the degree to which a bone marrow aspirate is diluted with peripheral blood, the admixture of peripheral blood leukocytes in the aspirate, and the true cellularity of bone marrow at the aspiration site can be estimated quantitatively. A highly significant correlation is shown to exist between the true bone marrow cellularity and the number of hemopoietic cells in the bone marrow aspirate. This correlation can be described by an exponential regression equation (r = 0.6997; p less than or equal to 0.001). The degree of bone marrow dilution with peripheral blood does not depend on the initial bone marrow cellularity and is devoid of diagnostic value. It fluctuates over a wide range (1.2- to 6.3-fold). The admixture of peripheral blood leukocytes has been found to be small (0.05%-9.7%) in most cases studied and much higher in those cases where the cellularity is low while the dilution is high.     

Rapid detection of malaria and other bloodstream parasites by fluorescence microscopy with 4'6 diamidino-2-phenylindole (DAPI).

DAPI is a fluorescent dye which appears to complex specifically with DNA. We have used this probe to detect and identify malarial infections by fluorescence microscopy. Experiments were conducted using Plasmodium berghei yoeli--infected mouse blood, P. lophurae--infected duck blood, and P. vivax--infected human blood. Infected avian blood was used to detect parasites within nucleated erythrocytes. Control blood smears from uninfected hosts revealed fluorescence only in the leukocytes of mammalian blood or in nuclei of leukocytes and erythrocytes of avian blood. Cytoplasmic staining of red blood cells was absent in all controls. In contrast, the cytoplasm of infected red blood cells was stippled with fluorescence centers. Ring forms, trophozoites, segmenters, and merozoites frequently were observed. This simple procedure can be applied directly to routine clinical analysis, as well as experimental procedures, DAPI can also be used to stain other parasites, including nuclei in microfilariae.     

The reorganization of the actin cytoskeleton of nuclear erythrocytes and leukocytes in fish, frogs and birds during cell migration

Changes in the spatial organization of actin filaments of nuclear erythrocytes and leukocytes during their migration in fish, frogs and birds have been studied by the method of confocal laser scanning microscopy. It has been shown that, during movement of cells, the reorganization of cytoskeleton microfilaments in erythrocytes is similar to that in leukocytes. During migration, red blood cells of amphibious and birds form pseudopodia filled with bunches in parallel laid actin filaments. Erythrocytes in fish do not form pseudopodia. Similar to leukocytes change in the structure of the actin cytoskeleton in nuclear erythrocytes determines the ability of red blood cells to reactions of migration and phagocytosis.     

Red blood cells augment leukocyte rolling in a virtual blood vessel

Leukocyte rolling and arrest on the vascular endothelium is a central event in normal and pathological immune responses. However, rigorous estimation of the fluid and surface forces involved in leukocyte-endothelial interactions has been difficult due to the particulate, non-Newtonian nature of blood. Here we present a Lattice-Boltzmann approach to quantify forces exerted on rolling leukocytes by red blood cells in a "virtual blood vessel." We report that the normal force imparted by erythrocytes is sufficient to increase leukocyte binding and that increases in tangential force and torque can promote rolling of previously adherent leukocytes. By simulating changes in hematocrit we show that a close "envelopment" of the leukocyte by the red blood cells is necessary to produce significant changes in the forces. This novel approach can be applied to a large number of biological and industrial problems involving the complex flow of particulate suspensions.     

Staining of rabbit eosinophil and pseudoeosinophil leukocytes.

Air-dried rabbit blood was stained by HE, PAS and a modification of the Undritz II method. Eosin stained granules red in the eosinophil leukocytes. PAS was negative and the modified Undritz method failed to give consistent results. Cells with eosinophilic granules appeared in the corneal stroma 1 h after removing the corneal epithelium. They were stained red consistently by both eosin and the modified Undritz II method. Electron micrographs failed to demonstrate crystalloids in the granules. Because of the staining characteristics and the lack of crystalloids in their granules these cells were classified as pseudoeosinophil leukocytes. The electron micrographs showed some glycogen 12 h after denuding the cornea, however, glycogen was not well stained by PAS until 18 h after denuding.     

Reorganization of actin cytoskeleton of nuclear erythrocytes and leukocytes in fish,frogs, and birds during migration

Using the method of confocal laser scanning microscopy, changes in the spatial organization of actin filaments of nuclear erythrocytes and leukocytes in fish, frogs, and birds during migration were studied. It has been shown that, during movement, in erythrocytes, like in leukocytes, reorganization of cytoskeleton microfilaments occurs. In the course of migration, in amphibians and birds, red blood cells form pseudopodia filled with bundles of actin filaments arranged in parallel, whereas no pseudopodia are formed in fish erythrocytes. The change in the structure of the actin cytoskeleton of nuclear erythrocytes, like in leukocytes, is responsible for the capability of red blood cells to undergo reactions of migration and phagocytosis.     

Improved flow cytometric method to enumerate residual cells: minimal linear detection limits for platelets,erythrocytes, and leukocytes

Increasing demand for quality control of blood products requires more sensitive methods to enumerate residual cells. Presently, the reported threshold (in cells per microliter) is 400 for red blood cells, 30-500 for platelets, and 1 for leukocytes. To examine precision and linearity in enumerating residual platelets and red blood cells, EDTA-anticoagulated blood from healthy donors was serially diluted with serum, stained in TruCount tubes using a no-lyse/no-wash procedure and a monoclonal antibody cocktail against the CD42a (FL1) and glycophorin-A (FL2) epitopes, and analyzed by flow cytometry. Leukocyte counts were determined in separate tubes. Cell preparation and analysis were performed once for 20 blood samples each and 20 times using the same specimen. Acquisition from the same tube was performed separately for platelets (threshold on FL1) and red blood cells (threshold on FL2). Multiparameter analysis was used for data evaluation. Linear results were obtained for platelets per microliter between 3,410 and 5 and for red blood cells per microliter between 54,000 and 3. For the lower cell concentrations, the coefficient of variation was 16.7% for platelets and 10.9% for red blood cells. The presented method allows the distinction between physiologically intact and ghost red blood cells. The method represents a reliable, sensitive, and accurate approach to quantify platelets and red blood cells in diluted blood. It can be applied to enumerate residual cells in plasma products and meets the increasing demand for quality control in blood components.     

The mechanism of removal of leukocytes by cellulose columns

Cellulose columns efficiently remove leukocytes from whole blood. Interaction of leukocytes with cellulose particles is not affected by glucose, galactose, fructose, mannose, or cellobiose. Although red cells normally pass through cellulose columns, they are retained after fixation in glutaraldehyde. We conclude that the leukocyte-removing activity of cellulose columns is due to mechanical filtration rather than to specific adherence of leukocytes to the cellulose particles.     

A Theory of Necrotaxis

A mathematical theory for the motion of polynuclear leukocytes after the laser irradiation of a red blood cell is derived and compared with the measured movement of a number of cells. An estimate is made on the diffusion constant and the molecular size for the unknown material which communicates the information on the irradiation of the red blood cell to the leukocyte.     

Phagocytosis of formol treated red blood cells

Formol treated red blood cells have been established to be phagocytized by human leukocytes in the presence of compatible AB0 standard test sera in case such red blood cells are stored continuously in water or in case they are incubated in saliva in advance for one hour at least at 37 degrees C. If formol treated red blood cells are continuously stored in 0.9% NaCl solution or if they are lyophilized in addition they will not be engulfed by human leukocytes in presence of compatible standard test sera of the AB0 system. The additional treatment with papain of formol treated red blood cells will cause a moderate phagocytosis.     

Red blood cells initiate leukocyte rolling in postcapillary expansions: a lattice Boltzmann analysis

Leukocyte rolling on the vascular endothelium requires initial contact between leukocytes circulating in the blood and the vessel wall. Although specific adhesion mechanisms are involved in leukocyte-endothelium interactions, adhesion patterns in vivo suggest other rheological mechanisms also play a role. Previous studies have proposed that the abundance of leukocyte rolling in postcapillary venules is due to interactions between red blood cells (RBCs) and leukocytes as they enter postcapillary expansions, but the details of the fluid dynamics have not been elucidated. We have analyzed the interactions of red and white blood cells as they flow from a capillary into a postcapillary venule using a lattice Boltzmann approach. This technique provides the complete solution of the flow field and quantification of the particle-particle forces in a relevant geometry. Our results show that capillary-postcapillary venule diameter ratio, RBC configuration, and RBC shape are critical determinants of the initiation of cell rolling in postcapillary venules. The model predicts that an optimal configuration of the trailing red blood cells is required to drive the white blood cell to the wall.     

The repopulating potential and differentiation capacity of hematopoietic stem cells from the blood and bone marrow of normal mice

Using the hematopoietic colony technique, we have investigated the repopulating potential of bone marrow cells and leukocytes of blood from normal mice and have demonstrated that the frequency of hematopoietic stem cells in bone marrow is 50 to 150 times that of stem cells in the circulating blood. The differentiation capacity of these stem cells has also been examined. Results of comparative studies of the serial sections of hematopoietic colonies formed from marrow and blood leukocytes indicate that the differentiation capacity of stem cells from marrow and blood is similar, and that at least 80% of these cells differentiate along a single cell line. Thus, peripheral blood stem cells can effect a complete hematopoietic graft, establishing in the host, donor red cells, granulocytes, and platelets. The possibility that blood leukocytes may serve as a potential source of stem cells for hematopoietic transplants has been considered. Although blood contains stem cells, their frequency is so low as to make it unlikely that they would become a useful source of precursor cells for transplantation purposes.     

不同全血过滤方法用于去白细胞血液制备的临床对照研究

目的:观察不同全血过滤方法用于去白细胞血液制备的效果。方法:采用两种全血过滤方法进行对比研究,对照组采用常规法,将采集后全血混匀后直接与白细胞滤器连接直接过滤;实验组采用湿润滤盘法,血液采集完成混匀后静置,先用上层血清10～20 m L湿润滤盘,再混匀与白细胞滤器连接后进行过滤。比较两组制备方法所用的过滤时间、血液回收率、过滤前后血液指标情况及24小时内溶血的发生情况。结果:两组全血过滤方法过滤前后白细胞、红细胞、血红蛋白、血小板及血浆游离血红蛋白水平比较差异均无明显统计学意义(P0.05)。而实验组过滤时间短于对照组,血液回收率高于对照组,且24小时内溶血比例明显低于对照组(P0.05)。结论:常规法与湿润滤盘法均能达到去白细胞血液标准,但湿润滤盘法较常规法能有效的降低过滤时间、增加血液回收率,减少去白细胞悬浮红细胞因溶血造成的血液不合格率,值得临床推广应用。     

Interpretation of atypical patterns encountered when using a flow cytometry-based method to detect residual leukocytes in leukoreduced red blood cell components

BACKGROUND: Universal leukoreduction of blood components is becoming the standard of care. Flow cytometry methods are being used for quality control of the leukoreduction process. METHODS: We provide an atlas of atypical flow cytograms generated by a commercial LeucoCOUNT assay that was used to enumerate residual leukocytes in leukoreduced red blood cell components. Numeric results are derived from a flow cytogram generated by the assay. RESULTS: Three types of atypical flow cytogram patterns were observed during process validation or routine quality control of leukoreduced red blood cell components. (a) Fixation artifact: Fixation of control or test samples can alter the staining intensity compared with fresh cells. (b) "Rain" pattern: Flow cytometry methods count slightly damaged leukocytes not removed during leukoreduction. Slightly damaged leukocytes appear on a flow cytogram like "rain" falling from a well-defined "cloud" of intact residual leukocytes. Discrepancies between automated flow cytometry results and subjective manual counting methods can occur. (c) Autofluorescence-debris pattern: Cell debris and age-related changes in the sample can cause shifts in the fluorescence staining pattern, resulting in erroneous test results. CONCLUSION: Review of flow cytograms is essential for accurate reporting of flow cytometry-based methods for enumerating residual leukocytes in leukoreduced blood components.     

Leukocyte-depleted blood: a comparison of available preparations.

Febrile nonhemolytic transfusion reactions due to leukoagglutinins are frequently seen in patients who have been given multiple blood transfusions. To prevent or reduce the severity of these reactions, leukocyte-poor blood (that containing fewer than 0.3 X 10(9) leukocytes per unit) is frequently requested by clinicians. Four methods commonly used in Canada to produce leukocyte-poor blood were examined for their relative effectiveness and appropriate use. The mean total leukocyte count per unit was reduced to 0.22 X 10(9) in buffy-coat-poor red blood cell preparations produced by centrifugation with the blood bag inverted, to 0.19 X 10(9) by perfusion through an Imugard filter, to 0.21 X 10(9) by the use of an IBM 2991 automated cell washer and to 0.13 X 10(9) with the use of frozen blood. The proportion of red cells recovered varied from 62% with the inverted-spin method to 85% with the use of frozen blood. Comparison of these data and the percentage of leukocytes removed, the shelf life of the product, the cost of supplies and the preparation time indicated that the use of sophisticated machinery, such as the IBM cell washer, or of glycerolization plus washing of frozen cells is not warranted for most patients. Instead, patients who have febrile nonhemolytic transfusion reactions should initially be treated with a leukocyte-poor red cell preparation produced by the inverted-spin method; only if such reactions recur should the blood bank be requested to provide filtered, washed or frozen red cells.     

Modulation of the human in vitro antibody response by human leukocyte interferon preparations

The ability of human leukocyte Interferon to modulate the plaque-forming-cell response of human peripheral blood leukocytes to horse red blood cells was examined. Human peripheral blood mononuclear cells were cultured in vitro with the addition of varying doses of human leukocyte interferon 24 hr prior to, simultaneously with, and 24 hr after sensitization of the cultures with horse red blood cells. Plaque-forming-cell responses were measured 5 days after sensitization with antigen using poly-L-lysine-coupled horse red blood cell monolayers. When human leukocyte interferon preparations were added 24 hr prior to sensitization with antigen, a significant enhancement of the plaque-forming-cell response was observed. When the interferon was added simultaneously with antigen, the plaque-forming-cell response was significantly suppressed. Therefore, human leukocyte interferon appears to have a time-dependent immunomodulatory activity. The kinetics of immunomodulation appear to be different from those of previously described mouse models.     

Forces on a Wall-Bound Leukocyte in a Small Vessel Due to Red Cells in the Blood Stream

As part of the inflammation response, white blood cells (leukocytes) are well known to bind nearly statically to the vessel walls, where they must resist the force exerted by the flowing blood. This force is particularly difficult to estimate due to the particulate character of blood, especially in small vessels where the red blood cells must substantially deform to pass an adhered leukocyte. An efficient simulation tool with realistically flexible red blood cells is used to estimate these forces. At these length scales, it is found that the red cells significantly augment the streamwise forces that must be resisted by the binding. However, interactions with the red cells are also found to cause an average wall-directed force, which can be anticipated to enhance binding. These forces increase significantly as hematocrit values approach 25% and decrease significantly as the leukocyte is made flatter on the wall. For a tube hematocrit of 25% and a spherical protrusion with a diameter three-quarters that of the vessel, the average forces are increased by ∼40% and the local forces are more than double those estimated with an effective-viscosity-homogenized blood. Both the enhanced streamwise and wall-ward forces and their unsteady character are potentially important in regard to binding mechanisms.     

The “telomereless” erythrocytes and telomere-length dependent erythropoiesis

Approximately 25 trillion erythrocytes (red blood cells) circulate in the bloodstream of an adult human, surpassing the number of circulating leukocytes (white blood cells) by a factor of about 1000. Moreover, the erythrocyte turnover rate accounts for approximately 76% of the turnover rate of all circulating blood cells. This simple math shows that the hematopoietic system principally spends its telomere length-dependent replicative capacity on building and maintaining the erythrocyte blood pool. Erythropoiesis (red blood cell production) is thus the principal cause of telomere shortening with age in hematopoietic cells (HCs), a conclusion that holds significant implications for linking telomere length dynamics in HCs to health and lifespan of modern humans.     

The effects of temperature on the filtration of diluted blood through 3 μm and 5 μm filters

The effect of temperature on the flow of diluted blood [Hct = 0.21], through 5 μm Nuclepore filters, is described by the Arrhenius equation with an energy of activation of 27.7 kJ/mol. Plasma, diluted with PBS, is almost three times less sensitive to temperature, with an energy of activation of 9.8 kJ/mol, while red cells are of intermediate sensitivity, with an energy of activation of 14.7 kJ/mol. The most sensitive elements to changes in temperature are leukocytes, with energies of activation of 31 kJ/mol and 35 kJ/mol for fast-flowing leukocytes (granulocytes and lymphocytes) and slow-flowing leukocytes (monocytes) respectively. Hence, the major determinants of the decline in filterability of blood through micropore filters are the leukocytes. This effect is compounded when blood is kept for 10 minutes or more at 10° C due to activation of granulocytes, which leads to permanent pore blocking when the affected blood is filtered at room temperature. The combination of increased passage time of leukocytes through peripheral areas at abnormally low temperatures and subsequent activation might influence the flow of blood in non-affected tissues. The effect of temperature on the filterability of red blood cells through 3 μm filters is not described by the Arrhenius equation and the deviations are seen as a gradual change of slope rather than a sharp break between two straight lines. The data are consistent with a gradual shift in rate limiting step away from the entry event into pores, which dominates at low temperature but becomes progressively less important at elevated temperatures. The changing parameter is probably the volume of the red cell, which is less important when flow is measured through 5 μm pores.