Phagocytosis, intracellular pH, and cell volume in the multifunctional analysis of granulocytes by flow cytometry

Phagocytosis of Escherichia coli K12 strain bacteria was used to measure by flow cytometry the functional activities of human granulocytes in whole blood or buffy coat preparations. In a first measurement, the increase in electric cell volume and acridine orange (AO) green and red fluorescence were used to quantify the degree of phagocytosis. In a second measurement, the intracellular pH and esterase activity of each cell were determined with 1,4-diacetoxy-2,3-dicyanobenzene to obtain information on the metabolic activities during phagocytosis and degradation of bacteria. The DNA of dead cells was simultaneously counterstained with propidium iodide in both assays. The volume, the AO green and red fluorescence, the internal pH, and esterase activity were automatically averaged for all granulocytes or lymphocytes of a measurement. The calculated mean values were transferred into the self-learning database of the DIAGNOS1-program system. The functional granulocyte parameters of normal healthy individuals can be used as reference values for the automated diagnosis of abnormal granulocytes in various infectious disease states. The assays require 1 ml of heparinized whole blood and the results are available within 1 hour.     

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.     

长薄鳅外周血细胞的显微结构和细胞化学特征研究

长薄鳅外周血细胞可分为红细胞、中性粒细胞、单核细胞、淋巴细胞和血栓细胞.在数量上,中性粒细胞、单核细胞、淋巴细胞和血栓细胞占白细胞总数的百分比分别是17.06%、5.83%、28.16%和48.94%.细胞化学染色显示所有白细胞均含有糖原物质,所有红细胞均不含酸性磷酸酶,中性粒细胞、单核细胞、淋巴细胞和血栓细胞均含有酸性磷酸酶.非特异件酯酶染色显示单核细胞呈阳性反应,中性粒细胞、淋巴细胞和血栓细胞均为部分呈阳性反应.所有细胞的碱性磷酸酶、过氧化物酶、苏丹黑显色反应均呈阴性.     

Survival in vivo of platelets stored for 48 hours in the buffycoat at 4 degrees C compared to platelet rich plasma stored at 22 degrees C

High speed centrifugation allows separation of whole blood into cell free plasma, a buffy coat and leukocyte poor red cells. The buffy coat can be used for the preparation of platelet concentrates. High lactate production at 22 degrees C requires storage of the buffy coat at 4 degrees C. Survival in vivo of platelet concentrates prepared from buffy coats stored at 4 degrees C for 48 h (BC-PC) was compared with the survival in vivo of platelet concentrates from platelet rich plasma stored at 22 degrees C for 48 h (PRP-PC). Both methods were studied in the same healthy volunteers (n = 8) using 51Cr labeled autologous platelets. The mean +/- SD recovery 15 min after reinfusion of the BC-PC was 30.5% +/- 13.3% and for PRP-PC 41.4% +/- 7.9% (p less than 0.0001). The survival in vivo for BC-PC was 2.4 days +/- 0.4 days and for PRP-PC 7.0 days +/- 1.4 days (p less than 0.0001). Since the survival in vivo is significantly less for platelets derived from the buffy coat stored at 4 degrees C, we advocate storage of platelets at 22 degrees C.     

Flow cytometric analysis of CD41-labeled platelets isolated by the rapid, one-step OptiPrep method from human blood.

BACKGROUND: Although platelet-rich plasma is relatively easy to produce by centrifugation of whole blood, yields of platelets may be variable because many of them are trapped within the erythrocyte layer. Although they can be recovered by washing these cells, it is a general rule that the number of centrifugations should be kept to a minimum to avoid activation of platelets. This work describes the rapid, one-step OptiPrep method for the isolation of highly purified platelets from human blood (buffy coat). METHODS: To provide a functionally intact and uncontaminated platelet fraction, a density gradient centrifugation was performed by using a density barrier prepared from OptiPrep. CD41 antibody staining was performed to assess the purity of the obtained platelet population by means of a FACScan flow cytometer. Platelets were identified by a morphologic gate in which events were further studied for CD41 expression. Data were analyzed by CellQuest (Becton Dickinson). RESULTS: Platelet-specific CD41 antibody staining showed that the purity of the platelet population recovered from this density barrier method was greater than 90%. The platelets showed an excellent morphologic state. CONCLUSION: The rapid, one-step OptiPrep density gradient centrifugation is a reliable method for obtaining highly purified platelets from human blood that are ready for further pharmacologic investigations.     

A Method for Separation of Granulocytes From Normal Human Blood Using Hidroxiethrl Starch

A comparison of the granulocyte yield obtained from normal buffy coats following the addition of different concentrations of hydrojtyethyl starch (H.E.S.) was made. Hydrcocyethyl starch proved to be at least as effective as Dextran both in regard to the percentage of granulocytes recovered and the speed of sedimentation of the red blood cells.     

Preparation and Pathogen Inactivation of Double Dose Buffy Coat Platelet Products using the INTERCEPT Blood System

Blood centers are faced with many challenges including maximizing production yield from the blood product donations they receive as well as ensuring the highest possible level of safety for transfusion patients, including protection from transfusion transmitted diseases. This must be accomplished in a fiscally responsible manner which minimizes operating expenses including consumables, equipment, waste, and personnel costs, among others.Several methods are available to produce platelet concentrates for transfusion. One of the most common is the buffy coat method in which a single therapeutic platelet unit (≥ 2.0 x10¹¹ platelets per unit or per local regulations) is prepared by pooling the buffy coat layer from up to six whole blood donations. A procedure for producing "double dose" whole blood derived platelets has only recently been developed.Presented here is a novel method for preparing double dose whole blood derived platelet concentrates from pools of 7 buffy coats and subsequently treating the double dose units with the INTERCEPT Blood System for pathogen inactivation. INTERCEPT was developed to inactivate viruses, bacteria, parasites, and contaminating donor white cells which may be present in donated blood. Pairing INTERCEPT with the double dose buffy coat method by utilizing the INTERCEPT Processing Set with Dual Storage Containers (the "DS set"), allows blood centers to treat each of their double dose units in a single pathogen inactivation processing set, thereby maximizing patient safety while minimizing costs. The double dose buffy coat method requires fewer buffy coats and reduces the use of consumables by up to 50% (e.g. pooling sets, filter sets, platelet additive solution, and sterile connection wafers) compared to preparation and treatment of single dose buffy coat platelet units. Other cost savings include less waste, less equipment maintenance, lower power requirements, reduced personnel time, and lower collection cost compared to the apheresis technique.     

史氏鲟外周血细胞的显微及超微结构

采用血细胞计数、光学显微及电子显微技术对二龄史氏鲟外周血细胞的数目、形态及结构进行了研究。二龄史氏鲟红细胞的数目为47.75×104个/mm3,白细胞数目为2.9万个/mm3,其中淋巴细胞所占比率最高。史氏鲟的外周血中除正常红细胞外,还有处于分裂状态及未成熟的红细胞。史氏鲟外周血中的白细胞有四种类型,分别为淋巴细胞、粒细胞、血栓细胞和单核细胞。其中粒细胞有两种,即嗜中性粒细胞和嗜酸性粒细胞。嗜中性粒细胞含有多种形状的核,其中分叶的核数目较多,粒细胞及淋巴细胞均类似于哺乳动物。对史氏鲟外周血细胞细微结构的观察显示:红细胞中具有少量的细胞器;淋巴细胞结构典型;单核细胞较粒细胞稍小且具有较多线粒体;血栓细胞具有梭形和圆形两种,胞质较少,其中梭形的血栓细胞胞质几乎透明;对粒细胞的颗粒按照形状和电子密度进行了分类。     

Gd(--) Abrami: a deficient G-6PD variant with hemizygous expression in blood cells of a woman with primary myelofibrosis.

A new deficient G-6PD variant, Gd(--) Abrami, was found in granulocytes, platelets and red blood cells of a 65-year-old woman with myelofibrosis. Enzyme and immunological titrations showed that only the deficient variant was present in blood cells whereas both the normal and abnormal enzymes were found in the fat cells of this patient. These results seem to indicate that the granulocytes, platelets and erythrocytes of this woman with myelofibrosis have arisen from a single abnormal precursor the functional X chromosome of which is the one carrying the abnormal G-6PD gene.     

Flow-cytometric characterization of stimulation, free radical formation, peroxidase activity and phagocytosis of human granulocytes with 2,7-dichlorofluorescein (DCF)

A standardized four-step assay for the flow cytometric determination of the oxidative activity of human polymorphonuclear leukocytes (PMNL) from normal human individuals and from septic patients was developed, using 2,7-dichlorofluorescin-diacetate (DCFH-DA) as indicator for the intracellular formation of H2O2 and free radicals. Spontaneous H2O2 and free radical formation was measured by preincubation of buffy coat PMNLs from fresh peripheral venous blood at 37 degrees C and pH 7.4 with 10 microM DCFH-DA. Intracellular peroxidase activity was determined by addition of 1 mM external H2O2 to this assay. A maximum of granulocyte oxidative burst activity was elicited by the addition of 150 nM phorbol-myristate-acetate (PMA). A physiological burst was generated by incubating buffy coat PMNLs together with E. coli bacteria. The DNA of dead cells was in all instances simultaneously counterstained with propidium iodide (PI). Quiescent or H2O2 or bacteria treated granulocytes moved as a single cell cluster to higher fluorescences. Stimulation with PMA, in contrast, generated always a bimodal distribution of granulocyte fluorescence with the high activity cell cluster being approximately sevenfold more active than the low activity cell cluster. Roughly half of the granulocytes in normal individuals had high fluorescence. An increase of the high activity granulocytes was observed in septic patients. Model experiments with the nonfluorescent DCFH-DA cleavage product DCFH (2,7-dichlorofluorescin) showed that DCFH was quickly photo-oxidized to fluorescent DCF (2,7-dichlorofluorescein) by UV-light and to a lower degree by daylight. DCFH even slowly autooxidized in the dark.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochemical and functional characterization of blood and inflammatory cells from the lizard Ameiva ameiva

The fine structure and differential cell count of blood and coelomic exudate leukocytes were studied with the aim to identify granulocytes from Ameiva ameiva, a lizard distributed in the tropical regions of the Americas. Blood leukocytes were separated with a Percoll cushion and coelomic exudate cells were obtained 24 h after intracoelomic thioglycollate injection. In the blood, erythrocytes, monocytes, thrombocytes, lymphocytes, plasma cells and four types of granulocytes were identified based on their morphology and cytochemistry. Types I and III granulocytes had round intracytoplasmic granules with the same basic morphology; however, type III granulocyte had a bilobued nucleus and higher amounts of heterochromatin suggesting an advance stage of maturation. Type II granulocytes had fusiformic granules and more mitochondria. Type IV granulocytes were classified as the basophil mammalian counterpart based on their morphology and relative number. Macrophages and granulocytes type III were found in the normal coelomic cavity. However, after the thioglycollate injection the number of type III granulocyte increased. Granulocytes found in the coelomic cavity were related to type III blood granulocyte based on the morphology and cytochemical localization of alkaline phosphatase and basic proteins in their intracytoplasmic granules. Differential blood leukocyte counts showed a predominance of type III granulocyte followed by lymphocyte, type I granulocyte, type II granulocyte, monocyte and type IV granulocyte. Taken together, these results indicate that types I and III granulocytes correspond to the mammalian neutrophils/heterophils and type II to the eosinophil granulocytes.     

Blood and inflammatory cells of the lungfish Lepidosiren paradoxa

A special interest exists concerning lungfish because they may possess characteristics of the common ancestor of land vertebrates. However, little is known about their blood and inflammatory cells; thus the fine structure, cytochemistry and differential cell counts of coelomic exudate and blood leucocytes were studied in Lepidosiren paradoxa. Blood smear analyses revealed erythrocytes, lymphocytes, monocytes, polymorphonuclear agranulocytes, thrombocytes and three different granulocytes. Blood monocytes and lymphocytes had typical vertebrate morphology. Thrombocytes had large vacuoles filled with a myelin rich structure. The polymorphonuclear agranulocyte had a nucleus morphologically similar to the human neutrophil with no apparent granules. Types I and II granulocytes had eosinophilic granules. Type I granulocytes had round or elongated granules heterogeneous in size, while type II had granules with an electron dense core. Type III granulocyte had many basophilic granules. The order of frequency was: type I granulocyte, followed by lymphocyte, type II granulocyte, monocyte, polymorphonuclear agranulocyte and type III granulocyte. Peroxidase localized mainly at the periphery of the granules from type II granulocytes, while no peroxidase expression was detected in type I granulocytes. Alkaline phosphatase was localized in the granules of type II granulocyte and acid phosphatase cytochemistry also labelled a few vacuoles of polymorphonuclear agranulocyte. About 85% of the coelomic inflammatory exudate cell population was type II granulocyte, 10% polymorphonuclear agranulocyte and 5% macrophages as judged by the nucleus and granule morphology. These results indicate that this lungfish utilises type II granulocytes as its main inflammatory granulocytes and that the polymorphonuclear agranulocyte may also be involved in the inflammatory response. The other two granulocytes appear similar to the mammalian eosinophil and basophil. In summary, this lungfish appears to possess the typical inflammatory granulocytes of teleosts, however, further functional studies are necessary to better understand the polymorphonuclear agranulocyte.     

Serological and genetic identification of a bovine B lymphocyte alloantigen system

A two-colour fluorescence micro cytotoxicity test was used to screen antisera for antibodies specific for bovine B lymphocytes. A total of 114 cattle alloantisera were screened against peripheral blood lymphocytes from 100 unrelated individuals. Anti-B lymphocyte activity was detected in 47 antisera. Cytotoxic antibodies to antigens other than B lymphocyte specific antigens were removed by absorbing the antisera with buffy coat cells or platelets isolated from whole blood. Selected antisera were used to type paternal half-sib families. The presence of a polymorphic, MHS-linked antigen system on B lymphocytes was demonstrated. The tissue distribution and MHS linkage of these antigens suggests this system is analogous to the class II or Ia antigens of other species.     

Serological and genetic identification of a bovine B lymphocyte alloantigen system

A two-colour fluorescence microcytotoxicity test was used to screen antisera for antibodies specific for bovine B lymphocytes. A total of 114 cattle alloantisera were screened against peripheral blood lymphocytes from 100 unrelated individuals. Anti-B lymphocyte activity was detected in 47 antisera. Cytotoxic antibodies to antigens other than B lymphocyte specific antigens were removed by absorbing the antisera with buffy coat cells or platelets isolated from whole blood. Selected antisera were used to type paternal half-sib families. The presence of a polymorphic, MHS-linked antigen system on B lymphocytes was demonstrated. The tissue distribution and MHS linkage of these antigens suggests this system is analogous to the class II or Ia antigens of other species.     

Mass spectrometric analysis of lipid species of human circulating blood cells

Circulating blood cell lipid composition may become increasingly important to provide new insights into cellular lipid abnormalities in diseases. Here we compared lipid species in monocytes, lymphocytes, granulocytes, platelets and red blood cells (RBC) of healthy volunteers using electrospray ionization tandem mass spectrometry and detected striking differences among the examined blood cells. The different cell types were characterized by unique lipid class and lipid species pattern. The predominant lipid classes were phosphatidylcholine (PC) and free cholesterol (FC) with cell type specific PC/FC ratios as markers of membrane fluidity which was 1.9 in monocytes, 1.3 in lymphocytes, 1.1 in granulocytes, 0.8 in platelets and 0.3 in RBC, respectively. Beside a three-fold elevated ceramide level of 2.6 mol%, granulocytes revealed the highest percentage of phosphatidylethanolamine-based plasmalogens and a decreased fraction of highly polyunsaturated (> or =3 double bonds) species compared to other cell types. Furthermore RBC showed a remarkable shift of glycerophospholipid chain length and platelets a nearly 4-fold increase of the cholesterol ester (CE) 18:2 (linoleic acid) fraction (55 mol% of total CE). In conclusion, the current study is a detailed comparison of lipid species in circulating blood cells of healthy human donors. This work could be a reference for studies in different patient cohorts directed towards discovery of novel lipid biomarkers in circulating blood cells.     

Cryopreservation of granulocytes for transfusion: Studies on human granulocyte isolation,the effect of glycerol on lysosomes,kinetics of glycerol uptake and cryopreservation with dimethyl sulfoxide and glycerol

Existing methods for the cryopreservation of granulocytes employ primarily dimethyl sulfoxide (Me₂SO) rather than glycerol as the cryoprotective additive of choice. Although Me₂SO has been demonstrated to be an effective cryoprotective additive for granulocyte preservation to yield viable cells (dye exclusion, phagocytosis, etc.), the inherent toxicity and clinical objections of Me₂SO as a cryoprotective additive for granulocyte preservation preclude its extensive and routine use in patients. Therefore, glycerol, with its important advantage of nontoxicity, has been investigated for its potential usefulness as a cryoprotective additive for preserving human granulocytes for transfusion.Granulocyte preparations were isolated from impure leukocyte concentrates obtained from the buffy coats of human whole blood. Studies on the isolation and purification of the granulocytes involved separation by sedimentation with dextran, removal of red cells by hypotonic shock with water, resuspension with Plasmatein and further purification by centrifugation. Intact viable granulocytes were obtained with a purity in excess of 90%.Lysosomes were studied as indicators of cryoinjury in granulocytes using β-glucuronidase as the key marker enzyme. This enzyme has been characterized as a sensitive indicator of damage to lysosomes and a direct linear relationship has been established between damage to granulocytes by freezing and amount of lysosomal enzyme released. Addition or presence of the cryoprotectant, glycerol, did not appear to have any adverse effect on lysosomes of intact granulocytes.Studies on the permeation kinetics of glycerol in granulocytes indicated that the additive was freely permeable and did not cause any potentially damaging osmotic changes in cell volume. Granulocytes in various concentrations of glycerol were then frozen at slow, moderate, and rapid cooling rates. Based on the small amount of β-glucuronidase released, good preservation of granulocyte lysosomes has been obtained with a slow cooling rate of 5 °C/min and a concentration of 15% glycerol. Further studies now are necessary to define those conditions of cooling rate and glycerol concentration required to develop a simple method for optimal preservation of granulocytes based on additional functional criteria of viability.     

Cell types in peripheral blood of the nurse shark: An approach to structure and function

Ultrastructural and functional studies were carried out on nurse shark (Ginglymostoma cirratum) peripheral blood cells in order to identify cells of definitive morphology and specific function. Along with erythrocytes and thrombocytes, four morphologically distinct leucocytes are recognized in peripheral blood: two types of granulocytes, the ‘eosiniphil’ and the ‘granulocyte’, and two mononuclear agranulocytic cells, one resembling mammalian macrophage and monocyte, the other resembling mammalian lymphocyte. Also present in peripheral circulation are blast-like cells and mitotic cells. In vitro phagocytosis was demonstrated by the monocyte-macrophage and the granulocyte while thrombocytes, eosinophils and lymphocytes showed no phagocytic activity in the system studied. It is stressed that care must be used in drawing functional analogies between blood cells of a mammal and an elasmobranch on the basis of morphological similarity alone.     

Effect of ultraviolet irradiation of autologous blood on cell volumes, cell adhesion and phagocytosis in normal probands and patients with multiple sclerosis

UVB induced changes of blood cell properties were investigated in 12 MS patients and in 10 healthy volunteers serving as normal controls. The mean cell volume (MCV) was determined by electronic sizing, the granulocyte and lymphocyte adherence was estimated in a capillary assay, and the phagocytic activity of granulocytes was measured in a test system based on the incorporation of opsonized baker's yeast (Saccharomyces cerevisiae). In MS patients the MCV of red cells and lymphocytes decreased rapidly within 6 UVB treatments. In contrast, the reduction of the granulocyte volume was delayed (between the 6th and 12th UVB). In the control group the mean value of the red cell and lymphocyte MCV remained rather unaffected. There was a slight rise of the granulocyte volume after the 6th UVB. The only significant change of adherence was an increase of granulocyte adherence in MS patients. Untreated patients had a significantly enhanced phagocytic activity in comparison to the control group. 6 UVB treatments included a significant reduction of the phagocytic activity in MS patients. However, subsequently the percentage of phagocytizing cells increased again, whereas the particle uptake per cell continued to decrease. In the control group only minor UVB induced changes of phagocytosis were observed. The in vitro UV irradiation caused an enhanced phagocytosis in the majority of cases in both controls and MS patients. In general, under the UVB treatment all parameters examined changed in the sense of a normalisation, in that the measured values reached a new level lying between the extreme pretreatment values accompanied by a reduced standard deviation. The effect of UVB was more pronounced in MS patients when compared with normal controls. This could result from an enhanced sensitivity to the influence of UVB of pathologically altered cells in MS patients. The monitoring of the MCV of red cells and lymphocytes as well as the repeated testing of granulocyte phagocytosis are recommended for supportion of therapy planning and follow-up of MS patients.     

Sedimentation behavior of activated human granulocytes

Human peripheral blood granulocytes (PMNs) obtained from normal adults were studied by an analytical gravity sedimentation system. Exposure of PMNs to endotoxin-activated serum (EAS) in a Ficoll density gradient containing Hank’s balanced salt solution with calcium and magnesium produced significantly different sedimentation patterns compared to those from granulocytes exposed to normal serum under the same conditions. Experiments were performed to determine whether changes in granulocyte density, volume, shape, or aggregation were responsible for the sedimentation pattern of granulocytes exposed to EAS. The altered gravity sedimentation behavior of endotoxin-activated granulocytes was abolished when calcium and magnesium were not present in the Ficoll density gradient. Granulocyte aggregation was inhibited by the absence of calcium and magnesium in the medium during granulocyte stimulation, whereas the changes in granulocyte shape and volume associated with granulocyte stimulation were not affected. The data indicate that the altered granulocyte sedimentation pattern in the presence of EAS and calcium and magnesium was produced by granulocyte aggregation and not by changes in granulocyte volume or shape.     

Compartmentation of hexokinase in human blood cells characterization of soluble and particulate enzymes

The isozyme distribution, kinetic properties and intracellular localization of hexokinase (ADP: D-hexose-6-phosphotransferase, EC 2.7.1.1) were studied in erythrocytes, blood platelets, lymphocytes and granulocytes. Soluble and particulate fractions were separated by a rapid density centrifugation method after controlled digitonin-induced cell lysis. In lymphocytes and platelets the major part of total activity was particle-bound (78 and 88%, respectively). In granulocytes and erythrocytes most of the hexokinase activity was found in the cytosol. All cell types, except granulocytes, contain mainly the type I isozyme. Platelets contain only type I hexokinase, while in lyphocytes a minor amount of type III is present in the soluble fraction (less than 10% of total activity). The major constituent of granulocytes is type III hexokinase (70–80% of total activity), the remaining 20–30% is type I hexokinase. Erythrocytes contain a multibanded type I hexokinase. The substrate affinities of the type I hexokinase do not differ significantly between the different cell types or between soluble, bound and solubilized fractions. Only soluble hexokinase from lymphocytes shows a slightly decreased K_m apparent for glucose (P < 0.05).