泥鳅外周血细胞的细胞化学染色特征及细胞化学谱

目的 研究泥鳅（Misgurnus anguillicaudatus）外周血细胞的细胞化学染色特征及细胞化学谱。方法 自泥鳅尾静脉取血,立即制作血涂片,进行瑞氏及7种细胞化学染色,包括酸性磷酸酶（ACP）、碱性磷酸酶（AKP）、氯乙酸AS-D萘酚酯酶（AS-D）、α-醋酸萘酚酯酶（α-NAE）、过碘酸-雪夫（PAS）、过氧化物酶（POX）和苏丹黑B(SBB)染色。结果 在泥鳅外周血中分辨出红细胞、4种白细胞和血栓细胞,4种白细胞分别为淋巴细胞、单核细胞、嗜中性粒细胞和嗜酸性粒细胞。红细胞PAS染色呈阳性,ACP、AKP、AS-D、α-NAE、POX和SBB染色均为阴性;淋巴细胞AS-D、α-NAE和PAS染色呈阳性,ACP、AKP、POX和SBB染色为阴性;单核细胞AS-D染色呈阳性,ACP、α-NAE和PAS染色为弱阳性,AKP、POX和SBB染色为阴性;嗜中性粒细胞ACP和AS-D呈阳性染色,α-NAE、PAS和SBB染色为弱阳性,AKP和POX染色为阴性;嗜酸性粒细胞ACP和AS-D染色呈强阳性,α-NAE、PAS和SBB染色为阳性,AKP和POX染色为阴性;血栓细胞AS-D染色...     

团头鲂外周血细胞的显微结构及细胞化学特征

采用常规瑞氏染色和细胞化学染色方法对团头鲂(Megalobrama amblycephala)外周血细胞的显微结构及细胞化学特征进行了观察。在团头鲂外周血细胞中可区分出六类细胞: 红细胞、淋巴细胞、单核细胞、嗜中性粒细胞、嗜酸性粒细胞和血栓细胞。其中淋巴细胞是除红细胞外含量最多的细胞, 其次分别为血栓细胞、单核细胞、嗜中性粒细胞、嗜酸性粒细胞。成熟红细胞多为卵圆形, 表面光滑, 胞核呈椭圆形或圆形, 染色质较为致密; 淋巴细胞多呈圆形, 胞质较少, 胞核常偏位; 单核细胞多为圆形, 胞核呈圆形或椭圆形, 胞质内可见空泡状结构; 嗜中性粒细胞近似圆形, 胞核常偏于细胞一侧, 呈分叶状、肾形或椭圆形, 核质界限清晰; 嗜酸性粒细胞一般为圆形, 胞核为肾形或椭圆形, 胞质中充满紫红色颗粒; 血栓细胞形态多样, 主要有椭圆形、纺锤形、长杆状和泪滴形, 核质比较大。淋巴细胞呈α-醋酸萘酚酯酶(ANAE)阳性, 呈过碘酸-雪夫(PAS)、氯乙酸AS-D萘酚酯酶(AS-DCE)弱阳性, 呈苏丹黑B(SBB)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)及过氧化物酶(POX)阴性; 单核细胞呈POX、ACP强阳性, PAS、SBB、AS-DCE和ANAE为阳性, 呈AKP阴性; 嗜中性粒细胞除PAS和ANAE为弱阳性外, 其他染色结果和单核细胞相同; 嗜酸性粒细胞呈POX、ANAE强阳性, SBB、ACP阳性, PAS及AS-DCE则为弱阳性, 呈AKP阴性; 血栓细胞呈PAS、AS-DCE及ANAE弱阳性, 呈SBB、ACP、AKP及POX阴性。团头鲂外周血细胞的显微结构及细胞化学特征与其他鱼类具有相似之处, 但亦有其明显的物种特异性。该研究结果可作为监测团头鲂健康状态的依据, 为其养殖及病理诊断提供基础资料。     

嗜中性粒细胞是人抵抗素表达的主要细胞

抵抗素(resistin)是小鼠白色脂肪组织大量表达的富含半胱氨酸的 分泌型蛋白.近年研究发现,人与啮齿类动物的抵抗素组织表达分布存在很 大差异．小鼠抵抗素主要在白色脂肪组织表达,而人抵抗素主要在单核细 胞/巨噬细胞表达,且在骨髓组织中大量表达,但目前骨髓中的细胞定位还 不清楚．本研究的目的是明确成人骨髓及外周血白细胞中抵抗素表达细胞 的类型．免疫荧光法检测骨髓中抵抗素表达细胞,结果显示,抵抗素主要表 达在细胞核呈杆状和分叶核状的成熟粒细胞中,其中杆状核粒细胞表达较 高,分叶核粒细胞表达减弱．Anti-hresistin IgG-Biotin-PE单色荧光流 式细胞术分选外周血白细胞中抵抗素表达细胞后经瑞氏化学染色,结果显 示,抵抗素表达细胞主要为杆状和分叶核状的嗜中性粒细胞,还有少量嗜酸 性粒细胞,且抵抗素蛋白分布在细胞质中. RT-qPCR结果在RNA水平上证明, 人抵抗素在嗜中性粒细胞中大量表达．Anti-hresistin IgG-FITC和anti- HNL IgG-Biotin-PE 双色荧光流式细胞术进一步证明,抵抗素的主要表达细 胞为成熟的嗜中性粒细胞．嗜中性粒细胞在机体免疫防御中起重要作用, 人骨髓及外周血中抵抗素主要在成熟嗜中性粒细胞中表达,这一研究结论 为人抵抗素与炎症反应的关联性及其功能的进一步研究奠定了基础.     

4种两栖爬行动物血细胞的显微结构及细胞化学特征观察

光镜下两栖动物中华蟾蜍Bufo gargarizans和牛蛙Rana catesbeiana、爬行动物铜蜓蜥Sphenomorphus indicus和青海沙蜥Phrynocephalus vlangalii4种动物外周血中均可观察到红细胞、血栓细胞、淋巴细胞、单核细胞、嗜中性粒细胞和嗜碱性粒细胞,中华蟾蜍和牛蛙的外周血中还观察到嗜酸性粒细胞。细胞化学染色显示:脂类存在于中华蟾蜍和牛蛙的单核细胞和粒细胞中,2种爬行动物的血细胞中未发现脂类。4种动物的血细胞中均未检测到碱性磷酸酶,但均检测到酸性磷酸酶,在中华蟾蜍和牛蛙血细胞中含量最多。过氧化物酶存在除青海沙蜥以外的3种动物的血细胞中,以在嗜中性粒细胞中居多;4种动物的外周血细胞中均检测到酸性-α-醋酸萘酯酶。     

血常规在头颈部鳞状细胞癌早期诊断中的作用

目的:探究血常规指标在头颈部鳞状细胞癌早期诊断中的作用,从血液学的角度为头颈部鳞状细胞癌的早期诊断提供参考依据。方法:对181例头颈部良恶性肿瘤患者的血常规结果进行回顾性分析,比较头颈部鳞状细胞癌患者组(实验组)和头颈部良性肿瘤组(对照组)患者的血小板计数(PLT)、血小板比积(PCT)、血小板平均容积(MPV)、血小板分布宽度(PDW)、大血小板比率(P-LCR)、中性粒细胞计数(NEU)、淋巴细胞计数(LYM)、单核细胞计数(MONO)、嗜酸性粒细胞计数(EOS)、嗜碱性粒细胞计数(BASO)、血小板计数/淋巴细胞计数比值(PLR)以及中性粒细胞计数/淋巴细胞计数比值(NLR)共12个指标。结果:实验组和对照组的PLT、PCT、MPV、PDW、P-LCR以及BASO计数值比较均没有统计学差异(p0.05)。对照组NEU、MONO、EOS、PLR和NLR均低于实验组,而LYM计数值高于实验组,其差异均具有统计学意义(p0.05),ROC曲线显示NLR对于早期诊断的意义要优于PLR。结论:血小板相关指标在头颈部鳞状细胞癌早期诊断中无明显意义,白细胞亚型计数具有重要的提示作用,而NLR的诊断意义要高于PLR,或许可以联合其他鳞癌标志物进行早期诊断。     

欧洲鳗鲡外周血细胞的显微和超微结构

利用光镜和透射电镜技术研究了欧洲鳗鲡（Anguilla anguilla）外周血细胞的显微和亚显微结构。结果表明：在外周血细胞中可以区分出红细胞、单核细胞、大淋巴细胞、小淋巴细胞、嗜中性粒细胞和血栓细胞;嗜中性粒细胞内的特殊颗粒包括A、B、C三型;嗜中性粒细胞分为Ⅰ型粒细胞（内含A、B、C三种特殊颗粒）、Ⅱ型粒细胞（内含A型和C型两种特殊颗粒）和Ⅲ型粒细胞（内含C型特殊颗粒）。还见到幼稚的和正在分裂的红细胞和单核细胞,提示示幼稚的红细胞能直接进入外周血流中,嗜酸性粒细胞和嗜碱性粒细胞无论在血涂片上或超薄切片上均未见到。描述了上述各种血细胞在光镜和电镜观察下的形态和细微结构。血栓细胞体积最小,嗜中性粒细胞体积最大;淋巴细胞数目最多,嗜中性粒细胞数目最少。     

假单胞菌对鳜鱼血液指标的影响

用假单胞菌接种健康鳜鱼引起发病,然后对发病鳜鱼进行红细胞、白细胞、血栓细胞计数;血涂片染色鉴别淋巴细胞、单核细胞、嗜中性粒细胞并分类计数;测定血红蛋白值及红细胞渗透脆性值并与对照组相应指标进行比较。结果表明:假单胞菌可引起鳜鱼的红细胞总数、小淋巴细胞和血红蛋白值下降,且与对照组相比,前两者存在极显著差异(p<0.01),后者存在显著差异(p<0.05);而白细胞总数显著增加(p<0.05);嗜中性粒细胞、单核细胞极显著增加(p<0.01);血栓细胞、大淋巴细胞和红细胞渗透脆性值变化不大。     

养殖齐口裂腹鱼外周血细胞显微观察

养殖齐口裂腹鱼Schizothorax prenanti外周血经瑞氏染色液染色,可鉴定出红细胞、嗜中性粒细胞、血栓细胞、淋巴细胞、单核细胞,一些未成熟的红细胞和少量进行无丝分裂的红细胞,未观察到嗜碱性粒细胞和嗜酸性粒细胞.在外周血中还可观察到3个阶段的嗜中性粒细胞:中性晚幼粒细胞胞体为圆形或近圆形,胞浆呈淡粉红色,量丰富,其中含有较多红色的细小颗粒,胞核呈肾形;中性杆状核粒细胞胞核较中性晚幼粒细胞的凹陷更强,呈"S"、"C"等形状;中性分叶核粒细胞占多数,胞体近圆形,核至少分成两叶,多达五或六叶.血栓细胞呈圆形、近圆形、梨形、纺锤形、杆状等多种形态,一个或多个聚集在一起.外周血细胞中,血栓细胞体积最小、数量最多,单核细胞体积最大、数量最少.     

日本白鲫外周血细胞显微及亚显微结构的研究

用光镜和电镜观察了日本白鲫外周血细胞的形态结构和过氧化物酶、糖原在血细胞内的分布。在日本白鲫外周血中，可见到红细胞、淋巴细胞、单核细胞、中性粒细胞和血栓细胞，未发现酸性粒细胞和碱性粒细胞，描述了上述各种血细胞在光镜和电镜观察下的形态和细微结构。对过氧化物酶，中性粒细胞呈阳性反应，对ＰＡＳ处理，淋巴细胞和中性粒细胞均呈阳性反应，中性粒细胞中有３种不同类型的特殊颗粒。     

斑鳢血细胞初步研究

对斑鳢的外周血细胞用常规Wright’s法染色并进行显微观察。结果显示,斑鳢外周血中具有红细胞、幼稚红细胞、小淋巴细胞、大淋巴细胞、血栓细胞、单核细胞和嗜中性粒细胞,未发现嗜酸性粒细胞和嗜碱性粒细胞。分别对各种血细胞的显微结构进行了描述。     

Differentiation stages of eosinophils characterized by hyaluronic acid binding via CD44 and responsiveness to stimuli

To characterize interleukin (IL)-5-induced eosinophils, we examined the expression of CD44, very late antigen (VLA)-4, and the IL-5 receptor alpha chain, as well as the levels of eosinophil peroxidase and the generation of superoxide. Eosinophils were prepared from IL-5-transgenic mice, then characterized using electron microscopy to determine their responses to stimuli. Whereas CD44 densities remained almost constant, the level of VLA-4 increased in parallel with eosinophil maturation. Although a subset of IL-5-induced eosinophils with high side scatter recovered from bone marrow and rare ones found in blood recognized hyaluronic acid (HA), most did not have this property. Bone marrow eosinophils with high side scatter and lower density contained eosinophil peroxidase, not only in granules, but also in membranous structures for 30% of this population. This population developed HA-binding ability in response to IL-3, IL-4, IL-5, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-2, monocyte chemotactic protein (MCP)-1, eotaxin, nerve growth factor (NGF), and opsonized zymosan (OZ). Peripheral blood eosinophils acquired HA-binding ability in response to the same stimuli, but their responses were less than those of bone marrow eosinophils with high levels of side scatter. However, splenic eosinophils did not respond to these stimuli. Although peripheral blood eosinophils did not proliferate when stimulated by IL-5, these were the only cells that released eosinophil peroxidase in response to IL-4, MIP-2, MCP-1, eotaxin, NGF, and OZ. With the exception of a subset of bone marrow eosinophils, the ability to acquire HA binding, but not the ability to generate superoxide, correlated with eosinophil peroxidase activity and major basic protein accumulation in the granules of maturing cells.     

Analysis of eosinophil turnover in vivo reveals their active recruitment to and prolonged survival in the peritoneal cavity

Eosinophils are potent effector cells associated with allergic inflammation and parasite infections. However, limited information exists about their turnover, migration, and survival in vivo. To address these important questions, we determined murine eosinophil turnover under steady state and inflammatory conditions by flow cytometric analysis of BrdU incorporation and analyzed their migration pattern and survival in different tissues after adoptive transfer into recipient mice. In naive mice approximately 50% of bone marrow eosinophils were labeled with BrdU during a 15-h pulse, whereas only 10% of splenic eosinophils were labeled within this time frame. Unexpectedly, the rate of eosinophil production did not change during acute infection with the helminth parasite Nippostrongylus brasiliensis despite massive eosinophilia in several tissues. Eosinophils present in lung and peritoneum remained largely BrdU negative, indicating that eosinophilia in end organs was mainly caused by increased survival of already existing eosinophils rather than increased production of new eosinophils in the bone marrow. Adoptive transfer experiments revealed that eosinophils preferentially migrated to the peritoneum in a macrophage-independent and pertussis toxin-sensitive manner, where they survived for several days. Peritoneal eosinophils expressed high levels of the inhibitory receptor Siglec-F, released less eosinophil peroxidase compared with eosinophils from the spleen, and could recirculate to other organs. These results demonstrate that the peritoneum serves as reservoir for eosinophils.     

Reactivity of rabbit antiserum to guinea pig eosinophils.

Although the eosinophil has been recognized as a distinctive cell type for almost 100 years, the major functions of these cells remain unknown. As an approach to defining these functions we have treated guinea pigs with rabbit antiserum to eosinophils (AES) in an attempt to ablate these cells from tissues. Rabbits were immunized thrice with purified eosinophils and the antisera were absorbed with peripheral blood cells from guinea pigs made eosinopenic with methyprednisolone to remove antibodies reactive with serum proteins and erythrocytes. The resulting sera reacted strongly with eosinophils in cytotoxicity tests and had weak or no reactivity with neutrophils. However, absorption of AES with purified neutrophils removed antieosinophil activity. Intraperitoneal injection of potent AES into guinea pigs resulted in complete absence of eosinophils from the peripheral blood and from the peritoneal cavity with only transient or no reduction in circulating neutrophils. Eosinophils were also reduced in bone marrow, spleen, and intestine. The ability of neutrophils to absorb AES activity in spite of weak reactivity in cytotoxicity tests may reflect a quantitative difference in antigenic determinants between eosinophils and neutrophils.     

The cytochemical demonstration of cyanide resistant peroxidase does not identify eosinophils selectively

Potassium cyanide (KCN) resistant peroxidase is generally accepted by hematologists as a selective stain for the eosinophilic cell line. However, it has been demonstrated biochemically that not only the peroxidases of neutrophils but also those of eosinophils can be inhibited by KCN. Therefore, bone marrow smears of hematologically normal patients were subjected to the peroxidase reaction in the presence of varying concentrations of KCN. It was found that with increasing concentrations of KCN not only neutrophils but also eosinophils were inhibited. Moreover, there were always neutrophilic promyelocytes that were still positive when a considerable number of the eosinophils was already inhibited. Therefore, it can be concluded from our results as well as from biochemical data that there is no concentration of KCN which demonstrates the total of the eosinophilic cell line selectively. The implications of these findings are discussed.     

Generation of rat eosinophils by recombinant rat interleukin-5 in vitro and in vivo

The addition of recombinant rat interleukin-5 (IL-5), which was purified from the hemolymph of silkworm Bombyx mori larvae infected with IL-5-expressing recombinant virus, to cultures of rat bone marrow cells resulted in an increase in the number of Luxol-fast-blue staining eosinophils in a time- and concentration-dependent manner. After 6 days culture with 100 pM recombinant rat IL-5, more than 90% of the bone marrow cells were eosinophil. The contents of major basic protein (MBP) in the bone marrow cells determined by Western blot analysis using a polyclonal antibody to rat MBP were also increased by recombinant rat IL-5 (100 pM). Furthermore, intravenous injections of recombinant rat IL-5 twice a day for six consecutive days increased the population of eosinophils in peripheral blood cells and in bone marrow cells. These findings indicate that rat IL-5 induces terminal differentiation and proliferation of progenitor cells to mature eosinophils in rats.     

Peroxidase Staining Combined with Autoradiography for Study of Eosinophilic Granules

Giemsa staining and a peroxidase reaction were applied to blood films in conjunction with autoradiography to establish the types of granulocytes that stain differentially with the benzidine-peroxidase reaction. Differential counts made on Ciemsa-stained and peroxidase-stained autoradiograms were compared. In T. spiralis-infected rats with an elevated eosinophil count, as judged by Giemsa staining, the percentage of granulocytes that stained more intensely with peroxidase was increased. The results suggested that the eosinophils were the intensely peroxidase-positive cells. Blood smears were stained for peroxidase before being coated with NTB₂ liquid emulsion. Although the blue color of the peroxidase reaction faded during photographic development, the color redeveloped when peroxidase-stained autoradiograms were stained once again after photographic development. It was found necessary to stain for peroxidase both before and after autoradiography. The correlation of Giemsa-stained and peroxidase-stained autoradiograms indicated that the peroxidase stain can be combined with autoradiography to obtain authentic results.     

Granulation staining and cytochemistry of peripheral blood leucocytes in healthy carp (Cyprinus carpio L.) I. Granulocytes

The cytochemistry and staining of granula in peripheral blood granulocytes in healthy carp ( Cyprinus carpio L.) are described. Blood smears were stained for periodic acid-Schiff (PAS), peroxidase, oxidase, alkaline and acid phosphatase, α-naphthyl-acetate esterase, α-naphthyl-butyrate esterase, naphthol-AS-chloroacetate esterase (AS-D), naphthol-AS-acetate esterase and β-glucuronidase. Different granula types were shown by triazid-staining (eosinophil and neutrophil granula) and methylenblue-staining for basophil granulation. Toluidinblue-staining was used for basophil granulocytes. Lipids were shown by the Sudan-black-reaction. Four granulocyte subpopulations are described: neutrophil, heterophil, basophil and eosinophil granulocytes. Neutrophils possess all tested granula types, whereas heterophil and basophil granulocytes show only basophil granula. Neutrophils and heterophils show no activity of the tested esterases with the exception of AS-D. Only neutrophils were peroxidase-positive. Alkaline phosphatase and β-glucuronidase were not detected in granulocytes. Basophils and especially eosinophils were rarely found in peripheral blood.     

Generation of Eosinophils from Cryopreserved Murine Bone Marrow Cells

Eosinophils are produced in the bone marrow from CD34⁺ eosinophil lineage–committed progenitors, whose levels in the bone marrow are elevated in a variety of human diseases. These findings suggest that increased eosinophil lineage–committed progenitor production is an important process in disease-associated eosinophilia. The pathways central to the biology of the eosinophil lineage–committed progenitor remain largely unknown. Thus, developing new methods to investigate the regulators of eosinophil lineage–committed progenitor differentiation is needed to identify potential therapeutic targets to specifically inhibit eosinophil production. We tested cytokine regimens to optimize liquid cultures for the study of eosinophil lineage–committed progenitor and eosinophil precursor differentiation into mature eosinophils. Stem cell factor (but not fms-related tyrosine kinase 3 ligand) was required for optimal yield of eosinophils. Furthermore, we evaluated the effects of cell preservation and scale on the culture, successfully culturing functional eosinophils from fresh and frozen murine bone marrow cells and in a standard-sized and 96-well culture format. In summary, we have developed an adaptable culture system that yields functionally competent eosinophils from murine low-density bone marrow cells and whose cytokine regime includes expansion of progenitors with stem cell factor alone with subsequent differentiation with interleukin 5.     

Bone marrow cell differential counts obtained by multidimensional flow cytometry.

Five-dimensional flow cytometric analysis of normal bone marrow aspirates was utilized to determine the frequency of neutrophils, eosinophils, monocytes, lymphocytes, nucleated erythrocytes, reticulocytes, platelets, and a cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils. Each of these bone marrow cell populations had unique features with respect to forward light scatter, orthogonal light scatter, and staining with Thiazole-Orange, LDS-751, and CD45 labeled with Phycoerythrin (PE). The identity of the cell populations was verified by sorting each of the cell populations and subsequent light microscopic examination of the cells. The frequencies of the nucleated bone marrow cell subpopulations of 50 normal donors were for neutrophils, mean 72.3%; SD +/- 5.1; 95% limits, 70.9-73.8%; eosinophils, mean 1.8%; SD +/- 1.3; 95% limits, 1.4-2.1%; monocytes, mean, 2.8%; SD +/- 1.2; 95% limits, 2.5-3.1%; lymphocytes, mean 12.1%; SD +/- 3.6; 95% limits 11.1-13.2%; nucleated erythrocytes, mean 8.9%; SD +/- 3.9; 95% limits, 7.8-10.1%; and the cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils, mean 1.6%; SD +/- 1.2; 95% limits, 1.3-1.9%. The percentage of reticulocytes in bone marrow aspirates from 50 normal donors correlated with the reticulocyte frequency in the peripheral blood of these donors. However, the mean frequency of reticulocytes was significantly greater (p < 0.0001) in bone marrow (mean 2.19%; SD +/- 0.88) than in peripheral blood (mean 1.71%; SD +/- 0.88). The technique could discriminate between immature and mature reticulocytes based on the brighter staining with both Thiazole-Orange and LDS-751 of the immature reticulocytes. This was confirmed by cell sorting of both reticulocyte populations, which revealed larger clumps of New Methylene Blue staining material in the brighter Thiazole-Orange and LDS-751 stained reticulocytes. The immature reticulocytes were present in normal bone marrow, but not in normal peripheral blood. As expected, a significantly greater frequency of nucleated cells was found in bone marrow aspirates (mean 0.85%; SD +/- 0.59) than in peripheral blood (mean 0.20%; SD +/- 0.11). The frequency of platelets was significantly lower in bone marrow (mean 1.24%; SD +/- 0.69) than in peripheral blood (mean 2.94%, SD +/- 1.14). Flow cytometric bone marrow analysis can provide clinical laboratories with a technique that generates quantitative bone marrow cell differentials and potentially can reduce the need for light microscopic examination of bone marrow smears.