An Advanced Protocol for the Quantification of Marine Sediment Viruses via Flow Cytometry

Viruses are highly abundant, diverse, and active components of marine environments. Flow cytometry has helped to increase the understanding of their impact on shaping microbial communities and biogeochemical cycles in the pelagic zone. However, to date, flow cytometric quantification of sediment viruses is still hindered by interference from the sediment matrix. Here, we developed a protocol for the enumeration of marine sediment viruses by flow cytometry based on separation of viruses from sediment particles using a Nycodenz density gradient. Results indicated that there was sufficient removal of background interference to allow for flow cytometric quantification. Applying this new protocol to deep-sea and tidal-flat samples, viral abundances enumerated by flow cytometry correlated well (R² = 0.899) with counts assessed by epifluorescence microscopy over several orders of magnitude from marine sediments of various compositions. Further optimization may be needed for sediments with low biomass or high organic content. Overall, the new protocol enables fast and accurate quantification of marine sediment viruses, and opens up the options for virus sorting, targeted viromics, and single-virus sequencing.     

Cell Surface Heterogeneity of Human Blood Neutrophils and Monocytes

Until recently, human blood neutrophils (PMN) and monocytes have been considered to be homogeneous cell populations. However, much evidence has accumulated on their functional heterogeneity. This functional heterogeneity suggests the existence of different subsets of myeloid cells analogous to T and B subsets of lymphoid cells. The goal of this study was to investigate this question of myeloid subsets by examining myeloid cells for cell surface reactivity for IgG and complement (C). Normal PMN and monocytes were examined from 60 subjects for the presence of two types of IgG-Fc receptors and two activated C components, C3b and C3d. Most PMN and monocytes showed Fc receptor activity for rabbit IgG (Fc-R). In addition, the majority of monocytes but very few PMN reacted with human IgG (anti-Rh0) coated Rh-positive erythrocytes (Fc-H). Most PMN and monocytes showed C receptor reactivity for C3b, but only a minor subpopulation of both myeloid cells had C3d receptors. These data provide evidence that human blood myeloid cells may be composed of subsets with different membrane marker reactivities.     

Demonstration of Histamine Receptors on Human Platelets by Flow Cytometry

Fluoresceinated human albumin conjugated with histamine (FHA-HIS) has been used for the demonstration of histamine receptors on human platelets. Such receptors were demonstrated on 40–63% of peripheral blood platelets in 4 healthy donors. The binding of FHA-HIS was inhibited on 35–79% of the platelets by the histamine H₁ receptor antagonists diphenhydramine and clemastine. The histamine H₂ receptor antagonist cimetidine blocked the FHA-HIS binding on 14–37% of the platelets. It is concluded that histamine H₁ as well as H₂ receptors occur on human platelets but the receptors are not equally distributed in the platelet population.     

流式细胞术检测T细胞亚群在2型糖尿病及糖尿病肾病患者中的应用

目的 观察分析流式细胞术检测T细胞亚群在2型糖尿病及糖尿病肾病患者中的应用价值.方法 选取2018年4月—2019年4月于该院收治的83例2型糖尿病患者作为研究对象,采用流式细胞术检测83例2型糖尿病患者,将其分为糖尿病组(均为2型糖尿病且无肾病患者,42例)和糖尿病肾病组(均为2型糖尿病且有肾病患者,41例),再选取...     

日本血吸虫感染鼠肝脏肉芽肿细胞分离条件的优化及分离效果的流式检测

目的 优化日本血吸虫感染小鼠肝脏肉芽肿细胞的分离方法,以获得足量和纯净的肉芽肿活细胞,流式检测确定细胞的分离效果。方法 血吸虫尾蚴经皮感染C57BL/6小鼠,于感染后6周、12周剖杀小鼠,取肝脏,采用Ⅳ型胶原酶两步消化法分离肉芽肿细胞,并对胶原酶浓度及消化时间进行探索;进行流式检测,分析粒细胞、淋巴细胞和CD4+T的分离效果。结果 感染6周小鼠肝脏的Ⅳ型胶原酶最佳浓度为0.25 mg/mL,最佳消化时间为30 min(第一步)和30 min(第二步),流式细胞仪检测该条件可获得较高比例CD4+T细胞;而感染12周小鼠的肝脏,Ⅳ型胶原酶最佳浓度为0.3 mg/mL,消化时间分别为40、30 min,流式检测此条件可获得更高比例的CD4+T细胞。结论 急慢性肝脏肉芽肿细胞的分离所需Ⅳ型胶原酶的浓度不同;采用本研究优化的胶原酶两步消化法可获得大量、纯净的肉芽肿活细胞,并且细胞表面标志保存完好,为研究日本血吸虫肝脏肉芽肿的细胞机制提供重要材料。     

Detection of the Human Erythrocyte Surface Antigen Gerbich by Flow Cytometry using Human Antibodies and Phycoerythrin for Extreme Immunofluorescence Sensitivity

Abstract. We used indirect immunofluorescence and flow cytometry to detect the human erythrocyte surface antigen Gerbich. The procedure consisted of sequential erythrocyte-labeling with human antibody to Gerbich, biotinylated goat anti-human IgG and finally phycoerythrin-conjugated streptavidin. With maximal excitation of phycoerythrin at 546 nm, an increase in the fluorescence sensitivity was achieved. All erythrocytes from normal controls had detectable Gerbich antigen with little variation in antigen density between individuals. Six Gerbich-negative patients had no detectable antigen. By diluting the erythrocytes, as few as 0.1% antigen-positive cells in an antigen-negative population could be detected. These studies indicate that flow cytometry is a useful technique for the detection of erythrocyte surface antigens.     

Detection of Platelet-Activating Antibodies Associated with Vaccine-Induced Thrombotic Thrombocytopenia by Flow Cytometry: An Italian Experience

Rare cases of thrombocytopenia and thrombosis after anti-COVID-19 adenovirus-associated mRNA vaccines (VITT) due to platelet-activating anti-platelet-factor 4 (PF4)/polyanion antibodies have been reported. VITT laboratory diagnosis, similarly to heparin-induced thrombocytopenia (HIT) diagnosis, requires immunoassays for anti-PF4/polyanion antibodies identification, such as ELISA assays and platelet-activating functional tests, such as heparin-induced platelet activation test (HIPA), to confirm their pathogenicity. We compared the flow cytometry (FC) measurement of platelet p-selectin exposure to the gold standard functional test HIPA for diagnosis confirmation in 13 patients with a clinical VITT syndrome (6M/7F; median age 56 (33–78)) who resulted positive to anti-PF4/polyanion antibodies ELISA assays (12/13). FC and HIPA similarly identified three different patterns: (1) a typical non-heparin-dependent VITT pattern (seven and six patients by FC and HIPA, respectively); (2) low/no platelet activation in patients under IvIg therapy (five out of five and two out of four patients by FC and HIPA, respectively); (3) a HIT pattern. Antibodies investigated by FC became negative after 7, 17, and 24 days of therapy in three patients. FC measurement of P-selectin exposure was as sensitive as HIPA but simpler to detect anti-PF4/polyanion antibodies in VITT patients. FC could reliably discriminate VITT from HIT, thus helping for the choice of the anticoagulant.     

87例骨髓增生异常综合征多参数流式细胞术免疫表型分析

目的：了解骨髓增生异常综合征（MDS）的免疫表型特征。方法：利用CD45／SSC参数散点图设门方法,对87例MDS患者的骨髓幼稚细胞群、成熟粒细胞群和成熟单核细胞群细胞表面分化抗原进行分析。结果：MDS的异常免疫表型可以分为抗原跨系列表达、抗原跨阶段表达、成熟粒细胞抗原分化异常、抗原表达缺失或表达增强、成熟粒细胞或幼稚细胞群CD45／SSC表达异常。78例（90．0％）MDS患者可以检测到明确的异常抗原表达,74例（85．1％）存在≥2个免疫表型异常,异常免疫表型不仅存在于幼稚细胞群（70．0％）,也存在于成熟粒细胞群（81．6％）和单核细胞群（39．1％）．常见的异常免疫表型包括：成熟粒细胞群CD13／CD16分化异常（41．3％）;幼稚细胞群CD45表达异常（34．5％）;成熟粒细胞群跨系列表达CD38（29．9％）;幼稚细胞群CD34＋CD11b＋跨阶段表达异常（24．1％）;成熟粒细胞群SSC减低（11．5％）;成熟粒细胞群跨系列表达CD56（8％）;单核细胞群跨系列表达CD56（8％）。随着患者骨髓幼稚细胞比例增高,异常免疫表型数量也逐渐增多。结论：在大多数MDS患者骨髓细胞中可以检测到明确的异常免疫表型,在此基础上应用多参数流式细胞仪可以有助于MDS的诊断和预后判断。     

Evaluating New Markers for Minimal Residual Disease Analysis by Flow Cytometry in Precursor B Lymphoblastic Leukemia

Minimal residual disease is currently the most powerful prognostic indicator in Precursor B lymphoblastic leukemia. Multiparameter flow cytometry is the most commonly used modality. Seventy three B ALL cases and 15 normal marrows were evaluated for expression patterns of leukemia markers (CD38, CD58, CD73) in all 73 cases and CD66c, CD86 and CD123 in 23 cases. CD73 was aberrantly expressed in 90.41% cases and CD86 in 60.87% B ALL cases. Thus addition of these markers in MRD panels can increase the sensitivity of the assay.     

Flow Cytometric Analysis of Lymphocyte Subsets of Mice Maintained on an Ethanol-Containing Liquid Diet

Alcoholic patients often have impaired immune function, yet little is known about the precise mechanism(s) of this impairment. We have previously shown that ethanol consumption by mice alters copolymer-specific humoral and cellular immune responses. In this study, we asked whether alcohol consumption by mice would phenotypically alter lymphocyte populations. Female C57BL/6 mice were fed a nutritionally complete liquid diet containing 35% ethanol-derived calories for up to 8 days. As controls, mice either were fed a liquid control diet that isocalorically substitutes sucrose for ethanol or remained on a standard solid diet and water ad libitum. Although mice fed ethanol-containing liquid or pair-fed control liquid diets have decreased numbers of spleen cells compared with solid diet controls, only the ethanol-containing diet allowed normally nonresponder C57BL/6 spleen cells to make antibody responses to the poly(Glu⁵⁰Tyr⁵⁰) synthetic copolymer antigen. Flow cytometric analysis of splenic lymphocyte populations of mice on the ethanol-containing diet shows an increase in the relative proportion of T-lymphocytes as compared with mice on either solid or liquid control diets. No such change is seen for either B-cell or natural killer cell populations in these same mice. Both liquid control and liquid ethanol diets caused a slight decrease in the CD4:CD8 ratios of splenic T-lymphocytes. We see the relative percentage of T-cells bearing the αβ-cell receptor (TcR) increases in the spleens of liquid ethanol diet mice; a smaller increase TcRαβ usage is seen in the spleens of liquid control mice, compared with solid diet mice. Flow cytometric analysis shows that little, if any, difference exists in TcRγδ expression between the liquid ethanol and either the liquid control or solid diet groups. Preliminary analysis of TcRαβ subsets suggest that ethanol increases the percentage of T-cells expressing Vβ5 and Vβ8, and decreases the percentage of Vβ11 expressing cells. These findings suggest that, in addition to modifying the immune response, ethanol alters the phenotypic expression of lymphocyte subsets.     

Immunologic Monitoring of T-Lymphocyte Subsets and Hla-Dr-Positive Monocytes in Kidney Transplant Recipients: A Prospective,Observational Cohort Study

The clinical significance of circulating T-lymphocyte subsets and human leukocyte antigen (HLA)-DR-positive monocytes in the peripheral blood of kidney transplant recipients (KTRs) remains unclear. We examined the efficacy of enumerating these cells for the immunologic monitoring of KTRs.Blood samples were obtained before transplantation, 2 weeks after transplantation and at diagnosis, and 2 weeks after treating biopsy-proven acute cellular rejection and cytomegalovirus (CMV) infection. Serial flow cytometric analysis was performed using peripheral blood obtained from 123 patients to identify the frequencies of HLA-DR⁺, CD3⁺, CD4⁺, CD8⁺, and CD25⁺ T-lymphocytes and HLA-DR-positive monocytes.Frequencies of CD4⁺CD25⁺/CD4⁺ T cells, CD8⁺CD25⁺/CD8⁺ T cells, and HLA-DR-positive monocytes were significantly lower at 2 weeks after transplantation than before transplantation (all P < 0.001). This decrease was not correlated with clinical parameters. The frequency of CD4⁺CD25⁺/CD4⁺ T cells was significantly higher in KTRs with acute rejection than in KTRs at 2 weeks after transplantation (9.10% [range 4.30–25.6%] vs 5.10% [range 0.10–33.3%]; P = 0.024). However, no significant differences were observed between stable KTRs and KTRs with CMV infection. Analysis of the receiver operating characteristic curve adjusted by covariates showed that acute rejection could be predicted with 75.0% sensitivity and 68.4% specificity by setting the cutoff value of CD4⁺CD25⁺/CD4⁺ T cell frequency as 5.8%.Circulating T-lymphocyte and monocyte subsets showed significant and consistent changes in their frequencies after immunosuppression. Of the various immune cells examined, circulating levels of CD4⁺CD25⁺ T cells might be a useful noninvasive immunologic indicator for detecting acute rejection.     

Flow Cytometry for Diagnosis and Assessment of Prognosis in Patients with Myelodysplastic Syndromes

The Myelodysplastic Syndrome (MDS) comprises a spectrum of hematopoietic stem cell disorders. Identification of additional parameters that might distinguish different risk groups or entities would be useful. Flow cytometric studies have begun to characterize individual or composite immunophenotypic abnormalities as well as light scatter properties that may be helpful for diagnosis and of prognostic value for the natural course of the disease and for outcome after therapy. Here we review the current state of the art of the use of flow cytometry in patients with MDS.     

慢性胃炎胃黏膜细胞DNA含量和增殖活性的流式细胞仪检测

背景慢性萎缩性胃炎是一种胃癌癌前状态,研究表明正常胃黏膜、癌前病变和胃癌细胞的DNA含量随病变的进展而逐渐增高。目的应用流式细胞仪检测慢性胃炎胃黏膜细胞的DNA含量和增殖活性,探讨两者在慢性胃炎发生、发展过程中的临床意义。方法选取90例经胃镜检查诊断为慢性胃炎者的胃黏膜活检标本,制备单细胞悬液,应用流式细胞仪进行细胞DNA含量和增殖活性检测。结果所有慢性胃炎胃黏膜细胞的DNA倍体类型均为二倍体,但慢性萎缩性胃炎和慢性萎缩性胃炎伴肠化生胃黏膜细胞的增殖指数(PI)较慢性非萎缩性胃炎显著增高(P<0.05)。除慢性非萎缩性胃炎外,其余慢性胃炎组幽门螺杆菌(H.pylori)阳性患者胃黏膜细胞的PI值均较阴性患者显著增高(P<0.05)。结论慢性萎缩性胃炎和H.pylori阳性慢性胃炎胃黏膜细胞的增殖活性显著增高。应用流式细胞仪检测胃黏膜细胞的DNA含量和增殖活性,也许能成为胃癌癌前状态和癌前病变病理诊断的参考指标。     

富含胆固醇的脂蛋白对兔主动脉平滑肌细胞脂类堆积的影响及机制

为探讨丙二醛对外周血单核细胞是否具有趋化作用,本实验采用改良的Ｂｏｙｄｅｎ 小室微孔滤膜法进行单核细胞的趋化试验。用Ｋｉｋｕｇａｗａ 报道的方法制备丙二醛。结果发现,低、中、高三种浓度（０ ．０５ ｍｍｏｌＬ、０ ．５ ｍｍｏｌＬ和１ ｍｍｏｌＬ） 的丙二醛所诱导的单核细胞平均移动距离分别为８５．３７ ±１０ ．４４ μｍ 、１０９．０３ ±７．８８ μｍ 及１２２ ．６７ ±６．２５ μｍ , 明显大于随机移动组（６９ ．８８ ±８ ．１９ μｍ）。方差分析表明,低浓度丙二醛组与随机移动组相比,差异有显著性意义（Ｐ＜０ ．０５） ,而中浓度和高浓度丙二醛组与随机移动组相比,差异有极显著性意义（Ｐ＜０．００１）。而且,丙二醛所诱导的单核细胞移动距离与其浓度呈剂量依赖关系。化学促动组单核细胞的平均移动距离（１１０．７２ ±７．３２ μｍ）亦明显大于随机移动组（ Ｐ＜ ０．００１） 。由此可见,丙二醛对外周血单核细胞有明显的趋化作用和化学促动作用。此结果提示,在动脉粥样硬化早期,丙二醛在动脉内膜中单核巨噬细胞的募集过程中起一定作用。