金胺O荧光染色法在病理特殊染色中的应用

目的 探讨金胺O荧光染色法应用在病理特殊染色上的可行性.方法 采用金胺O配制成的荧光染色液,用于考虑为结核的病理活检组织中,并以经典的抗酸染色法为对照.结果 在荧光镜下,金胺O荧光染色法将结核杆菌染成金黄色.结论 金胺O荧光染色法在病理特殊染色工作中有一定价值.     

肉芽肿性小叶性乳腺炎106例临床病理特征及病因分析

目的探讨肉芽肿性小叶性乳腺炎(granulomatous lobular mastitis,GLM)的临床病理学特征,并分析其与非结核分枝杆菌(nontuberculous mycobacteria,NTM)感染的相关性。方法对106例已经确诊的GLM重新阅片,并行金胺O、PASM、PAS特殊染色及荧光定量PCR检测。结果106例GLM均为女性,平均年龄33岁,有生育史及哺乳史104例,97例距末次妊娠3年发病,平均病程3个月。镜下组织学特征以乳腺终末导管小叶单位为中心的慢性化脓性肉芽肿性炎症。荧光定量PCR检测结核/NTM均阴性,PASM、PAS特殊染色未查见真菌,金胺O特殊染色1分15例、2分3例,金胺O染色阴性88例,阳性率为16.98%。18例金胺O染色阳性患者随访发现伴窦道形成5例,手术切口愈合差者2例,在1年内复发5例;88例金胺O染色阴性患者伴窦道形成14例,手术切口愈合差者5例,在3个月内复发5例。结论GLM是少见的良性炎症性乳腺疾病,NTM感染可能是GLM的病因之一,将金胺O、PASM、PAS特殊染色及荧光定量PCR联合检测可作为GLM的常规检查,为治疗GLM提供病理学依据。     

提高抗酸染色阳性率的几点细节

正在临床病理诊断中遇到肉芽肿性病变伴干酪样坏死或结核样结节常常应用抗酸染色作诊断与鉴别诊断。作为基层医院,在没有荧光显微镜无法进行金胺O染色且无条件开展PCR定量检测技术的实验室,Ziehl-Neelsen苯酚碱性品红法检测抗酸杆菌是最常用也是最传统、经典的染色法,由于其成本低,操作简单、直观准确、费用少且无需专门仪器设备,在病理实验室中广泛应用,但由于该方法特异性高、阳性率较低,为了提高抗酸杆菌的检出率,作者对染液的配制、染     

规范化特殊染色技术对肺真菌感染性疾病病理诊断的意义

目的探讨规范化特殊染色技术在肺真菌感染性疾病病理诊断中应用的意义。方法选取2011年9月~2016年3月北京朝阳医院最终病理诊断为肺真菌感染性疾病104例,行HE染色、HE染色联合传统手工特殊染色法染过碘酸雪夫氏染色(PAS)及六胺银和HE染色联合全自动特殊染色法染PAS及六胺银,将两种特染技术规范化比较,镜下观察分析结果(均为初次染色结果,不包括复染结果),以最终病理诊断结果为标准,与临床初诊结果比较。结果肺真菌感染性疾病确诊率和真菌分型率,从低到高顺序一致,为临床初步诊断(29.8%和19.2%)、HE染色法(32.7%和32.7%)、HE染色联合传统手工特殊染色法染PAS和六胺银(90.4%和87.5%)、HE染色联合全自动特殊染色法染PAS和六胺银(98.1%和94.2%)。这4种方法从两方面比较差异有统计学意义(P0.01,P0.01),第4种方法与前两种比较方法差异有统计学意义(P0.01,P0.01),与第3种方法比较确诊率差异有统计学意义(P0.05),分型率差异无统计学意义(P0.05)。但全自动特殊染色法染PAS及六胺银操作步骤更简单,化学试剂规范化,无人工和环境因素影响,耗时短,脱片和复染张数更少。结论 HE染色法联合全自动特殊染色法染PAS和六胺银更加规范化,有助于提高肺真菌感染性疾病的确诊率和真菌分型率。     

活检组织中真菌Calcofluor White荧光染色法的改良

目的：探索对Calcofluor White荧光染色法的改良,为活检诊断真菌感染提供一种快速、经济、对比更清晰的染色方法。方法：采用Calcofluor White M2R和苏木素、伊红染液,组合成3种染色步骤,对32例病例进行染色,筛选出适用于活检组织,并且在光镜与荧光镜下能同时观察的改良染色法步骤。结果：光镜下,组织呈现HE染色固有的形态。切换至荧光显微镜下,真菌则被显示为亮蓝色,组织背景呈黄绿色,结果清晰、醒目,对比强烈。结论：改良Calcofluor White荧光染色法能使同一张切片在光镜和荧光显微镜下同时观察,相互对照和定位,是提高真菌检出的好方法,值得在真菌的病理活检工作中广泛使用。     

应用HE、P/VB、AB染色法观察心脏瓣膜疾病的病理特征

心脏瓣膜疾病是临床常见的心脏病，近20年来心脏瓣膜病的发病原因及病理改变呈现多元化，以往仅用HE染色，形态结构虽清晰可见，但组织成分不甚清楚。为了能摸索出针对心脏瓣膜组织的特殊染色方法，经过长期的染色实践，应用P／VB和AB特殊染色法，能清楚地显示瓣膜内各种组织成分的改变，从而为研究心脏瓣膜病的病理改变特点提供了可靠的技术方法。     

6种不同染色方法在检测肺组织中新型隐球菌的应用比较

目的探讨6种检测肺组织中新型隐球菌(cryptococcus neoformans,CN)不同的染色方法及其意义。方法采用常规HE染色法、PAS染色法、六胺银染色法、阿尔辛蓝染色法、亚甲蓝染色法、改良Hale胶体铁组合染色法,对20例病理诊断为肺隐球菌病的石蜡样本进行染色。结果 6种方法均可显示肺组织中有CN,其中在HE染色中CN孢子着色不佳,轮廓不清晰,较难辨认; PAS法检测的CN细胞壁呈紫红色,胞质呈浅红色;六胺银法检测的CN呈黑褐色,菌壁四周深黑,菌体中间空白;阿尔辛蓝法检测的CN荚膜呈蓝色,细胞核呈红色;亚甲蓝法检测的CN呈蓝色,菌壁四周浅蓝色,菌体中间透亮,对比和背景较差;改良Hale胶体铁组合染色法检测的CN在肺泡腔、肺间质的结缔组织中成堆或散在分布,呈深蓝色,圆形或卵圆形,轮廓清晰,单核细胞及多核巨细胞中亦可见较多吞噬的CN,细胞核呈红褐色,胶原纤维呈紫红色,背景淡黄色,镜下极易辨认。结论改良Hale胶体铁组合染色法是检测CN效果最佳的染色方法。     

用于胃幽门螺杆菌检查的改良 Giemsa 染色法

幽门螺杆菌（ＨＰ）是一种革兰氏阴性菌，已经证明ＨＰ与一些胃炎和消化性溃疡有较密切的关系。ＨＰ也是胃窦炎最常见的病因之一，并与远端胃癌和低度恶性胃淋巴瘤的发生有关。所以，越来越引起人们的重视。在临床病理工作中，胃粘膜活检不要仅局限于胃粘膜本身的病变检查，还需要进一步检查有无ＨＰ感染。ＨＰ是一种较弱的噬苏木素菌，对ＨＥ染色着色较淡，在观察时有一定的困难。为了明确有无ＨＰ感染常借助于特殊染色。用于ＨＰ特殊染色的方法有Ｇｉｅｍ－ｓａ染色法和银染色法等。我们采用改良的微波Ｇｉｅｍｓａ染色法，即在原Ｇｉｅｍｓａ染色法的基础上，加入了表面活性剂ＴｒｉｔｏｎＸ－１００，在染色过程中经微波炉加温处理。应用改良的Ｇｉｅｍｓａ染色法，可缩短染色时间。染色结果表明，胃粘膜组织和ＨＰ着色良好，细微结构清楚，结果明确。微波Ｇｉｅｍｓａ染色法用于胃粘膜石蜡切片染色，具有常规ＨＥ染色和特殊染色的双重效果，能同时清楚地显示出胃粘膜组织和ＨＰ的结构。     

介绍一种真菌的染色方法

真菌中含有醛基 ,能将银分子还原为黑色的金属银而显色。日常所用的Ｇｒｏｔｔ Ｇｏｍｏｒｉ六胺银染色法就是利用这一原理进行染色的。但是Ｇｒｏｔｔ Ｇｏｍｏｒｉ六胺银染色法步骤多、要求高、耗时长、银液的浪费也较大。笔者在日常染色中 ,用Ｇｏｍｏｒｉ氨银液代替六胺银液配合微波进行染色 ,两者染色效果差异不大 ,但Ｇｏｍｏｒｉ氨银 微波法省时、省料 ,也较易着色 ,取得了满意的工作效果。一、材料与方法1.试剂及其配制 :(1)Ｇｏｍｏｒｉ氨银液 :用小量杯取 3ｍｌ10 %硝酸银水溶液 ,逐滴加入 10 %ＫＯＨ水溶液 1ｍｌ,产生棕黑色沉…     

病理特殊染色联合病理免疫组化在鉴别肿瘤病理中的临床意义

目的:探讨病理特殊染色联合病理免疫组化在肿瘤病理诊断中的临床价值.方法:选取86例我院2019年8月至2021年8月就诊的肿瘤患者,所有患者入院后均进行穿刺活检,采用病理特殊染色法、病理免疫组化对穿刺采集的组织进行检查,以术后病理诊断结果为"金标准",比较病理特殊染色法、病理免疫组化单独、联合检查诊断价值.结果:经术后病理诊断结果显示,恶性62例;经病理特殊染色、免疫组化检查结果显示,恶性分别50例、48例;经联合诊断显示,恶性61例;与病理特殊染色、免疫组化单独诊断比较,联合诊断灵敏度、准确率较高,漏诊率较低(P<0.05).结论:病理特殊染色、病理免疫组化联合诊断准确率显著高于单独检查,有助于临床提高早期诊断效能,为临床制定治疗方案提供依据.     

Phenolic acridine orange fluorescent stain for mycobacteria.

A new fluorescence acid-fast staining method with acridine orange as the specific stain is presented. Only two reagents are required: the acridine orange-specific stain and a destaining-counterstaining reagent. Compared with auramine fluorescence acid-fast staining, there was less nonspecific staining of non-acid-fast debris which fluoresced a pale green contrasting color to provide a background in which to search for the red-to-orange fluorescing acid-fast bacilli. The results of the study indicate that the acridine orange method is superior to the auramine method in detecting acid-fast bacilli in specimen smears.     

Reticulated platelet and its clinical significance

Reticulated platelets (RP) retain some residual mRNA in their cytoplasm and are thought to be the most recently produced platelets in circulation. They can be visualized on a blood film with new methylene blue staining. RP is a flow cytometric assay utilizing a fluorescent dye, either thiazole orange or auramine O. There is a difference in reference values for RP between thiazole orange and auramine O. From the analytic results of RP in patients with thrombocytopenic disorders, RP measurement is considered useful for estimating thrombopoiesis in bone marrow and for differential diagnosis and elucidating the pathophysiology of thrombocytopenic disorders. In the future, test for RP might have a possibility of routine examination because RP can be rapidly and simply measured using whole blood stained with auramine O using an automated reticulocyte counter modified to determine RP. Furthermore, it is anticipated that standardization and morphological definition for RP are established and accuracy for RP is improved.     

Use of lipophilic fluorescent probes for the isolation of hybrid cells in flow cytometry

Flow cytometry was used for the isolation of hybrid cells immediately after fusion. Precursor cells were stained by two lipophilic fluorescent probes: perylenoyl-labeled triglyceride (perylenoyl-TG, green fluorescence, 520 nm) and rhomdaminyl-labeled triglyceride (rhodaminyl-TG, red fluorescence, greater than 580 nm). Since the maximum emission of perylenoyl-TG coincides with the maximum absorbance of rhodaminyl-TG, the two fluorescent dyes form an effective donor-acceptor pair. Cells stained by perylenoyl-TG (0.25-1 microgram/ml) at the excitation wavelength of 457 nm displayed high intensity of fluorescence in the green region (520 nm), and low intensity of fluorescence in the red region (greater than 580 nm). Using the same conditions, cells that were stained by rhodaminyl-TG displayed a low intensity of fluorescence in both regions. When cells were simultaneously labeled by perylenoyl-TG and rhodaminyl-TG (used in a concentration ratio of 1:10, respectively) essentially total energy transfer was observed, and the cells exhibited a high intensity of red fluorescence. After the fusion of cells which had been separated stained by perylenoyl-TG and rhodaminyl-TG, the hybrid cells containing the two fluorescent probes had a high intensity of red fluorescence. Resonance exitation energy transfer between the two fluorescent dyes permits effective sorting of hybrid cells by flow cytofluorometry.     

Localization of unesterified cholesterol in human atherosclerotic lesions. Demonstration of filipin-positive, oil-red-O-negative particles.

Both unesterified and esterified cholesterol accumulate in human atherosclerotic lesions. Whereas previous studies have established that esterified cholesterol deposits intra- and extracellularly, less is known concerning the distribution of lesion unesterified cholesterol. The objective of this study was to establish the location and in what structures unesterified cholesterol accumulates in lesions. The fluorescent probe filipin has been used to detect unesterified cholesterol. In addition, the lipid-soluble dye oil red O (which does not stain unesterified cholesterol) was used to stain hydrophobic lipids, including esterified cholesterol. Filipin staining occurred in association with three extracellular structures: spherical particles, elongated crystals, and granular or amorphous calcium deposits. These structures were not stained by oil red O. Filipin-stained particles sometimes accumulated within cells, which did not contain any oil-red-O-stained lipid. Interestingly, extracellular filipin-stained particles occurred in loci separate from extracellular oil-red-O-stained particles. The results of this study suggest that accumulation of unesterified and esterified cholesterol occurs within many diverse structures in atherosclerotic lesions. Extracellular filipin-stained particles constituted a significant component of accumulated cholesterol. These cholesterol-rich particles have not been previously observed because they are not stained by lipid-soluble dyes such as oil red O.     

Early detection of Chlamydia trachomatis using fluorescent, DNA binding dyes.

HeLa 229 cells were infected with genital tract strains of Chlamydia trachomatis. After incubation for varying times the infected cells were fixed and stained with the fluorescent DNA binding dyes Hoechst 33258 or DAPI for comparison with conventional Giemsa stain. Fluorochrome-treated preparations were examined by incident ultraviolet fluorescence microscopy and the Giemsa-stained preparations by dark-ground light microscopy. Chlamydial inclusion bodies could be identified unambiguously as early as 18 hours after infection of HeLa 229 cells using either Hoechst 33258 or DAPI but not until some 48 hours in Giemsa-stained preparations. The DNA rich chlamydial elementary bodies in infected egg yolk suspension were readily detected using Hoechst 33258. The fluorescent dye technique was simpler and more rapid than Giemsa staining. Using Hoechst 33258 it is possible to speed up the identification of chlamydial isolates growing in tissue culture.     

Evaluation of a safe sputum processing method for detecting tuberculosis.

AIMS--To evaluate a safe sputum processing method for detection of tuberculosis in developing countries. METHODS--A sample processing method was developed in which acid fast bacilli were killed with 1% sodium hypochlorite and concentrated by flotation on a layer of xylene before staining by the Ziehl Neelsen or auramine O methods. RESULTS--Best results were obtained by auramine O staining after flotation. Staining by the Ziehl Neelsen method after flotation gave better results than direct Ziehl Neelsen staining without flotation. CONCLUSIONS--The flotation method with Ziehl Neelsen staining offers advantages for smear preparation in the tuberculosis control programmes of developing countries.     

Fish cast NETs: neutrophil extracellular traps are released from fish neutrophils

Neutrophil extracellular traps (NETs), which are extracellular DNA structures released from neutrophils, are described and characterized for the first time in fish using fluorescent confocal microscopy. Confocal images of fish neutrophil suspensions stained with 6'-diamino-2-phenylindole, dihydrochloride DNA fluorescent stain (DAPI) revealed the presence of NETs which appeared as fibrous structures connecting several cells. Co-localization of NETs with neutrophil granular proteins and actin was investigated using specific antibodies and probes. Double staining of neutrophils with SYTOX green and DAPI revealed that SYTOX stain applied to living cells stained extracellular DNA, but not nuclei. NETs are actively released from stimulated living cells, associated with granular proteins, but not with cytoskeleton, and are not a product of nuclear degradation seen in late apoptotic stages. Additionally, a fluorometric microtiter plate assay to quantify the release of NETs was adopted for use with fish neutrophils, and the effect of stress on NETs release was studied. This assay detected the inhibition of DNA release during stress conditions. In summary, NETs were released from living fish kidney neutrophils upon stimulation, characterized using fluorescence DNA-binding dyes, specific antibodies and probes, and quantified using a microtiter plate fluorometric assay that can rapidly measure a large number of samples. Detection of NETs can be used as an additional assay to an existing battery of functional tests, and as a new research model to study the effects of stress, immunomodulators, and diseases.     

A sensitive assay of cytotoxicity applicable to mixed cell populations

The fluorescent vital dye, Hoechst 33342, was used to stain cultured cells prior to assay of antibody dependent complement mediated cytotoxicity. The fluorescence of nonviable dye stained cells is quenched by cellular uptake of trypan blue, but trypan blue excluding cells remain intensely fluorescent. Detection by fluorescence microscopy of one viable prestained cell per 10(5) unstained cells was accurate and reliable. The technique was found to have sensitivity equal to a clonogenic assay for measuring cytotoxicity. The dye stained cell assay may be used to measure depletion of a selected cell type, when those cells are stained prior to mixing with another cell population. This technique may prove useful to study model systems for depletion of tumor cells or T-cells from bone marrow.     

An automated optoelectronic reticulocyte counter

Microscopic reticulocyte counting is time consuming and imprecise. A new reticulocyte counter has been developed, and the authors evaluated its utility for laboratory use. The counter, R-1000 of Sysmex-TOA Medical Electronics Company, Kobe, Japan, is based on the principles of flow cytometry. Reticulocytes are detected as fluorescent cells stained with a basic dye, auramine O, under argon-laser light. The automated count had high correlation to the manual count (r = 0.941). Linearity and reproducibility were both high. About 60 specimens were tested in one hour. Not only the reticulocyte percentage and count but also the maturity of reticulocytes was found from the intensity of the fluorescence, whether high, moderate, or slight. Normal reference values were 0.007 +/- 0.0055 (0.70 +/- 0.55%) for the reticulocytes, (4.63 +/- 1.09) X 10(9)/L for the reticulocyte count, 2.3 +/- 1.9% for highly fluorescent cells, 18.7 +/- 5.1% for moderately fluorescent cells, and 78.8 +/- 6.6% for cells with slight fluorescence. In patients with suppressed bone marrow function, such as is caused by chemotherapy, the reticulocyte fraction and count were low, and cells with slight fluorescence increased. In patients in whom bone marrow function was stimulated, such as with hemolytic anemia, the reticulocyte percentage, reticulocyte count, and highly fluorescent cells were high. Patients with chronic renal failure being treated by hemodialysis had a similar reticulocyte pattern to that in hemolytic anemia except that the reticulocyte count was decreased. Results for elderly patients were not different from those of healthy young controls. Some patients with a normal reticulocyte count and percentage had numerous highly fluorescent cells, perhaps because of hemolytic anemia not yet identified. Automated reticulocyte counting provides reliable data, so such measurement should be useful for analysis of the kinetics of red blood cells and for the study of the pathogenesis of anemia.     

Spectral characteristics of metachromatically stained cartilage: effects of enzymatic degradation and dehydration.

When a microspectrophotometer was used to study cartilage, stained metachromatically with Azure A and Safranin O, the following 3 points were made: 1. the absorption maximum of stained cartilage matrix is identical to that reported for solutions of these dyes containing chondroitin sulfate; 2. partial enzymatic degradation of the cartilage matrix has no effect on the position of the absorption peak (although the intensity is greatly diminished); 3. ethanolic dehydration shifts the absorption peak somewhat toward the orthochromatic position, and decreases the intensity of the stain.