肺泡蛋白沉积症9例临床病理特征与诊断

目的探讨肺泡蛋白沉积症（PAP）的临床病理特征及其诊断方法。方法对9例PAP进行常规HE染色,光镜观察,并用淀粉酶消化后过碘酸希夫（D-PAS）及黏液卡红进行组织化学染色。结果PAP显示肺泡腔内及部分小支气管腔内充满嗜伊红性细颗粒状蛋白性物质;蛋白性物质中杂有多少不等退变及脱落的肺泡上皮细胞;肺泡Ⅱ型上皮细胞增生;肺泡间隔毛细血管充血;周围肺组织代偿性肺气肿。此外,肺泡腔内嗜伊红性细颗粒状磷脂类蛋白性物质D—PAS（＋）（呈紫红色）,黏液卡红（-）。结论PAP临床罕见,易误诊或被忽视。典型PAP为肺泡腔内出现嗜伊红性细颗粒状蛋白性物质,蛋白性物质D—PAS（＋）,黏液卡红（-）,是确诊PAP的主要方法。纤支镜及胸腔镜肺活检是获取标本的主要途径。     

Ag—NOR银染技术的改进

本文介绍一种改良的AgNOR银染技术方法。该法将现行的滴染技术改为用立式或卧式染缸染色,并调整了硝酸银溶液的浓度,摸索了不同组织在15％～50％硝酸银溶液的缸染时间及温度。改良后的方法在消除非特异性银沉淀颗粒及降低背景染色等方面有较好效果,染色结果可靠,操作更为方便。     

55例非特异性慢性肉芽肿性炎中抗酸菌L型感染的探讨

目的 探讨非特异性慢性肉芽肿性炎的病因。方法 利用HE染色、改良抗酸染色（IK）和BCG免疫组化染色对55例该种病变进行检测。结果 55例中54例（98%）抗酸染色阳性,51例（92%）BCG免疫组化染色阳性,两者的符合率为94.5%。结论 非特异性慢性肉芽肿性炎很可能与抗酸菌L型感染有关。     

3D imaging of biofilms on implants by detection of scattered light with a scanning laser optical tomograph

Biofilms - communities of microorganisms attached to surfaces - are a constant threat for long-term success in modern implantology. The application of laser scanning microscopy (LSM) has increased the knowledge about microscopic properties of biofilms, whereas a 3D imaging technique for the large scale visualization of bacterial growth and migration on curved and non-transparent surfaces is not realized so far.Towards this goal, we built a scanning laser optical tomography (SLOT) setup detecting scattered laser light to image biofilm on dental implant surfaces. SLOT enables the visualization of living biofilms in 3D by detecting the wavelength-dependent absorption of non-fluorescent stains like e.g. reduced triphenyltetrazolium chloride (TTC) accumulated within metabolically active bacterial cells. Thus, the presented system allows the large scale investigation of vital biofilm structure and in vitro development on cylindrical and non-transparent objects without the need for fluorescent vital staining. We suggest SLOT to be a valuable tool for the structural and volumetric investigation of biofilm formation on implants with sizes up to several millimeters.     

Modified Field's staining—a rapid stain for Trichomonas vaginalis

Trichomonas vaginalis, a flagellate protozoan parasite commonly found in the human genitourinary tract, is transmitted primarily by sexual intercourse. Diagnosis is usually by in vitro culture method and staining with Giemsa stain. There are laboratories that use Gram stain as well. We compared the use of modified Field's (MF), Giemsa, and Gram stains on 2 axenic and xenic isolates of T. vaginalis, respectively. Three smears from every sediment of spun cultures of all 4 isolates were stained, respectively, with each of the stains. We showed that MF staining, apart from being a rapid stain (20 s), confers sharper staining contrast, which differentiates the nucleus and the cytoplasm of the organism when compared to Giemsa and Gram staining especially on parasites from spiked urine samples. The alternative staining procedure offers in a diagnostic setting a rapid stain that can easily visualize the parasite with sharp contrasting characteristics between organelles especially the nucleus and cytoplasm. Vacuoles are more clearly visible in parasites stained with MF than when stained with Giemsa.     

鲍曼不动杆菌生物被膜与耐药性的相关性分析

目的探讨鲍曼不动杆菌生物被膜与其耐药性的相关性。方法对2011年6月至2014年8月该院分离的鲍曼不动杆菌临床分布、生物膜形成情况及生物膜形成与耐药性的关系进行研究。结果标本来源主要为痰液、分泌物、腹腔积液等;强度为"+"生物膜菌株56株,占66.67%(56/84);强度为"-"生物膜菌株3株,占3.57%(3/84),所占比例最低,96.43%鲍曼不动杆菌具有较强的生物膜形成能力;具有生物膜的菌株耐药性明显高于不具有生物膜的菌株,差异有统计学意义(P0.05),不同强度生物膜菌株的耐药性比较差异无统计学意义(P0.05)。结论鲍曼不动杆菌生物膜与其耐药性密切相关,但不同强度生物膜与其耐药性无明显相关性,值得对生物膜与其耐药性的关系进行更加深入的研究。     

载超顺磁性氧化铁和DiI荧光高分子微球巨噬细胞MR成像

目的 制备载超顺磁氧化铁(SPIO)纳米粒和DiI荧光高分子微球(DiI-SPIO-PLGA),探讨其作为MRI对比剂体外巨噬细胞成像的效果和作为荧光示踪剂体外示踪巨噬细胞的可行性。 方法 采用双乳化法制备DiI-SPIO-PLGA微球,并检测其理化性质。培养小鼠RAW264.7巨噬细胞,与DiI-SPIO-PLGA微球共孵育12 h后行普鲁士蓝染色和荧光显微镜观察,将吞噬了微球的细胞和空白细胞分别重悬于0.5 ml 1%琼脂糖Eppendof管中,行MR扫描。 结果 所得样品为外壳装载SPIO颗粒和DiI荧光的微球,粒径(868.00±68.73)nm。普鲁士蓝染色结果显示,几乎所有细胞内都有蓝染颗粒分布,部分区域聚集成堆;倒置荧光显微镜下可见细胞内有大量红色微球。MRI显示吞噬了DiI-SPIO-PLGA微球的实验组管内信号显著降低,管内信号值/背景信号值明显低于对照组(P<0.05)。 结论 DiI-SPIO-PLGA微球能有效加强MR成像效果,荧光信号强烈,可同时作为MRI阴性对比剂和荧光示踪剂。     

Use of a blocking antibody method for the flow cytometric measurement of ZAP-70 in B-CLL

BACKGROUND: In this study we developed a method to measure the amount of ZAP-70 [zeta accessory protein] in B-CLL cells without relying on the ZAP-70 expression of patient B or T cells to normalize fluorescence intensity. METHODS: B-CLL cells were fixed with formaldehyde before surface staining with gating antibodies CD19PC5 and CD5FITC. The cells were permeabilized with saponin, and the ZAP-70 antigen was blocked in one tube with unlabeled antibody to ZAP-70 [clone 1E7.2]. Zap-70-PE was then added to this tube. ZAP-70-PE was added to a second tube without unlabeled antibody to ZAP-70. The mean fluorescence intensity of the ZAP-70 in the tube without unlabeled antibody divided by the mean fluorescence intensity of the ZAP-70 in the tube with unlabeled antibody equals the RATIO of total fluorescence to non-specific ZAP-70 fluorescence in the B-CLL cells. In a second method of analysis, a region is created in the histogram showing ZAP-70 fluorescence intensity in the tube with unlabeled antibody to ZAP-70. This region is set to 0.9% positive cells. This same region is then used to measure the % positive [%POS] ZAP-70 cells in the tube without unlabeled antibody to ZAP-70. The brighter the ZAP-70 fluorescence above the non-specific background, the higher the %POS. RESULTS: Due to the varying amount of non-specific staining between patient B-CLL cells and other cells, the blocking antibody method yielded a more quantitative and reproducible measure of ZAP-70 in B-CLL cells than other methods, which use the ratio of B-CLL fluorescence to normal B or T-cell fluorescence. Using this improved method, ZAP-70 was determined to be negative if the RATIO was less than 2:1 and positive if the RATIO was greater than 2:1. ZAP-70 was determined to be negative if the %POS was less than 5% and positive if the %POS was greater than 5%, a cut-off value lower than previous values published, due to exclusion of non-specific staining. Both cut-offs were based upon patient specimen distribution profiling. CONCLUSIONS: Use of a blocking antibody resulted in a robust, reproducible clinical B-CLL assay that is not influenced by the need to measure the amount of ZAP-70 in other cells. ZAP-70 results segre gate patients into indolent and aggressive groups suggested by published clinical outcomes.     

Lysosomal and ultrastructural changes in human "toxic" neutrophils during bacterial infection

"Toxic" neutrophils from humans with severe bacterial infections, identified by the presence of Döhle bodies, "toxic" granules, and vacuoles were shown to differ from normal neutrophils both in ultrastructure and in lysosome activity. Döhle bodies were identified as lamellar aggregates of rough endoplasmic reticulum. Toxic granules corresponded to the azurophilic granules usually identified by Romanowsky stains only in neutrophil precursors. By electron microscopy such granules were large, electron-dense, and peroxidase positive; they could usually be distinguished from the smaller, less dense, "specific" granules also present in control neutrophils, but in the latter they became visible by light microscopy only after prolonged staining or following fixation with glutaraldehyde. These observations suggest that toxic granules represent an abnormal staining reaction of the large dense granules in the toxic cells, and not phagocytized material, newly formed abnormal granules or autophagic bodies. Alkaline phosphatase activity was significantly greater in toxic neutrophils than in normal ones; 80% of the activity of both was located in the lysosome fraction. Beta glucuronidase was normal. Total acid phosphatase was normal, but the percentage located in the nonlysosome fraction of toxic neutrophils was increased, suggesting that lysosomes were "labilized." Formation of neutral red vacuoles in supravitally stained preparations, an index of lysosome activity, occurred more rapidly in toxic neutrophils. This reaction paralleled degranulation and the formation of clear vacuoles in unstained wet mounts and could be blocked by colchicine, a lysosome stabilizer, or enhanced by procedures which activate lysosomes. "Autophagic" vacuoles were observed by electron microscopy in some toxic neutrophils. These observations are discussed in relation to the concept that the "toxic" neutrophils in severe bacterial infection reflect cellular immaturity and/or stimulation or degeneration.     

Laser Induced Fluorescence Identification of Sinoatrial and Atrioventricular Nodal Conduction Tissue

Transcatheter ahlation of nodal tissue is used for the treatment of arrhythmia resistant to medical therapy. We have investigated the use of laser induced fluorescence spectroscopy for the in vitro recognition of nodal conduction tissue. Twelve fresh human necropsy specimens (< 48 hours)were obtained from sinoatrial node and atrioventricular node areas. Spectra were recorded during excitation at 308 nm (XeCl excimer iaser, 1.5–2.0 mJ/puJse, 10 Hz). Each area examined was marked for subsequent histoiogic examination. Four hundred eleven spectra were obtained, of which 37 contained nodai conduction tissue (21 sinoatrial, 16 atrioventricular node). Normalized fluorescence emission intensity from these areas was compared with that of surrounding endomyocardial tissue at 18 wavelengths and 35 ratios of fluorescence intensity at selected wavelengths. Spectra recorded from nodal tissue could be clearly distinguished hy a visible decrease in fluorescence emission intensity at wavelengths from 440 to 500 nm (P < 0.0006 at 450 nm), peak area, and peak width when compared to that of adjacent atrial endomyocardial tissue. Nodal conduction tissue was also distinguished from ventricular endocardium (14 spectra) by an increase in fluorescence emission at 430 to 550 nm (P < 0.0001). The specificity was 73% and 88% and the sensitivity was 73% and 60% for sinus nodal and atrioventricular nodal conduction tissue identification, respectively. A ratio of fluorescence emission intensity > 1.3 for 380/475 nm was able to detect nodal conducfion tissue (P < 0.001). Conclusion. Laser induced fluorescence can differentiate nodal conduction tissue from atrial and ventricular endocardium and may provide a new diagnostic tool for the recognition and subsequent ablation of nodal conduction tissue.     

Role of basement membrane collagen and elastin in the autofluorescence spectra of the colon.

BACKGROUND: Autofluoresence can be used to detect neoplasia in the colon. Two known fluorophores, collagen and elastin, are probably partly responsible for colonic emission spectra. Their contribution to colonic autofluorescence was investigated. METHODS: Autofluorescence spectra of normal, dysplastic, and malignant colonic tissue were studied by using excitation wavelengths from 280 nm to 350 nm. The wavelengths of peak emission and their widths at half maximum intensity were measured. Similar measurements were performed on collagen types I, III, IV, V, IX, and elastin. Colonic spectra were compared to those of collagen and elastin. Spectral differences between collagen types IV (basement membrane) I, III, V, and IX were studied. RESULTS: Four major emission peaks were noted whose wavelength of peak emission and full widths at half maximum intensity were independent of tissue histology. The emission spectra of type IV collagen differed markedly from that of nonbasement membrane collagens and elastin. CONCLUSIONS: Type IV (basement membrane) collagen is most likely responsible for the emission peak at 365 nm. The spectra of basement membrane collagen and not other types of collagen should be used in studies of epithelial tissue spectra. Elastin did not appear to be responsible for any of the four autofluorescence peaks observed in colonic tissue.     

Quantified coronary frequency domain optical coherence tomography signal analysis for the evaluation of erythrocyte-rich thrombus: ex-vivo validation study

Previous study has demonstrated that erythrocyte-rich thrombi contain more inflammatory cells and reflect high thrombus burden, leading to impaired myocardial reperfusion in myocardial infarction. The aim of this study is to investigate the utility of quantified frequency domain optical coherence tomography (FD-OCT) signal analysis in evaluating the erythrocyte-rich thrombus with ex-vivo materials. We evaluated 54 specimens of coronary artery thrombus obtained by thrombectomy catheter from 8 patients who underwent primary percutaneous coronary intervention. The thrombi were immersed in saline immediately after thrombectomy and FD-OCT image acquisition was performed ex-vivo. Quantitative analysis for all contiguous frames was performed by the dedicated automated software (OCT system software, Light Lab Inc.). For the maximum thrombus area, mean signal intensity (MSI) and normalized standard deviation of signal (NSD) was evaluated. All thrombi were stained using double staining of phosphotungstic acid—hematoxylin and eosin to enable automatic extraction of erythrocyte from fibrin. Computer-assisted analysis was performed using dedicated image processing software (WinROOF, Mitani Corp., Tokyo, Japan) for color identification of the erythrocyte area. Erythrocyte-rich thrombus, defined as % erythrocyte [(erythrocyte area/total thrombus area)?×?100]?≥?10%, showed significantly lower MSI [4.39?±?0.24 vs. 4.74?±?0.35, p?=?0.002] than that of <10%. The cut-off point for prediction of erythrocyte-rich thrombus was defined as MSI?≤?4.56, sensitivity: 87.5%, specificity: 82.9%, area under the curve: 0.836, respectively). The present ex-vivo study suggested the utility of quantified FD-OCT signal analysis on the detection of erythrocyte-rich thrombus.     

New methodology for viability testing in environmental samples

Environmental samples can be complex and are comprised of microorganisms and a matrix of decaying organic matter as well as an inorganic phase such as sand or precipitated material (waste water, sludge, soils, etc.). Nucleic acid dyes have recently been developed to address the growing need for environmental analyses (cell staining, counting, viability testing and specific organism identification). However, certain dyes may not be ideally suited for testing of environmental samples, because they readily adhere to the substrate material as well as their target molecule, resulting in increased non-specific binding and background fluorescence. The aim of this study was to address the limitations of the widely used and commercially available Live/Dead BacLight Bacterial Viability kit (Molecular Probes, Eugene, OR). A new combination of nucleic acid dyes, i.e. SYTO13 and SYTOX Orange (Molecular Probes, Eugene, OR), was proposed as an alternative. The dyes were carefully chosen for their spectral separation and increase of fluorescence quantum yield. A protocol for this combination was first designed and optimized and the two staining assays were compared against suspensions of live and dead E. coli, mixed in different proportions and it was shown that both protocols performed equally on pure cultures. However, when testing activated sludge samples, the commercial kit showed greater background fluorescence and non-specific binding than the alternate combination. Therefore, the proposed dye combination and its corresponding protocol are deemed more suitable for use on complex environmental samples than the Live/Dead BacLight Bacterial Viability kit.     

Comparison of the antimicrobial effects of chlorine, silver ion, and tobramycin on biofilm

The systematic understanding of how various antimicrobial agents are involved in controlling biofilms is essential in order to establish an effective strategy for biofilm control, since many antimicrobial agents are effective against planktonic cells but are ineffective when they are used against the same bacteria growing in a biofilm state. Three different antimicrobial agents (chlorine, silver, and tobramycin) and three different methods for the measurement of membrane integrity (plate counts, the measurement of respiratory activity with 5-cyano-2,3-ditolyl tetrazolium chloride [CTC] staining, and BacLight Live/Dead staining) were used along with confocal laser scanning microscopy (CLSM) and epifluorescence microscopy to examine the activities of the antimicrobials on biofilms in a comparative way. The three methods of determining the activities of the antimicrobials gave very different results for each antimicrobial agent. Among the three antimicrobials, tobramycin appeared to be the most effective in reducing the respiratory activity of biofilm cells, based upon CTC staining. In contrast, tobramycin-treated biofilm cells maintained their membrane integrity better than chlorine- or silver-treated ones, as evidenced by imaging by both CLSM and epifluorescence microscopy. Combined and sequential treatments with silver and tobramycin showed an enhanced antimicrobial efficiency of more than 200%, while the antimicrobial activity of either chlorine or tobramycin was antagonized when the agents were used in combination. This observation makes sense when the different oxidative reactivities of chlorine, silver, and tobramycin are considered.     

THE RELATIVE REACTION WITHIN LIVING MAMMALIAN TISSUES : V. (b) INFLUENCE OF LYMPH-INSOLUBLE TISSUE MATERIALS ON THE SIGNIFICANCE OF THE COLORATION WITH SOME PHTHALEIN INDICATORS.

The influence of lymph-insoluble tissue materials upon the colors manifested by phthalein indicators has been tested by comparing the hue of sections of various organs, stained by immersion in colored lymph, with the hue of the surrounding fluid. The reaction has been brought to approximate that of life by a greater or less saturation of the material with carbon dioxide. No evidence has been obtained of indicator errors referable to the association of the phthaleins with tissue substances. The inferences to be derived from the experiments, and from others detailed in a preceding paper, are discussed. The results of them all are in essential agreement. They demonstrate that the observed hues in tissues vitally stained with phenol red, brom cresol purple, and chlor phenol red cannot be laid to indicator errors resulting from an association of the phthalein with tissue materials. The fact is of special note in connection with organs which exhibit, when stained, the colors indicative of an outspoken acidity. The findings constitute a control upon the influence of the tissue materials on the colors manifested by the phthalein indicators when used as vital stains; but they leave untouched the problem of the influence of tissue activities upon the coloration.     

A method for assaying biofilm capacity on polyurethane-coated slides.

Isolates of coagulase-negative Staphylococci (CNS) were examined for their ability to form biofilms on polyurethane-coated slides. These slides provided a smooth plastic coating simulating polymeric plastic surfaces of medical grade catheter tubing. Slides were placed into plastic conical tubes containing tryptic soy broth inoculated with 10(6) bacteria per mL. The tubes were then incubated at 37 degrees for 48 hours. After incubation, 1 of the slides was stained with a fluorescent acridine orange stain and the other with a safranin stain. The incubation tubes were also stained with safranin. Forty-eight percent of the 65 CNS isolates were found to form a biofilm using acridine orange staining. Forty percent of the 65 CNS isolates were found to form a biofilm using the safranin stain on slides, whereas only 34% were found to adhere on sides of plastic tubes. Increased sensitivity of the fluorescent stain was probably due to enhanced visualization of smaller numbers of bacteria on the plastic. This method using fluorescent stained plastic-coated slides was easier to visualize and interpret than the tube method.     

Quality control of azure B preparations by liquid chromatography and standardization with azure B tetrafluoroborate

A liquid chromatographic procedure for the quantitative determination of the thiazine dye azure B, the principal constituent of Romanowsky stains, is presented. Unlike previous methods relying on peak area normalization, the present approach involves real quantitation through calibration with the reference standard azure B tetrafluoroborate. The method has been used for the quality control of commercial azure B preparations and to study their stability in stock and staining solutions, either or not in the presence of eosin Y. Results suggest that highly pure azure B perchlorate meets the requirements of a reference material, useful for standardization of Romanowsky-Giemsa staining in haematology.     

骨髓活检塑料包埋切片铁染色在血液病病理诊断中的意义

目的　研究铁染色在血液病骨髓活检病理诊断中的意义。方法　骨髓活检塑料包埋 ,半薄切片 ,Perls蓝染色 ,半定量分析。结果　 584例血液病中铁染色阳性率最低者为缺铁性贫血 (0 / 7) ,此后依次为特发性血小板减少性紫癜 (6 5 % ,4/ 62 ) ,真性红细胞增多症 (9 1 % ,1 / 1 1 ) ;阳性率较高者依次为纯红细胞再生障碍性贫血 (1 0 0 % ,8/ 8) ,骨髓增生异常综合征 (75 5 % ,40 / 53) ,巨幼细胞性贫血 (60 % ,9/ 1 5)。骨髓增生异常综合征铁染色阳性率显著高于慢性再生障碍性贫血和特发性血小板减少性紫癜 (P <0 0 1 ) ,纯红细胞再生障碍性贫血铁染色阳性率显著高于慢性粒细胞白血病 (P <0 0 1 ) ,骨髓增生异常综合征铁染色阳性率显著高于骨髓增殖性疾病 (不能分类 ) (P <0 0 1 )。结论　骨髓活检铁染色在某些血液病骨髓病理诊断及鉴别诊断中具有重要参考价值     

改良Marsland法与LFB双重染色方法及其应用

目的介绍一种改良的Marsland法与Luxol fast blue(LFB)双重染色方法及其应用。方法用10%硝酸银溶液和0.2%Luxol fast blue溶液在一张石蜡切片上同时显示两种不同的神经组织成分。结果神经纤维呈黑色,神经元、轴索、树突呈黑色或紫黑色,髓鞘呈鲜蓝色。结论改良的Marsland法与LFB双重染色方法,使神经纤维染成黑色,髓鞘呈鲜艳蓝色,两种颜色对比明显,背晃清晰。     

石棉沉积症伴肺间质纤维化1例误诊原因分析

目的 阐明石棉沉积性肺间质纤维化的临床病理特征，提高对其临床及病理表现的认识，避免漏诊和误诊。方法 报道1例石棉沉积性肺间质纤维化的临床和病理改变，并结合文献对该疾病的临床表现及病理学特征进行讨论。结果 组织学表现为胸膜、肺泡间隔和支气管血管周围呈慢性炎症及纤维化，病变不连续，与相对正常的肺组织相间隔。肺间质中可见散在分布的串珠样石棉小体，铁染色（＋），呈蓝黑色。结论 肺石棉沉积症的病理改变主要为肺间质纤维化，病变类似普通型间质性肺炎（UIP），其诊断和鉴别诊断主要依据在肺间质纤维化背景中找到石棉小体，可使用铁染色加以证实。