改良Pereira髓过氧化物酶快速染色法及应用

目的　为了使髓过氧化物酶 (MPO)染色快速准确、安全可靠 ,对Pereira碘化钾MPO染色方法做了进一步改进。方法　采用将碘化钾溶于Wright Giemsa染色液中的新配方 ,使试剂更稳定、保存时间长 ,并简化了操作、缩短了染色时间。结果　此法与Washburn联苯胺法比较 ,两者阳性率十分相近 ,差异无显著性意义 (P >0 0 5 )。结论　改良Pereira髓过氧化物酶法阳性反应标本存放多年不褪色 ,是目前众多碘化钾法中较理想的MPO染色方法之一。     

PDGF-D、MPO与CD15在大肠癌中的表达及临床相关性研究

目的：探讨血小板源性生长因子D（PDGF-D）、髓过氧化物酶（MPO）YL粒细胞相关抗原（CD15）在大肠癌组织中的表达及其与临床特征之间的关系。方法：采用免疫组化染色方法检测88例大肠癌组织、72例大肠腺瘤组织及50例正常大肠粘膜组织中PDGF-D、MPO及CD15的表达情况。结果：PDGF—D、MPO、CD15在大肠癌组织中的阳性表达率分别为85．23％、63．64％、61．36％。PDGF—D、MPO在正常组、大肠腺瘤组和大肠癌组三者之间的表达均有显著性差异（P〈0．05）。CD15在正常组、大肠腺瘤组中的阳性表达率与大肠癌组中的阳性表达率有显著性差异（P〈0．05）,但在正常组与大肠腺瘤组中的阳性表达率无显著性差异（P〉0．05）。PDGF—D、MPO、CD15的表达在有淋巴结转移组织中的阳性率分别为92_31％、75．00％,73．08％;在无淋巴结转移组织中的阳性率分别为75．00％、52．78％,47．22％,三者在有无淋巴结转移组织中的阳性率均有显著性差异（P〈0．05）。PDGF．D、MPO、CD15在大肠癌中的表达与性别、年龄及组织分化程度均无相关（P〉0．05）。经Spearman相关性分析,PDGF—D及MPO在大肠癌的表达具有相关性（P〈O．05）。大肠癌中CD15与PDGF—D、MPO的表达无明显相关性（P〉0．05）：结论：PDGF—D、MPO与CDl5在大肠癌中的高表达,提示均参与了大肠癌的发生发展,可作为大肠癌恶性程度和侵袭转移的分子生物学标志物。     

消化道组织块黏液细胞的组织化学染色

目的：探索中华蟾蜍、黑斑蛙消化道组织块黏液细胞的组织化学染色。方法：利用阿辛蓝-过碘酸Schiff（AB-PAS）反应,对消化道组织块黏液细胞进行染色和观察。结果：对消化道壁较薄的组织块（食管、小肠、大肠）,采用3％乙酸3min、1％阿尔新蓝30min、3％乙酸3min、3％过碘酸氧化10min、Schiff反应20min染色,即可达到良好染色效果;对消化道壁较厚的组织块（胃）,则应采用3％乙酸9min、1％阿尔新蓝70min、3％乙酸9min、3％过碘酸氧化30min、Schiff反应60min染色,将能达到良好染色效果。结论：经过染色可将黏液细胞分为四个类型：Ⅰ型红色,PAS染色阳性,AB染色阴性;Ⅱ型蓝色,PAS染色阴性,AB染色阳性;Ⅲ型紫红色,PAS染色强阳性,AB染色弱阳性;Ⅳ型蓝紫色,PAS染色弱阳性,AB染色强阳性。     

抗中性粒细胞胞浆抗体、抗核抗体联合检测对类风湿关节炎的诊断价值

目的：探讨抗中性粒细胞胞浆抗体（ANCA）与抗核抗体（ANA）联合检测对类风湿关节炎的临床意义。方法：采用IIF法对82例RA患者（RA组）、74例非RA自身免疫疾病患者（非RA组）和52例健康体检者（正常对照组）的血清ANCA和ANA谱进行了检测分析,并用ELISA法进行抗丝氨酸蛋白酶3（PR3）、抗髓过氧化物酶（MPO）、ANA谱的定量检测。结果：RA组82例患者中,64例ANCA阳性,阳性率为78．08％,其中核周型（PANCA）37例,阳性率为45．1％,胞浆型（CANCA）27例,阳性率为32．9％;非RA组74例患者中有7例ANCA阳性率分别为9．4％;正常对照组50例中没有一例ANCA阳性。利用Elisa法对患者血清进行检测,分别能够特异的检测到PR3、MPO、抗双链DNA抗体（抗ds—DNA抗体）、抗SS—A等抗体、抗ss—A抗体、抗PM—SCL抗体的存在。结论：联合ANCA、ANA检测有助于提高类风湿关节炎的诊断。     

两种方法对上海及周边地区实验大小鼠螺杆菌携带情况的调查

目的了解上海及周边地区实验小鼠、大鼠螺杆菌携带情况,为我国实验动物等级及监测标准的制定提供参考和依据。方法PCR法共检测了352只小鼠（清洁级101只,SPF级251只）,101只大鼠（清洁级69只,SPF级32只）;ELISA法共检测了88只小鼠（清洁级26只,SPF级62只）,165只大鼠（清洁级84只,SPF级81只）;并对其中88只小鼠、101只大鼠的PCR和ELISA法阳性检测率进行比较。结果PCR法检测小鼠平均阳性率为35．8％（126／352）,清洁级阳性率为51．5％（52／101）,SPF级阳性率为29．5％（74／251）;大鼠平均阳性率为70．3％（71／101）,清洁级阳性率为69．6％（48／69）,SPF级阳性率为71．9％（23／32）;ELISA法检测小鼠平均阳性率为15．9％（14／88）,清洁级阳性率为19．2％（5／26）,SPF级阳性率为14．5％（9／62）;大鼠平均阳性率为52．7％（87／165）,清洁级53．6％（45／84）,SPF级51．9％（42／81）;88只小鼠PCR法阳性检测率为72．7％（64／88）,ELISA法阳性检测率为15．9％（14／88）;101只大鼠PCR法阳性检测率为70．3％（71／101）,ELISA法阳性检测率为49．5％（50／101）。结论上海及周边地区实验大鼠、小鼠中皆存在着不同程度的螺杆菌感染,两种方法阳性检出率比较结果表明回盲部内容物PCR法较检测血清中抗螺杆菌抗体ELISA法更为敏感。     

几种染色剂对桔小实蝇产卵孔的染色效果

为寻找水果上桔小实蝇产卵情况的快速检测方法,本文测定了甲基蓝、龙胆紫、品红、曙红、藏红和刚果红等6种染色剂对水果上桔小实蝇Bactrocera dorsalis（Hendel）产卵孔的染色效果。结果表明染色剂对可疑水果进行染色,若有产卵孔,产卵孔可迅速被染色,染色率最高可达100％。筛选出甲基蓝、藏红和刚果红作为芒果、番石榴和夏橙的最适染色剂,各种染色剂在浓度为0．5％时染色效果较佳,同时对持续冷藏保存后的水果上的产卵孔仍然具有较高的染色率。     

PCR法快速检测临床标本中结核杆菌DNA

应用聚合酶链反应（ＰＣＲ）快速检测临床标本（脑脊液、胸水、腹水、血、痰液）中的结核杆菌ＤＮＡ,特异性扩增片段１２３ｂｐ,为结核杆菌的特异性重复序列ＩＳ６１１０部分基因。ＰＣＲ检测人型结核杆菌的敏感性达１０ｆｇＤＮＡ。临床标本的ＰＣＲ检测阳性率（２３．３％）明显高于抗酸染色涂片（２．９％）和细菌培养（５．７％）的阳性率（Ｐ〈０．０５）。通过设立对照系统及对扩增产物酶切分析,表明该法无假阴性结果（特异     

面部原发性皮肤隐球菌病1例

报道1例自身免疫功能正常的原发性皮肤隐球菌病。患者因"面部红斑进行性加重半年,破溃伴结痂2个月余"来我科就诊,皮肤科检查可见左侧面颊部3 cm×5 cm大小红斑,边界清楚,中央可见数片浅溃疡,覆盖灰褐色痂皮。病理检查示真皮浅层及深层可见大量炎性细胞浸润,PAS染色见大量紫红色孢子聚集在多核巨细胞内外;阿申蓝染色阳性;粘蛋白卡红染色阳性;免疫组化染色(抗体为兔抗隐球菌抗体)可见棕色圆形孢子。根据其临床症状、病理检查、特殊染色及免疫组化染色,确诊为原发性皮肤隐球菌病。经伊曲康唑治疗痊愈。     

改良PAS染色法在急性肝损伤中的应用

目的：观察改良肝脏糖原PAS染色法,并观察肝脏糖原染色在急性肝损伤中的应用。方法：复制CCl4急性肝损伤模型,首次100％CCl43 mL／kg皮下注射,此后50％CCl4橄榄油溶液2 mL／kg每周2次共4次皮下注射,诱导大鼠急性肝损伤模型。计算大鼠肝体比;HE染色观察肝组织炎症病理;试剂盒检测血清丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）、总胆红素（TBil）、白蛋白（Alb）。肝脏常规PAS染色与改良PAS染色观察肝糖原染色。结果：与正常组相比,模型组ALT、AST活性与TBil含量明显升高（P＜0．05）,Alb含量明显降低（P＜0．05）;HE染色示,模型组肝小叶结构排列紊乱,肝细胞脂肪变、气球样变明显。常规PAS染色,正常组肝组织PAS染色阳性占肝脏面积为32．38％±5．50％;与正常组相比,模型组肝组织PAS阳性染色明显减少（P＜0．01）,占肝脏面积为8．60％±3．34％。改良PAS染色提示,正常组肝脏可见大量PAS阳性染色,占肝脏面积为75．50％±9．02％;与正常组相比,模型组肝组织PAS阳性染色明显减少（P＜0．01）,占肝脏面积为17．61％±3．53％。在空白对照组与模型肝组织中,肝糖原改良PAS染色阳性率明显高于常规PAS染色法（P＜0．01）。改良PAS染色肝糖原阳性染色面积更真实反映急性肝损伤程度。结论：改良肝脏糖原PAS染色法有助于急性肝损伤程度评估。     

一期梅毒实验室诊断差异性研究

目的通过梅毒螺旋体初筛试验、确认试验和鉴别诊断试验,探讨一期梅毒实验室诊断差异性,最大限度减少漏诊与误诊,为深入研发新型早期梅毒诊断试剂奠定基础。方法依据2000年中国卫生部防疫司颁布的性病诊断标准,临床筛选一期梅毒患者86例（研究组）和非梅毒患者100例（对照组）,对患者血清进行甲苯胺红不加热血清试验（TRUST）初筛和梅毒螺旋体明胶颗粒凝集试验（TPPA）确认。筛选临床体征、TRUST法和TPPA法三者结果有差异的患者进一步鉴别诊断,鉴别诊断主要应用荧光定量PCR（FQ-PCR）法、免疫PCR法与自身抗体检测等试验。结果初筛TRUST法灵敏度和特异性分别为62．8％、93．0％;确认TPPA法灵敏度与特异性分别为66．3％、100％。TRUST法和TPPA法两者结果差异占12．8％;临床体征诊断、TRUST法和TPPA法三者结果差异占41．9％。TPPA法与TRUST法两者均阴性的一期梅毒患者中,FQ-PCR阳性率达88．0％,免疫PCR阳性率占40．0％。TPPA法阳性、TRUST法阴性的一期梅毒患者免疫PCR法与TPPA法结果一致;TPPA法阴性、TRUST法阳性11例患者中结核抗体阳性2例,类风湿因子阳性3例与抗Sm抗体结果阳性6例。结论一期梅毒患者实验室诊断结果差异性较大,漏诊与误诊的比例较高,有待研发新型的诊断试剂和提高诊断水平。     

Cytochemical identification of turbot myeloperoxidase-positive granulocytes by potassium iodide and oxidized pyronine Y staining

Cytomorphological and cytochemical staining are important methods for the identification of cell types, in particular in fish which often lack biological tools such as specific antibodies. Myeloperoxidase (MPO) is usually used as an intracellular marker of neutrophil accumulation in tissues and a marker of neutrophil activity in plasma. In this study, we reported a potassium iodide and oxidized pyronine Y (KI-PyY) staining method for rapid and highly sensitive detection of MPO-positive cells in turbot blood, peritoneum, and tissues. MPO-positive cells, which mostly represented neutrophils, were stained brown and clearly distinguished from other cells, such as lymphocytes, monocytes, and macrophages, which were stained pink. Following bacterial stimulation, the proportions of neutrophils were 27.49% and 38.05% in peripheral blood leukocytes and peritoneum, respectively, judging by the stained MPO. Kidney granulocytes contained abundant MPO-positive cells which were probably immature neutrophils with low expression of MPO. It is noteworthy that MPO-positive cells were detected in the tissue sections of kidney, spleen, and gut, with distribution profiles specific to each tissue. However, the cell morphology was not distinct in the stained tissue sections. These results indicate that the KI-PyY staining method is highly sensitive, applicable to different types of samples, and will be useful for the study of neutrophils in different compartments of fish.     

An investigation of new commercial samples of Methyl Green and Pyronin Y

Summary New commercial samples of Methyl Green (Gurr Certistain), Pyronine G (Gurr Certistain) and Pyronin Y (Polysciences) have been investigated using spectrophotometry, thin layer chromatography and nuclear magnetic resonance, in addition to standardized simultaneous and sequential staining methods using purified Ethyl Green and pure Pyronin Y as reference dyes.The Methyl Green was found to be Ethyl Green contaminated with Crystal Violet. It did not have any advantages compared with Ethyl Green supplied by American dye companies. The Pyronine G sample was Pyronin Y with a high dye content that gave good staining results when used with purified Ethyl Green. Pyronin Y from Polysciences was found to be essentially pure Pyronin Y.     

Disruption of the aspartate to heme ester linkage in human myeloperoxidase: impact on ligand binding, redox chemistry, and interconversion of redox intermediates

In human heme peroxidases the prosthetic group is covalently attached to the protein via two ester linkages between conserved glutamate and aspartate residues and modified methyl groups on pyrrole rings A and C. Here, monomeric recombinant myeloperoxidase (MPO) and the variants D94V and D94N were produced in Chinese hamster ovary cell lines. Disruption of the Asp(94) to heme ester bond decreased the one-electron reduction potential E'(0) [Fe(III)/Fe(II)] from 1 to -55 mV at pH 7.0 and 25 degrees C, whereas the kinetics of binding of low spin ligands and of compound I formation was unaffected. By contrast, in both variants rates of compound I reduction by chloride and bromide (but not iodide and thiocyanate) were substantially decreased compared with the wild-type protein. Bimolecular rates of compound II (but not compound I) reduction by ascorbate and tyrosine were slightly diminished in D94V and D94N. The presented biochemical and biophysical data suggest that the Asp(94) to heme linkage is no precondition for the autocatalytic formation of the other two covalent links found in MPO. The findings are discussed with respect to the known active site structure of MPO and its complexes with ligands.     

The Preparation of Vital Neutral Red

Neutral red iodide suitable for vital staining was prepared by condensing nitrosodimethylanilin hydrochloride with m-toluylenediamine and the indamine, toluylene blue, was obtained. This was subjected to air oxidation and converted to the eurhodine, neutral red. The purification of this dye was brought about by converting it into its comparatively insoluble stannous chloride double salt, filtering, dissolving in water, and precipitating the neutral red iodide with potassium iodide solution. This was re-dissolved in water, reprecipitated with potassium iodide solution and crystallized from 95% ethanol. The uncrystallized dye was also found satisfactory for vital staining. Several other preparations of neutral red iodide were made, using a somewhat different procedure than that given above, and it was generally found that satisfactory stains were obtained only when the preparation was free from toluylene blue.

The chloride of the color base was prepared by continuing the air oxidation of the toluylene blue until a test sample indicated its complete conversion into neutral red. The color was salted out with sodium chloride and crystallized from 95% ethanol. Both the crystallized and uncrystallized products were found to be excellent stains.     

The Preparation of Vital Neutral Red

Neutral red iodide suitable for vital staining was prepared by condensing nitrosodimethylanilin hydrochloride with m-toluylenediamine and the indamine, toluylene blue, was obtained. This was subjected to air oxidation and converted to the eurhodine, neutral red. The purification of this dye was brought about by converting it into its comparatively insoluble stannous chloride double salt, filtering, dissolving in water, and precipitating the neutral red iodide with potassium iodide solution. This was re-dissolved in water, reprecipitated with potassium iodide solution and crystallized from 95% ethanol. The uncrystallized dye was also found satisfactory for vital staining. Several other preparations of neutral red iodide were made, using a somewhat different procedure than that given above, and it was generally found that satisfactory stains were obtained only when the preparation was free from toluylene blue.

The chloride of the color base was prepared by continuing the air oxidation of the toluylene blue until a test sample indicated its complete conversion into neutral red. The color was salted out with sodium chloride and crystallized from 95% ethanol. Both the crystallized and uncrystallized products were found to be excellent stains.     

The application of propidium iodide staining to the study of the macronucleus and micronuclei in the suctorian, Heliophrya sp

Morphological changes in the macronucleus and micronuclei of the ciliated protozoon Heliophrya chapmani were investigated using the nucleic acid-specific stain propidium iodide. The fluorescence patterns of nuclei observed in propidium iodide preparations correspond well with those observed using more conventional DNA-specific methods, such as the Feulgen stain. The advantages of propidium iodide staining (minimal cell loss during staining, rapidity of the staining process, and the avoidance of cell damage during hydrolysis) make this method a quick and efficient alternative in the cytochemical study of the protozoan nucleus.     

Further studies on the mechanism involved in differential staining of BUdR-substituted Vicia faba chromosomes

In a preceding publication we reported that photolysis of BUdR-substituted Vicia faba chromatids occurs during observation with a fluorescence microscope when chromosomes were mounted in a solution containing trypsin and a photosensitive dye (Hoechst 33258 or acridine orange). The present investigations support the hypothesis that the rapid dissolving of the double BUdR-substituted (BB) chromatids observed with our method is due to single-strand breaks induced by a photosensitive dye-visible light system. The agents cysteamine and potassium iodide which reduce BUdR radicals and in this way may inhibit single-strand breaks modify the rate of chromosomes showing differential staining. It was totally suppressed by high cysteamine concentrations and markedly reduced by potassium iodide. Several acridine dyes were tested concerning their ability to induce differential staining. Some of them, e.g. aurophosphine and coriphosphine O, yield good results, others, e.g. acriflavine and acridine yellow, give poor differential staining. In an experiment in which the trypsin concentration was varied to induce approximately optimum and non-optimum digestion conditions the necessity of trypsin treatment in our method was confirmed.     

The Application of Propidium Iodide Staining to the Study of the Macronucleus and Micronuclei in the Suctorian, Heliophrya Sp

Morphological change in the macronucleus and micronuclei of the ciliated protozoon Heliophrya chapmani were investigated using the nucleic acid-specific stain presidium iodide. The fluorescence patterns of nuclei observed in propidium iodide preparations correspond well with those observed using more conventional DNA-specific methods, such as the Feulgen stain. The advantages of propidium iodide staining (minimal cell loss during staining, rapidity of the staining process, and the avoidance of cell damage during hydrolysis) make this method a quick and efficient alternative in the cytochemical study of the protozoan nucleus.     

The Gram Staining Mechanism of Cat Tongue Keratin. II. The Role of Potassium Iodide-Iodine Solution

The aniline-xylene decolorizer of the Gram-Weigert staining procedure failed to remove crystal violet dye from stained sections of cat tongue without prior treatment of the sections with potassium iodide-iodine solution. The potassium iodide of the iodide-iodine solution was found to release the major part of the crystal violet dye bound by the tongue sections. Iodine appeared also to play a role in dye release, but only to a slight degree. The amount of Gram-positive staining was increased both by alkaline treatment of the tissue prior to staining, and by increasing the pH of the iodide-iodine solution.