冷冻切片常遇到的问题及解决对策

目的 提高制作冷冻切片质量，确保实验结果科学性。方法 通过对切片机工作室温度设置选择，防卷板的调整应用及切片常遇到的问题分析，阐述提高制作高质量冷冻切片的方法。结果 通过对冷冻切片机有关参数的设定，提高了切片质量。结论 冷冻切片常遇到的问题，通过及时调整切片机相应设置，可以便捷解决。     

Identification of mast cells in bronchoalveolar lavage fluid

The present study was carried out to compare the effectiveness of different fixation and staining methods in the identification of mast cells obtained by bronchoalveolar lavage from patients with interstitial lung diseases. Cell preparations were fixed with formaldehyde or methanol. Mast cells were identified by metachromatic staining with May Grünwald Giemsa, Toluidine blue or Gallamine blue Giemsa. After formaldehyde fixation only a few mast cells were identified, regardless of the stain method used. In contrast, a significantly higher number of mast cells were observed after methanol fixation. Using this fixative, Toluidine blue stain showed a higher number of mast cells than May Grünwald Giemsa. However, there was no difference between Toluidine and Gallamine blue Giemsa in the number of cells observed. The easy identification of mast cells after staining with toluidine, combined with its easy application, suggest that Toluidine blue stain after methanol fixation is the most useful method for determining the presence of mast cells in lavage fluid.     

声带鳞状细胞癌早期改变的病理学观察

目的探讨声带鳞状细胞癌早期病理学的特点，提高病理诊断水平。方法总结89例声带鳞状细胞癌早期改变病例的病理资料，对其石蜡切片进行HE染色、PAS染色及p53、Ki-67免疫组化染色；以59例声带角化症（分为单纯增生组40例和异型增生组19例）和30例声带浸润癌（浸润深度〉3mm的癌）作为对照。结果在HE染色下，声带鳞状细胞癌的早期改变可区分为两种类型：Ⅰ型为上皮全层癌变型，占67．4％（60／89）；Ⅱ型为上皮基底层及副基底层癌变型，占32．6％（29／89），又可分为Ⅱa和Ⅱb两个亚型。HE染色显示有可疑微小浸润者52例，PAS染色示其中的43例（83％）的可疑病灶周边基膜样物质消失，有微浸润，Ⅰ型微浸润的比例较Ⅱ型明显偏低（P=0．007）。HE染色下3例（3．4％，3／89）认为无微浸润者经深切证实有浸润，并经PAS染色确认。Ⅰ型和Ⅱ型的p53表达率差异无显著性（P=0．445），而Ki-67阳性率Ⅰ型高于Ⅱ型（P=0．048）。癌早期改变组的p53阳性率高于声带角化症伴单纯增生组（P=0．008），而与声带角化症异型增生组和声带进展癌组之间的差异无统计学意义（P=0．240，P=0．268）。癌早期改变组的Ki-67阳性率明显低于浸润癌组（P=0．000），并明显高于角化症伴单纯增生组（P=0．001），但与角化症伴异型增生组之间差异无显著性（P=0．248）。结论声带鳞状细胞癌早期改变可区分为Ⅰ型和Ⅱ型，Ⅱ型癌变可在不累及上皮全层的情况下，由上皮的基底层和（或）副基底层细胞直接向固有膜内增生及癌变，此型占全部病例的近1／3，早期浸润是Ⅱ型诊断的可靠依据；Ⅱ型的存在提示声带鳞状细胞癌的早期发生和演进可能存在不同的机制；PAS染色和p53、Ki-67免疫组化染色有助于声带鳞状细胞癌Ⅱ型早期的诊断。     

Cell populations of the ganglion cell layer: displaced amacrine and matching amacrine cells in the pigeon retina

Summary The proportion and size distribution of ganglion and non-ganglion cells in the ganglion cell layer of different areas of the pigeon retina was examined in whole-mounts of the retina by retrograde axonal transport of horseradish peroxidase (HRP) from large brain injections. A maximum of 98% of cells were labelled in the red field and a maximum of 77% in the peripheral yellow field. Unlabelled cell bodies were 30% smaller than labelled ganglion cells and had a mean diameter of 6.2 m and a size range of 4 to 9 m. The morphology of cells in the ganglion cell layer was examined by Golgi staining of retinal whole-mounts. Small glia, displaced amacrine and ganglion cells were found. Displaced amacrine cell bodies were about 30% smaller than ganglion cells and their size distribution was similar to the unlabelled cells in HRP preparations. Displaced amacrine cells had small rounded cell bodies (mean diameter 6.2 m) increasing in size with eccentricity, and a unistratified dendritic tree of fine, nearly radial, varicose dendrites in sublamina 4 of the inner plexiform layer. They had elliptical dendritic fields (mean diameter 66 m) aligned parallel to the retina's horizontal meridian. A population of amacrine cells was found with somas at the inner margin of the inner nuclear layer and soma and dendritic morphology matching those of displaced amacrines. These amacrine cells had unistratified dendritic trees at the junction of sublaminae 1 and 2 of the inner plexiform layer. Pigeon displaced amacrine cells and their matching amacrines are similar to starburst cells of the rabbit retina. They may participate in on and off pathways to ganglion cells and their lamination suggests that they are cholinergic.     

Localization of [F]fluorodeoxyglucose in mouse brain neurons with micro-autoradiography

This is the first study of micro-autoradiography (micro-ARG) for [¹⁸F]2-fluoro-2-deoxy- -glucose ([¹⁸F]FDG). The localization of [¹⁸F]FDG was demonstrated in dendrites of neuron and also in the myelinated axon in mouse normal brain in vivo. The nucleolus was relatively free of label. The counted silver grain numbers in autoradiogram were linearly correlated to the ¹⁸F radioactivities in the specimen. The micro-ARG using positron emitting ¹⁸F is a very time-saving technique with 4 hours exposure compared with the conventional method using ³H- or ¹⁴C-labeled tracers.     

Assessment of different methods for staining Helicobacter pylori in endoscopic gastric biopsies

The recent implication of Helicobacter pylori in the pathogenesis of gastritis-peptic ulcer syndrome and its relevance for the development of upper gastrointestinal malignancy warrant efficient methods for the detection and demonstration of the organism in biopsy specimens. We have compared 5 staining methods, namely, haematoxylin and eosin (H & E), immunohistochemistry (IHC), the silver staining HpSS, the alcian yellow-toluidine blue (Leung) method (A-Y) and Genta staining, for the demonstration of the organism in gastric biopsies taken from antrum, body and fundus of 118 patients who presented to our hospital with upper gastrointestinal symptoms. We found no significant differences in the efficacy of H & E, IHC, HpSS and A-Y in the demonstration of H. pylori in all 3 gastric sites. The least reproducible stain in our hands was the Genta stain. We conclude that H & E is adequate for the initial assessment of gastric biopsies in symptomatic upper gastrointestinal patients. This is because it is a well-tested, cheap and easy staining method, requiring a relatively short period of time to perform, with highly reproducible results. It has an added advantage of enabling simultaneous assessment of morphological changes accompanying H. pylori infection. When the density of the organism is expected to be low, we recommend addition of HpSS staining because of its high sensitivity and low cost. The disadvantages of the other staining methods (IHC, A-Y and Genta) are discussed.     

Fc epsilon receptors on human cell lines and peripheral blood lymphocytes detected by binding of IgE immune complexes

To identify Fc epsilon receptors on human cell lines and peripheral blood lymphocytes, we developed a new method which relies on the binding of constructed immune complexes to Fc epsilon receptor-positive cells. Cell suspensions from either cell lines or peripheral blood lymphocytes were incubated with complexes of human myeloma IgE and murine monoclonal anti-human IgE at various ratios prior to cytocentrifugation. The complexes bound to the cells were subsequently visualized by immunoperoxidase staining. The specificity of this assay to detect cell surface Fc epsilon receptors was shown by the ability of human myeloma IgE to block the binding of the IgE complexes, resulting in unstained cells, whereas IgM, IgG, and IgA were unable to block the binding of the complexes (stained cells). This method is reproducible, allows quantification of a single sample at different times, and provides a record of the results. It can also be adapted to identify any cell surface receptor for which the ligand is known.     

“Dark” (compacted) neurons may not die through the necrotic pathway

Dark neurons were produced in the cortex of the rat brain by hypoglycemic convulsions. In the somatodendritic domain of each affected neuron, the ultrastructural elements, except for disturbed mitochondria, were remarkably preserved during the acute stage, but the distances between them were reduced dramatically (ultrastructural compaction). Following a 1-min convulsion period, only a few neurons were involved and their environment appeared undamaged. In contrast, 1-h convulsions affected many neurons and caused swelling of astrocytic processes and neuronal dendrites (excitotoxic neuropil). A proportion of dark neurons recovered the normal structure in 2 days. The non-recovering dark neurons were removed from the brain cortex through two entirely different pathways. In the case of 1-h convulsions, their organelles swelled, then disintegrated and finally dispersed into the neuropil through large gaps in the plasma membrane (necrotic-like removal). Following a 1-min convulsion period, the non-recovering dark neurons fell apart into membrane-bound fragments that retained the compacted interior even after being engulfed by astrocytes or microglial cells (apoptotic-like removal). Consequently, in contrast to what is generally accepted, the dark neurons produced by 1-min hypoglycemic convulsions do not die as a consequence of necrosis. As regards the case of 1-h convulsions, it is assumed that a necrotic-like removal process is imposed, by an excitotoxic environment, on dark neurons that previously died through a non-necrotic pathway. Apoptotic neurons were produced in the hippocampal dentate gyrus by intraventricularly administered colchicine. After the biochemical processes had been completed and the chromatin condensation in the nucleus had reached an advanced phase, the ultrastructural elements in the somatodendritic cytoplasm of the affected cells became compacted. If present in an apparently undamaged environment such apoptotic neurons were removed from the dentate gyrus through the apoptotic sequence of morphological changes, whereas those present in an impaired environment were removed through a necrotic-like sequence of morphological changes. This suggests that the removal pathway may depend on the environment and not on the death pathway, as also assumed in the case of the dark neurons produced by hypoglycemic convulsions.     

CD4 responses to conserved HIV-1 T helper epitopes show both negative and positive associations with virus load in chronically infected subjects

Characterization of immune responses to immunodominant CD4 epitopes in HIV-1 that are associated with control of HIV infection could be used to strengthen the efficacy of polyepitope HIV vaccines. We measured both the proliferative and the CD4 interferon (IFN)-gamma and interleukin (IL)-2 cytokine responses specific for 11 previously identified HIV-1 T helper epitopes in 10 HIV-infected non-progressors (LTNPs) (infected for a median of 15 years with a stable CD4 count of >500 cells x 10(6)/l), and seven slow progressors (SPs) (infected for a median of 15 years with a CD4 count that had declined to <500 cells x 10(6)/l). Both groups were antiretroviral treatment-naive at the time of evaluation. The median virus load of SP group was higher than that of the LTNP group (P = 0.0002). The CD4 response to a peptide pool representing all potential CD4 Gag epitopes and to Gag p24 protein was also studied. Compared to SPs, LTNPs had higher numbers of Gag-specific IFN-gamma+IL-2+ CD4s (P = 0.0059). The Gag-specific cytokine and proliferative responses correlated inversely with virus load (P = 0.03 and 0.0002, respectively), highlighting the potential importance of this response in immunity to HIV. A direct correlation was noted between proliferation and the Gag-specific IL-2 (P = 0.0053) rather than IFN-gamma response (P = 0.1336), demonstrating that the proliferation assay reflected the IL-2 rather than the IFN-gamma secreting capacity of CD4 cells. Several subjects with diverse class II DRB1 alleles responded, confirming the 11 selected peptides to be both antigenic and conserved. CD4 cytokine responses to one Gag and two conserved Pol peptides correlated negatively with virus load. The cytokine response to two additional Pol peptides correlated positively with virus load. The data indicate that there is not an absolute correlation between the CD4 immune response to conserved and broadly antigenic helper T cell epitopes in HIV non-progression.