Chromosome preparations of human whole blood lymphocytes: an improved technique

A new modification of currently utilized methods of processing cell cultures of whole peripheral blood for obtaining chromosome preparations necessary for differential staining (Q, G and C) is proposed. After the usual hypotonic treatment of cultures, a 3%-5% aquaeous solution of glacial acetic acid is added to the cell suspension prior to fixation in order to completely destroy and wash out red cells, as well as for selective fixation of lymphocytes.     

Measurement of hemoglobin in long-term fixed erythroid cells: application development for cell science experiments in microgravity

Studying the effects of microgravity on cell differentiation will enhance our understanding of fundamental biology and is indispensable for a sustained space program. Rauscher murine erythroleukemic cells were chosen as a model system to study erythroid cell differentiation aboard the International Space Station because these cells undergo differentiation in response to the natural inducer, erythropoietin, as well as various chemical inducers. We have now developed a method to quantify hemoglobin in Rauscher cells after weeks of fixation and storage required for such space biology experiments. By exploiting the pseudoperoxidase activity of hemoglobin and by using reagents that yield a soluble chromophore that freely passes out of fixed cells, we developed a highly specific and sensitive assay applicable to cells fixed as long as 4 months.     

A Shorter Fixation Protocol for Transmission Electron Microscopy: An Alternative to Spend Less Time

ABSTRACT

The performance of a moderately shorter fixation protocol for transmission electron microscopy (TEM) was evaluated by analyzing the cell structure quality after the processing. The relevance of this experimental technique is mainly based on reducting time of the steps of conventional protocols: fixation, washes, dehydration, and epoxy resin infiltration. Two sources of murine cells were used, the peritoneal and mesenteric lymph node cells. A fixation and material processing faster than usual methods can save time and improve results. Samples analysis indicated good preservation of different cell structures and organelles after this protocol.     

A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry

A sensitive method for quantification of cells undergoing apoptisis that permits the simultaneous measurement of dual-color cell surface immunofluorescence is presented. Unfixed cells are stained with 7-amino-actinomycin D (7-AAD) for discrimination of live from early apoptotic cells and from cells which have lost membrane integrity (late apoptotic or necrotic, dead cells). Owing to its spectral characteristics 7-AAD can be combined with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) cell surface staining. After staining, the samples can be treated with paraformaldehyde (PF) solution to eliminate the risk for exposure of laboratory personnel to biohazardous agents and to preserve the cells through fixation for later analysis on the flow cytometer. The value of the method is shown on the measurement of apoptosis in human thymocytes and in human peripheral blood mononuclear cells (PBMC) exposed to various inducers of active cell death. The method is validated by fluorescent activated cell sorting in combination with morphologic examination of the sorted cells. The technique we are presenting is particularly valuable in a clinical setting because it allows rapid multiparameter analysis of apoptosis in combination with cell surface phenotype on biohazardous samples with single laser instrumentation.     

Delay in fixation does not affect the immunoreactivity of proliferating cell nuclear antigen (PCNA).

The effect of delayed fixation on the immunoreactivity of proliferating cell nuclear antigen (PCNA) was investigated using eight breast carcinomas. Topologically shuffled samples of each tumour were immersed in fixative at times of 0.5, 1, 2, 4, 6, 18, and 24 h after surgical removal. In addition to a PCNA index (percentage of positive cells per 1200 tumour cells), a semi-quantitative PCNA grading system was used, based on estimates of more than or less than 50 per cent of positive tumour cells at each time interval. The PCNA index of six tumours increased by a mean of 10 per cent with a fixation delay of 24 h. The PCNA grade of all eight tumours showed no change with delayed fixation.     

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.     

微波固定组织与福尔马林固定组织的比较

报告利用微波固定组织与常规福尔马林固定组织的比较研究。将同一小鼠的不同脏器组织分成二部份,一部份用微波固定,另一部份用常规福尔马林固定,常规组织学石蜡包埋切片,进行光镜观察。微波组与福尔马林组的组织学结构及细胞结构均显示清楚。结果表明:微波固定可有效地用来处理脏器标本,由于微波固定时间极短,故此法不但可用于临床病理学常规组织处理,也可用于手术或其它快速诊断的要求中。     

Estrogen,progesterone, and HER‐2 receptor immunostaining in cytology: The effect of varied fixation on human breast cancer cells

The ASCO/CAP Expert Panel recommends that all invasive breast carcinomas and breast cancer recurrences be tested for ER, PR and HER‐2 expression. The guidelines for testing of surgical specimens by immunohistochemistry (IHC) are well defined, whereas they are lacking for cytological samples. We evaluated various fixation protocols for optimal receptor testing by immunohistochemistry and immunocytochemistry (ICC) of human breast cancer cell lines MCF‐7 (ER/PR positive) and SKBR‐3 (overexpressing HER‐2). The cells were fixed in 10% neutral buffered formalin or Saccomanno Fixative (SF) for various time points, and either embedded in paraffin as cell blocks or prepared as cytospins. ER and PR slides were assigned a proportion score (PS; 0–5), an intensity score (IS; 0–3) and a total score (TS = PS + IS). Standard DAKO scoring system ranging from 0 to 3+ was used for the evaluation of HER‐2 staining. Human breast cancer cells stained successfully for ER, PR and HER‐2 when fixed in formalin and prepared as cell blocks. The optimal fixation time for formalin‐fixed cells ranged from 2 to 96 hours. Cells fixed in SF from 2 to 96 hours also stained well for ER and PR. However, SF produced variable results for HER‐2 staining; particularly, SF fixation beyond 24 hours caused false negative results. The interpretation of HER‐2 staining on cytospins was not feasible irrespective of the fixative and fixation time. In summary, formalin fixation from 2 to 96 hours and preparation of cells as cell blocks produces optimal results for ER, PR, and HER‐2 testing in human breast cancer cells. Diagn. Cytopathol. 2013;41:864–870. © 2013 Wiley Periodicals, Inc.     

Discharge patterns of neurons in the rostral superior colliculus of cat: activity related to fixation of visual and auditory targets

Neurons in the rostral superior colliculus (SC) of alert cats exhibit quasi-sustained discharge patterns related to the fixation of visual targets. Because some SC neurons also respond to auditory stimuli, we investigated whether there is a population of neurons in the rostral SC which is active in relation to fixation of both auditory and visual targets. We identified cells which were active with visual fixation and which continued to discharge if the fixation stimulus was briefly extinguished. The population of neurons exhibited similar discharge characteristics when the fixation stimulus was auditory. Few neurons were significantly more active during fixation of visual targets than during fixation of auditory targets. Most fixation neurons showed a diminished discharge rate during spontaneous (self-generated) saccadic eye movements away from a visual fixation stimulus, regardless of the direction of the saccade. this diminished discharge rate (or pause) typically began, on average, 12.2 ms before saccade onset and the duration of the pause was Ionger than the duration of the saccade. These observations are consistent with the hypothesis that increased discharge of these neurons is related to active fixation and that reductions in their activity are important for the generation of saccades. However, the lack of a precise relationship between pause duration and saccade duration implies that these neurons would be unlikely to project directly to the saccadic burst generator. The mean interval from the beginning of the pauses of fixation neurons to be beginning of the saccades away from fixation targets is also shorter than has been found in brainstem omnipause neurons. By analogy with the concept of a receptive field, agaze position error field depicts the range of gaze position error for which a cell is active. Although fixation neurons appear to encode the magnitude and direction of the error between visual targets and the visual axis, visual error fields at the end of fixating eye movements were significantly larger than those at stimulus onset. For auditory stimuli, this difference was not significant. These observations are compatible with a number of recent experiments indicating that neural signals of eye position are damped or delayed with respect to current eye position.     

Neuroimmunological electron microscopy with microwave-accelerated fixation

Immunoelectron microscopy is an important tool used to determine the precise location of immune complexes. Standard concentrations of glutaraldehyde destroy these complexes. This paper describes a method in which the period of glutaraldehyde fixation is shortened by concomitant microwave treatment. Using 1.25% glutaraldehyde and microwave fixation ideal preservation and demonstration of MHC class I antigen on Schwann cells was obtained by the peroxidase method.     

万向式骨外固定器治疗桡骨远端不稳定性骨折的临床应用

目的评价万向式骨外固定器治疗桡骨远端不稳定骨折的疗效。方法应用万向式桡骨远端骨外固定器治疗159例（145人次）桡骨远端不稳定骨折,53例因骨折严重外加克氏针固定,11例因关节面塌陷骨缺损同时采取植骨。结果随访时间12～28个月,平均随访18．12个月,按Dienst等推荐的测量方法制定的解剖评估标准、功能评估标准及影像学评估标准,解剖复位优良者147例,优良率92．45％;功能优良者148例,优良率93．08％。结论应用万向式骨外固定器治疗桡骨远端不稳定骨折是目前理想的手术方法。同时验证了此万向式骨外固定器的灵活性、可操作性及固定后的稳定性,尤其适合于广大基层医院推广。     

Morphology of human neutrophils: A comparison of cryofixation,routine gluteraldehyde fixation,and the effects of dimethyl sulfoxide

Cell shape and density are critical to the evaluation of neutrophil function and/or activation. Dimethyl sulfoxide-cryofixation-freeze-substitution processing (DCF) instantly preserves cell processes and ultrastructural elements with fewer artifacts than routine chemical fixation with glutaraldehyde and postfixation osmium tetroxide (GO). This study morphometrically examined density-separated neutrophils to assess differences in DCF and GO processing procedures and studied the effect of dimethyl sulfoxide followed by GO fixation (DGO) on morphology. Fifteen consecutive neutrophils were analyzed using computerized planimetry for differences in DCF v. GO treatments (n = 4) and DGO v. GO treatments (n = 4). Cryofixed and DGO-fixed cells were significantly rounder than GO cells which had a more irregular surface with membrane projections. The cell volume of GO cells was 27–30% smaller than in DCF or DGO processing, while the surface area was similar. The increased volume in DCF and DGO cells did not appear to be due to abnormal cell swelling, since membranes, nuclear envelope, and mitochondrial cristae were more intact than in GO cells. Preservation of mitochondria as well as endocytic caveolae with a subplasmalemmal coating was best in DCF samples, moderate in DGO, and poorest in GO. Morphometric data showed that the nuclear compartment was 22% smaller, while the cytoplasm (and its associated compartments) was 29% smaller in GO compared to DCF-processed neutrophils. This was consistent with the more dense cytoplasm in GO cells. Pretreatment of neutrophils with dimethyl sulfoxide (DMSO) resulted in volume preservation and improved the morphology of GO fixation. In summary, DCF appears to be an excellent method for preserving neutrophil membranes and cytoplasmic organelles (particularly mitochondria), and prevents a number of artifacts caused by routine GO fixation. Morphology can also be improved by using DMSO in conjunction with GO. Anat. Rec. 252:254–263, 1998. © 1998 Wiley-Liss, Inc.     

The utility of vapor fixation for cell block preparation in fine needle aspiration of malignant lesions of lymph nodes

Cell blocks overcome certain limitations of fine needle aspiration cytology (FNAC), such as study of tissue architecture and application of immunohistochemistry (IHC). The main disadvantage of cell block by conventional method is cell loss. This study compares formaldehyde vapor-fixed cell blocks with conventional formalin-fixed blocks obtained from aspirates of malignant lesions of lymph nodes. Seventy-nine patients with suspected primary or metastatic malignancy in lymph nodes were studied. Cytologic smears were prepared in all the cases. Conventional formalin-fixed cell blocks were made in 34 cases, vapor-fixed cell blocks in 34 cases, and in the remaining 11 cases, both methods were employed. Cell block sections were compared for cellularity, fixation artifacts, morphologic details, and crispness of immunohistochemical staining using defined subjective criteria. Cellular yield was slightly less with vapor-fixed cell blocks in comparison with conventional formalin-fixed cell blocks. Staining artifacts were more frequent with vapor fixation (33.3%) as compared to conventional formalin fixation (20%). Also, vapor fixation required more time (6 h) as compared to conventional formalin fixation (1 h). There was no difference in the two techniques with respect to immunohistochemical staining. Cell blocks prepared by vapor fixation are comparable to conventional formalin-fixed cell blocks.     

Fixation and permanent mounting of fluorescent probes after vital labelling of cultured cells

The use of fluorescent probes for the visualization of organelles in living cells and assessment of live/dead cells has an increasing importance in cell biology. However, rapid and irreversible morphological changes of labelled cells (due to the photosensitizing effect of most fluorescent probes) make prolonged observation and detailed analysis of living cells under continuous excitation difficult. In this study, we describe a method for fixing and mounting cultured HeLa and 3T3 cells labelled with acridine orange (lysosomes), rhodamine 123 (mitochondria), Hoechst 33342 (nuclei), and propidium iodide/acridine orange (live/dead HeLa cells subjected to nutrient deprivation). Fixation is performed with vapours of a commercially available formaldehyde solution for 0.5-1 min followed by air drying and permanent mounting in DPX. After this procedure, both the general morphology and selective fluorescent labelling of cells are well preserved. The method of vapour fixation and DPX mounting is simple, rapid and reproducible, allowing definitive preservation of the fluorescence pattern observed in unfixed cell cultures.     

Improved structural preservation and immunolocalization of the microtubule cytoskeleton in plant and animal cells by freeze substitution/fixation

Immunofluorescence localization is widely used to determine the distribution and organization of the microtubule (Mt) cytoskeleton in cells, with most protocols using conventional chemical fixation (CCF). However, concerns associated with CCF are slow rate of fixation, alteration of cell morphology, and redistribution of target antigens. Freeze substitution/fixation (FSF) is a method commonly used in electron microscopy to overcome these problems, but has not been exploited at the light microscopic level. Thus, techniques using FSF were investigated in three divergent cell types (tobacco suspension cultures, Tradescantia pollen tubes, and mouse myeloma cultures) and evaluations were made of cell morphology and immunofluorescence localization of the Mt cytoskeleton. Tobacco cells prepared by FSF exhibited an extensive network of Mts with consistent and strong labeling, while CCF preparations had disorganized and fuzzy Mts. Pollen prepared with FSF retained cytoplasmic details, and had continuous longitudinal arrays of cortical Mts. In contrast, CCF induced structural abnormalities in pollen characterized by swollen and ruptured tube tips with sinuous, nonuniformly labeled Mts. Likewise, in myeloma cells, Mt labeling was more uniform and stronger with less background with FSF versus CCF. CCF also caused myelomas to shrink. Advantages of FSF over CCF are that rapid freezing attains an instantaneous cessation of metabolism with better preservation of cellular structures, and enhanced and more consistent Mt labeling. The absence of numerous amendments used in CCF (e.g., buffers and osmotic reagents) may avoid interaction with target antigens. FSF preparations should be useful in critical immunofluorescence localizations of other antigens, especially those sensitive to CCF manipulations.     

Methodological aspects of the immunostaining of proliferating cell nuclear antigen (PCNA) in cytospin preparations of MCF-7 cell line

Cytospins of a human breast cancer cell line (MCF-7) were studied for the expression of PCNA, a cell cycle-related protein, using a variety of fixation and immunostaining procedures. The best fixative for PCNA was found to be buffered formaldehyde solution at 4°C followed by methanol at 20°C, whereas alcoholic fixatives decreased greatly the PCNA immunoreactivity. Air-drying procedures of cytospins prior to and after fixation greatly undermined the PCNA immunostaining. A modified immunoperoxidase method provided a stronger staining of the PCNA-reactive cells than the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. PCNA immunoreactivity could be maintained up to 2 mo, putting slides in methanol at ? 2°C. In conclusion, our report indicates that PCNA is a labile antigen, which may critically be affected by temperature and air-drying procedures. © 1994 Wiley-Liss, Inc.     

腰椎单侧与双侧内固定后的生物力学特征比较

文题释义： 椎弓根螺钉内固定：主要应用于脊柱退变性疾病,可以在术后维持脊柱的稳定性,促进骨质的融合。对于存在脊柱不稳或者是脊柱骨折的患者,椎弓根钉内固定可以起到复位、维持脊柱稳定性的作用。对于存在脊柱侧弯畸形或者是后凸畸形的患者可以进行矫形。 应力分析：分析机械部件内的应力值和应力分布的方法,在此文中利用该方法分析不同螺钉固定方法对腰椎应力的影响。 背景：椎弓根螺钉固定椎体是目前临床治疗腰椎退行性疾病的主要手段,从生物力学的角度可以很好地评价椎弓根固定系统稳定性,利用有限元分析法进行椎弓根螺钉固定椎体的应力分析成为越来越多研究者的选择。 目的：从生物力学角度分析单侧及双侧椎弓根螺钉固定时人体腰椎弯曲运动时的应力、位移变化,为实际临床应用提供理论借鉴。 方法：基于志愿者腰椎CT数据建立腰椎三维模型,与志愿者签署了知情同意书,且得到医院伦理委员会批准。利用Abaqus软件模拟实际腰椎受力情况,对单侧及双侧椎弓根螺钉固定时腰椎的弯曲运动进行有限元分析,观察2种固定方式下腰椎椎体、椎间盘、椎弓根钉的受力大小和位移情况。 结果与结论：①双侧固定下左侧螺钉受力为22.2 MPa,右侧螺钉受力为21.14 MPa,远远小于单侧固定时螺钉所受应力79.19 MPa;椎间盘在单侧固定下应力值比双侧固定时大87%;椎体在双侧固定时,要比单侧固定所受应力小72%;②从位移情况来看,双侧固定时,螺钉、椎间盘与椎体的位移要比单侧固定分别小53%,55%以及62%;③因此从力学角度分析,双侧椎弓根螺钉固定时受力小,要比单侧固定对人体更加友好,更有利于患者的恢复。 ORCID: 0000-0002-4813-066X(陆向东) 中国组织工程研究杂志出版内容重点：人工关节;骨植入物;脊柱;骨折;内固定;数字化骨科;组织工程     

踝部骨折的骨片钉内固定治疗的临床观察

目的探讨对踝部骨折行骨片钉内固定的临床应用价值。方法回顾性分析48例踝部骨折患者行骨片钉内固定的应用情况。每位患者术前均行X线、CT等影像学检查,术后均摄X线片观察复位情况及螺钉位置。结果术后随访4~36个月,平均13个月.采用Leeds标准评定:优良40例,踝关节功能完全恢复;可6例,踝关节功能部分丧失,背伸运动>10°,或与对侧相比不小于40％,运动后轻微痉挛;差2例,踝关节功能大部分丧失。结论骨片钉内固定是治疗踝部骨折较简单、有效的方法。     

股骨转子间骨折内固定器械研究进展

股骨转子间骨折是常见的骨折，亦是当今创伤骨科的研究热点。随着对股骨转子间骨折认识的深入及手术固定材料技术的发展，目前手术内固定治疗已成为首选治疗方式，现行的手术内固定治疗方式主要有髓外固定和髓内固定两种，二者各有优势，但也存在不足。髓内固定因微创和生物力学的优势逐渐成为主流内固定治疗方式。本文就股骨转子间骨折内固定器械的设计、优缺点、临床应用及其发展等方面进行综述，以期为临床内固定选择提供参考。     

指骨粉碎骨折的不同固定方法——生物力学研究

本实验对指骨粉碎骨折的五种固定方法的刚度进行了研究,每种固定方法进行了压缩、弯曲和扭转,得出了一些有参考价值的数据.带6孔螺钉的侧方钢板似乎提供了更坚强的固定.假如存在蝶形骨片而不能进行钢板固定(如在多块骨折的情况下,没有足够的皮质供螺钉固定),选用克氏针固定也可达到满意的效果.本结果将为临床上手术医师选择固定方法提供参考.