Differential uptake of systemic fluorochrome Hoechst 33342 in lung and liver metastasis of B16 melanoma

Summary The growth and vascularization patterns of B16 melanoma colonies in the liver and lungs were measured and compared by histological techniques and dye diffusion patterns after injection of the fluorochrome Hoechst 33342. In the liver, the fluorescent pattern of dye diffusion revealed that uninodular tumours measuring up to 146 n in diameter were not functionally vascularized. However, when the nodules fused to give rise to multinodular tumours measuring between 256 and 366 n in diameter, a reticular dye diffusion pattern revealed functional tumour vascularization. In the lungs, subpleural, parenchymal and peritubular (i.e. surrounding blood vessels and airways) tumours were observed. The two former classes were vascularized down to thicknesses and diameters of 49 and 24 m respectively. In contrast, dye diffusion was never seen in peritubular tumour cuffs up to 609 m in thickness. The results indicate differences in vascularization patterns in B16 tumours in the liver and lungs, and differences between tumours growing in different sites within the lungs. If these results are applicable to metastases in these two organs, they indicate potential diffusion-mediated resistance to chemotherapy, and potential hypoxia-mediated resistance to radiotherapy of both metastases and micrometastases.     

Enhanced polymer one-step staining (EPOS) for proliferating cell nuclear antigen (PCNA) and Ki-67 antigen: Application to intra-operative frozen diagnosis

Enhanced polymer one-step staining (EPOS) is a novel, highly sensitive one-step immunostaining method. This simple and rapid technlque was applied to intra-operattve frozen diagnosis. The markers of choice were proliferating cell nuclear anmen (PCNA) and Ki-67 antigen. These cell prollferation markers were both identifiable in fresh frozen see tions of the human tonsil In approximately 7 min. The suitable staining sequences are as follows. Frozen sections prepared using 3-aminopropyitimethoxysilane-cpated glass slides are immediately fixed, without air drying, for 15s in a mixture of 50% formalin and 50% methanol for PCNA, and in 10% formalln for Ki-67 antigen. After a brief rinse in phosphate-buffered saline (PSS), sections are incubated with the EPOS antibody for 3 min, followed by PBS rinse for 1 min. The peroxidase activity is visualized in diaminobenzidine-H₂O₂ solution containing 10mmol/L imidazole for 2 min. After a light rinse in tap water, the nuclei are briefly counterstained with 5% methyl green. When necessary, endogenous peroxi-dase blockage in 1% periodic acid solution for 1 min is added before the EPOS antibody incubation. This procedure is applicable to frozen sections of gastric cancers, malignant lymphomas, and brain, liver and peritoneal lesions in which differential diagnosis between benignancy and malignancy was required.     

Branches of the thoracic sympathetic trunk in the human fetus

Summary The segmental organization of the thoracic sympathetic trunk and all its ramifications was studied in 6 human fetuses (16–22 weeks) by means of the acetylcholinesterase in toto staining method. Each trunk was divided into 12 sympathetic segments. A segment is defined as that part of the sympathetic trunk which is connected via its rami communicates with one spinal nerve, without discriminating between grey and white rami. The diameter of the rami communicantes and their direction towards the spinal nerves are variable. The number of peripheral segmental ramifications of the trunk is much larger than assumed previously. Each thoracic sympathetic segment gives off at least 4–5 nerves. Three categories of nerves are discerned: (1) large splanchnic rootlets confined to the greater, lesser and least thoracic splanchnic nerves, (2) medium-sized splanchnic nerves directed towards thoracic viscera, some of which give off branches towards costovertebral joint plexuses and, described for the first time in man, (3) small nerves which ramify extensively and form nerve plexuses in the capsule of the costovertebral joints. The majority of the ramifications is formed by the nerves of the third category. The existence of Kuntz's nerve, connecting the 2nd intercostal nerve and 1st thoracic spinal nerve, is confirmed in four specimens. The nerve plexuses of the costovertebral joints receive a segmentally organized innervation: they receive their input from the neighbouring sympathetic segment and the one cranial to it.It is concluded that the thoracic sympathetic branches in man show a complex, segmentally organized pattern and may have a considerable component of somatosensory nerve fibers. The complex relationships must be taken into account in surgical sympathectomies.     

多种金属离子与单宁酸反应媒染微血管的对比研究

目的　镜下观察单宁酸与金属盐溶液中Ca2 + 、Au+ 、Ag+ 、Pb2 + 、Cu2 + 、Al3 + 、U6+ 、K+ 联用显示大脑微血管的效果。方法　用单宁酸媒染固定液灌流大鼠 ,取脑切片 ,入氯化钙、氯化金、硝酸银、硝酸铅、硫酸铜、硫酸铝钾、醋酸双氧铀、高锰酸钾和重铬酸钾等溶液中呈色。结果　单宁酸与Ca2 + 、Cu2 + 、Ag+ 、Al3 + 结合显示血管清晰 ,与Au+ 、U6+ 、Pb2 + 、K+ 联用媒染血管效果欠佳。结论　含Ca2 + 、Cu2 + 、Ag+ 、Al3 + 的金属盐类可替代氯化铁媒染微血管 ,氯化金、醋酸双氧铀、硝酸铅、重铬酸钾和高锰酸钾不宜与单宁酸联用来显示血管     

Morphogenesis of the primary arterial trunks of the forelimb in the rat embryos: the trunks originate from the lateral surface of the dorsal aorta independently of the intersegmental arteries

It has been believed that the primary arterial trunk of the mammalian forelimb is derived from the 7th intersegmental artery. Here we examined the early morphogenesis of the arteries and nerves in the forelimb region by adopting a method that combined intravascular dye-injection with nerve staining to whole mounted rat embryos. The study was carried out on greater numbers of specimens at smaller intervals of embryonic stages and from earlier stages than those in previous reports. We report that: (1) The multiple primary arterial trunks in the forelimb region (primary subclavians) originate directly from the lateral surface of the dorsal aorta independently of the intersegmental arteries, previous to the formation of limb buds. (2) The tips of the 8th (and the 9th) primary subclavians that originate from the aorta near the origin of the 8th (or the 9th) intersegmental artery bend cranially and/or caudally. With the formation of limb bud, they extend to form the longitudinal trunks in the presumptive axillary region. The primary arteries in the free arm region branch off from this longitudinal trunk, and one of them develops into the axial artery. (3) The origins of the primary subclavians shift their positions on the surface of the dorsal aorta and approach the origins of the neighboring intersegmental arteries to join them, and then replace the latter. Consequently, the primary subclavians appear to be ”the lateral branches of the in tersegmental arteries.” (4) The 8th primary subclavian is dominant at first, but is replaced by the 7th primary subclavian, which develops into the definitive subclavian artery. (5) With the brachial nerve plexus formation, the axillary arterial plexus derived from the longitudinal trunk develops to form two stems of the axillary artery. Accepted: 15 April 1999     

Noninvasive prenatal diagnosis of chromosomal aneuploidies by isolation and analysis of fetal cells from maternal blood

The isolation and analysis of nucleated fetal cells (NFCs) from maternal blood may represent a new approach to noninvasive prenatal diagnosis. Although promising, these techniques require highly accurate separation of NFCs from nucleated cells of maternal origin; the two major problems limiting these techniques are the relative rarity of fetal cells in maternal blood and the need to establish their fetal origin. We now report a novel procedure that has allowed accurate separation of NFCs from maternal cells. The technique reported involves direct micromanipulator isolation of histochemically identified hemoglobin F‐positive nucleated cells to obtain fetal nucleated red blood cells (FNRBCs) of high yield and purity. Using this technique, followed by cell‐by‐cell multicolor fluorescence in situ hybridization (FISH) analysis of purified FNRBCs, we were able to detect some of the most common human aneuploidies (including Down syndrome, Klinefelter syndrome, and trisomy 13) in 33 pregnant women referred for amniocentesis. The procedure used, which can be completed in <72 hrs, produced complete concordance with the results of amniocentesis. We also confirm findings of prior studies suggesting that the number of FNRBCs in maternal circulation is remarkably higher in abnormal pregnancies than in normal pregnancies, especially in women carrying a fetus with trisomy 21. © 2001 Wiley‐Liss, Inc.     

Comparison of the unlabeled and labeled pre-mRNA splicing assays in vitro

Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome that catalyzes the removal of non-coding intron sequences to ligate exons into mature mRNA prior to transport and translation. The purpose of our study is to explore whether the in vitro unlabeled pre-mRNA splicing assay could be performed as an alternative method of splicing reaction other than the radiolabeled one. Two different splicing methods in vitro , P labeled and unlabeled pre-mRNA as the substrates in the reaction, were investigated. The radiolabeled products were visualized by autoradiography while the unlabeled products were observed by Ethidium Bromide (EB) staining. As a result, although there are more unspecific bands in the EB staining assay than 32P labeled one, the RNA products of in vitro splicing could be observed clearly. This suggests that the unlabeled pre-mRNA splicing assay can be an optional substitution for the isotope-labeled assay.     

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。