Refractile mycobacteria in Romanowsky-stained bone marrow smears. A comparison of acid-fast-stained tissue sections and Romanowsky-stained smears.

The appearance of mycobacteria was studied in Wright-stained bone marrow preparations of human immunodeficiency virus-infected patients and compared with acid-fast-stained trephine biopsy sections and culture results. Mycobacterium avium complex in Romanowsky-stained preparations may be seen as extracellular and intracellular clear or red refractile beaded rods and nonrefractile "negative images." Refractile mycobacteria were seen in 17 of 20 culture-positive cases. Acid-fast stain of the trephine biopsy demonstrated organisms in only 11 of the 20 cases. Thus, six cases were culture positive and contained refractile rods but had no acid-fast organisms on the trephine biopsy. No false-positive results were seen with Romanowsky stain; the three false-negative results for refractility also were negative with acid-fast stain. Examination of Romanowsky-stained smears or imprints for refractile mycobacteria provides a reliable and sensitive method to identify mycobacteria in this population. Romanowsky-stained bone marrow aspirate and imprint smears should be examined for refractile bacilli when mycobacterial infection is suspected.     

DXA、力学测试和矿盐含量评估大鼠股骨性能的精确性比较

目的 为深入了解DXA、力学测试和矿盐含量在航天骨丢失研究的应用,本文比较了这三种方法评估大鼠股骨性能的精确性,并比较了大鼠股骨在活体、离体和冷冻三种情况下的DXA测量差别.方法 选择3只雄性SD大鼠,首先对其两侧股骨进行DXA测量,得到活体、离体和冷冻三种情况的骨矿密度(BMD),然后进行力学测试和矿盐含量测试.结果 DXA、力学测试和矿盐含量的变异系数(CV)分别为7.5%～8.5%,10.1%～17.1%和0.8%.对于DXA测量,活体股骨BMD值与离体和冷冻股骨BMD值都有极其显著差别(P＜0.001),而离体与冷冻股骨BMD值无显著性差别.结论 矿盐含量精确性好于DXA和力学测试.DXA测量活体与离体骨骼差别很大,冷冻不会影响DXA测量.     

Detection of bone marrow infiltration by non-Hodgkin's lymphoma — Comparison of histological findings,analysis of gene rearrangements,and examination by magnetic resonance imaging

Summary The histological examination of bone marrow specimens is one of the standard procedures in staging non-Hodgkin's lymphoma.To investigate the validity of a conventional unilateral iliac crest biopsy, we performed a prospective study comparing histological findings with analysis of gene rearrangements in bone marrow samples and magnetic resonance imaging of bone marrow. Twenty-seven consecutive patients with non-Hodgkin's lymphoma (ten with high grade, seventeen with low grade) were studied. In twelve patients, histological examination revealed bone marrow infiltration. Results of histology and magnetic resonance imaging were discordant in three of the twenty-seven patients. With magnetic resonance imaging, suspected infiltration was found in two patients without histological evidence for bone marrow involvement in the disease. In one patient, an infiltration was described by histology but MRI revealed no pathological findings. In this case, DNA analysis confirmed bone marrow infiltration by detection of a clonal rearrangement of the immunoglobulin heavy chain gene.Analysis of gene rearrangements was performed in ten patients. As examined by histology, five of them had bone marrow involvement in the disease and five had not. In all these cases, analysis of gene rearrangements confirmed the histological findings.Our data show that, despite the small volume of bone marrow specimens, the sensitivity of an iliac lymphoma.     

Comparative evaluation of bone marrow aspirate particle smears, imprints and biopsy sections

Comparative evaluation of bone marrow aspirate particle smears, imprints and biopsy sections was done on 30 haematological problems. Core needle biopsy of the bone marrow is a safe and useful procedure. It is a valuable diagnostic aid for measurement of marrow cellularity, metastatic tumours and fibrosis. It should not be taken as a substitute for examination of the marrow by aspiration smear but is a complementary procedure which affords several advantages. Bone marrow biopsy was of maximum utility in myelofibrosis which was diagnosed on biopsy alone. There were three additional cases with normal bone marrow aspiration in which specific diagnosis could only be made from bone marrow biopsy sections. New methodologies i.e. plastic embedding and semi thin sections of undecalcified bone marrow, can be expected to improve the cytological details of tissue obtained by biopsy. Imprint preparations obtained from biopsy can be useful in patients of malignancy but we have found them to be of limited value except in cases of dry tap.     

Rapid preparation of fresh frozen tissue-engineered bone sections for histological, histomorphological and histochemical analyses

Fresh frozen sections are the best materials to assess tissue-engineered bone using cells/ceramic complexes. However, there are a lot of technical difficulties in obtaining serial sections suitable for microscopic examinations. Kawamoto et al. developed a method for the production of fresh frozen sections using new adhesive tape, and showed that sections were very useful for histological and histochemical studies. However, no study reported that the method was useful for tissue-engineered bone from histochemical and histomorphological points of view. This study aimed to determine the efficacy of fresh frozen sectioning for evaluating tissue-engineered bone. We revealed that fresh frozen sections retained the original morphology of tissue-engineered bone, and their biochemical characteristics. Therefore, rapid preparation of fresh frozen sections using adhesive tape is extremely useful for research of tissue-engineered bone, and serial sections can be assessed from both histomorphological and biochemical point of views. It is expected that this method will become a powerful tool in tissue-engineering of hard tissues.     

A comparison of flow cytometry,bone marrow biopsy,and bone marrow aspirates in the detection of lymphoid infiltration in B cell disorders

AIMS: To evaluate the diagnostic value of bone marrow aspirates, trephine biopsies (BMB), and flow cytometry (FC) in the assessment of bone marrow infiltration in chronic lymphoid disorders. METHODS: Investigations were carried out in 110 diagnostic and follow up specimens from B cell disorders, namely: chronic lymphocytic leukaemia (CLL; 65), non-Hodgkin's lymphoma (NHL; 39), and hairy cell leukaemia (HCL; 6). A selected panel of monoclonal antibodies was used both for FC and immunohistochemistry. RESULTS: In CLL there was agreement between the three investigations in 71% of samples and in 88% when only FC and BMB were compared. In nine of 65 samples, FC and BMB were positive, although the aspirate was reported as negative. Four BMB negative samples had minimal residual disease (MRD) detected by FC, whereas two samples were positive both on BMB and aspirate but showed no evidence of disease on FC. In NHL, there was agreement between the three investigations in 22 of 39 cases, and in 27 of 39 cases there was agreement between FC and BMB. In eight of 39 NHL cases, FC was negative but the BMB was either positive (five) or uncertain (three), whereas in three of 39, FC was positive but BMB was either negative (one) or uncertain (two). In three of five uncertain BMB, no clonal population was detected by the polymerase chain reaction, whereas in the remaining two cases the nodular aggregates disappeared on further sectioning. CONCLUSIONS: Both BMB and FC are better than bone marrow aspirates for the detection of infiltration in B cell disorders. FC might be slightly more sensitive than BMB to detect MRD in CLL, whereas BMB may be slightly better than FC in NHL.     

Subjective and objective assessment of image quality—A comparison

Forced-choice just noticeable difference (JND) studies are extremely sensitive to image quality variations that are below the threshold at which the differences are apparent to or definable by the observer. Paired comparisons of 4K and 2K laser-printed posteroanterior chest images consistently demonstrated that although images are viewed as comparable by radiologists, when forced to choose the better (“sharper”) image, they actually select the higher-resolution images in 83% of the paired observations. We conclude that small differences in image quality may be detectable even in image sets which are considered to be comparable by subjective assessments.     

Effect of exogenous DNA on content and biosynthesis of endogenous DNA in rat bone marrow

The effect of exogenous DNA on the content and rate of biosynthesis of endogenous DNA in rat bone marrow was studied. After injection of high-polymer homologous DNA into intact rats the endogenous DNA content per gram bone marrow was found to be reduced for the first 3 days (the decrease was most marked after 3 days, namely by 36%) and it returned to normal by the 6th day. The rate of DNA biosynthesis in rat bone marrow was increased after 18 h (when it was twice as high as in the control) and after 6 days (by 58%), and it was close to normal again 1–3 days after injection of DNA.(Presented by Academician of the Academy of Medical Sciences of the USSR P. D. Gorizontov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 8, pp. 952–953, August, 1976.     

Comparative assessment of proliferation and DNA content in breast carcinoma by image analysis and flow cytometry.

Although tumor DNA content and proliferation are usually determined by flow cytometry (FCM), quantitative microscopic image analysis is a viable alternative technique that also provides important histologic correlations. To compare these methods, we measured DNA content and proliferation in 54 consecutive breast cancers and 15 benign breast lesions by FCM and IA. DNA content determination was concordant in 49 of 54 cancers measured by FCM and IA. Four of the discordant cases were aneuploid by IA and diploid by FCM. There was good correlation between the DNA index (DI) measured by FCM and IA (r = 0.89, P less than 0.0001). Proliferation was assessed by IA quantitation of Ki-67 and PCNA/Cyclin antibody staining, as well as by flow cytometric S-phase fraction (SPF). Ki-67 positivity was greater in breast cancer than in benign controls (21.6% +/- 13.1% vs. 7.9% +/- 5.6% [P less than 0.0001]), as was PCNA/Cyclin positivity (10.2 +/- 6.7% vs. 2.7 +/- 2.5% [P less than 0.0001]). S-phase fraction measured by FCM was 7.9% +/- 5.7% for carcinomas and 3.17% +/- 2.1% for benign controls (P less than 0.003). Ki-67 and Cyclin staining, as well as SPF, were significantly increased in aneuploid compared to diploid tumors, and increased staining was associated with worsening nuclear grade. There were significant correlations between SPF and Ki-67 staining (r = 0.48, P less than 0.0001) and SPF and Cyclin staining (r = 0.48, P less than 0.0001). We conclude that FCM and IA provide comparable measurements of DNA content, although occasional discrepancies occur. Image analysis provides a valuable alternative method for assessing tumor cell proliferation and may offer certain advantages over FCM.     

Changes with aging in the bone mineral content of the lumbar spine and femoral neck in healthy women in Hungary

The bone mineral content (BMC) of the lumbar spine and femoral neck was studied in relation to aging in healthy Hungarian women by duel photon-absorptiometric method. The data were compared with North-American and West-European values. The Hungarian values are lower than these for reported from North-America or France, but similar to the BMC values for Sweden. The observed vertebral and femoral bone loss could be well represented by cubic equations. The acceleration of bone loss seems to begin around 40 years. The rate of bone loss was similar to the published values but the decrease in bone loss in the 6th and 7th decades was more suggestive. With regard to the fracture-threshold below which the risk for non-traumatic fractures of vertebrae increases, about 60% of Hungarian women at age 50-59 and about 84% at age 60-69 are considered to be at risk. It is concluded that geographical and habitual differences might be important factors in the development and change of BMC for different populations.     

Osteomyelofibrosis/-sclerosis: A histological and cytogenetic study on core biopsies of the bone marrow

Summary A combined histological and cytogenetic study was performed on the bone marrow in 33 patients with overt osteomyelofibrosis/-sclerosis (MF/OMS) and so called agnogenic myeloid metaplasia including blast crisis. Histopathology of the plastic embedded samples of bone marrow showed an abnormal proliferation of megakaryopoiesis with conspicuous atypias of growth and maturation in addition to a neoplastic neutrophilic granulopoiesis, particularly in the early stages of MF. Thus a biphasic population of neoplastic hematopoiesis is postulated and this lesion is called chronic megakaryocytic-granulocytic myelosis (CMGM) with myelofibrosis — CMGM stage III — or with osteomyelosclerosis — CMGM stage IV. Initiation of fibrillogenesis, the most striking alteration of this disorder, is partially attributed to disorganization of megakaryopoiesis with abnormal proliferation and clustering around the sinuses and intraluminal growth, with subsequent obliteration of the vascular compartment. Cytogenetic evaluation demonstrated the Philadelphia chromosome (Ph'-chromosome) in 93% of CGL and in 67% of MF/OMS, including cases with blast crisis. Unlike CGL and MF/OMS where a Ph'-chromosome is common, myelofibrosis of non-neoplastic origin and AML displayed no Ph'-chromosome. Further aberrations such as aneuploidy involved the C/D group chromosomes predominantly and were especially prominent in blast crisis (about 50%) with no significant differences in CGL and MF/OMS or in AML. Our results of chromosomal analysis, evaluated in close context with histopathology, show no fundamental differences between CGL and myeloproliferative disorders of mixed cellularity, i.e., chronic megakaryocytic-granulocytic myelosis (CMGM). For this reason the terminal stages of fibrotic and osteosclerotic lesions belong into these categories of CMGM or CGL respectively. In conclusion MF/OMS are final stages or subtypes of CML, carrying the same chromosomal marker and demonstrating remarkable atypias of the hematopoietic tissue suggestive of malignancy. The fibrotic/ osteosclerotic alteration itself is thought to represent a secondary nonneoplastic feature.Supported by the Deutsche Forschungsgemeinschaft (grant GE 121/20)     

Endothelial cells and hematopoiesis: A light microscopic study of fetal,normal, and pathologic human bone marrow in plastic-embedded sections

The origin and morphological identity of hematopoietic progenitor cells, as well as their precursor, the pleuripotential hematopoietic stem cell (HSC), has not been established. Our studies of 2 μm sectioned undecalcified plastic-embedded bone marrow (BM) from healthy human fetuses; normal adults; patients with acute myeloblastic leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic granulocytic leukemia (CGL) in various stages (chronic, accelerated, acute blastic phase, and after autografting); and patients recovering from therapy-induced marrow hypoplasia suggest that proliferative hematopoietic zones exist near the endosteum (endosteal marrow) and the vascular endothelium (capillary and sinus-lining endothelium) and a maturational zone distal to these regions. In some of these areas, morphologically recognizable hematopoietic cells were seen and interpreted as emerging and maturing in a sequential progression, suggesting an origin from the endosteal or endothelial progenitors. In other loci, early hematopoietic cells were seen in close contact with the endosteal or vascular endothelial (VE) cells. This latter relationship suggested that these areas of cellular contact were important and represented sites of cell to cell interaction that may be associated with the liberation of growth factors by endosteal and endothelial cells and their action on hematopoietic progenitor cells. Following treatmentinduced hypoplasia, the endosteal and VE cells were seen to modulate, transform, and migrate into the surrounding empty and edematous marrow space as fibroblasts. Later, as hemopoietic regeneration began, clusters of regenerating hematopoietic cells were seen adjacent to bone trabecule (BT) and near the vascular endothelium. We postulate that endosteal and VE cells are the equivalent of embryonal-stage, undifferentiated mesenchyme and, under the appropriate regulatory influence, are capable of modulation and transformation (differentiation) into stromal (fibroblast-like) cells and precursors of hematopoietic cells in normal (physiologic) and stressed (pathologic) conditions. Recently, human endothelial cells have been shown to express a large number of cell surface antigens in common with hematopoietic (myeloid and lymphoid) cells. It is also possible that, in some situations, the VE cells act to establish a microenvironment and liberate growth factor (s), enabling pleuripotential and progenitor cell differentiation into mature hematopoietic cells adjacent to the vascular endothelium. Indeed, vascular endothelium has been shown to elaborate growth factors that participate in normal hematopoiesis. © 1992 Wiley-Liss, Inc.     

全骨髓贴壁法分离培养大鼠骨髓间充质干细胞及其生物学特性

背景：培养出生长状态良好、数量足够多的大鼠骨髓间充质干细胞是其作为种子细胞在组织工程等领域广泛应用的重要前提。 目的：寻求快捷有效的大鼠骨髓间充质干细胞体外培养条件,观察培养的间充质干细胞生物学特性。 方法：全骨髓法体外分离培养大鼠骨髓间充质干细胞,贴壁筛选法进行纯化,倒置相差显微镜下观察细胞形态及生长特征,免疫荧光分析细胞骨架,MTT法绘制细胞生长曲线,流式细胞仪检测其表面标记。 结果与结论：分离培养的细胞呈长梭形或多边形;细胞生长曲线呈S形;微丝免疫荧光染色结果显示,培养的骨髓间充质干细胞具有良好的细胞骨架系统。第3代骨髓间充质干细胞CD29、CD44、CD71、CD90均呈阳性表达,而CD13、CD34、CD45、CD133呈阴性。提示,经全骨髓贴壁法体外分离培养的细胞在形态学和细胞表面标志物表达方面具有干细胞生物学特性,经鉴定为骨髓间充质干细胞,其第3代活性最佳,可用于后续实验。     

大鼠骨髓间充质干细胞的纯化、冻存及成瘤性分析

背景：骨髓间充质干细胞在骨髓中的含量极低,体外的长期培养过程中易丧失干细胞潜能以及体内移植后安全性等问题的存在,限制了骨髓间充质干细胞在临床上的广泛应用。 目的：探索体外分离纯化、冻存大鼠骨髓间充质干细胞的最适方法,并观察以此方法培养的骨髓间充质干细胞移植体内后是否具有成瘤性。 方法：分别采用差速贴壁结合24 h首次换液、24 h首次换液、48 h首次换液的方法纯化培养骨髓间充质干细胞,筛选最适纯化方法进行后续实验。配制含体积分数为10%,20%,30%,40%,50%胎牛血清的细胞冻存液冻存细胞,复苏后计算细胞存活率,测定复苏后细胞的生长曲线及成脂诱导能力。将第3,15代骨髓间充质干细胞进行裸鼠肌肉、肝脏局部注射体内移植,45 d后取注射部位行病理组织标本检查。 结果与结论：差速贴壁结合24 h首次换液法所获得骨髓间充质干细胞纯度最高,而细胞增殖能力与其他两组无明显差别,因此选用该方法纯化骨髓间充质干细胞,然后进行传代培养;含体积分数为30%血清冻存液既可保证细胞活性与增殖能力,又可保证干细胞特性及多向分化潜能。骨髓间充质干细胞传至15代仍保持间充质干细胞特性,骨髓间充质干细胞移植入裸鼠肝脏45 d后仍可在肝脏局部存活,生长状态与体外培养相似,无异型性及向周围浸润生长,提示体外长时间培养15代以内的骨髓间充质干细胞可在裸鼠体内存活且无成瘤性。     

Macroporous hydroxyapatite scaffolds for bone tissue engineering applications: physicochemical characterization and assessment of rat bone marrow stromal cell viability

In this work, a new methodology is reported for developing hydroxyapatite (HA) scaffolds using an organic sacrifice template. The novelty of work consists of possibility of obtaining porous and highly interconnected scaffolds mimicking the sacrificial component. Our purpose consisted of evaluating the physicochemical properties of the HA scaffolds by means of Fourier transform infra-red spectroscopy, X-ray diffraction analysis, and scanning electron microscopy (SEM) attached with an X-ray detector. The HA scaffolds obtained possess a porosity of approximately 70%, and macropores diameter in the range of 50-600 microm. In contrast, results regarding the microcomputed tomography analysis have demonstrated both high pore uniformity and interconnectivity across the scaffolds. The compressive strength of the HA scaffolds was found to be 30.2 +/- 6.0 MPa. Bioactivity of the HA scaffolds was assessed by immersion into a simulated body fluid solution, in vitro. SEM observations have showed a deposition of apatite on the surface of the HA scaffolds, with a "cauliflower-like" morphology after 1 day, and tend to be more pronounced with the immersion time. The changes in calcium and phosphorus concentration were monitored by inductively-coupled plasma optical emission spectrometry. Cytotoxicity of the HA scaffolds was preliminarily investigated by carrying direct observation of mouse fibroblasts cells (L929 cell-line) death in the inverted microscope, and then cell viability was determined by means of carrying out a MTS assay. Complementarily, a luminescent cell viability assay based on the quantification of adenosine triphosphate was performed using rat bone marrow stromal cells (RBMSCs). A LIVE/DEAD assay and SEM analysis allowed the visualization of the RBMSCs adhesion and proliferation on the surface of the HA scaffolds. According to the results obtained from 3D architecture, mechanical properties, biocompatibility, and adhesion tests, it is suggested that HA scaffolds has potential to find applications in bone tissue engineering scaffolding.     

Forced choice and ordinal discrete rating assessment of image quality: A comparison

This study compared a five-category ordinal scale and a two-alternative forced-choice subjective rating of image quality preferences in a multiabnormality environment. 140 pairs of laser-printed posteroanterior (PA) chest images were evaluated twice by three radiologists who were asked to select during a side-by-side review which image in each pair was the “better” one for the determination of the presence or absence of specific abnormalities. Each pair included one image (the digitized film at 100 μm pixel resolution and laser printed onto film) and a highly compressed (～60∶1) and decompressed version of the digitized film that was laser printed onto film. Ratings were performed once with a five-category ordinal scale and once with a two-alternative forced-choice scale. The selection process was significantly affected by the rating scale used. The “comparable” or “equivalent for diagnosis” category was used in 88.5% of the ratings with the ordinal scale. When using the two-alternative forced-choice approach, noncompressed images were selected 66.8% of the time as being the “better” images. This resulted in a significantly lower ability to detect small differences in perceived image quality between the noncompressed and compressed images when the ordinal rating scale is used. Observer behavior can be affected by the type of question asked and the rating scale used. Observers are highly sensitive to small differences in image presentation during a side-by-side review.