Application of Pop-off Method to Bone Marrow and Peripheral Blood Specimens for Purposes of Electron Microscopy

In this paper, we describe a pop-off method applicable to the hematological field. Bone marrow or peripheral blood specimens from patients were placed on a clean glass slide and fixed immediately in 2% glutaraldehyde solution for 10 minutes. For the DAB reaction, the slide was immersed in the DAB reagent for 30 minutes, and post fixed with 1 % OsO₄ solution for 1 hour. Specimens on the slide were washed with buffer solution, dehydrated and polymerized directly on the slide. A gelatin capsule filled with Epon mixed monomer was then reversed over the specimen. After polymerization was completed, the specimen was popped off from the slide to the capsule and trimmed carefully to prepare for ultrathin sectioning. This method allows the entire sequence of tissue preparation to be carried out on the slide, from fixation to embedding, and even, especially in scanty specimens, including the DAB reaction. Electron microscopic findings in specimens prepared by this technique show excellent preservation and the absence of specific artifacts.     

A Simple Technique for Preparation of Bone Marrow or Peripheral Blood Buffy Coat Cells for Electron Microscopy

A simple and rapid method of processing ethylene diamine-tetra-acetic acid anticoagulated peripheral blood or aspirated bone marrow for electron microscopy is described. The resultant buffy coat pellet is easily processed into epoxy resin. Semithin sections (1 μ) stained with 1% toluidine blue reveal the various stratified cell layers allowing convenient selection for ultramicrotomy and ultrastructural evaluation.     

A Simple Technique for Preparation of Bone Marrow or Peripheral Blood Buffy Coat Cells for Electron Microscopy

A simple and rapid method of processing ethylene diamine-tetra-acetic acid anticoagulated peripheral blood or aspirated bone marrow for electron microscopy is described. The resultant buffy coat pellet is easily processed into epoxy resin. Semithin sections (1 μ) stained with 1% toluidine blue reveal the various stratified cell layers allowing convenient selection for ultramicrotomy and ultrastructural evaluation.     

Simple Technique to Facilitate Tissue Sampling for Electron Microscopy

The need to examine certain components of a specimen with the electron microscope is frequently recognized only after study of the paraffin sections, by which time the total specimen has usually been fixed in Formalin. Such specimens are frequently large, with the result that representative samples for electron microscopy may be difficult to isolate. A simple, quick, and inexpensive method for overcoming this sampling problem is described. This technique is best employed in laboratories that routinely use a single tissue fixative suitable for both light and electron microscopy. The specimen is taken direct from the Formalin, and very thin slices are prepared by hand with a blade, stained with methylene blue, and examined in the wet state under the microscope. On identification, representative tissue is removed by microdissection and processed further for electron microscopy.     

Simple Technique to Facilitate Tissue Sampling for Electron Microscopy

The need to examine certain components of a specimen with the electron microscope is frequently recognized only after study of the paraffin sections, by which time the total specimen has usually been fixed in Formalin. Such specimens are frequently large, with the result that representative samples for electron microscopy may be difficult to isolate. A simple, quick, and inexpensive method for overcoming this sampling problem is described. This technique is best employed in laboratories that routinely use a single tissue fixative suitable for both light and electron microscopy. The specimen is taken direct from the Formalin. and very thin slices are prepared by hand with a blade, stained with methylene blue, and examined in the wet state under the microscope. On identification, representative tissue is removed by microdissection and processed further for electron microscopy.     

Diagnostic Electron Microscopy Laboratory of the Future

The future of diagnostic electron microscopy is intimately related to the introduction of techniques presently used in basic research, the introduction of new technologies and instrumentation, the availability of expertise, and monetary constraints. It is suggested that a certain degree of centralization of diagnostic electron microscopy is inevitable if one wishes to introduce state-of-the-art technologies and maintain a high degree of expertise.     

Peripheral Blood versus Bone Marrow from Unrelated Donors: Bone Marrow Allografts Have Improved Long-Term Overall and Graft-versus-Host Disease-Free,Relapse-Free Survival

Peripheral blood (PB) and bone marrow (BM) from unrelated donors can serve as a graft source for hematopoietic cell transplantation (HCT). Currently, PB is most commonly used in roughly 80% of adult recipients. Determining the long-term impact of graft source on outcomes would inform this decision. Data collected by the Center for International Blood and Marrow Transplant Research from 5200 adult recipients of a first HCT from an 8/8 or 7/8 HLA antigen-matched unrelated donor for treatment of acute leukemia, chronic myelogenous leukemia, or myelodysplastic syndrome between 2001 and 2011 were analyzed to determine the impact of graft source on graft-versus-host disease (GVHD) relapse-free survival (GRFS), defined as freedom from grade III/IV acute GVHD, chronic GVHD requiring immunosuppressive therapy, relapse, and death, and overall survival. GRFS at 2 years was superior in BM recipients compared with PB recipients (16%; 95% confidence interval [CI], 14% to 18% versus 10%; 95% CI, 8% to 11%; P <.0001) in the 8/8 HLA-matched cohort and 7/8 HLA-matched cohort (11%; 95% CI, 8% to 14% versus 5%; 95% CI, 4% to 7%; P?=?.001). With 8/8 HLA-matched unrelated donors, overall survival at 5 years was superior in recipients of BM (43%; 95% CI, 40% to 46% versus 38%; 95% CI, 36% to 40%; P?=?.014). The inferior 5-year survival in the PB cohort was attributable to a higher frequency of deaths while in remission compared with the BM cohort. For recipients of 7/8 HLA-matched grafts, survival at 5 years was similar in BM recipients and PB recipients (32% versus 29%; P?=?.329). BM grafts are associated with improved long-term GRFS and overall survival in recipients of matched unrelated donor HCT and should be considered the unrelated allograft of choice, when available, for adults with acute leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome.     

New Techniques: A Fast Method for Processing Biopsy Material for Electron Microscopy

A rapid method for fixation and embedding of biopsy material for electron microscopic examination has been developed. The procedure was simplified by fix ing the biopsies with fixative in ready-to-use ampules and hastened by chemical dehydration in 2.2-dimethoxypropane. The procedure takes 4 h and yields excellent results.     

A Rapid and Simple Method for Electron Microscopy of Paraffin-Embedded Tissue

A new rapid and simple method for electron microscopy of paraffin-embedded material is described. The paraffin-embedded tissue is deparaffinized in xylene, stained in a 0.01% toluidine blue/absolute ethanol solution, infiltrated in a propylene oxide/resin mixture, and embedded in epoxy resin. This quick method, which excludes rehydration, postosmification, and dehydration, takes about 3 h and produces results equal to previously described more laborious re-embedding techniques.     

A Rapid and Simple Method for Electron Microscopy of Paraffin-Embedded Tissue

A new rapid and simple method for electron microscopy of paraffin-embedded material is described. The paraffin-embedded tissue is deparaffinized in xylene, stained in a 0.01% toluidine blue/absolute ethanol solution, infiltrated in a propylene oxide/resin mixture, and embedded in epoxy resin. This quick method, which excludes rehydration, postosmification, and dehydration, takes about 3 h and produces results equal to previously described more laborious re-embedding techniques.     

Immunogold-Silver Staining Method for Lymphocyte Cell Surface Antigens with Light,Transmission Electron,and Scanning Electron Microscopy

Abstract

The immunogold-silver staining (IGSS) method combined with light, transmission electron, and scanning electron microscopy (LM, TEM and SEM, respectively) was used for detecting lymphocyte surface antigens. Two different sizes of colloidal gold particles (5 nrn and 15 nm) were applied as markers and IntenSEII kit as a physical developer for gold particles.

The silver enhanced gold particles were clearly observed on cell surfaces as black dots in LM and TEM and as white dots in SEM equipped with a mixed signal of secondary electron and back-scattered electron (SE/BE) signals. Monoclonal antibody (MAb)-positive cells possessing the complexes on their well preserved surfaces were easily identified among other lymphoid cells at low magnifications of LM or SEM equipped with SE/BE signals. Thus, the IGSS method has a great advantage for a qualitative screening such as the percentage of lymphocyte subsets in a cell suspension. However, the IGSS method was inadequate for semiquantitative study with antigen density on cell surfaces because gold particles enhanced with the physical developer were considerably enlarged, and a silver-gold complex was not considered to show one antigen site on cell surface. (The J Histotechnol 16:217, 1993)     

Microcomputer Application: An Image Database System for Electron Microscopy

An electron microscopist in a small electron microscopy laboratory that processes 200 cases per year can generate at least 2000 electron micrographs within this time period. These electron micrographs are stored in filing cabinets, which slowly encroach on existing laboratory space. Related image data are often located in separate filing systems. This type of storage system is inefficient for rapid retrieval of sets of electron micrographs for research, teaching, or retrospective review of patient material. An electron microscopy image base, which is implemented on a microcomputer, can provide flexible and simultaneous access to both digitized electron micrographs and their relevant textual data. In addition to the advantage of more efficient retrieval, electronic storage of textual data and digitized electron micrographs also offers the advantage of decreased storage space for this type of data. Implementation of the initial version of the electron microscopy image base demonstrated that future versions must provide improved resolution of the digitized electron micrograph and sufficient electronic mass storage to support such a large image base. Ongoing developments in database technology and mass storage make it worthwhile to pursue additional refinements of the electron microscopy image base.     

Electron Microscopic Characteristics of Peripheral Blood Lymphocytes in Children with Infectious Mononucleosis

Infectious mononucleosis is associated with pronounced changes in surface architectonics of peripheral blood lymphocytes persisting during convalescence and remote period after the disease. The degree of these changes depends on the disease agent and age-specific characteristics of the body. The most pronounced and sustained disorders in the morphostructural organization of lymphocytes are caused by Epstein-Barr virus (in comparison with agents of other etiology); these disorders are more pronounced in children aged 7-14 years than in those aged 3-6 years.     

Value of a Simple Method to Assess Chronic Rejection in Renal Allograft on Electron Microscopy

Abstract

Electron microscopy is a powerful tool for the assessment of complex lesions in nontumor renal pathology, however it is a time-consuming procedure. We evaluated a simple method to assess morphological signs of chronic rejection in renal allograft that seems to have prognostic significance.     

血液病患者骨髓微环境和外周血部分细胞因子水平的对比

目的 探讨骨髓微环境中细胞因子IL-Iβ、IL-2、IL-4、IL-10、INF-γ、TNF-α水平与外周血中相应细胞因子的差异。方法选取2018年1月~2019年2月云南省第一人民医院血液科诊断的缺铁性贫血（IDA）、骨髓增生异常综合征（MDS-EB-1）、慢性粒细胞白血病慢性期（CML-CP）及急性髓系白血病（AML）患者各20例。应用流式细胞仪分析IDA、MDS-EB-1、CML-CP和AML患者骨髓及外周血IL-Iβ、IL-2、IL-4、IL-10、INF-γ、TNF-α的水平。结果 IDA患者骨髓微环境和外周血中细胞因子水平比较,差异无统计学意义（P＞0.05）;MDS-EB-1、CML-CP及AML患者骨髓微环境中IL-Iβ、IL-2、IL-4、IL-10、INF-γ、TNF-α水平高于外周血,差异有统计学意义（P＜0.05）。结论 IDA、MDS-EB-1、CML-CP及AML患者骨髓微环境中细胞因子IL-Iβ、IL-2、IL-4、IL-10、INF-γ、TNF-α的水平高于外周血中相应的细胞因子水平。在研究血液系统恶性肿瘤细胞因子时,应检测骨髓微环境中的细胞因子。     

Techniques a Simple Method for the Collection of Cells from Cerebrospinal Fluid for Electron Microscopy

A simple membrane filter technique permits the collection of cells from cerebrospinal fluid for electron microscopy. The method is universally applicable for the concentration of small number of cells from free-floating cell suspensions and has several advantages: (1) Only a small fluid sample (1 ml) is needed. (2) Excellent cell preservation is obtainable. (3) High cell recovery rates are achieved. (4) Quantitative classification of cell types is possible.     

Techniques a Simple Method for the Collection of Cells from Cerebrospinal Fluid for Electron Microscopy

A simple membrane filter technique permits the collection of cells from cerebrospinal fluid for electron microscopy. The method is universally applicable for the concentration of small number of cells from free-floating cell suspensions and has several advantages: (1) Only a small fluid sample (1 ml) is needed. (2) Excellent cell preservation is obtainable. (3) High cell recovery rates are achieved. (4) Quantitative classification of cell types is possible.     

Diagnosis of Neuronal Ceroid Lipofuscinosis (Batten Disease) by Electron Microscopy in Peripheral Blood Specimens

Neuronal ceroid lipopofuscinosis (Batten disease, NCL) represents a group of common childhood neurodegenerative diseases with a shared feature of deposition of abnormal metabolic products in neurons and other tissues, including peripheral blood lymphocytes. In most forms of NCL no specific enzyme defect is known and the diagnosis relies primarily on ultrastructural identification of characteristic membrane-bound inclusions containing the abnormal metabolic product. All buffy-coat specimens examined during a 7-year period (1997–2004) for the exclusion or confirmation of the diagnosis NCL were reviewed. From a total of 265 samples, 9 were inadequate and NCL was diagnosed in 56. Five showed granular osmophilic deposits of infantile Batten disease (NCL1), 10 showed curvilinear profiles of classical late infantile Batten disease (NCL2), and 17 showed vacuolated lymphocytes with fingerprint profiles, indicating classical juvenile Batten disease (NCL3). 24 samples (43%) demonstrated compact electron-dense deposits with fingerprint profiles in the absence of vacuolated lymphocytes, indicative of variant forms NCL. Ultrastructual examination of peripheral blood allows reliable and specific diagnosis of subtypes of Batten disease, including variants, and is a useful, minimally invasive test for the diagnosis of NCL in childhood.